Wieland Huttner

"What makes us human?" is a central question of many research fields, notably anthropology. In this review, we focus on the development of the human neocortex, the part of the brain with a key role in cognition, to gain neurobiological insight toward answering this question. We first discuss cortical stem and progenitor cells and human-specific genes that affect their behavior. We thus aim to understand the molecular foundation of the expansion of the neocortex that occurred in the course of human evolution, as this expansion is generally thought to provide a basis for our unique cognitive abilities. We then review the emerging evidence pointing to differences in the development of the neocortex between present-day humans and Neanderthals, our closest relatives. Finally, we discuss human-specific genes that have been implicated in neuronal circuitry and offer a perspective for future studies addressing the question of what makes us human.

Cell metabolism is a key regulator of human neocortex development and evolution. Several lines of evidence indicate that alterations in neural stem/progenitor cell (NPC) metabolism lead to abnormal brain development, particularly brain size-associated neurodevelopmental disorders, such as microcephaly. Abnormal NPC metabolism causes impaired cell proliferation and thus insufficient expansion of NPCs for neurogenesis. Therefore, the production of neurons, which is a major determinant of brain size, is decreased and the size of the brain, especially the size of the neocortex, is significantly reduced. This review discusses recent progress understanding NPC metabolism, focusing in particular on glucose metabolism, fatty acid metabolism and amino acid metabolism (e.g., glutaminolysis and serine metabolism). We provide an overview of the contributions of these metabolic pathways to brain development and evolution, as well as to the etiology of neurodevelopmental disorders. Furthermore, we discuss the advantages and disadvantages of various experimental models to study cell metabolism in the developing brain.

Metabolism has recently emerged as a major target of genes implicated in the evolutionary expansion of human neocortex. One such gene is the human-specific gene ARHGAP11B. During human neocortex development, ARHGAP11B increases the abundance of basal radial glia, key progenitors for neocortex expansion, by stimulating glutaminolysis (glutamine-to-glutamate-to-alpha-ketoglutarate) in mitochondria. Here we show that the ape-specific protein GLUD2 (glutamate dehydrogenase 2), which also operates in mitochondria and converts glutamate-to-αKG, enhances ARHGAP11B's ability to increase basal radial glia abundance. ARHGAP11B + GLUD2 double-transgenic bRG show increased production of aspartate, a metabolite essential for cell proliferation, from glutamate via alpha-ketoglutarate and the TCA cycle. Hence, during human evolution, a human-specific gene exploited the existence of another gene that emerged during ape evolution, to increase, via concerted changes in metabolism, progenitor abundance and neocortex size.

The development of the neocortex involves an interplay between neural cells and the vasculature. However, little is known about this interplay at the ultrastructural level. To gain a 3D insight into the ultrastructure of the developing neocortex, we have analyzed the embryonic mouse neocortex by serial block-face scanning electron microscopy (SBF-SEM). In this study, we report a first set of findings that focus on the interaction of blood vessels, notably endothelial tip cells (ETCs), and the neural cells in this tissue. A key observation was that the processes of ETCs, located either in the ventricular zone (VZ) or subventricular zone (SVZ)/intermediate zone (IZ), can enter, traverse the cytoplasm, and even exit via deep plasma membrane invaginations of the host cells, including apical progenitors (APs), basal progenitors (BPs), and newborn neurons. More than half of the ETC processes were found to enter the neural cells. Striking examples of this ETC process "invasion" were (i) protrusions of apical progenitors or newborn basal progenitors into the ventricular lumen that contained an ETC process inside and (ii) ETC process-containing protrusions of neurons that penetrated other neurons. Our observations reveal a - so far unknown - complexity of the ETC-neural cell interaction.

The generation of neurons in the developing neocortex is a major determinant of neocortex size. Crucially, the increase in cortical neuron numbers in the primate lineage, notably in the upper-layer neurons, contributes to increased cognitive abilities. Here, we review major evolutionary changes affecting the apical progenitors in the ventricular zone and focus on the key germinal zone constituting the foundation of neocortical neurogenesis in primates, the outer subventricular zone (OSVZ). We summarize characteristic features of the OSVZ and its key stem cell type, the basal (or outer) radial glia. Next, we concentrate on primate-specific and human-specific genes, expressed in OSVZ-progenitors, the ability of which to amplify these progenitors by targeting the regulation of the cell cycle ultimately underlies the evolutionary increase in upper-layer neurons. Finally, we address likely differences in neocortical development between present-day humans and Neanderthals that are based on human-specific amino acid substitutions in proteins operating in cortical progenitors.

In this review, we focus on human-specific features of neocortical neurogenesis in development and evolution. Two distinct topics will be addressed. In the first section, we discuss the expansion of the neocortex during human evolution and concentrate on the human-specific gene ARHGAP11B. We review the ability of ARHGAP11B to amplify basal progenitors and to expand a primate neocortex. We discuss the contribution of ARHGAP11B to neocortex expansion during human evolution and its potential implications for neurodevelopmental disorders and brain tumors. We then review the action of ARHGAP11B in mitochondria as a regulator of basal progenitor metabolism, and how it promotes glutaminolysis and basal progenitor proliferation. Finally, we discuss the increase in cognitive performance due to the ARHGAP11B-induced neocortical expansion. In the second section, we focus on neocortical development in modern humans versus Neanderthals. Specifically, we discuss two recent findings pointing to differences in neocortical neurogenesis between these two hominins that are due to a small number of amino acid substitutions in certain key proteins. One set of such proteins are the kinetochore-associated proteins KIF18a and KNL1, where three modern human-specific amino acid substitutions underlie the prolongation of metaphase during apical progenitor mitosis. This prolongation in turn is associated with an increased fidelity of chromosome segregation to the apical progenitor progeny during modern human neocortical development, with implications for the proper formation of radial units. Another such key protein is transketolase-like 1 (TKTL1), where a single modern human-specific amino acid substitution endows TKTL1 with the ability to amplify basal radial glia, resulting in an increase in upper-layer neuron generation. TKTL1's ability is based on its action in the pentose phosphate pathway, resulting in increased fatty acid synthesis. The data imply greater neurogenesis during neocortical development in modern humans than Neanderthals due to TKTL1, in particular in the developing frontal lobe.

Integrating independent but converging lines of research on brain function and neurodevelopment across scales, this article proposes that serotonin 2A receptor (5-HT2AR) signalling is an evolutionary and developmental driver and potent modulator of the macroscale functional organization of the human cerebral cortex. A wealth of evidence indicates that the anatomical and functional organization of the cortex follows a unimodal-to-transmodal gradient. Situated at the apex of this processing hierarchy-where it plays a central role in the integrative processes underpinning complex, human-defining cognition-the transmodal cortex has disproportionately expanded across human development and evolution. Notably, the adult human transmodal cortex is especially rich in 5-HT2AR expression and recent evidence suggests that, during early brain development, 5-HT2AR signalling on neural progenitor cells stimulates their proliferation-a critical process for evolutionarily-relevant cortical expansion. Drawing on multimodal neuroimaging and cross-species investigations, we argue that, by contributing to the expansion of the human cortex and being prevalent at the apex of its hierarchy in the adult brain, 5-HT2AR signalling plays a major role in both human cortical expansion and functioning. Owing to its unique excitatory and downstream cellular effects, neuronal 5-HT2AR agonism promotes neuroplasticity, learning and cognitive and psychological flexibility in a context-(hyper)sensitive manner with therapeutic potential. Overall, we delineate a dual role of 5-HT2ARs in enabling both the expansion and modulation of the human transmodal cortex.

As is evident from the theme of the Research Topic “Small Size, Big Problem: Understanding the Molecular Orchestra of Brain Development from Microcephaly,” the pathomechanisms leading to mirocephaly in human are at best partially understood. As molecular cell biologists and developmental neurobiologists, we present here a treatise with theoretical considerations that systematically dissect possible causes of microcephaly, which we believe is timely. Our considerations address the cell types affected in microcephaly, that is, the cortical stem and progenitor cells as well as the neurons and macroglial cell generated therefrom. We discuss issues such as progenitor cell types, cell lineages, modes of cell division, cell proliferation and cell survival. We support our theoretical considerations by discussing selected examples of factual cases of microcephaly, in order to point out that there is a much larger range of possible pathomechanisms leading to microcephaly in human than currently known.

Cortical neurogenesis depends on the balance between self-renewal and differentiation of apical progenitors (APs). Here, we study the epigenetic control of AP's division mode by focusing on the enzymatic activity of the histone methyltransferase DOT1L. Combining lineage tracing with single-cell RNA sequencing of clonally related cells, we show at the cellular level that DOT1L inhibition increases neurogenesis driven by a shift of APs from asymmetric self-renewing to symmetric neurogenic consumptive divisions. At the molecular level, DOT1L activity prevents AP differentiation by promoting transcription of metabolic genes. Mechanistically, DOT1L inhibition reduces activity of an EZH2/PRC2 pathway, converging on increased expression of asparagine synthetase (ASNS), a microcephaly associated gene. Overexpression of ASNS in APs phenocopies DOT1L inhibition, and also increases neuronal differentiation of APs. Our data suggest that DOT1L activity/PRC2 crosstalk controls AP lineage progression by regulating asparagine metabolism.

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders. As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

Herai et al. discuss the known fact that a low percentage of modern humans who lack any overt phenotypes carry the ancestral TKTL1 allele. Our paper demonstrates that the amino acid substitution in TKTL1 increases neural progenitor cells and neurogenesis in the developing brain. It is another question if, and to what extent, this has consequences for the adult brain.

WNT signaling is important in development, stem cell maintenance, and disease. WNT ligands typically signal via receptor activation across the plasma membrane to induce β-catenin-dependent gene activation. Here, we show that in mammalian primary cilia, WNT receptors relay a WNT/GSK3 signal that β-catenin-independently promotes ciliogenesis. Characterization of a LRP6 ciliary targeting sequence and monitoring of acute WNT co-receptor activation (phospho-LRP6) support this conclusion. Ciliary WNT signaling inhibits protein phosphatase 1 (PP1) activity, a negative regulator of ciliogenesis, by preventing GSK3-mediated phosphorylation of the PP1 regulatory inhibitor subunit PPP1R2. Concordantly, deficiency of WNT/GSK3 signaling by depletion of cyclin Y and cyclin-Y-like protein 1 induces primary cilia defects in mouse embryonic neuronal precursors, kidney proximal tubules, and adult mice preadipocytes.

The human-specific gene ARHGAP11B has been implicated in human neocortex expansion. However, the extent of ARHGAP11B's contribution to this expansion during hominid evolution is unknown. Here we address this issue by genetic manipulation of ARHGAP11B levels and function in chimpanzee and human cerebral organoids. ARHGAP11B expression in chimpanzee cerebral organoids doubles basal progenitor levels, the class of cortical progenitors with a key role in neocortex expansion. Conversely, interference with ARHGAP11B's function in human cerebral organoids decreases basal progenitors down to the chimpanzee level. Moreover, ARHGAP11A or ARHGAP11B rescue experiments in ARHGAP11A plus ARHGAP11B double-knockout human forebrain organoids indicate that lack of ARHGAP11B, but not of ARHGAP11A, decreases the abundance of basal radial glia-the basal progenitor type thought to be of particular relevance for neocortex expansion. Taken together, our findings demonstrate that ARHGAP11B is necessary and sufficient to ensure the elevated basal progenitor levels that characterize the fetal human neocortex, suggesting that this human-specific gene was a major contributor to neocortex expansion during human evolution.

Neanderthal brains were similar in size to those of modern humans. We sought to investigate potential differences in neurogenesis during neocortex development. Modern human transketolase-like 1 (TKTL1) differs from Neanderthal TKTL1 by a lysine-to-arginine amino acid substitution. Using overexpression in developing mouse and ferret neocortex, knockout in fetal human neocortical tissue, and genome-edited cerebral organoids, we found that the modern human variant, hTKTL1, but not the Neanderthal variant, increases the abundance of basal radial glia (bRG) but not that of intermediate progenitors (bIPs). bRG generate more neocortical neurons than bIPs. The hTKTL1 effect requires the pentose phosphate pathway and fatty acid synthesis. Inhibition of these metabolic pathways reduces bRG abundance in fetal human neocortical tissue. Our data suggest that neocortical neurogenesis in modern humans differs from that in Neanderthals.

Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals.

Comparing the biology of humans to that of other primates, and notably other hominids, is a useful path to learn more about what makes us human. Some of the most interesting differences among hominids are closely related to brain development and function, for example behaviour and cognition. This makes it particularly interesting to compare the hominid neural cells of the neocortex, a part of the brain that plays central roles in those processes. However, well-preserved tissue from great apes is usually extremely difficult to obtain. A variety of new alternative tools, e.g. brain organoids, are now beginning to make it possible to search for such differences and analyse their potential biological and biomedical meaning. Here we present an overview of recent findings from comparisons of the neural stem and progenitor cells (NSPCs) and neurons of hominids. In addition to differences in proliferation and differentiation of NSPCs, and maturation of neurons, we highlight that the regulation of the timing of these processes is emerging as a general foundational difference in the development of the neocortex of hominids.

Although the neurogenesis-enhancing effects of exercise have been extensively studied, the molecular mechanisms underlying this response remain unclear. Here, we propose that this is mediated by the exercise-induced systemic release of the antioxidant selenium transport protein, selenoprotein P (SEPP1). Using knockout mouse models, we confirmed that SEPP1 and its receptor low-density lipoprotein receptor-related protein 8 (LRP8) are required for the exercise-induced increase in adult hippocampal neurogenesis. In vivo selenium infusion increased hippocampal neural precursor cell (NPC) proliferation and adult neurogenesis. Mimicking the effect of exercise through dietary selenium supplementation restored neurogenesis and reversed the cognitive decline associated with aging and hippocampal injury, suggesting potential therapeutic relevance. These results provide a molecular mechanism linking exercise-induced changes in the systemic environment to the activation of quiescent hippocampal NPCs and their subsequent recruitment into the neurogenic trajectory.

Extracellular matrix (ECM) has long been known to regulate many aspects of neural development in many different species. However, the role of the ECM in the development of the human neocortex is not yet fully understood. In this review we discuss the role of the ECM in human neocortex development and the different model systems that can be used to investigate this. In particular, we will focus on how the ECM regulates human neural stem and progenitor cell proliferation and differentiation, how the ECM regulates the architecture of the developing human neocortex and the effect of mutations in ECM and ECM-associated genes in neurodevelopmental disorders.

When considering what makes us human, the development of the neocortex, the seat of our higher cognitive abilities, is of central importance. Throughout this complex developmental process, neocortical stem and progenitor cells (NSPCs) exert a priming role in determining neocortical tissue fate, through a series of cellular and molecular events. In this Perspective article, we address five questions of relevance for potentially human-specific aspects of NSPCs, (i) Are there human-specific NSPC subtypes? (ii) What is the functional significance of the known temporal differences in NSPC dynamics between human and other great apes? (iii) Are there functional interactions between the human-specific genes preferentially expressed in NSPCs? (iv) Do humans amplify certain metabolic pathways for NSPC proliferation? and finally (v) Have differences evolved during human evolution, notably between modern humans and Neandertals, that affect the performance of key genes operating in NSPCs? We discuss potential implications inherent to these questions, and suggest experimental approaches on how to answer them, hoping to provide incentives to further understand key issues of human cortical development.

During development, the decision of stem and progenitor cells to switch from proliferation to differentiation is of critical importance for the overall size of an organ. Too early a switch will deplete the stem/progenitor cell pool, and too late a switch will not generate the required differentiated cell types. With a focus on the developing neocortex, a six-layered structure constituting the major part of the cerebral cortex in mammals, we discuss here the cell biological features that are crucial to ensure the appropriate proliferation vs. differentiation decision in the neural progenitor cells. In the last two decades, the neural progenitor cells giving rise to the diverse types of neurons that function in the neocortex have been intensely investigated for their role in cortical expansion and gyrification. In this review, we will first describe these different progenitor types and their diversity. We will then review the various cell biological features associated with the cell fate decisions of these progenitor cells, with emphasis on the role of the radial processes emanating from these progenitor cells. We will also discuss the species-specific differences in these cell biological features that have allowed for the evolutionary expansion of the neocortex in humans. Finally, we will discuss the emerging role of cell cycle parameters in neocortical expansion.

The mammalian neocortex is the seat of higher cognitive functions, such as thinking and language in human. A hallmark of the neocortex are the cortical neurons, which are generated from divisions of neural progenitor cells (NPCs) during development, and which constitute a key feature of the well-organized layered structure of the neocortex. Proper formation of neocortex structure requires an orchestrated cellular behavior of different cortical NPCs during development, especially during the process of cortical neurogenesis. Here, we review the great diversity of NPCs and their contribution to the development of the neocortex. First, we review the categorization of NPCs into different classes and types based on their cell biological features, and discuss recent advances in characterizing marker expression and cell polarity features in the different types of NPCs. Second, we review the different modes of cell divisions that NPCs undergo and discuss the importance of the balance between proliferation and differentiation of NPCs in neocortical development. Third, we review the different proliferative capacities among different NPC types and among the same type of NPC in different mammalian species. Dissecting the differences between NPC types and differences among mammalian species is beneficial to further understand the development and the evolutionary expansion of the neocortex and may open up new therapeutic avenues for neurodevelopmental and psychiatric disorders.

The role of WNT/β-catenin signalling in mouse neocortex development remains ambiguous. Most studies demonstrate that WNT/β-catenin regulates progenitor self-renewal but others suggest it can also promote differentiation. Here we explore the role of WNT/STOP signalling, which stabilizes proteins during G2/M by inhibiting glycogen synthase kinase (GSK3)-mediated protein degradation. We show that mice mutant for cyclin Y and cyclin Y-like 1 (Ccny/l1), key regulators of WNT/STOP signalling, display reduced neurogenesis in the developing neocortex. Specifically, basal progenitors, which exhibit delayed cell cycle progression, were drastically decreased. Ccny/l1-deficient apical progenitors show reduced asymmetric division due to an increase in apical-basal astral microtubules. We identify the neurogenic transcription factors Sox4 and Sox11 as direct GSK3 targets that are stabilized by WNT/STOP signalling in basal progenitors during mitosis and that promote neuron generation. Our work reveals that WNT/STOP signalling drives cortical neurogenesis and identifies mitosis as a critical phase for neural progenitor fate.

Increase in the size of human neocortex―acquired in evolution―accounts for the unique cognitive capacity of humans. This expansion reflects the evolutionarily enhanced proliferative ability of basal progenitors (BPs), including the basal radial glia and basal intermediate progenitors (bIPs) in mammalian cortex, which may have been acquired through epigenetic alterations in BPs. However, how the epigenome in BPs differs across species is not known. Here, we report that histone H3 acetylation is a key epigenetic regulation in bIP amplification and cortical expansion. Through epigenetic profiling of sorted bIPs, we show that histone H3 lysine 9 acetylation (H3K9ac) is low in murine bIPs and high in human bIPs. Elevated H3K9ac preferentially increases bIP proliferation, increasing the size and folding of the normally smooth mouse neocortex. H3K9ac drives bIP amplification by increasing expression of the evolutionarily regulated gene, Trnp1, in developing cortex. Our findings demonstrate a previously unknown mechanism that controls cortical architecture.

The development of the cerebral cortex relies on the controlled division of neural stem and progenitor cells. The requirement for precise spatiotemporal control of proliferation and cell fate places a high demand on the cell division machinery, and defective cell division can cause microcephaly and other brain malformations. Cell-extrinsic and -intrinsic factors govern the capacity of cortical progenitors to produce large numbers of neurons and glia within a short developmental time window. In particular, ion channels shape the intrinsic biophysical properties of precursor cells and neurons and control their membrane potential throughout the cell cycle. We found that hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel subunits are expressed in mouse, rat, and human neural progenitors. Loss of HCN channel function in rat neural stem cells impaired their proliferation by affecting the cell-cycle progression, causing G1 accumulation and dysregulation of genes associated with human microcephaly. Transgene-mediated, dominant-negative loss of HCN channel function in the embryonic mouse telencephalon resulted in pronounced microcephaly. Together, our findings suggest a role for HCN channel subunits as a part of a general mechanism influencing cortical development in mammals.

Neocortex expansion during human evolution provides a basis for our enhanced cognitive abilities. Yet, which genes implicated in neocortex expansion are actually responsible for higher cognitive abilities is unknown. The expression of human-specific ARHGAP11B in embryonic/foetal mouse, ferret and marmoset neocortex was previously found to promote basal progenitor proliferation, upper-layer neuron generation and neocortex expansion during development, features commonly thought to contribute to increased cognitive abilities. However, a key question is whether this phenotype persists into adulthood and if so, whether cognitive abilities are indeed increased. Here, we generated a transgenic mouse line with physiological ARHGAP11B expression that exhibits increased neocortical size and upper-layer neuron numbers persisting into adulthood. Adult ARHGAP11B-transgenic mice showed altered neurobehaviour, notably increased memory flexibility and a reduced anxiety level. Our data are consistent with the notion that neocortex expansion by ARHGAP11B, a gene implicated in human evolution, underlies some of the altered neurobehavioural features observed in the transgenic mice, such as the increased memory flexibility, a neocortex-associated trait, with implications for the increase in cognitive abilities during human evolution.

Interspecies chimerism is a useful tool to study interactions between cells of different genetic makeup in order to elucidate the mechanisms underlying non-cell-autonomous processes, including evolutionary events. However, generating interspecies chimeras with high efficiency and chimerism level remains challenging. Here, we describe a protocol for generating chimeras between mouse and rat. Donor embryonic stem cells of one species are microinjected into early embryos of the other species (recipient), which are implanted into host foster mothers of the recipient species. For complete details on the use and execution of this protocol, please refer to Stepien et al. (2020).

The evolutionary expansion of the neocortex, the seat of higher cognitive functions in humans, is primarily due to an increased and prolonged proliferation of neural progenitor cells during development. Basal progenitors, and in particular basal radial glial cells, are thought to have a key role in the increased generation of neurons that constitutes a foundation of neocortex expansion. Recent studies have identified primate-specific and human-specific genes and changes in gene expression that promote increased proliferative capacity of cortical progenitors. In many cases, the cell biological basis underlying this increase has been uncovered. Model systems such as mouse, ferret, nonhuman primates, and cerebral organoids have been used to establish the relevance of these genes for neocortex expansion.

The size of the neocortex and its morphology are highly divergent across mammalian species. Several approaches have been utilized for the analysis of neocortical development and comparison among different species. In the present protocol (Note: This protocol requires basic knowledge of brain anatomy), we describe three ex vivo neocortical slice/tissue culture methods: (i) organotypic slice culture (mouse, ferret, human); (ii) hemisphere rotation culture (mouse, ferret); and (iii) free-floating tissue culture (mouse, ferret, human). Each of these three culture methods offers distinct features with regard to the analyses to be performed and can be combined with genetic manipulation by electroporation and treatment with specific inhibitors. These three culture methods are therefore powerful techniques to examine the function of genes involved in neocortical development.

Over the past few years, human-specific genes have received increasing attention as potential major contributors responsible for the 3-fold difference in brain size between human and chimpanzee. Accordingly, mutations affecting these genes may lead to a reduction in human brain size and therefore, may cause or contribute to microcephaly. In this review, we will concentrate, within the brain, on the cerebral cortex, the seat of our higher cognitive abilities, and focus on the human-specific gene ARHGAP11B and on the gene family comprising the three human-specific genes NOTCH2NLA, -B, and -C. These genes are thought to have significantly contributed to the expansion of the cerebral cortex during human evolution. We will summarize the evolution of these genes, as well as their expression and functional role during human cortical development, and discuss their potential relevance for microcephaly. Furthermore, we will give an overview of other human-specific genes that are expressed during fetal human cortical development. We will discuss the potential involvement of these genes in microcephaly and how these genes could be studied functionally to identify a possible role in microcephaly.

The human brain has unique features that are difficult to study in animal models, including the mechanisms underlying neurodevelopmental and psychiatric disorders. Despite recent advances in human primary brain tissue culture systems, the use of these models to elucidate cellular disease mechanisms remains limited. A major reason for this is the lack of tools available to precisely manipulate a specific area of the tissue in a reproducible manner. Here we report an easy-to-use tool for site-specific manipulation of human brain tissue in culture. We show that line-shaped cryogel scaffolds synthesized with precise microscale dimensions allow the targeted delivery of a reagent to a specific region of human brain tissue in culture. 3-sulfopropyl acrylate (SPA) was incorporated into the cryogel network to yield a negative surface charge for the reversible binding of molecular cargo. The fluorescent dyes BODIPY and DiI were used as model cargos to show that placement of dye loaded scaffolds onto brain tissue in culture resulted in controlled delivery without a burst release, and labelling of specific regions without tissue damage. We further show that cryogels can deliver tetrodotoxin to tissue, inhibiting neuronal function in a reversible manner. The robust nature and precise dimensions of the cryogel resulted in a user-friendly and reproducible tool to manipulate primary human tissue cultures. These easy-to-use cryogels offer an innovate approach for more complex manipulations of ex-vivo tissue.

Cellular redox states regulate the balance between stem cell maintenance and activation. Increased levels of intracellular reactive oxygen species (ROS) are linked to proliferation and lineage specification. In contrast to this general principle, we here show that in the hippocampus of adult mice, quiescent neural precursor cells (NPCs) maintain the highest ROS levels (hiROS). Classifying NPCs on the basis of cellular ROS content identified distinct functional states. Shifts in ROS content primed cells for a subsequent state transition, with lower ROS content marking proliferative activity and differentiation. Physical activity, a physiological activator of adult hippocampal neurogenesis, recruited hiROS NPCs into proliferation via a transient Nox2-dependent ROS surge. In the absence of Nox2, baseline neurogenesis was unaffected, but the activity-induced increase in proliferation disappeared. These results provide a metabolic classification of NPC functional states and describe a mechanism linking the modulation of cellular ROS by behavioral cues to the activation of adult NPCs.

The neocortex, the seat of our higher cognitive abilities, has expanded in size during the evolution of certain mammals such as primates, including humans. This expansion occurs during development and is linked to the proliferative capacity of neural stem and progenitor cells (NPCs) in the neocortex. A number of cell-intrinsic and cell-extrinsic factors have been implicated in increasing NPC proliferative capacity. However, NPC metabolism has only recently emerged as major regulator of NPC proliferation. In this Perspective, we summarize recent insights into the role of NPC metabolism in neocortical development and neurodevelopmental disorders and its relevance for neocortex evolution. We discuss certain human-specific genes and microcephaly-implicated genes that operate in, or at, the mitochondria of NPCs and stimulate their proliferation by promoting glutaminolysis. We also discuss other metabolic pathways and develop a perspective on how metabolism mechanistically regulates NPC proliferation in neocortical development and how this contributed to neocortex evolution.

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

During mammalian brain development, neural stem and progenitor cells generate the neurons for the six-layered neocortex. The proliferative capacity of the different types of progenitor cells within the germinal zones of the developing neocortex is a major determinant for the number of neurons generated. Furthermore, the various modes of progenitor cell divisions, for which the orientation of the mitotic spindle of progenitor cells has a pivotal role, are a key parameter to ensure the appropriate size and proper cytoarchitecture of the neocortex. Here, we review the roles of primary cilia and centrosomes of progenitor cells in these processes during neocortical development. We specifically focus on the apical progenitor cells in the ventricular zone. In particular, we address the alternating, dual role of the mother centriole (i) as a component of one of the spindle poles during mitosis, and (ii) as the basal body of the primary cilium in interphase, which is pivotal for the fate of apical progenitor cells and their proliferative capacity. We also discuss the interactions of these organelles with the microtubule and actin cytoskeleton, and with junctional complexes. Centriolar appendages have a specific role in this interaction with the cell cortex and the plasma membrane. Another topic of this review is the specific molecular composition of the ciliary membrane and the membrane vesicle traffic to the primary cilium of apical progenitors, which underlie the ciliary signaling during neocortical development; this signaling itself, however, is not covered in depth here. We also discuss the recently emerging evidence regarding the composition and roles of primary cilia and centrosomes in basal progenitors, a class of progenitors thought to be of particular importance for neocortex expansion in development and evolution. While the tight interplay between primary cilia and centrosomes makes it difficult to allocate independent roles to either organelle, mutations in genes encoding ciliary and/or centrosome proteins indicate that both are necessary for the formation of a properly sized and functioning neocortex during development. Human neocortical malformations, like microcephaly, underpin the importance of primary cilia/centrosome-related processes in neocortical development and provide fundamental insight into the underlying mechanisms involved.

The neocortex, a six-layer neuronal brain structure that arose during the evolution of, and is unique to, mammals, is the seat of higher order brain functions responsible for human cognitive abilities. Despite its recent evolutionary origin, it shows a striking variability in size and folding complexity even among closely related mammalian species. In most mammals, cortical neurogenesis occurs prenatally, and its length correlates with the length of gestation. The evolutionary expansion of the neocortex, notably in human, is associated with an increase in the number of neurons, particularly within its upper layers. Various mechanisms have been proposed and investigated to explain the evolutionary enlargement of the human neocortex, focussing in particular on changes pertaining to neural progenitor types and their division modes, driven in part by the emergence of human-specific genes with novel functions. These led to an amplification of the progenitor pool size, which affects the rate and timing of neuron production. In addition, in early theoretical studies, another mechanism of neocortex expansion was proposed-the lengthening of the neurogenic period. A critical role of neurogenic period length in determining neocortical neuron number was subsequently supported by mathematical modeling studies. Recently, we have provided experimental evidence in rodents directly supporting the mechanism of extending neurogenesis to specifically increase the number of upper-layer cortical neurons. Moreover, our study examined the relationship between cortical neurogenesis and gestation, linking the extension of the neurogenic period to the maternal environment. As the exact nature of factors promoting neurogenic period prolongation, as well as the generalization of this mechanism for evolutionary distinct lineages, remain elusive, the directions for future studies are outlined and discussed.

Neurogenesis in the adult hippocampus declines with age, a process that has been implicated in cognitive and emotional impairments. However, the mechanisms underlying this decline have remained elusive. Here, we show that the age-dependent downregulation of lamin B1, one of the nuclear lamins in adult neural stem/progenitor cells (ANSPCs), underlies age-related alterations in adult hippocampal neurogenesis. Our results indicate that higher levels of lamin B1 in ANSPCs safeguard against premature differentiation and regulate the maintenance of ANSPCs. However, the level of lamin B1 in ANSPCs declines during aging. Precocious loss of lamin B1 in ANSPCs transiently promotes neurogenesis but eventually depletes it. Furthermore, the reduction of lamin B1 in ANSPCs recapitulates age-related anxiety-like behavior in mice. Our results indicate that the decline in lamin B1 underlies stem cell aging and impacts the homeostasis of adult neurogenesis and mood regulation.

A hallmark of the evolutionary expansion of the neocortex is a specific increase in the number of neurons generated for the upper neocortical layers during development. The cause underlying this increase is unknown. Here, we show that lengthening the neurogenic period during neocortical development is sufficient to specifically increase upper-layer neuron generation. Thus, embryos of mouse strains with longer gestation exhibited a longer neurogenic period and generated more upper-layer, but not more deep-layer, neurons than embryos with shorter gestation. Accordingly, long-gestation embryos showed a greater abundance of neurogenic progenitors in the subventricular zone than short-gestation embryos at late stages of cortical neurogenesis. Analysis of a mouse-rat chimeric embryo, developing inside a rat mother, pointed to factors in the rat environment that influenced the upper-layer neuron generation by the mouse progenitors. Exploring a potential maternal source of such factors, short-gestation strain mouse embryos transferred to long-gestation strain mothers exhibited an increase in the length of the neurogenic period and upper-layer neuron generation. The opposite was the case for long-gestation strain mouse embryos transferred to short-gestation strain mothers, indicating a dominant maternal influence on the length of the neurogenic period and hence upper-layer neuron generation. In summary, our study uncovers a hitherto unknown link between embryonic cortical neurogenesis and the maternal gestational environment and provides experimental evidence that lengthening the neurogenic period during neocortical development underlies a key aspect of neocortical expansion.

Evolutionary expansion of the mammalian neocortex (Ncx) has been linked to increased abundance and proliferative capacity of basal progenitors (BPs) in the subventricular zone during development. BP proliferation is governed by both intrinsic and extrinsic signals, several of which have been identified. However, a role of neurotransmitters, a canonical class of extrinsic signaling molecules, in BP proliferation remains to be established. Here, we show that serotonin (5-HT), via its receptor HTR2A, promotes BP proliferation in an evolutionarily relevant manner. HTR2A is not expressed in embryonic mouse Ncx; accordingly, 5-HT does not increase mouse BP proliferation. However, ectopic HTR2A expression can increase mouse BP proliferation. Conversely, CRISPR/Cas9-mediated knockout of endogenous HTR2A in embryonic ferret Ncx reduces BP proliferation. Pharmacological activation of endogenous HTR2A in fetal human Ncx ex vivo increases BP proliferation via HER2/ERK signaling. Hence, 5-HT emerges as an important extrinsic pro-proliferative signal for BPs, which may have contributed to evolutionary Ncx expansion.

The evolutionary expansion of the mammalian neocortex is widely considered to be a basis of increased cognitive abilities. This expansion is a consequence of the enhanced production of neurons during the fetal/embryonic development of the neocortex, which in turn reflects an increased proliferative capacity of neural progenitor cells; in particular basal progenitors (BPs). The remarkable heterogeneity of BP subtypes across mammals, notably their various morphotypes and molecular fingerprints, which has recently been revealed, corroborates the importance of BPs for neocortical expansion. Here, we argue that the morphology of BPs is a key cell biological basis for maintaining their high proliferative capacity and therefore plays crucial roles in the evolutionary expansion of the neocortex.

The neocortex has expanded during mammalian evolution. Overexpression studies in developing mouse and ferret neocortex have implicated the human-specific gene ARHGAP11B in neocortical expansion, but the relevance for primate evolution has been unclear. Here, we provide functional evidence that ARHGAP11B causes expansion of the primate neocortex. ARHGAP11B expressed in fetal neocortex of the common marmoset under control of the gene's own (human) promoter increased the numbers of basal radial glia progenitors in the marmoset outer subventricular zone, increased the numbers of upper-layer neurons, enlarged the neocortex, and induced its folding. Thus, the human-specific ARHGAP11B drives changes in development in the nonhuman primate marmoset that reflect the changes in evolution that characterize human neocortical development.

During development, starting from a pool of pluripotent stem cells, tissue-specific genetic programs help to shape and develop functional organs. To understand the development of an organ and its disorders, it is important to understand the spatio-temporal dynamics of the gene expression profiles that occur during its development. Modifications in existing genes, the de-novo appearance of new genes, or, occasionally, even the loss of genes, can greatly affect the gene expression profile of any given tissue and contribute to the evolution of organs or of parts of organs. The neocortex is evolutionarily the most recent part of the brain, it is unique to mammals, and is the seat of our higher cognitive abilities. Progenitors that give rise to this tissue undergo sequential waves of differentiation to produce the complete sets of neurons and glial cells that make up a functional neocortex. We will review herein our understanding of the transcriptional regulators that control the neural precursor cells (NPCs) during the generation of the most abundant class of neocortical neurons, the glutametergic neurons. In addition, we will discuss the roles of recently-identified human- and primate-specific genes in promoting neurogenesis, leading to neocortical expansion.

During primate, and notably human, evolution, the neocortex increased massively in size. This increase forms the basis of our higher cognitive abilities in comparison to other mammals. In recent years, major advances in understanding this evolutionary expansion have been achieved. Here, we would like to discuss the genetic foundation, principles and mechanisms underlying neocortical expansion.

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.

Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin-Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor-like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.

Neocortex expansion is largely based on the proliferative capacity of basal progenitors (BPs), which is increased by extracellular matrix (ECM) components via integrin signaling. Here we show that the transcription factor Sox9 drives expression of ECM components and that laminin 211 increases BP proliferation in embryonic mouse neocortex. We show that Sox9 is expressed in human and ferret BPs and is required for BP proliferation in embryonic ferret neocortex. Conditional Sox9 expression in the mouse BP lineage, where it normally is not expressed, increases BP proliferation, reduces Tbr2 levels and induces Olig2 expression, indicative of premature gliogenesis. Conditional Sox9 expression also results in cell-non-autonomous stimulation of BP proliferation followed by increased upper-layer neuron production. Our findings demonstrate that Sox9 exerts concerted effects on transcription, BP proliferation, neuron production, and neurogenic vs. gliogenic BP cell fate, suggesting that Sox9 may have contributed to promote neocortical expansion.

The human-specific gene ARHGAP11B is preferentially expressed in neural progenitors of fetal human neocortex and increases abundance and proliferation of basal progenitors (BPs), which have a key role in neocortex expansion. ARHGAP11B has therefore been implicated in the evolutionary expansion of the human neocortex, but its mode of action has been unknown. Here, we show that ARHGAP11B is imported into mitochondria, where it interacts with the adenine nucleotide translocase (ANT) and inhibits the mitochondrial permeability transition pore (mPTP). BP expansion by ARHGAP11B requires its presence in mitochondria, and pharmacological inhibition of ANT function or mPTP opening mimic BP expansion by ARHGAP11B. Searching for the underlying metabolic basis, we find that BP expansion by ARHGAP11B requires glutaminolysis, the conversion of glutamine to glutamate for the tricarboxylic acid (TCA) cycle. Hence, an ARHGAP11B-induced, mitochondria-based effect on BP metabolism that is a hallmark of highly mitotically active cells appears to underlie its role in neocortex expansion.

Neurodevelopmental disorders are heterogeneous and identifying shared genetic aetiologies and converging signalling pathways affected could improve disease diagnosis and treatment. Truncating mutations of the abnormal spindle-like microcephaly associated (ASPM) gene cause autosomal recessive primary microcephaly (MCPH) in humans. ASPM is a positive regulator of Wnt/β-Catenin signalling and controls symmetric to asymmetric cell division. This process balances neural progenitor proliferation with differentiation during embryogenesis, the malfunction of which could interfere with normal brain development. ASPM mutations may play a role also in other neurodevelopmental disorders, nevertheless, we lack the details of how or to what extent. We therefore assessed neurodevelopmental disease and circuit endophenotypes in mice with a truncating Aspm1-7 mutation. Aspm1-7 mice exhibited impaired short- and long-term object recognition memory and markedly enhanced place learning in the IntelliCage®. This behaviour pattern is reminiscent of a cognitive phenotype seen in mouse models and patients with a rare form of autism spectrum disorder (ASD) as well as in mouse models of altered Wnt signalling. These alterations were accompanied by ventriculomegaly, corpus callosum dysgenesis and decreased parvalbumin (PV)+ interneuron numbers in the hippocampal Cornu Ammonis (CA) region and thalamic reticular nucleus (TRN). PV+ cell number correlated to object recognition (CA and TRN) and place learning (TRN). This opens the possibility that, as well as causing MCPH, mutant ASPM potentially contributes to other neurodevelopmental disorders such as ASD through altered parvalbuminergic interneuron development affecting cognitive behaviour. These findings provide important information for understanding the genetic overlap and improved treatment of neurodevelopmental disorders associated with ASPM.

Open spina bifida (OSB) is one of the most prevalent congenital malformations of the central nervous system that often leads to severe disabilities. Previous studies reported the volume and thickness of the neocortex to be altered in children and adolescents diagnosed with OSB. Until now, the onset and the underlying cause of the atypical neocortex organization in OSB patients remain largely unknown. To examine the effects of OSB on foetal neocortex development, we analysed human foetuses of both sexes diagnosed with OSB between 11-15 weeks of gestation by immunofluorescence for established neuronal and neural progenitor marker proteins and compared the results with healthy controls of the same, or very similar, gestational age. Our data indicate that neocortex development in OSB foetuses is altered as early as 11 weeks of gestation. We observed a marked reduction in the radial thickness of the OSB neocortex, which appears to be attributable to a massive decrease in the number of deep- and upper-layer neurons per field, and found a marked reduction in the number of basal progenitors (BPs) per field in the OSB neocortex, consistent with an impairment of cortical neurogenesis underlying the neuronal decrease in OSB foetuses. Moreover, our data suggest that the decrease in BP number in the OSB neocortex may be associated with BPs spending a lesser proportion of their cell cycle in M-phase. Together, our findings expand our understanding of the pathophysiology of OSB and support the need for an early foetal therapy, i.e. in the first trimester of pregnancy.SIGNIFICANCE STATEMENTOpen spina bifida (OSB) is one of the most prevalent congenital malformations of the central nervous system. This study provides novel data on neocortex development of human OSB foetuses. Our data indicate that neocortex development in OSB foetuses is altered as early as 11 weeks of gestation. We observed a marked reduction in the radial thickness of the OSB neocortex, which appears to be attributable a decrease in the number of deep- and upper-layer neurons per field, and found a marked reduction in the number of basal progenitors per field, indicating that impaired neurogenesis underlies the neuronal decrease in OSB foetuses. Our findings support the need for an early foetal therapy and expand our understanding of the pathophysiology of OSB.

Neural progenitor cells (NPCs) play a central role during the development and evolution of the mammalian neocortex. Precise temporal and spatial control of NPC proliferation by a concert of cell-intrinsic and cell-extrinsic factors is essential for the correct formation and proper function of the neocortex. In this review, we focus on the regulation of NPC proliferation by neurotransmitters, which act as a group of cell-extrinsic factors during mammalian neocortex development. We first summarize, from both in vivo and in vitro studies, our current knowledge on how γ-aminobutyric acid (GABA), glutamate and serotonin modulate NPC proliferation in the developing neocortex and the potential involvements of different receptors in the underlying mechanisms. Another focus of this review is to discuss future perspectives using conditionally gene-modified mice and human brain organoids as model systems to further our understanding on the contribution of neurotransmitters to the development of a normal neocortex, as well as how dysregulated neurotransmitter signaling leads to developmental and psychiatric disorders.

Cortical folding is a key feature of the evolutionary expansion of the neocortex. This folding is thought to allow the increase of the cortical surface area within the confinement of the developing skull. Despite its functional importance, the mechanisms that regulate the development of cortical folds have remained elusive. This chapter will discuss several important aspects of folding, including the timing of cortical fold development, the evolution of cortical folding, the cellular and mechanical mechanisms suggested to regulate folding, the model systems being used to study these mechanisms, and, finally, the neurodevelopmental disorders that affect it. While key advances have recently been made in the field, it is clear that several questions remain open, especially those regarding the function of cortical folding and mechanisms that regulate it.

The formation of the neocortex relies on intracellular and extracellular signaling molecules that are involved in the sequential steps of corticogenesis, ranging from the proliferation and differentiation of neural progenitor cells to the migration and dendrite formation of neocortical neurons. Abnormalities in these steps lead to disruption of the cortical structure and circuit, and underly various neurodevelopmental diseases, including dyslexia and autism spectrum disorder (ASD). In this review, we focus on the axon guidance signaling Slit-Robo, and address the multifaceted roles of Slit-Robo signaling in neocortical development. Recent studies have clarified the roles of Slit-Robo signaling not only in axon guidance but also in progenitor cell proliferation and migration, and the maturation of neocortical neurons. We further discuss the etiology of neurodevelopmental diseases, which are caused by defects in Slit-Robo signaling during neocortical formation.

Neurons forming the central nervous system are generated by neural stem and progenitor cells, via a process called neurogenesis (Gotz and Huttner, Nat Rev Mol Cell Biol, 6:777-788, 2005). In this book chapter, we focus on neurogenesis in the dorsolateral telencephalon, the rostral-most region of the neural tube, which contains the part of the central nervous system that is most expanded in mammals (Borrell and Reillo, Dev Neurobiol, 72:955-971, 2012; Wilsch-Brauninger et al., Curr Opin Neurobiol 39:122-132, 2016). We will discuss recent advances in the dissection of the cell biological mechanisms of neurogenesis, with particular attention to the organization and function of the Golgi apparatus and its relationship to the centrosome.

Adult neurogenesis in the mammalian hippocampus is a well-known phenomenon. However, it remains controversial as to what extent adult neurogenesis actually occurs in the adult human hippocampus, and how brain diseases, such as epilepsy, affect human adult neurogenesis. To address these questions, we analyzed immature neuronal marker-expressing (PSA-NCAM+) cells and proliferating neuronal progenitor (Ki67+/HuB+/DCX+) cells in the surgically removed hippocampus of epileptic patients. In control patients, a substantial number of PSA-NCAM+ cells were distributed densely below the granule cell layer. In epileptic patients with granule cell dispersion, the number of PSA-NCAM+ cells was reduced, and aberrant PSA-NCAM+ cells were found. However, the numbers of Ki67+/HuB+/DCX+ cells were very low in both control and epileptic patients. The large number of PSA-NCAM+ cells and few DCX+/HuB+/Ki-67+ cells observed in the controls suggest that immature-type neurons are not recently generated neurons, and that the level of hippocampal neuronal production in adult humans is low. These results also suggest that PSA-NCAM is a useful marker for analyzing the pathology of epilepsy, but different interpretations of the immunohistochemical results between humans and rodents are required.

Microinjection into single cells in brain tissue is a powerful technique to study and manipulate neural stem cells. However, such microinjection requires expertise and is a low-throughput process. We developed the "Autoinjector", a robot that utilizes images from a microscope to guide a microinjection needle into tissue to deliver femtoliter volumes of liquids into single cells. The Autoinjector enables microinjection of hundreds of cells within a single organotypic slice, resulting in an overall yield that is an order of magnitude greater than manual microinjection. The Autoinjector successfully targets both apical progenitors (APs) and newborn neurons in the embryonic mouse and human fetal telencephalon. We used the Autoinjector to systematically study gap-junctional communication between neural progenitors in the embryonic mouse telencephalon and found that apical contact is a characteristic feature of the cells that are part of a gap junction-coupled cluster. The throughput and versatility of the Autoinjector will render microinjection an accessible high-performance single-cell manipulation technique and will provide a powerful new platform for performing single-cell analyses in tissue for bioengineering and biophysics applications.

The human brain has undergone substantial change since humans diverged from chimpanzees and the other great apes1,2. However, the genetic and developmental programs that underlie this divergence are not fully understood. Here we have analysed stem cell-derived cerebral organoids using single-cell transcriptomics and accessible chromatin profiling to investigate gene-regulatory changes that are specific to humans. We first analysed cell composition and reconstructed differentiation trajectories over the entire course of human cerebral organoid development from pluripotency, through neuroectoderm and neuroepithelial stages, followed by divergence into neuronal fates within the dorsal and ventral forebrain, midbrain and hindbrain regions. Brain-region composition varied in organoids from different iPSC lines, but regional gene-expression patterns remained largely reproducible across individuals. We analysed chimpanzee and macaque cerebral organoids and found that human neuronal development occurs at a slower pace relative to the other two primates. Using pseudotemporal alignment of differentiation paths, we found that human-specific gene expression resolved to distinct cell states along progenitor-to-neuron lineages in the cortex. Chromatin accessibility was dynamic during cortex development, and we identified divergence in accessibility between human and chimpanzee that correlated with human-specific gene expression and genetic change. Finally, we mapped human-specific expression in adult prefrontal cortex using single-nucleus RNA sequencing analysis and identified developmental differences that persist into adulthood, as well as cell-state-specific changes that occur exclusively in the adult brain. Our data provide a temporal cell atlas of great ape forebrain development, and illuminate dynamic gene-regulatory features that are unique to humans.

Normal long-term repopulating somatic stem cells (SSCs) preferentially divide asymmetrically, with one daughter cell remaining in the niche and the other going on to be a transient amplifying cell required for generating new tissue in homeostatic maintenance and repair processes, whereas cancer stem cells (CSCs) favor symmetric divisions. We have previously proposed that differential β-catenin modulation of transcriptional activity via selective interaction with either the Kat3 coactivator CBP or its closely related paralog p300, regulates symmetric versus asymmetric division in SSCs and CSCs. We have previously demonstrated that SSCs that divide asymmetrically per force retain one of the dividing daughter cells in the stem cell niche, even when treated with specific CBP/β-catenin antagonists, whereas CSCs can be removed from their niche via forced stochastic symmetric differentiative divisions. We now demonstrate that loss of p73 in early corticogenesis biases β-catenin Kat3 coactivator usage and enhances β-catenin/CBP transcription at the expense of β-catenin/p300 transcription. Biased β-catenin coactivator usage has dramatic consequences on the mode of division of neural stem cells (NSCs), but not neurogenic progenitors. The observed increase in symmetric divisions due to enhanced β-catenin/CBP interaction and transcription leads to an immediate increase in NSC symmetric differentiative divisions. Moreover, we demonstrate for the first time that the complex phenotype caused by the loss of p73 can be rescued in utero by treatment with the small-molecule-specific CBP/β-catenin antagonist ICG-001. Taken together, our results demonstrate the causal relationship between the choice of β-catenin Kat3 coactivator and the mode of stem cell division.

Malformations of the human neocortex in development constitute a heterogeneous group of complex disorders, resulting in pathologies such as intellectual disability and abnormal neurological/psychiatric conditions such as epilepsy or autism. Advances in genomic sequencing and genetic techniques have allowed major breakthroughs in the field, revealing the molecular basis of several of these malformations. Here, we focus on those malformations of the human neocortex, notably microcephaly, and macrocephaly, where an underlying basis has been established at the level of the neural stem/progenitor cells (NPCs) from which neurons are directly or indirectly derived. Particular emphasis is placed on NPC cell biology and NPC markers. A second focus of this review is on experimental model systems used to dissect the underlying mechanisms of malformations of the human neocortex in development at the cellular and molecular level. The most commonly used model system have been genetically modified mice. However, although basic features of neocortical development are conserved across the various mammalian species, some important differences between mouse and human exist. These pertain to the abundance of specific NPC types and/or their proliferative capacity, as exemplified in the case of basal radial glia. These differences limit the ability of mouse models to fully recapitulate the phenotypes of malformations of the human neocortex. For this reason, additional experimental model systems, notably the ferret, non-human primates and cerebral organoids, have recently emerged as alternatives and shown to be of increasing relevance. It is therefore important to consider the benefits and limitations of each of these model systems for studying malformations of the human neocortex in development.

The dramatic evolutionary expansion of the neocortex, together with a proliferation of specialized cortical areas, is believed to underlie the emergence of human cognitive abilities. In a broader phylogenetic context, however, neocortex evolution in mammals, including humans, is remarkably conservative, characterized largely by size variations on a shared six-layered neuronal architecture. By contrast, the telencephalon in non-mammalian vertebrates, including reptiles, amphibians, bony and cartilaginous fishes, and cyclostomes, features a great variety of very different tissue structures. Our understanding of the evolutionary relationships of these telencephalic structures, especially those of basally branching vertebrates and invertebrate chordates, remains fragmentary and is impeded by conceptual obstacles. To make sense of highly divergent anatomies requires a hierarchical view of biological organization, one that permits the recognition of homologies at multiple levels beyond neuroanatomical structure. Here we review the origin and diversification of the telencephalon with a focus on key evolutionary innovations shaping the neocortex at multiple levels of organization.

The neocortex, the seat of higher cognitive functions, exhibits a key feature across mammalian species-a highly variable degree of folding. Within the neocortex, two distinct subtypes of cortical areas can be distinguished, the isocortex and the proisocortex. Here, we have compared specific spatiotemporal aspects of folding between the proisocortex and the isocortex in 13 primates, including human, chimpanzee, and various Old World and New World monkeys. We find that folding at the boundaries of the dorsal isocortex and the proisocortex, which gives rise to the cingulate sulcus (CiS) and the lateral fissure (LF), is conserved across the primates studied and is therefore referred to as conserved folding. In contrast, the degree of folding within the dorsal isocortex exhibits huge variation across these primates, indicating that this folding, which gives rise to gyri and sulci, is subject to major changes during primate evolution. We therefore refer to the folding within the dorsal isocortex as evolved folding. Comparison of fetal neocortex development in long-tailed macaque and human reveals that the onset of conserved folding precedes the onset of evolved folding. Moreover, the analysis of infant human neocortex exhibiting lissencephaly, a developmental malformation thought to be mainly due to abnormal neuronal migration, shows that the evolved folding is perturbed more than the conserved folding. Taken together, our study presents a two-step model of folding that pertains to primate neocortex development and evolution. Specifically, our data imply that the conserved folding and the evolved folding constitute two distinct, sequential events.

Neocortex expansion during mammalian evolution has been linked to an increase in proliferation of basal progenitors in the subventricular zone. Here, we explored a potential role of YAP, the major downstream effector of the Hippo pathway, in proliferation of basal progenitors. YAP expression and activity are high in ferret and human basal progenitors, which exhibit high proliferative capacity, but low in mouse basal progenitors, which lack such capacity. Conditional expression of a constitutively active YAP in mouse basal progenitors resulted in increased proliferation of basal progenitor and promoted production of upper-layer neurons. Pharmacological and genetic interference with YAP function in ferret and human developing neocortex resulted in decreased abundance of cycling basal progenitors. Together, our data indicate that YAP is necessary and sufficient to promote the proliferation of basal progenitors and suggest that increases in YAP levels and presumably activity contributed to the evolutionary expansion of the neocortex.

The evolutionary expansion of the mammalian neocortex (Ncx) is thought to be linked to increased proliferative capacity of basal progenitors (BPs) and their neurogenic capacity. Here, by quantifying BP morphology in the developing Ncx of mouse, ferret, and human, we show that increased BP proliferative capacity is linked to an increase in BP process number. We identify human membrane-bound PALMDELPHIN (PALMD-Caax) as an underlying factor, and we show that it drives BP process growth and proliferation when expressed in developing mouse and ferret Ncx. Conversely, CRISPR/Cas9-mediated disruption of PALMD or its binding partner ADDUCIN-γ in fetal human Ncx reduces BP process numbers and proliferation. We further show that PALMD-induced processes enable BPs to receive pro-proliferative integrin-dependent signals. These findings provide a link between BP morphology and proliferation, suggesting that changes in BP morphology may have contributed to the evolutionary expansion of the Ncx.

Ever since the discovery of thyroid hormone deficiency as the primary cause of cretinism in the second half of the 19th century, the crucial role of thyroid hormone (TH) signaling in embryonic brain development has been established. However, the biological understanding of TH function in brain formation is far from complete, despite advances in treating thyroid function deficiency disorders. The pleiotropic nature of TH action makes it difficult to identify and study discrete roles of TH in various aspect of embryogenesis, including neurogenesis and brain maturation. These challenges notwithstanding, enormous progress has been achieved in understanding TH production and its regulation, their conversions and routes of entry into the developing mammalian brain. The endocrine environment has to adjust when an embryo ceases to rely solely on maternal source of hormones as its own thyroid gland develops and starts to produce endogenous TH. A number of mechanisms are in place to secure the proper delivery and action of TH with placenta, blood-brain interface, and choroid plexus as barriers of entry that need to selectively transport and modify these hormones thus controlling their active levels. Additionally, target cells also possess mechanisms to import, modify and bind TH to further fine-tune their action. A complex picture of a tightly regulated network of transport proteins, modifying enzymes, and receptors has emerged from the past studies. TH have been implicated in multiple processes related to brain formation in mammals-neuronal progenitor proliferation, neuronal migration, functional maturation, and survival-with their exact roles changing over developmental time. Given the plethora of effects thyroid hormones exert on various cell types at different developmental periods, the precise spatiotemporal regulation of their action is of crucial importance. In this review we summarize the current knowledge about TH delivery, conversions, and function in the developing mammalian brain. We also discuss their potential role in vertebrate brain evolution and offer future directions for research aimed at elucidating TH signaling in nervous system development.

The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.

During development, both cells and tissues must acquire the correct shape to allow their proper function. This is especially relevant in the nervous system, where the shape of individual cell processes, such as the axons and dendrites, and the shape of entire tissues, such as the folding of the neocortex, are highly specialized. While many aspects of neural development have been uncovered, there are still several open questions concerning the mechanisms governing cell and tissue shape. In this review, we discuss the role of the extracellular matrix (ECM) in these processes. In particular, we consider how the ECM regulates cell shape, proliferation, differentiation and migration, and more recent work highlighting a key role of ECM in the morphogenesis of neural tissues.

Proper temporal and spatial activation of stem cells relies on highly coordinated cell signaling. The primary cilium is the sensory organelle that is responsible for transmitting extracellular signals into a cell. Primary cilium size, architecture, and assembly-disassembly dynamics are under rigid cell cycle-dependent control. Using mouse incisor tooth epithelia as a model, we show that ciliary dynamics in stem cells require the proper functions of a cholesterol-binding membrane glycoprotein, Prominin-1 (Prom1/CD133), which controls sequential recruitment of ciliary membrane components, histone deacetylase, and transcription factors. Nuclear translocation of Prom1 and these molecules is particularly evident in transit amplifying cells, the immediate derivatives of stem cells. The absence of Prom1 impairs ciliary dynamics and abolishes the growth stimulation effects of sonic hedgehog (SHH) treatment, resulting in the disruption of stem cell quiescence maintenance and activation. We propose that Prom1 is a key regulator ensuring appropriate response of stem cells to extracellular signals, with important implications for development, regeneration, and diseases.

Prominin-1 is a cell surface biomarker that allows the identification of stem and cancer stem cells from different organs. It is also expressed in several differentiated epithelial and non-epithelial cells. Irrespective of the cell type, prominin-1 is associated with plasma membrane protrusions. Here, we investigate its impact on the architecture of membrane protrusions using microvilli of Madin-Darby canine kidney cells as the main model. Our high-resolution analysis revealed that upon the overexpression of prominin-1 the number of microvilli and clusters of them increased. Microvilli with branched and/or knob-like morphologies were observed and stimulated by mutations in the ganglioside-binding site of prominin-1. The altered phenotypes were caused by the interaction of prominin-1 with phosphoinositide 3-kinase and Arp2/3 complex. Mutation of tyrosine 828 of prominin-1 impaired its phosphorylation and thereby inhibited the aforementioned interactions abolishing altered microvilli. This suggests that the interplay of prominin-1-ganglioside membrane complexes, phosphoinositide 3-kinase and cytoskeleton components regulates microvillar architecture. Lastly, the expression of prominin-1 and its mutants modified the structure of filopodia emerging from fibroblast-like cells and silencing human prominin-1 in primary hematopoietic stem cells resulted in the loss of uropod-associated microvilli. Altogether, these findings strengthen the role of prominin-1 as an organizer of cellular protrusions.

Brain organoids have become increasingly used systems allowing 3D-modeling of human brain development, evolution, and disease. To be able to make full use of these modeling systems, researchers have developed a growing toolkit of genetic modification techniques. These techniques can be applied to mature brain organoids or to the preceding embryoid bodies (EBs) and founding cells. This review will describe techniques used for transient and stable genetic modification of brain organoids and discuss their current use and respective advantages and disadvantages. Transient approaches include adeno-associated virus (AAV) and electroporation-based techniques, whereas stable genetic modification approaches make use of lentivirus (including viral stamping), transposon and CRISPR/Cas9 systems. Finally, an outlook as to likely future developments and applications regarding genetic modifications of brain organoids will be presented.

The evolutionary increase in size and complexity of the primate neocortex is thought to underlie the higher cognitive abilities of humans. ARHGAP11B is a human-specific gene that, based on its expression pattern in fetal human neocortex and progenitor effects in embryonic mouse neocortex, has been proposed to have a key function in the evolutionary expansion of the neocortex. Here, we study the effects of ARHGAP11B expression in the developing neocortex of the gyrencephalic ferret. In contrast to its effects in mouse, ARHGAP11B markedly increases proliferative basal radial glia, a progenitor cell type thought to be instrumental for neocortical expansion, and results in extension of the neurogenic period and an increase in upper-layer neurons. Consequently, the postnatal ferret neocortex exhibits increased neuron density in the upper cortical layers and expands in both the radial and tangential dimensions. Thus, human-specific ARHGAP11B can elicit hallmarks of neocortical expansion in the developing ferret neocortex.

A specific subpopulation of neural progenitor cells, the basal radial glial cells (bRGCs) of the outer subventricular zone (OSVZ), are thought to have a key role in the evolutionary expansion of the mammalian neocortex. In the developing lissencephalic mouse neocortex, bRGCs exist at low abundance and show significant molecular differences from bRGCs in developing gyrencephalic species. Here, we demonstrate that the developing mouse medial neocortex (medNcx), in contrast to the canonically studied lateral neocortex (latNcx), exhibits an OSVZ and an abundance of bRGCs similar to that in developing gyrencephalic neocortex. Unlike bRGCs in developing mouse latNcx, the bRGCs in medNcx exhibit human bRGC-like gene expression, including expression of Hopx, a human bRGC marker. Disruption of Hopx expression in mouse embryonic medNcx and forced Hopx expression in mouse embryonic latNcx demonstrate that Hopx is required and sufficient, respectively, for bRGC abundance as found in the developing gyrencephalic neocortex. Taken together, our data identify a novel bRGC subpopulation in developing mouse medNcx that is highly related to bRGCs of developing gyrencephalic neocortex.

The six-layered neocortex of the mammalian pallium has no clear homolog in birds or non-avian reptiles. Recent research indicates that although these extant amniotes possess a variety of divergent and nonhomologous pallial structures, they share a conserved set of neuronal cell types and circuitries. These findings suggest a principle of brain evolution: that natural selection preferentially preserves the integrity of information-processing pathways, whereas other levels of biological organization, such as the three-dimensional architectures of neuronal assemblies, are less constrained. We review the similarities of pallial neuronal cell types in amniotes, delineate candidate gene regulatory networks for their cellular identities, and propose a model of developmental evolution for the divergence of amniote pallial structures.

Neocortical expansion, thought to underlie the cognitive traits unique to humans, is accompanied by cortical folding. This folding starts around gestational week (GW) 20, but what causes it remains largely unknown. Extracellular matrix (ECM) has been previously implicated in neocortical expansion and here we investigate the potential role of ECM in the formation of neocortical folds. We focus on three specific ECM components localized in the human fetal cortical plate (CP): hyaluronan and proteoglycan link protein 1 (HAPLN1), lumican and collagen I (collectively, HLC). Addition of HLC to cultures of human fetal neocortex (11-22 GW) caused local changes in tissue stiffness, induced CP folding, increased CP hyaluronic acid (HA), and required the HA-receptor CD168 and downstream ERK signaling. Importantly, loss of HA reduced HLC-induced and 22 GW physiological nascent folds. This was altered in samples with neurodevelopmental disorders, indicating it may be a useful system to study such disorders.

Since their recent development, organoids that emulate human brain tissue have allowed in vitro neural development studies to go beyond the limits of monolayer culture systems, such as neural rosettes. We present here a review of organoid studies that focuses on cortical wall development, starting with a technical comparison between pre-patterning and self-patterning brain organoid protocols. We then follow neocortex development in space and time and list those aspects where organoids have succeeded in emulating in vivo development, as well as those aspects that continue to be pending tasks. Finally, we present a summary of medical and evolutionary insight made possible by organoid technology.

Delamination of neural progenitor cells (NPCs) from the ventricular surface is a crucial prerequisite to form the subventricular zone, the germinal layer linked to the expansion of the mammalian neocortex in development and evolution. Here, we dissect the molecular mechanism by which the transcription factor Insm1 promotes the generation of basal progenitors (BPs). Insm1 protein is most highly expressed in newborn BPs in mouse and human developing neocortex. Forced Insm1 expression in embryonic mouse neocortex causes NPC delamination, converting apical to basal radial glia. Insm1 represses the expression of the apical adherens junction belt-specific protein Plekha7. CRISPR/Cas9-mediated disruption of Plekha7 expression suffices to cause NPC delamination. Plekha7 overexpression impedes the intrinsic and counteracts the Insm1-induced, NPC delamination. Our findings uncover a novel molecular mechanism underlying NPC delamination in which a BP-genic transcription factor specifically targets the integrity of the apical adherens junction belt, rather than adherens junction components as such.

Understanding the molecular basis that underlies the expansion of the neocortex during primate, and notably human, evolution requires the identification of genes that are particularly active in the neural stem and progenitor cells of the developing neocortex. Here, we have used existing transcriptome datasets to carry out a comprehensive screen for protein-coding genes preferentially expressed in progenitors of fetal human neocortex. We show that fifteen human-specific genes exhibit such expression, and many of them evolved distinct neural progenitor cell-type expression profiles and levels compared to their ancestral paralogs. Functional studies on one such gene,NOTCH2NL, demonstrate its ability to promote basal progenitor proliferation in mice. An additional 35 human genes with progenitor-enriched expression are shown to have orthologs only in primates. Our study provides a resource of genes that are promising candidates to exert specific, and novel, roles in neocortical development during primate, and notably human, evolution.

Neurogenesis is the process through which neural stem and progenitor cells generate neurons. During the development of the mouse neocortex, stem and progenitor cells sequentially give rise to neurons destined to different cortical layers and then switch to gliogenesis resulting in the generation of astrocytes and oligodendrocytes. Precise spatial and temporal regulation of neural progenitor differentiation is key for the proper formation of the complex structure of the neocortex. Dynamic changes in gene expression underlie the coordinated differentiation program, which enables the cells to generate the RNAs and proteins required at different stages of neurogenesis and across different cell types. Here, we review the contribution of epigenetic mechanisms, with a focus on Polycomb proteins, to the regulation of gene expression programs during mouse neocortical development. Moreover, we discuss the recent emerging concept of epigenetic and transcriptional pre-patterning in neocortical progenitor cells as well as post-transcriptional mechanisms for the fine-tuning of mRNA abundance.

There is growing evidence that both peripheral and resident immune cells play an important part in regulating adult neural stem cell proliferation and neurogenesis, although the contribution of the various immune cell types is still unclear. Mast cells, a population of immune cells known for their role in the allergic response, have been implicated in the regulation of adult hippocampal neurogenesis. Mast cell-deficient c-kitW-sh/W-sh mice have previously been shown to exhibit significantly decreased adult hippocampal neurogenesis and associated learning and memory deficits. However, given that numerous other cell types also express high levels of c-kit, the utility of these mice as a reliable model of mast cell-specific depletion is questionable. We show here, using a different model of mast cell deficiency (Mcpt5CreR26DTA/DTA), that precursor proliferation and adult neurogenesis are not influenced by mast cells in vivo. Interestingly, when applied at supraphysiological doses, mast cells can activate latent hippocampal precursor cells and increase subventricular zone precursor proliferation in vitro, an effect that can be blocked with specific histamine-receptor antagonists. Thus, we conclude that while both mast cells and their major chemical mediator histamine have the potential to affect neural precursor proliferation and neurogenesis, this is unlikely to occur under physiological conditions.

The expansion of the neocortex during human evolution is due to changes in our genome that result in increased and prolonged proliferation of neural stem and progenitor cells during neocortex development. Three principal types of such genomic changes can be distinguished, first, novel gene regulation in human, second, novel function in human of genes existing in both human and non-human species, and third, novel, human-specific genes. The latter comprise both, increases in the copy number of genes existing also in non-human species, and the emergence of genes giving rise to unique, human-specific gene products. Examples of all these types of changes in the human genome have been identified, with ARHGAP11B constituting a paradigmatic example of a unique, human-specific protein.

The generation of neocortical neurons from neural progenitor cells (NPCs) is primarily controlled by transcription factors binding to DNA in the context of chromatin. To understand the complex layer of regulation that orchestrates different NPC types from the same DNA sequence, epigenome maps with cell type resolution are required. Here, we present genomewide histone methylation maps for distinct neural cell populations in the developing mouse neocortex. Using different chromatin features, we identify potential novel regulators of cortical NPCs. Moreover, we identify extensive H3K27me3 changes between NPC subtypes coinciding with major developmental and cell biological transitions. Interestingly, we detect dynamic H3K27me3 changes on promoters of several crucial transcription factors, including the basal progenitor regulator Eomes We use catalytically inactive Cas9 fused with the histone methyltransferase Ezh2 to edit H3K27me3 at the Eomes locus in vivo, which results in reduced Tbr2 expression and lower basal progenitor abundance, underscoring the relevance of dynamic H3K27me3 changes during neocortex development. Taken together, we provide a rich resource of neocortical histone methylation data and outline an approach to investigate its contribution to the regulation of selected genes during neocortical development.

Neurodegeneration is a common starting point of reactive gliosis, which may have beneficial and detrimental consequences. It remains incompletely understood how distinctive pathologies and cell death processes differentially regulate glial responses. Müller glia (MG) in the retina are a prime model: Neurons are regenerated in some species, but in mammals there may be proliferative disorders and scarring. Here, we investigated the relationship between retinal damage and MG proliferation, which are both induced in a reproducible and temporal order in organotypic culture of EGF-treated mouse retina: Hypothermia pretreatment during eye dissection reduced neuronal cell death and MG proliferation; stab wounds increased both. Combined (but not separate) application of defined cell death signaling pathway inhibitors diminished neuronal cell death and maintained MG mitotically quiescent. The level of neuronal cell death determined MG activity, indicated by extracellular signal-regulated kinase (ERK) phosphorylation, and proliferation, both of which were abolished by EGFR inhibition. Our data suggest that retinal cell death, possibly either by programmed apoptosis or necrosis, primes MG to be able to transduce the EGFR-ERK activity required for cell proliferation. These results imply that cell death signaling pathways are potential targets for future therapies to prevent the proliferative gliosis frequently associated with certain neurodegenerative conditions.

In light microscopy, refractive index mismatches between media and sample cause spherical aberrations that often limit penetration depth and resolution. Optical clearing techniques can alleviate these mismatches, but they are so far limited to fixed samples. We present Iodixanol as a non-toxic medium supplement that allows refractive index matching in live specimens and thus substantially improves image quality in live-imaged primary cell cultures, planarians, zebrafish and human cerebral organoids.

The evolutionary expansion of the mammalian brain, notably the neocortex, provides a platform for the higher cognitive abilities that characterize humans. Cortical expansion is accompanied by increased folding of the pial surface, which gives rise to a gyrencephalic (folded) rather than lissencephalic (unfolded) neocortex. This expansion reflects the prolonged and increased proliferation of neural stem and progenitor cells (NPCs). Distinct classes of NPCs can be distinguished based on either cell biological criteria (apical progenitors [APs], basal progenitors [BPs]) or lineage (primary progenitors and secondary progenitors). Cortical expansion in development and evolution is linked to an increased abundance and proliferative capacity of BPs, notably basal radial glial cells, a recently characterized type of secondary progenitor derived from apical radial glial cells, the primary progenitors. To gain insight into the molecular basis underlying the prolonged and increased proliferation of NPCs and in particular BPs, comparative genomic and transcriptomic approaches, mostly for human versus mouse, have been employed and applied to specific NPC types and subpopulations. These have revealed two principal sets of molecular changes. One concerns differences in the expression of common genes between species with different degrees of cortical expansion. The other comprises human-specific genes or genomic regulatory sequences. Various systems that allow functional testing of these genomic and gene expression differences between species have emerged, including transient and stable transgenesis, genome editing, cerebral organoids, and organotypic slice cultures. These provide future avenues for uncovering the molecular basis of cortical expansion. For further resources related to this article, please visit the WIREs website.

Neocortex evolutionary expansion is primarily due to increased proliferative capacity of neural progenitor cells during cortical development. Exploiting insights into the cell biology of cortical progenitors gained during the past two decades, recent studies uncovered a variety of gene expression differences that underlie differential cortical progenitor behavior. These comprise both, differences between cortical areas that likely provide a molecular basis for cortical folding, and differences across species thought to be responsible for increases in neocortex size. Human-specific signatures have been identified for gene regulatory elements, non-coding gene products, and protein-encoding genes, and have been functionally examined in in vivo as well as novel in vitro model systems.

Neural stem and progenitor cells in the developing cerebral cortex, but also when grown in culture, display a range of distinct phenomena during cytokinesis. Cleavage furrow ingression in neural progenitor cells can bisect their basal processes and, later on, result in midbody formation at the apical surface. After abscission, these midbodies are released as membrane-bound particles into the extracellular space, in contrast to uptake and degradation of postabscission midbodies in other cell types. Whether these cellular dynamics are unique to neural stem cells, or more ubiquitously found, and what biological significance these processes have for cell differentiation or cell-cell communication, are open questions that require a combination of approaches. Here, we discuss techniques to study the specific membrane dynamics underlying the basal process splitting and postabscission midbody release in neural stem cells. We provide some basic concepts and protocols to isolate, enrich and stain released midbodies, and follow midbody dynamics over time. Moreover, we discuss techniques to prepare cortical sections for high-voltage electron microscopy to visualize the fine basal processes of progenitor cells.

The pentaspan membrane glycoprotein prominin-1 (CD133) is widely used in medicine as a cell surface marker of stem and cancer stem cells. It has opened new avenues in stem cell-based regenerative therapy and oncology. This molecule is largely used with human samples or the mouse model, and consequently most biological tools including antibodies are directed against human and murine prominin-1. Although the general structure of prominin-1 including its membrane topology is conserved throughout the animal kingdom, its primary sequence is poorly conserved. Thus, it is unclear if anti-human and -mouse prominin-1 antibodies cross-react with their orthologs in other species, especially dog. Answering this issue is imperative in light of the growing number of studies using canine prominin-1 as an antigenic marker. Here, we address this issue by cloning the canine prominin-1 and use its overexpression as a green fluorescent protein fusion protein in Madin-Darby canine kidney cells to determine its immunoreactivity with antibodies against human or mouse prominin-1. We used immunocytochemistry, flow cytometry and immunoblotting techniques and surprisingly found no cross-species immunoreactivity. These results raise some caution in data interpretation when anti-prominin-1 antibodies are used in interspecies studies.

Human neocortex expansion likely contributed to the remarkable cognitive abilities of humans. This expansion is thought to primarily reflect differences in proliferation versus differentiation of neural progenitors during cortical development. Here, we have searched for such differences by analysing cerebral organoids from human and chimpanzees using immunohistofluorescence, live imaging, and single-cell transcriptomics. We find that the cytoarchitecture, cell type composition, and neurogenic gene expression programs of humans and chimpanzees are remarkably similar. Notably, however, live imaging of apical progenitor mitosis uncovered a lengthening of prometaphase-metaphase in humans compared to chimpanzees that is specific to proliferating progenitors and not observed in non-neural cells. Consistent with this, the small set of genes more highly expressed in human apical progenitors points to increased proliferative capacity, and the proportion of neurogenic basal progenitors is lower in humans. These subtle differences in cortical progenitors between humans and chimpanzees may have consequences for human neocortex evolution.

Correct orientation of cell division is considered an important factor for the achievement of normal brain size, as mutations in genes that affect this process are among the leading causes of microcephaly. Abnormal spindle orientation is associated with reduction of the neuronal progenitor symmetric divisions, premature cell cycle exit, and reduced neurogenesis. This mechanism has been involved in microcephaly resulting from mutation of ASPM, the most frequently affected gene in autosomal recessive human primary microcephaly (MCPH), but it is presently unknown how ASPM regulates spindle orientation. In this report, we show that ASPM may control spindle positioning by interacting with citron kinase (CITK), a protein whose loss is also responsible for severe microcephaly in mammals. We show that the absence of CITK leads to abnormal spindle orientation in mammals and insects. In mouse cortical development, this phenotype correlates with increased production of basal progenitors. ASPM is required to recruit CITK at the spindle, and CITK overexpression rescues ASPM phenotype. ASPM and CITK affect the organization of astral microtubules (MT), and low doses of MT-stabilizing drug revert the spindle orientation phenotype produced by their knockdown. Finally, CITK regulates both astral-MT nucleation and stability. Our results provide a functional link between two established microcephaly proteins.

Neocortex expansion in development and evolution reflects an increased and prolonged activity of neural progenitor cells. Insight into key aspects of the underlying cell biology has recently been obtained. First, the restriction of apical progenitors to undergo mitosis at the ventricular surface is overcome by generation of basal progenitors, which are free to undergo mitosis at abventricular location, typically the subventricular zone. This process involves basolateral ciliogenesis, delamination from the apical adherens junction belt, and loss of apical cell polarity. Second, proliferative capacity of basal progenitors is supported by self-produced extracellular matrix constituents, which in turn promote growth factor signalling. Humans amplify these processes by characteristic alterations in expression of key regulatory genes (PAX6), and via human-specific genes (ARHGAP11B).

Blood vessels are part of the stem cell niche in the developing cerebral cortex, but their in vivo role in controlling the expansion and differentiation of neural stem cells (NSCs) in development has not been studied. Here, we report that relief of hypoxia in the developing cerebral cortex by ingrowth of blood vessels temporo-spatially coincided with NSC differentiation. Selective perturbation of brain angiogenesis in vessel-specific Gpr124 null embryos, which prevented the relief from hypoxia, increased NSC expansion at the expense of differentiation. Conversely, exposure to increased oxygen levels rescued NSC differentiation in Gpr124 null embryos and increased it further in WT embryos, suggesting that niche blood vessels regulate NSC differentiation at least in part by providing oxygen. Consistent herewith, hypoxia-inducible factor (HIF)-1α levels controlled the switch of NSC expansion to differentiation. Finally, we provide evidence that high glycolytic activity of NSCs is required to prevent their precocious differentiation in vivo Thus, blood vessel function is required for efficient NSC differentiation in the developing cerebral cortex by providing oxygen and possibly regulating NSC metabolism.

The generation of multiciliated cells (MCCs) is required for the proper function of many tissues, including the respiratory tract, brain, and germline. Defects in MCC development have been demonstrated to cause a subclass of mucociliary clearance disorders termed reduced generation of multiple motile cilia (RGMC). To date, only two genes, Multicilin (MCIDAS) and cyclin O (CCNO) have been identified in this disorder in humans. Here, we describe mice lacking GEMC1 (GMNC), a protein with a similar domain organization as Multicilin that has been implicated in DNA replication control. We have found that GEMC1-deficient mice are growth impaired, develop hydrocephaly with a high penetrance, and are infertile, due to defects in the formation of MCCs in the brain, respiratory tract, and germline. Our data demonstrate that GEMC1 is a critical regulator of MCC differentiation and a candidate gene for human RGMC or related disorders.

Cytokinesis, the physical separation of daughter cells at the end of cell cycle, is commonly considered a highly stereotyped phenomenon. However, in some specialized cells this process may involve specific molecular events that are still largely unknown. In mammals, loss of Citron-kinase (CIT-K) leads to massive cytokinesis failure and apoptosis only in neuronal progenitors and in male germ cells, resulting in severe microcephaly and testicular hypoplasia, but the reasons for this specificity are unknown. In this report we show that CIT-K modulates the stability of midbody microtubules and that the expression of tubulin β-III (TUBB3) is crucial for this phenotype. We observed that TUBB3 is expressed in proliferating CNS progenitors, with a pattern correlating with the susceptibility to CIT-K loss. More importantly, depletion of TUBB3 in CIT-K-dependent cells makes them resistant to CIT-K loss, whereas TUBB3 overexpression increases their sensitivity to CIT-K knockdown. The loss of CIT-K leads to a strong decrease in the phosphorylation of S444 on TUBB3, a post-translational modification associated with microtubule stabilization. CIT-K may promote this event by interacting with TUBB3 and by recruiting at the midbody casein kinase-2α (CK2α) that has previously been reported to phosphorylate the S444 residue. Indeed, CK2α is lost from the midbody in CIT-K-depleted cells. Moreover, expression of the nonphosphorylatable TUBB3 mutant S444A induces cytokinesis failure, whereas expression of the phospho-mimetic mutant S444D rescues the cytokinesis failure induced by both CIT-K and CK2α loss. Altogether, our findings reveal that expression of relatively low levels of TUBB3 in mitotic cells can be detrimental for their cytokinesis and underscore the importance of CIT-K in counteracting this event.

We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock-in mice, expression of GFP, which occurs specifically in neural stem cells committed to neurogenesis, was found to be nearly completely (≈90%) abolished in the progeny of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, near-maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation or Cas9 protein complex microinjection to disrupt the expression of Eomes/Tbr2, a gene fundamental for neocortical neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the present in vivo application of the CRISPR/Cas9 system in neural stem cells provides a rapid, efficient and enduring disruption of expression of specific genes to dissect their role in mammalian brain development.

Mota and Herculano-Houzel (Reports, 3 July 2015, p. 74) assign power functions to neuroanatomical data and present a model to account for evolutionary patterns of cortical folding in the mammalian brain. We detail how the model assumptions are in conflict with experimental and observational work and show that the model itself does not accurately fit the data.

The evolutionary expansion of the neocortex primarily reflects increases in abundance and proliferative capacity of cortical progenitors and in the length of the neurogenic period during development. Cell cycle parameters of neocortical progenitors are an important determinant of cortical development. The ferret (Mustela putorius furo), a gyrencephalic mammal, has gained increasing importance as a model for studying corticogenesis. Here, we have studied the abundance, proliferation, and cell cycle parameters of different neural progenitor types, defined by their differential expression of the transcription factors Pax6 and Tbr2, in the various germinal zones of developing ferret neocortex. We focused our analyses on postnatal day 1, a late stage of cortical neurogenesis when upper-layer neurons are produced. Based on cumulative 5-ethynyl-2'-deoxyuridine (EdU) labeling as well as Ki67 and proliferating cell nuclear antigen (PCNA) immunofluorescence, we determined the duration of the various cell cycle phases of the different neocortical progenitor subpopulations. Ferret neocortical progenitors were found to exhibit longer cell cycles than those of rodents and little variation in the duration of G1 among distinct progenitor types, also in contrast to rodents. Remarkably, the main difference in cell cycle parameters among the various progenitor types was the duration of S-phase, which became shorter as progenitors progressively changed transcription factor expression from patterns characteristic of self-renewal to those of neuron production. Hence, S-phase duration emerges as major target of cell cycle regulation in cortical progenitors of this gyrencephalic mammal. J. Comp. Neurol. 524:456-470, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

Apical radial glia (aRG), the stem cells in developing neocortex, are unique bipolar epithelial cells, extending an apical process to the ventricle and a basal process to the basal lamina. Here, we report novel features of the Golgi apparatus, a central organelle for cell polarity, in mouse aRGs. The Golgi was confined to the apical process but not associated with apical centrosome(s). In contrast, in aRG-derived, delaminating basal progenitors that lose apical polarity, the Golgi became pericentrosomal. The aRG Golgi underwent evolutionarily conserved, accordion-like compression and extension concomitant with cell cycle-dependent nuclear migration. Importantly, in line with endoplasmic reticulum but not Golgi being present in the aRG basal process, its plasma membrane contained glycans lacking Golgi processing, consistent with direct ER-to-cell surface membrane traffic. Our study reveals hitherto unknown complexity of neural stem cell polarity, differential Golgi contribution to their specific architecture, and fundamental Golgi re-organization upon cell fate change.

Autosomal recessive primary microcephaly results from abnormal brain development linked to proliferation defects in neural progenitors. The most frequent form, caused by ASPM mutations, is usually defined by a reduced brain volume and is associated with intellectual disability. Although many ASPM cases have now been reported, structural brain abnormalities and their link with cognitive disabilities have rarely been investigated. In this study, we used high resolution T1-weighted magnetic resonance imaging in seven patients with ASPM mutations and 39 healthy age-matched controls to quantify regional volumes, thickness, surface area, gyrification index and white matter volumes of 30 cortical regions. We observed a consistent reduction of 50% or more in the volume and surface area of all cortical regions except for the hippocampus and surrounding medial temporal structures, which were significantly less reduced. Neuropsychologic assessment indicated significant impairments of cognitive abilities. However, these impairments were associated with normal mnesic abilities, in keeping with the relative preservation of the hippocampus and medial temporal structures. These results show that, contrary to current opinion, the cortical volume and surface area of patients with ASPM mutations is reduced depending on a regionally specific fashion and their cognitive profile reflects this heterogeneity. The precise characterization of the cortical map and cognitive abilities of patients with ASPM mutations should allow developing more focused reeducative interventions well-suited to their real abilities.

Cerebral organoids-3D cultures of human cerebral tissue derived from pluripotent stem cells-have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.

During stem cell divisions, mitotic microtubules do more than just segregate the chromosomes. They also determine whether a cell divides virtually symmetrically or asymmetrically by establishing spindle orientation and the plane of cell division. This can be decisive for the fate of the stem cell progeny. Spindle defects have been linked to neurodevelopmental disorders, yet the role of spindle orientation for mammalian neurogenesis has remained controversial. Here we explore recent advances in understanding how the microtubule cytoskeleton influences mammalian neural stem cell division. Our focus is primarily on the role of spindle microtubules in the development of the cerebral cortex. We also highlight unique characteristics in the architecture and dynamics of cortical stem cells that are tightly linked to their mode of division. These features contribute to setting these cells apart as mitotic "rule breakers," control how asymmetric a division is, and, we argue, are sufficient to determine the fate of the neural stem cell progeny in mammals.

In animal cells, supernumerary centrosomes, resulting from centriole amplification, cause mitotic aberrations and have been associated with diseases, including microcephaly and cancer. To evaluate how centriole amplification impacts organismal development at the cellular and tissue levels, we used the in vivo imaging potential of the zebrafish. We demonstrate that centriole amplification can induce multipolar anaphase, resulting in binucleated cells. Such binucleation causes substantial apoptosis in the neuroepithelium. Interestingly, not all epithelia are similarly sensitive to binucleation, as skin cells tolerate it without entering apoptosis. In the neuroepithelium, however, binucleation leads to tissue degeneration and subsequent organismal death. Notably, this tissue degeneration can be efficiently counterbalanced by compensatory proliferation of wild-type cells. Because the risk for generating a binucleated daughter recurs at every cell division, centriole amplification in the neuroepithelium is especially deleterious during progenitor proliferation. Once cells reach the differentiation phase, however, centriole amplification does not impair neuronal differentiation.

The evolutionary expansion of the neocortex in mammals has been linked to enlargement of the subventricular zone (SVZ) and increased proliferative capacity of basal progenitors (BPs), notably basal radial glia (bRG). The transcription factor Pax6 is known to be highly expressed in primate, but not mouse, BPs. Here, we demonstrate that sustaining Pax6 expression selectively in BP-genic apical radial glia (aRG) and their BP progeny of embryonic mouse neocortex suffices to induce primate-like progenitor behaviour. Specifically, we conditionally expressed Pax6 by in utero electroporation using a novel, Tis21-CreERT2 mouse line. This expression altered aRG cleavage plane orientation to promote bRG generation, increased cell-cycle re-entry of BPs, and ultimately increased upper-layer neuron production. Upper-layer neuron production was also increased in double-transgenic mouse embryos with sustained Pax6 expression in the neurogenic lineage. Strikingly, increased BPs existed not only in the SVZ but also in the intermediate zone of the neocortex of these double-transgenic mouse embryos. In mutant mouse embryos lacking functional Pax6, the proportion of bRG among BPs was reduced. Our data identify specific Pax6 effects in BPs and imply that sustaining this Pax6 function in BPs could be a key aspect of SVZ enlargement and, consequently, the evolutionary expansion of the neocortex.

Evolutionary expansion of the human neocortex reflects increased amplification of basal progenitors in the subventricular zone, producing more neurons during fetal corticogenesis. In this work, we analyze the transcriptomes of distinct progenitor subpopulations isolated by a cell polarity-based approach from developing mouse and human neocortex. We identify 56 genes preferentially expressed in human apical and basal radial glia that lack mouse orthologs. Among these, ARHGAP11B has the highest degree of radial glia-specific expression. ARHGAP11B arose from partial duplication of ARHGAP11A (which encodes a Rho guanosine triphosphatase-activating protein) on the human lineage after separation from the chimpanzee lineage. Expression of ARHGAP11B in embryonic mouse neocortex promotes basal progenitor generation and self-renewal and can increase cortical plate area and induce gyrification. Hence, ARHGAP11B may have contributed to evolutionary expansion of human neocortex.

The mammalian neocortex has undergone repeated selection for increases and decreases in size and complexity, often over relatively short evolutionary time. But because probing developmental mechanisms across many species is experimentally unfeasible, it is unknown whether convergent morphologies in distantly related species are regulated by conserved developmental programs. In this work, we have taken advantage of the abundance of available mammalian genomes to find evidence of selection on genomic regions putatively regulating neurogenesis in large- versus small-brained species. Using published fetal human RNA-seq data, we show that the gene-neighborhood (i.e., microsynteny) of long intergenic non-coding RNAs (lincRNAs) implicated in cortical development is differentially conserved in large-brained species, lending support to the hypothesis that lincRNAs regulating neurogenesis are selectively lost in small-brained species. We provide evidence that this is not a phenomenon attributable to lincRNA expressed in all tissue types and is therefore likely to represent an adaptive function in the evolution of neurogenesis. A strong correlation between transcription factor-adjacency and lincRNA sequence conservation reinforces this conclusion.

Midbody release from proliferative neural progenitor cells is tightly associated with the neuronal commitment of neural progenitor cells during the progression of neurogenesis in the mammalian cerebral cortex. While the central portion of the midbody, a cytoplasmic bridge between nascent daughter cells, is engulfed by one of the daughter cell by most cells in vitro, it is shown to be released into the extracellular cerebrospinal fluid (CF) in vivo in mouse embryos. Several proteins have been involved in midbody release; however, few studies have addressed the participation of the plasma membrane's lipids in this process. Here, we show by Shotgun Lipidomic analysis that phosphatydylserine (PS), among other lipids, is enriched in the released midbodies compared to lipoparticles and cellular membranes, both collected from the CF of the developing mouse embryos. Moreover, the developing mouse embryo neural progenitor cells released two distinct types of midbodies carrying either internalized PS or externalized PS on their membrane. This strongly suggests that phagocytosis and an alternative fate of released midbodies exists. HeLa cells, which are known to mainly engulf the midbody show almost no PS exposure, if any, on the outer leaflet of the midbody membrane. These results point toward that PS exposure might be involved in the selection of recipients of released midbodies, either to be engulfed by daughter cells or phagocytosed by non-daughter cells or another cell type in the developing cerebral cortex.

Stem and progenitor cells in the developing mammalian brain are highly polarized cells that carry a primary cilium protruding into the brain ventricles. Here, cilia detect signals present in the cerebrospinal fluid that fills the ventricles. Recently, striking observations have been made regarding the dynamics of primary cilia in mitosis and cilium reformation after cell division. In neural progenitors, primary cilia are not completely disassembled during cell division, and some ciliary membrane remnant can be inherited by one daughter cell that tends to maintain a progenitor fate. Furthermore, newborn differentiating cells grow a primary cilium on their basolateral plasma membrane, in spite of them possessing apical membrane and adherens junctions, and thus change the environment to which the primary cilium is exposed. These phenomena are proposed to be involved in cell fate determination and delamination of daughter cells in conjunction with the production of neurons. Here, we describe several methods that can be used to study the structure, localization, and dynamics of primary cilia in the developing mouse brain; these include time-lapse imaging of live mouse embryonic brain tissues, and analysis of primary cilia structure and localization using correlative light- and electron- and serial-block-face scanning electron microscopy.

Expansion of the neocortex is a hallmark of human evolution. However, determining which adaptive mechanisms facilitated its expansion remains an open question. Here we show, using the gyrencephaly index (GI) and other physiological and life-history data for 102 mammalian species, that gyrencephaly is an ancestral mammalian trait. We find that variation in GI does not evolve linearly across species, but that mammals constitute two principal groups above and below a GI threshold value of 1.5, approximately equal to 109 neurons, which may be characterized by distinct constellations of physiological and life-history traits. By integrating data on neurogenic period, neuroepithelial founder pool size, cell-cycle length, progenitor-type abundances, and cortical neuron number into discrete mathematical models, we identify symmetric proliferative divisions of basal progenitors in the subventricular zone of the developing neocortex as evolutionarily necessary for generating a 14-fold increase in daily prenatal neuron production, traversal of the GI threshold, and thus establishment of two principal groups. We conclude that, despite considerable neuroanatomical differences, changes in the length of the neurogenic period alone, rather than any novel neurogenic progenitor lineage, are sufficient to explain differences in neuron number and neocortical size between species within the same principal group.

Neural stem and progenitor cells have a central role in the development and evolution of the mammalian neocortex. In this review, we first provide a set of criteria to classify the various types of cortical stem and progenitor cells. We then discuss the issue of cell polarity, as well as specific subcellular features of these cells that are relevant for their modes of division and daughter cell fate. In addition, cortical stem and progenitor cell behavior is placed into a tissue context, with consideration of extracellular signals and cell-cell interactions. Finally, the differences across species regarding cortical stem and progenitor cells are dissected to gain insight into key developmental and evolutionary mechanisms underlying neocortex expansion.

Prominin-1 (CD133) is physiologically expressed at the apical membranes of secretory (serous and mucous) and duct cells of major salivary glands. We investigated its expression in various human salivary gland lesions using two distinct anti-prominin-1 monoclonal antibodies (80B258 and AC133) applied on paraffin-embedded sections and characterized its occurrence in saliva. The 80B258 epitope was extensively expressed in adenoid cystic carcinoma, in lesser extent in acinic cell carcinoma and pleomorphic adenoma, and rarely in mucoepidermoid carcinoma. The 80B258 immunoreactivity was predominately detected at the apical membrane of tumor cells showing acinar or intercalated duct cell differentiation, which lined duct- or cyst-like structures, and in luminal secretions. It was observed on the whole cell membrane in non-luminal structures present in the vicinity of thin-walled blood vessels and hemorrhagic areas in adenoid cystic carcinoma. Of note, AC133 labeled only a subset of 80B258-positive structures. In peritumoral salivary gland tissues as well as in obstructive sialadenitis, an up-regulation of prominin-1 (both 80B258 and AC133 immunoreactivities) was observed in intercalated duct cells. In most tissues, prominin-1 was partially co-expressed with two cancer markers: carcinoembryonic antigen (CEA) and mucin-1 (MUC1). Differential centrifugation of saliva followed by immunoblotting indicated that all three markers were released in association with small membrane vesicles. Immuno-isolated prominin-1-positive vesicles contained CEA and MUC1, but also exosome-related proteins CD63, flotillin-1, flotillin-2 and the adaptor protein syntenin-1. The latter protein was shown to interact with prominin-1 as demonstrated by its co-immunoisolation. A fraction of saliva-associated prominin-1 appeared to be ubiquitinated. Collectively, our findings bring new insights into the biochemistry and trafficking of prominin-1 as well as its immunohistochemical profile in certain types of salivary gland tumors and inflammatory diseases.

Mitotic spindle orientation is crucial for symmetric vs. asymmetric cell division and depends on astral microtubules. Here, we show that distinct subpopulations of astral microtubules exist, which have differential functions in regulating spindle orientation and division symmetry. Specifically, in polarized stem cells of developing mouse neocortex, astral microtubules reaching the apical and basal cell cortex, but not those reaching the central cell cortex, are more abundant in symmetrically than asymmetrically dividing cells and reduce spindle orientation variability. This promotes symmetric divisions by maintaining an apico-basal cleavage plane. The greater abundance of apical/basal astrals depends on a higher concentration, at the basal cell cortex, of LGN, a known spindle-cell cortex linker. Furthermore, newly developed specific microtubule perturbations that selectively decrease apical/basal astrals recapitulate the symmetric-to-asymmetric division switch and suffice to increase neurogenesis, in vivo. Thus, our study identifies a novel link between cell polarity, astral microtubules and spindle orientation in morphogenesis.

The neocortex is the seat of higher cognitive functions and, in evolutionary terms, is the youngest part of the mammalian brain. Since its origin, the neocortex has expanded in several mammalian lineages, and this is particularly notable in humans. This expansion reflects an increase in the number of neocortical neurons, which is determined during development and primarily reflects the number of neurogenic divisions of distinct classes of neural progenitor cells. Consequently, the evolutionary expansion of the neocortex and the concomitant increase in the numbers of neurons produced during development entail interspecies differences in neural progenitor biology. Here, we review the diversity of neocortical neural progenitors, their interspecies variations and their roles in determining the evolutionary increase in neuron numbers and neocortex size.

Fate decisions in neural progenitor cells are orchestrated via multiple pathways, and the role of histone acetylation in these decisions has been ascribed to a general function promoting gene activation. Here, we show that the histone acetyltransferase (HAT) cofactor transformation/transcription domain-associated protein (Trrap) specifically regulates activation of cell-cycle genes, thereby integrating discrete cell-intrinsic programs of cell-cycle progression and epigenetic regulation of gene transcription in order to control neurogenesis. Deletion of Trrap impairs recruitment of HATs and transcriptional machinery specifically to E2F cell-cycle target genes, disrupting their transcription with consequent cell-cycle lengthening specifically within cortical apical neural progenitors (APs). Consistently, Trrap conditional mutants exhibit microcephaly because of premature differentiation of APs into intermediate basal progenitors and neurons, and overexpressing cell-cycle regulators in vivo can rescue these premature differentiation defects. These results demonstrate an essential and highly specific role for Trrap-mediated histone regulation in controlling cell-cycle progression and neurogenesis.

This microinjection protocol allows the manipulation and tracking of neural stem and progenitor cells in tissue at single-cell resolution. We demonstrate how to apply microinjection to organotypic brain slices obtained from mice and ferrets; however, our technique is not limited to mouse and ferret embryos, but provides a means of introducing a wide variety of membrane-impermeable molecules (e.g., nucleic acids, proteins, hydrophilic compounds) into neural stem and progenitor cells of any developing mammalian brain. Microinjection experiments are conducted by using a phase-contrast microscope equipped with epifluorescence, a transjector and a micromanipulator. The procedure normally takes ∼2 h for an experienced researcher, and the entire protocol, including tissue processing, can be performed within 1 week. Thus, microinjection is a unique and versatile method for changing and tracking the fate of a cell in organotypic slice culture.

During vertebrate development, a wide variety of cell types and tissues emerge from a single fertilized oocyte. One of these tissues, the central nervous system, contains many types of neurons and glial cells that were born during the period of embryonic and post-natal neuro- and gliogenesis. As to neurogenesis, neural progenitors initially divide symmetrically to expand their pool and switch to asymmetric neurogenic divisions at the onset of neurogenesis. This process involves various mechanisms involving intrinsic as well as extrinsic factors. Here, we discuss the recent advances and insights into regulation of neurogenesis in the developing vertebrate central nervous system. Topics include mechanisms of (a)symmetric cell division, transcriptional and epigenetic regulation, and signaling pathways, using mostly examples from the developing mammalian neocortex.

Mammalian neocortex size primarily reflects the number and mode of divisions of neural stem and progenitor cells. Cortical stem cells (apical progenitors) switching from symmetric divisions, which expand their population, to asymmetric divisions, which generate downstream neuronal progenitors (basal progenitors), start expressing Tis21, a so-called antiproliferative/prodifferentiative gene. Tis21 encodes a small (17.5 kDa), functionally poorly characterized protein and a relatively large (2 kb), highly conserved 3' UTR. Here, we show that mice lacking the Tis21 3' UTR develop a microcephalic neocortex with fewer neurons, notably in the upper layers. This reflects a progressive decrease in basal progenitors, which in turn is due to a fraction of apical progenitors prematurely switching from asymmetric self-renewing to symmetric self-consuming divisions. This switch is caused by the markedly increased Tis21 protein level resulting from lack of microRNA-, notably miR-92-, dependent restriction of Tis21 expression. Our data show that a premature onset of consumptive neural stem cell divisions can lead to microcephaly.

Neocortex expansion during evolution is associated with the enlargement of the embryonic subventricular zone, which reflects an increased self-renewal and proliferation of basal progenitors. In contrast to human, the vast majority of mouse basal progenitors lack self-renewal capacity, possibly due to lack of a basal process contacting the basal lamina and downregulation of cell-autonomous production of extracellular matrix (ECM) constituents. Here we show that targeted activation of the ECM receptor integrin αvβ3 on basal progenitors in embryonic mouse neocortex promotes their expansion. Specifically, integrin αvβ3 activation causes an increased cell cycle re-entry of Pax6-negative, Tbr2-positive intermediate progenitors, rather than basal radial glia, and a decrease in the proportion of intermediate progenitors committed to neurogenic division. Interestingly, integrin αvβ3 is the only known cell surface receptor for thyroid hormones. Remarkably, tetrac, a thyroid hormone analog that inhibits the binding of thyroid hormones to integrin αvβ3, completely abolishes the intermediate progenitor expansion observed upon targeted integrin αvβ3 activation, indicating that this expansion requires the binding of thyroid hormones to integrin αvβ3. Convergence of ECM and thyroid hormones on integrin αvβ3 thus appears to be crucial for cortical progenitor proliferation and self-renewal, and hence for normal brain development and the evolutionary expansion of the neocortex.

During brain development, radial glial cells possess an apico-basal polarity and are coupled by adherens junctions (AJs) to an F-actin belt. To elucidate the role of the actin, we conditionally deleted the key component α-E-catenin in the developing cerebral cortex. Deletion at early stages resulted in severe disruption of tissue polarity due to uncoupling of AJs with the intracellular actin fibers leading to the formation of subcortical band heterotopia. Interestingly, this phenotype closely resembled the phenotype obtained by conditional RhoA deletion, both in regard to the macroscopic subcortical band heterotopia and the subcellular increase in G-actin/F-actin ratio. These data therefore together corroborate the role of the actin cytoskeleton and its anchoring to the AJs for neuronal migration disorders.

MicroRNAs (miRNAs) are 20-25 nucleotide long, noncoding, and single-strand RNAs that have been found in almost all organisms and shown to exert essential roles by regulating the stability and translation of target mRNAs. In mammals most miRNAs show tissue specific and developmentally regulated expression. Approximately 70 % of all miRNAs are expressed in the brain and a growing number of studies have shown that miRNAs can modulate both brain development function and dysfunction. Moreover, miRNAs have been involved in a variety of human pathologies, including cancer and diabetes and are rapidly emerging as new potential drug targets. In order to further characterize miRNA functions, it is therefore crucial to develop techniques enabling their detection in tissues (both fixed and in vivo) with single-cell resolution. Here, we describe methods for the detection/monitoring of miRNA expression, that can be applied in both developing embryos and fixed samples, which we and others have applied to the investigation of both embryonal and postnatal neurogenesis in mice, but also in zebrafish, and cell cultures.

Basal radial glia (bRG) is a recently identified major type of neural stem cell in fetal primate, notably human, neocortex. In this issue of Neuron, Betizeau et al. (2013) now demonstrate that four morphologically distinct bRG subtypes exist in the outer subventricular zone of fetal macaque neocortex, and reveal an unexpected complexity of lineages generating neurons.

Primary cilia are key sensory organelles that are thought to be disassembled prior to mitosis. Inheritance of the mother centriole, which nucleates the primary cilium, in relation to asymmetric daughter cell behavior has previously been studied. However, the fate of the ciliary membrane upon cell division is unknown. Here, we followed the ciliary membrane in dividing embryonic neocortical stem cells and cultured cells. Ciliary membrane attached to the mother centriole was endocytosed at mitosis onset, persisted through mitosis at one spindle pole, and was asymmetrically inherited by one daughter cell, which retained stem cell character. This daughter re-established a primary cilium harboring an activated signal transducer earlier than the noninheriting daughter. Centrosomal association of ciliary membrane in dividing neural stem cells decreased at late neurogenesis when these cells differentiate. Our data imply that centrosome-associated ciliary membrane acts as a determinant for the temporal-spatial control of ciliogenesis.

Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β3-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds.

as it expands, so does it fold. The degree to which it folds, however, cannot strictly be attributed to its expansion. Across species, cortical volume does not keep pace with cortical surface area, but rather folds appear more rapidly than expected. As a result, larger brains quickly become disproportionately more convoluted than smaller brains. Both the absence (lissencephaly) and presence (gyrencephaly) of cortical folds is observed in all mammalian orders and, while there is likely some phylogenetic signature to the evolutionary appearance of gyri and sulci, there are undoubtedly universal trends to the acquisition of folds in an expanding neocortex. Whether these trends are governed by conical expansion of neocortical germinal zones, the distribution of cortical connectivity, or a combination of growth- and connectivity-driven forces remains an open question. But the importance of cortical folding for evolution of the uniquely mammalian neocortex, as well as for the incidence of neuropathologies in humans, is undisputed. In this hypothesis and theory article, we will summarize the development of cortical folds in the neocortex, consider the relative influence of growth- vs. connectivity-driven forces for the acquisition of cortical folds between and within species, assess the genetic, cell-biological, and mechanistic implications for neocortical expansion, and discuss the significance of these implications for human evolution, development, and disease. We will argue that evolutionary increases in the density of neuron production, achieved via maintenance of a basal proliferative niche in the neocortical germinal zones, drive the conical migration of neurons toward the cortical surface and ultimately lead to the establishment of cortical folds in large-brained mammal species.

The importance of thyroid hormones during brain development has been appreciated for many decades. In humans, low levels of circulating maternal thyroid hormones, e.g., caused by maternal hypothyroidism or lack of iodine in diet, results in a wide spectrum of severe neurological defects, including neurological cretinism characterized by profound neurologic impairment and mental retardation, underlining the importance of the maternal thyroid hormone contribution. In fact, iodine intake, which is essential for thyroid hormone production in the thyroid gland, has been related to the expansion of the brain, associated with the increased cognitive capacities during human evolution. Because thyroid hormones regulate transcriptional activity of target genes via their nuclear thyroid hormone receptors (THRs), even mild and transient changes in maternal thyroid hormone levels can directly affect and alter the gene expression profile, and thus disturb fetal brain development. Here we summarize how thyroid hormones may have influenced human brain evolution through the adaptation to new habitats, concomitant with changes in diet and, therefore, iodine intake. Further, we review the current picture we gained from experimental studies in rodents on the function of maternal thyroid hormones during developmental neurogenesis. We aim to evaluate the effects of maternal thyroid hormone deficiency as well as lack of THRs and transporters on brain development and function, shedding light on the cellular behavior conducted by thyroid hormones.

Gyrencephaly (the folding of the surface of the neocortex) is a mammalian-specific trait present in almost all mammalian orders. Despite the widespread appearance of the trait, little is known about the mechanism of its genesis or its adaptive significance. Still, most of the hypotheses proposed concentrated on the pattern of connectivity of mature neurons as main components of gyri formation. Recent work on embryonic neurogenesis in several species of mammals revealed different progenitor and stem cells and their neurogenic potential as having important roles in the process of gyrification. Studies in the field of comparative neurogenesis revealed that gyrencephaly is an evolutionarily labile trait, and that some species underwent a secondary loss of a convoluted brain surface and thus reverted to a more ancient form, a less folded brain surface (lissencephaly). This phenotypic reversion provides an excellent system for understanding the phenomenon of secondary loss. In this review, we will outline the theory behind secondary loss and, as specific examples, present species that have undergone this transition with respect to neocortical folding. We will also discuss different possible pathways for obtaining (or losing) gyri. Finally, we will explore the potential adaptive consequence of gyrencephaly relative to lissencephaly and vice versa.

In mammals, embryonic neural progenitors as well as adult neural stem cells can be prospectively isolated based on the cell surface expression of prominin-1 (CD133), a plasma membrane glycoprotein. In contrast, characterization of neural progenitors in non-mammalian vertebrates endowed with significant constitutive neurogenesis and inherent self-repair ability is hampered by the lack of suitable cell surface markers. Here, we have investigated whether prominin-1-orthologues of the major non-mammalian vertebrate model organisms show any degree of conservation as for their association with neurogenic geminative zones within the central nervous system (CNS) as they do in mammals or associated with activated neural progenitors during provoked neurogenesis in the regenerating CNS.

Around a century ago, the midbody (MB) was described as a structural assembly within the intercellular bridge during cytokinesis that served to connect the two future daughter cells. The MB has become the focus of intense investigation through the identification of a growing number of diverse cellular and molecular pathways that localize to the MB and contribute to its cytokinetic functions, ranging from selective vesicle trafficking and regulated microtubule (MT), actin, and endosomal sorting complex required for transport (ESCRT) filament assembly and disassembly to post-translational modification, such as ubiquitination. More recent studies have revealed new and unexpected functions of MBs in post-mitotic cells. In this review, we provide a historical perspective, discuss exciting new roles for MBs beyond their cytokinetic function, and speculate on their potential contributions to pluripotency.

Coordination of multiple kinesin and myosin motors is required for intracellular transport, cell motility and mitosis. However, comprehensive resources that allow systems analysis of the localization and interplay between motors in living cells do not exist. Here, we generated a library of 243 amino- and carboxy-terminally tagged mouse and human bacterial artificial chromosome transgenes to establish 227 stably transfected HeLa cell lines, 15 mouse embryonic stem cell lines and 1 transgenic mouse line. The cells were characterized by expression and localization analyses and further investigated by affinity-purification mass spectrometry, identifying 191 candidate protein-protein interactions. We illustrate the power of this resource in two ways. First, by characterizing a network of interactions that targets CEP170 to centrosomes, and second, by showing that kinesin light-chain heterodimers bind conventional kinesin in cells. Our work provides a set of validated resources and candidate molecular pathways to investigate motor protein function across cell lineages.

MicroRNAs (miRNAs) are rapidly emerging as a new layer of regulation of mammalian brain development. However, most of the miRNA target genes remain unidentified. Here, we explore gene expression profiling upon miRNA depletion and in vivo target validation as a strategy to identify novel miRNA targets in embryonic mouse neocortex. By this means, we find that Foxp2, a transcription factor associated with speech and language development and evolution, is a novel miRNA target. In particular, we find that miR-9 and miR-132 are able to repress ectopic expression of Foxp2 protein by targeting its 3' untranslated region (3'UTR) in vivo. Interestingly, ectopic expression of Foxp2 in cortical projection neurons (a scenario that mimics the absence of miRNA-mediated silencing of Foxp2 expression) delays neurite outgrowth in vitro and impairs their radial migration in embryonic mouse neocortex in vivo. Our results uncover a new layer of control of Foxp2 expression that may be required for proper neuronal maturation.

The expansion of the neocortex during mammalian brain evolution results primarily from an increase in neural progenitor cell divisions in its two principal germinal zones during development, the ventricular zone (VZ) and the subventricular zone (SVZ). Using mRNA sequencing, we analyzed the transcriptomes of fetal human and embryonic mouse VZ, SVZ, and cortical plate. In mouse, the transcriptome of the SVZ was more similar to that of the cortical plate than that of the VZ, whereas in human the opposite was the case, with the inner and outer SVZ being highly related to each other despite their cytoarchitectonic differences. We describe sets of genes that are up- or down-regulated in each germinal zone. These data suggest that cell adhesion and cell-extracellular matrix interactions promote the proliferation and self-renewal of neural progenitors in the developing human neocortex. Notably, relevant extracellular matrix-associated genes include distinct sets of collagens, laminins, proteoglycans, and integrins, along with specific sets of growth factors and morphogens. Our data establish a basis for identifying novel cell-type markers and open up avenues to unravel the molecular basis of neocortex expansion during evolution.

The precise polarization and orientation of developing neurons is essential for the correct wiring of the brain. In pyramidal excitatory neurons, polarization begins with the sprouting of opposite neurites, which later define directed migration and axo-dendritic domains. We here show that endogenous N-cadherin concentrates at one pole of the newborn neuron, from where the first neurite subsequently emerges. Ectopic N-cadherin is sufficient to favour the place of appearance of the first neurite. The Golgi and centrosome move towards this newly formed morphological pole in a second step, which is regulated by PI3K and the actin/microtubule cytoskeleton. Moreover, loss of function experiments in vivo showed that developing neurons with a non-functional N-cadherin misorient their cell axis. These results show that polarization of N-cadherin in the immediate post-mitotic stage is an early and crucial mechanism in neuronal polarity.

A challenge in the field of neural stem cell biology is the mechanistic dissection of single stem cell behavior in tissue. Although such behavior can be tracked by sophisticated imaging techniques, current methods of genetic manipulation do not allow researchers to change the level of a defined gene product on a truly acute time scale and are limited to very few genes at a time. To overcome these limitations, we established microinjection of neuroepithelial/radial glial cells (apical progenitors) in organotypic slice culture of embryonic mouse brain. Microinjected apical progenitors showed cell cycle parameters that were indistinguishable to apical progenitors in utero, underwent self-renewing divisions and generated neurons. Microinjection of single genes, recombinant proteins or complex mixtures of RNA was found to elicit acute and defined changes in apical progenitor behavior and progeny fate. Thus, apical progenitor microinjection provides a new approach to acutely manipulating single neural stem and progenitor cells in tissue.

Subventricular zone (SVZ) progenitors are a hallmark of the developing neocortex. Recent studies described a novel type of SVZ progenitor that retains a basal process at mitosis, sustains expression of radial glial markers, and is capable of self-renewal. These progenitors, referred to here as basal radial glia (bRG), occur at high relative abundance in the SVZ of gyrencephalic primates (human) and nonprimates (ferret) but not lissencephalic rodents (mouse). Here, we analyzed the occurrence of bRG cells in the embryonic neocortex of the common marmoset Callithrix jacchus, a near-lissencephalic primate. bRG cells, expressing Pax6, Sox2 (but not Tbr2), glutamate aspartate transporter, and glial fibrillary acidic protein and retaining a basal process at mitosis, occur at similar relative abundance in the marmoset SVZ as in human and ferret. The proportion of progenitors in M-phase was lower in embryonic marmoset than developing ferret neocortex, raising the possibility of a longer cell cycle. Fitting the gyrification indices of 26 anthropoid species to an evolutionary model suggested that the marmoset evolved from a gyrencephalic ancestor. Our results suggest that a high relative abundance of bRG cells may be necessary, but is not sufficient, for gyrencephaly and that the marmoset's lissencephaly evolved secondarily by changing progenitor parameters other than progenitor type.

Delamination of neural progenitors from the apical adherens junction belt of the neuroepithelium is a hallmark of cerebral cortex development and evolution. Specific cell biological processes preceding this delamination are largely unknown. Here, we identify a novel, pre-delamination state of neuroepithelial cells in mouse embryonic neocortex. Specifically, in a subpopulation of neuroepithelial cells that, like all others, exhibit apical-basal polarity and apical adherens junctions, the re-establishing of the primary cilium after mitosis occurs at the basolateral rather than the apical plasma membrane. Neuroepithelial cells carrying basolateral primary cilia appear at the onset of cortical neurogenesis, increase in abundance with its progression, selectively express the basal (intermediate) progenitor marker Tbr2, and eventually delaminate from the apical adherens junction belt to become basal progenitors, translocating their nucleus from the ventricular to the subventricular zone. Overexpression of insulinoma-associated 1, a transcription factor known to promote the generation of basal progenitors, increases the proportion of basolateral cilia. Basolateral cilia in cells delaminating from the apical adherens junction belt are preferentially found near spot-like adherens junctions, suggesting that the latter provide positional cues to basolateral ciliogenesis. We conclude that re-establishing a basolateral primary cilium constitutes the first known cell biological feature preceding neural progenitor delamination.

Successful brain development requires tight regulation of sequential symmetric and asymmetric cell division. Although Pax6 is known to exert multiple roles in the developing nervous system, its role in the regulation of cell division is unknown. Here, we demonstrate profound alterations in the orientation and mode of cell division in the cerebral cortex of mice deficient in Pax6 function (Pax6(Sey/Sey)) or after acute induced deletion of Pax6. Live imaging revealed an increase in non-vertical cellular cleavage planes, resulting in an increased number of progenitors with unequal inheritance of the apical membrane domain and adherens junctions in the absence of Pax6 function. This phenotype appears to be mediated by the direct Pax6 target Spag5, a microtubule-associated protein, reduced levels of which result in the replication of the Pax6 phenotype of altered cell division orientation. In addition, lack of Pax6 also results in premature delamination of progenitor cells from the apical surface due to an overall decrease in proteins mediating anchoring at the ventricular surface. Moreover, continuous long-term imaging in vitro revealed that Pax6-deficient progenitors generate daughter cells with asymmetric fates at higher frequencies. These data demonstrate a cell-autonomous role for Pax6 in regulating the mode of cell division independently of apicobasal polarity and cell-cell interactions. Taken together, our work reveals several direct effects that the transcription factor Pax6 has on the machinery that mediates the orientation and mode of cell division.

The differentiation of stem cells is a fundamental process in cell biology and understanding its mechanism might open a new avenue for therapeutic strategies. Using an ex vivo co-culture system consisting of human primary haematopoietic stem and progenitor cells growing on multipotent mesenchymal stromal cells as a feeder cell layer, we describe here the exosome-mediated release of small membrane vesicles containing the stem and cancer stem cell marker prominin-1 (CD133) during haematopoietic cell differentiation. Surprisingly, this contrasts with the budding mechanism underlying the release of this cholesterol-binding protein from plasma membrane protrusions of neural progenitors. Nevertheless, in both progenitor cell types, protein-lipid assemblies might be the essential structural determinant in the release process of prominin-1. Collectively, these data support the concept that prominin-1-containing lipid rafts may host key determinants necessary to maintain stem cell properties and their quantitative reduction or loss may result in cellular differentiation.

BACKGROUND: Rodent and human prominin-1 are expressed in numerous adult epithelia and somatic stem cells. A report has shown that human PROMININ-1 carrying the AC133 epitope can be used to identify rare prostate basal stem cells (Richardson et al., J Cell Sci 2004; 117:3539-3545). Here we re-investigated its general expression in male reproductive tract including mouse and human prostate and in prostate cancer samples using various anti-prominin-1 antibodies. METHODS: The expression was monitored by immunohistochemistry and blotting. Murine tissues were stained with 13A4 monoclonal antibody (mAb) whereas human samples were examined either with the AC133 mAb recognizing the AC133 glycosylation-dependent epitope or 80B258 mAb directed against the PROMININ-1 polypeptide. RESULTS: Mouse prominin-1 was detected at the apical domain of epithelial cells of ductus deferens, seminal vesicles, ampullary glands, and all prostatic lobes. In human prostate, immunoreactivity for 80B258, but not AC133 was revealed at the apical side of some epithelial (luminal) cells, in addition to the minute population of AC133/80B258-positive cells found in basal compartment. Examination of prostate adenocarcinoma revealed the absence of 80B258 immunoreactivity in the tumor regions. However, it was found to be up-regulated in luminal cells in the vicinity of the cancer areas. CONCLUSIONS: Mouse prominin-1 is widely expressed in prostate whereas in human only some luminal cells express it, demonstrating nevertheless that its expression is not solely associated with basal stem cells. In pathological samples, our pilot evaluation shows that PROMININ-1 is down-regulated in the cancer tissues and up-regulated in inflammatory regions. Prostate © 2010 Wiley-Liss, Inc.

Neural stem and progenitor cells giving rise to neurons in developing mammalian neocortex fall into two principal classes with regard to location of mitosis-apical and basal, and into three principal classes in terms of cell polarity during mitosis-bipolar, monopolar, and nonpolar. Insight has been gained into how inheritance of polarized, apical and basal, cell constituents is related to symmetric versus asymmetric divisions of these progenitors, and how this inheritance is linked to their expansion, self-renewal, and neurogenesis. Retention and inheritance of the basal process emerge as key for self-renewal, notably for the monopolar progenitors of prospective gyrencephalic neocortex that undergo asymmetric mitoses at basal locations. The resulting expansion of the neocortex during evolution is proposed to be associated with an increased cone-shape of radial units.

The central portion of the midbody, a cytoplasmic bridge between nascent daughter cells at the end of cell division, has generally been thought to be retained by one of the daughter cells, but has, recently, also been shown to be released into the extracellular space. The significance of midbody-retention versus -release is unknown. Here we show, by quantitatively analysing midbody-fate in various cell lines under different growth conditions, that the extent of midbody-release is significantly greater in stem cells than cancer-derived cells. Induction of cell differentiation is accompanied by an increase in midbody-release. Knockdown of the endosomal sorting complex required for transport family members, Alix and tumour-suppressor gene 101, or of their interaction partner, centrosomal protein 55, impairs midbody-release, suggesting mechanistic similarities to abscission. Cells with such impaired midbody-release exhibit enhanced responsiveness to a differentiation stimulus. Taken together, midbody-release emerges as a characteristic feature of cells capable of differentiation.

During mammalian cerebral cortex development, the G1-phase of the cell cycle is known to lengthen, but it has been unclear which neural stem and progenitor cells are affected. In this paper, we develop a novel approach to determine cell-cycle parameters in specific classes of neural stem and progenitor cells, identified by molecular markers rather than location. We found that G1 lengthening was associated with the transition from stem cell-like apical progenitors to fate-restricted basal (intermediate) progenitors. Unexpectedly, expanding apical and basal progenitors exhibit a substantially longer S-phase than apical and basal progenitors committed to neuron production. Comparative genome-wide gene expression analysis of expanding versus committed progenitor cells revealed changes in key factors of cell-cycle regulation, DNA replication and repair and chromatin remodelling. Our findings suggest that expanding neural stem and progenitor cells invest more time during S-phase into quality control of replicated DNA than those committed to neuron production.

Besides being a marker of various somatic stem cells in mammals, prominin-1 (CD133) plays a role in maintaining the photoreceptor integrity since mutations in the PROM1 gene are linked with retinal degeneration. In spite of that, little information is available regarding its distribution in eyes of non-mammalian vertebrates endowed with high regenerative abilities. To address this subject, prominin-1 cognates were isolated from axolotl, zebrafish and chicken, and their retinal compartmentalization was investigated and compared to that of their mammalian orthologue. Interestingly, prominin-1 transcripts--except for the axolotl--were not strictly restricted to the outer nuclear layer (i.e., photoreceptor cells), but they also marked distinct subdivisions of the inner nuclear layer (INL). In zebrafish, where the prominin-1 gene is duplicated (i.e., prominin-1a and prominin-1b), a differential expression was noted for both paralogues within the INL being localized either to its vitreal or scleral subdivision, respectively. Interestingly, expression of prominin-1a within the former domain coincided with Pax-6-positive cells that are known to act as progenitors upon injury-induced retino-neurogenesis. A similar, but minute population of prominin-1-positive cells located at the vitreal side of the INL was also detected in developing and adult mice. In chicken, however, prominin-1-positive cells appeared to be aligned along the scleral side of the INL reminiscent of zebrafish prominin-1b. Taken together our data indicate that in addition to conserved expression of prominin-1 in photoreceptors, significant prominin-1-expressing non-photoreceptor retinal cell populations are present in the vertebrate eye that might represent potential sources of stem/progenitor cells for regenerative therapies.

Interkinetic nuclear migration (INM), the movement of neuroepithelial and radial glial cell nuclei along the apical-basal axis in concert with the cell cycle, underlies the pseudostratification of the ventricular zone (VZ). Recent studies provide insight into the molecular mechanisms of INM and its effects on neural progenitor cell fate determination. Moreover, INM not only has a key role in increasing the VZ progenitor pool, but also may have set the stage for the evolution of subventricular zone progenitors implicated in cortical expansion.

Mutations in ASPM (abnormal spindle-like microcephaly associated) cause primary microcephaly in humans, a disorder characterized by a major reduction in brain size in the apparent absence of nonneurological anomalies. The function of the Aspm protein in neural progenitor cell expansion, as well as its localization to the mitotic spindle and midbody, suggest that it regulates brain development by a cell division-related mechanism. Furthermore, evidence that positive selection affected ASPM during primate evolution has led to suggestions that such a function changed during primate evolution. Here, we report that in Aspm mutant mice, truncated Aspm proteins similar to those causing microcephaly in humans fail to localize to the midbody during M-phase and cause mild microcephaly. A human ASPM transgene rescues this phenotype but, interestingly, does not cause a gain of function. Strikingly, truncated Aspm proteins also cause a massive loss of germ cells, resulting in a severe reduction in testis and ovary size accompanied by reduced fertility. These germline effects, too, are fully rescued by the human ASPM transgene, indicating that ASPM is functionally similar in mice and humans. Our findings broaden the spectrum of phenotypic effects of ASPM mutations and raise the possibility that positive selection of ASPM during primate evolution reflects its function in the germline.

A major cause of the cerebral cortex expansion that occurred during evolution is the increase in subventricular zone (SVZ) progenitors. We found that progenitors in the outer SVZ (OSVZ) of developing human neocortex retain features of radial glia, in contrast to rodent SVZ progenitors, which have limited proliferation potential. Although delaminating from apical adherens junctions, OSVZ progenitors maintained a basal process contacting the basal lamina, a canonical epithelial property. OSVZ progenitor divisions resulted in asymmetric inheritance of their basal process. Notably, OSVZ progenitors are also found in the ferret, a gyrencephalic nonprimate. Functional disruption of integrins, expressed on the basal process of ferret OSVZ progenitors, markedly decreased the OSVZ progenitor population size, but not that of other, process-lacking SVZ progenitors, in slice cultures of ferret neocortex. Our findings suggest that maintenance of this epithelial property allows integrin-mediated, repeated asymmetric divisions of OSVZ progenitors, providing a basis for neocortical expansion.

Prominin-1 (CD133) is a cholesterol-interacting pentaspan membrane protein concentrated in plasma membrane protrusions. In epithelial cells, notably neuroepithelial stem cells, prominin-1 is found in microvilli, the primary cilium and the midbody. These three types of apical membrane protrusions are subject to remodeling during (neuro)epithelial cell differentiation. The protrusion-specific localization of prominin involves its association with a distinct cholesterol-based membrane microdomain. Moreover, the three prominin-1-containing plasma membrane protrusions are the origin of at least two major subpopulations of prominin-1-containing extracellular membrane particles. Intriguingly, the release of these particles has been implicated in (neuro)epithelial cell differentiation.

Prominin-1 (CD133) and its paralogue, prominin-2, are pentaspan membrane glycoproteins that are strongly expressed in the kidney where they have been originally cloned from. Previously, we have described the localization of prominin-1 in proximal tubules of the nephron. The spatial distribution of prominin-2, however, has not yet been documented in the kidney. We therefore examined the expression of this molecule along distinct tubular segments of the human and murine nephron using in situ hybridization and immunohistochemistry. Our findings indicated that human prominin-2 transcripts and protein were confined to distal tubules of the nephron including the thick ascending limb of Henle's loop and the distal convoluted tubule, the connecting duct and to the collecting duct system. Therein, this glycoprotein was enriched at the basolateral plasma membrane of the tubular epithelial cells with exception of the thick ascending limb where it was also found in the apical domain. This is in contrast with the exclusive apical localization of prominin-1 in epithelial cells of proximal nephron tubules. The distribution of murine prominin-2 transcripts was reminiscent of its human orthologue. In addition, a marked enrichment in the epithelium covering the papilla and in the urothelium of the renal pelvis was noted in mice. Finally, our biochemical analysis revealed that prominin-2 was released into the clinically healthy human urine as a constituent of small membrane vesicles. Collectively our data show the distribution and subcellular localization of prominin-2 within the kidney in situ and its release into the urine. Urinary detection of this protein might offer novel diagnostic approaches for studying renal diseases affecting distal segments of the nephron.

During embryonic cortical development, expression of Tis21 is associated with cell cycle lengthening and neurogenic divisions of progenitor cells. We here investigated if the expression pattern of Tis21 also correlates with the generation of new neurons in the adult hippocampus. We used Tis21 knock-in mice expressing green fluorescent protein (GFP) and studied Tis21-GFP expression together with markers of adult hippocampal neurogenesis in newly generated cells. We found that Tis21-GFP 1) was absent from the radial glia-like putative stem cells (type-1 cells), 2) first appeared in transient amplifying progenitor cells (type-2 and 3 cells), 3) did not colocalize with markers of early postmitotic maturation stage, 4) was expressed again in maturing neurons, and 5) finally decreased in mature granule cells. Our data show that, in the course of adult neurogenesis, Tis21 is expressed in a phase additional to the one of the embryonic neurogenesis. This additional phase of expression might be associated with a new and different function of Tis21 than during embryonic brain development, where no Tis21 is expressed in mature neurons. We hypothesize that this function is related to the final functional integration of the newborn neurons. Tis21 can thus serve as new marker for key stages of adult neurogenesis.

Chromogranins/secretogranins (Cgs) are the major soluble proteins of large dense-core secretory vesicles (LDCVs). We have recently reported that the absence of chromogranin A (CgA) caused important changes in the accumulation and in the exocytosis of catecholamines (CAs) using a CgA-knock-out (CgA-KO) mouse. Here, we have analyzed a CgB-KO mouse strain that can be maintained in homozygosis. These mice have 36% less adrenomedullary epinephrine when compared to Chgb(+/+) [wild type (WT)], whereas the norepinephrine content was similar. The total evoked release of CA was 33% lower than WT mice. This decrease was not due to a lower frequency of exocytotic events but to less secretion per quantum (approximately 30%) measured by amperometry; amperometric spikes exhibited a slower ascending but a normal decaying phase. Cell incubation with L-DOPA increased the vesicle CA content of WT but not of the CgB-KO cells. Intracellular electrochemistry, using patch amperometry, showed that L-DOPA overload produced a significantly larger increase in cytosolic CAs in cells from the KO animals than chromaffin cells from the WT. These data indicate that the mechanisms for vesicular accumulation of CAs in the CgB-KO cells were saturated, while there was ample capacity for further accumulation in WT cells. Protein analysis of LDCVs showed the overexpression of CgA as well as other proteins apparently unrelated to the secretory process. We conclude that CgB, like CgA, is a highly efficient system directly involved in monoamine accumulation and in the kinetics of exocytosis from LDCVs.

The apical plasma membrane of polarized epithelial cells is composed of distinct subdomains, that is, planar regions and protrusions (microvilli, primary cilium), each of which are constructed from specific membrane microdomains. Assemblies containing the pentaspan glycoprotein prominin-1 and certain membrane lipids, notably cholesterol, are characteristic features of these microdomains in apical membrane protrusions. Here we highlight the recent findings concerning the molecular architecture of the apical plasma membrane of epithelial cells and its dynamics. The latter is illustrated by the budding and fission of prominin-1-containing membrane vesicles from apical plasma membrane protrusions, which is controlled, at least in part, by the level of membrane cholesterol and the cholesterol-dependent organization of membrane microdomains.

Granins are major constituents of dense-core secretory granules in neuroendocrine cells, but their function is still a matter of debate. Work in cell lines has suggested that the most abundant and ubiquitously expressed granins, chromogranin A and B (CgA and CgB), are involved in granulogenesis and protein sorting. Here we report the generation and characterization of mice lacking chromogranin B (CgB-ko), which were viable and fertile. Unlike neuroendocrine tissues, pancreatic islets of these animals lacked compensatory changes in other granins and were therefore analyzed in detail. Stimulated secretion of insulin, glucagon and somatostatin was reduced in CgB-ko islets, in parallel with somewhat impaired glucose clearance and reduced insulin release, but normal insulin sensitivity in vivo. CgB-ko islets lacked specifically the rapid initial phase of stimulated secretion, had elevated basal insulin release, and stored and released twice as much proinsulin as wildtype (wt) islets. Stimulated release of glucagon and somatostatin was reduced as well. Surprisingly, biogenesis, morphology and function of insulin granules were normal, and no differences were found with regard to beta-cell stimulus-secretion coupling. We conclude that CgB is not required for normal insulin granule biogenesis or maintenance in vivo, but is essential for adequate secretion of islet hormones. Consequentially CgB-ko animals display some, but not all, hallmarks of human type-2 diabetes. However, the molecular mechanisms underlying this defect remain to be determined.

Chromaffin cells of the adrenal medulla are neural crest-derived cells of the sympathoadrenal lineage. Unlike the closely-related sympathetic neurons, a subpopulation of proliferation-competent cells exists even in the adult. Here, we describe the isolation, expansion, and in vitro characterization of proliferation-competent progenitor cells from the bovine adrenal medulla. Similar to neurospheres, these cells, when prevented from adherence to the culture dish, grew in spheres, which we named chromospheres. These chromospheres were devoid of mRNA specific for smooth muscle cells (MYH11) or endothelial cells (PECAM1). During sphere formation, markers for differentiated chromaffin cells, such as phenylethanolamine-N-methyl transferase, were downregulated while neural progenitor markers nestin, vimentin, musashi 1, and nerve growth factor receptor, as well as markers of neural crest progenitor cells such as Sox1 and Sox9, were upregulated. Clonal analysis and bromo-2'-deoxyuridine-incorporation analysis demonstrated the self-renewing capacity of chromosphere cells. Differentiation protocols using NGF and BMP4 or dexamethasone induced neuronal or endocrine differentiation, respectively. Electrophysiological analyses of neural cells derived from chromospheres revealed functional properties of mature nerve cells, such as tetrodotoxin-sensitive sodium channels and action potentials. Our study provides evidence that proliferation and differentiation competent chromaffin progenitor cells can be isolated from adult adrenal medulla and that these cells might harbor the potential for the treatment of neurodegenerative diseases, such as Parkinson's disease.

The developing cerebral cortex contains apical and basal types of neurogenic progenitor cells. Here, we investigated the cellular properties and neurogenic output of basal progenitors, also called intermediate neuronal progenitors (INPs). We found that basal mitoses expressing transcription factor Tbr2 (an INP marker) were present throughout corticogenesis, from embryonic day 10.5 through birth. Postnatally, Tbr2(+) progenitors were present in the dentate gyrus, subventricular zone (SVZ), and posterior periventricle (pPV). Two morphological subtypes of INPs were distinguished in the embryonic cortex, "short radial" in the ventricular zone (VZ) and multipolar in the SVZ, probably corresponding to molecularly defined INP subtypes. Unexpectedly, many short radial INPs appeared to contact the apical (ventricular) surface and some divided there. Time-lapse video microscopy suggested that apical INP divisions produced daughter INPs. Analysis of neurogenic divisions (Tis21-green fluorescent protein [GFP](+)) indicated that INPs may produce the majority of projection neurons for preplate, deep, and superficial layers. Conversely, proliferative INP divisions (Tis21-GFP(-)) increased from early to middle corticogenesis, concomitant with SVZ growth. Our findings support the hypothesis that regulated amplification of INPs may be an important factor controlling the balance of neurogenesis among different cortical layers.

Interkinetic nuclear migration (INM) is a hallmark of the polarized stem and progenitor cells in the ventricular zone (VZ) of the developing vertebrate CNS. INM is responsible for the pseudostratification of the VZ, a crucial aspect of brain evolution. The nuclear migration toward the apical centrosomes in G2 is thought to be a dynein-microtubule-based process. By contrast, the cytoskeletal machinery involved in the basally directed nuclear translocation away from the centrosome in G1 has been enigmatic. Studying the latter aspect of INM requires manipulation of the cytoskeleton without impairing mitosis and cytokinesis. To this end, we have established a culture system of mouse embryonic telencephalon that reproduces cortical development, and have applied it to explore a role of actomyosin in INM. Using the nonmuscle myosin II inhibitor blebbistatin at a low concentration at which neither cell cycle progression nor cytokinesis is impaired, we show that myosin II is required for the apical-to-basal (ap-->bl), ab-centrosomal INM. Myosin II activity is also necessary for the nuclear translocation during delamination of subventricular zone (SVZ) cells, a second, telencephalon-specific type of neural progenitor. Moreover, the inhibition of ab-centrosomal INM changes the balance between VZ and SVZ progenitor cell fate. Our data suggest a unifying concept in which the actomyosin contraction underlying ab-centrosomal INM sets the stage for the evolutionary increase in VZ pseudostratification and for SVZ progenitor delamination, a key process in cortical expansion.

During mouse embryonic development, neural progenitors lengthen the G1 phase of the cell cycle and this has been suggested to be a cause, rather than a consequence, of neurogenesis. To investigate whether G1 lengthening alone may cause the switch of cortical progenitors from proliferation to neurogenesis, we manipulated the expression of cdk/cyclin complexes and found that cdk4/cyclinD1 overexpression prevents G1 lengthening without affecting cell growth, cleavage plane, or cell cycle synchrony with interkinetic nuclear migration. Specifically, overexpression of cdk4/cyclinD1 inhibited neurogenesis while increasing the generation and expansion of basal (intermediate) progenitors, resulting in a thicker subventricular zone and larger surface area of the postnatal cortex originating from cdk4/cyclinD1-transfected progenitors. Conversely, lengthening of G1 by cdk4/cyclinD1-RNAi displayed the opposite effects. Thus, G1 lengthening is necessary and sufficient to switch neural progenitors to neurogenesis, and overexpression of cdk4/cyclinD1 can be used to increase progenitor expansion and, perhaps, cortical surface area.

We have investigated whether the precursor cells that give rise to the neurons of the neocortex during mouse embryonic development segregate sister chromatids nonrandomly upon mitosis, as would be predicted by the immortal strand hypothesis. Using various protocols of 5-bromo-2-deoxyuridine (BrdU) labeling and chase, we were unable to detect BrdU label-retaining neocortical precursor cells at any of the embryonic stages analyzed, even when the entire brain was analyzed by serial sectioning. Analysis of mitotic neuroepithelial and radial glial cells revealed BrdU-labeled sister chromatid segregation to both nascent daughter cells, which showed a mirror-symmetrical pattern in the first and a non-mirror-symmetrical pattern in the second division after BrdU labeling. Taken together, our data are incompatible with embryonic neocortical precursor cells segregating the sister chromatids selectively to one daughter cell upon mitosis and hence argue against the existence of immortal DNA strands in these cells. In light of the previously reported existence of immortal DNA strands in adult neural stem cells, we discuss that either 1) embryonic and adult neural stem cells in the cortex are distinct or 2) that most, if not all, of the embryonic precursor cells to neocortical neurons are progenitor cells rather than true neural stem cells.

High spatial and temporal resolution of conditional gene expression is typically difficult to achieve in whole tissues or organisms. We synthesized two reversibly inhibited, photoactivatable ('caged') doxycycline derivatives with different membrane permeabilities for precise spatial and temporal light-controlled activation of transgenes based on the 'Tet-on' system. After incubation with caged doxycycline or caged cyanodoxycycline, we induced gene expression by local irradiation with UV light or by two-photon uncaging in diverse biological systems, including mouse organotypic brain cultures, developing mouse embryos and Xenopus laevis tadpoles. The amount of UV light needed for induction was harmless as we detected no signs of toxicity. This method allows high-resolution conditional transgene expression at different spatial scales, ranging from single cells to entire complex organisms.

Prominin-1 (CD133) is a cholesterol-interacting pentaspan membrane glycoprotein specifically associated with plasma membrane protrusions. Prominin-1 is expressed by various stem and progenitor cells, notably neuroepithelial progenitors found in the developing embryonic brain. Here, we further investigated its expression in the murine brain. Biochemical analyses of brain membranes at early stages of development revealed the expression of two distinct splice variants of prominin-1, s1 and s3, which have different cytoplasmic C-terminal domains. The relative abundance of the s3 variant increased toward adulthood, whereas the opposite was observed for the s1 variant. Our combined in situ hybridization and immunohistochemistry revealed the expression of prominin-1 in a subpopulation of Olig-2-positive oligodendroglial cells present within white matter tracts of postnatal and adult brain. Furthermore, immunohistological and biochemical characterization suggested strongly that the s3 variant is a novel component of myelin. Consistent with this, the expression of prominin-1.s3 was significantly reduced in the brain of myelin-deficient mice. Finally, oligodendrocytes expressed selectively the s3 variant whereas GFAP-positive astrocytes expressed the s1 variant in primary glial cell cultures derived from embryonic brains. Collectively, our data demonstrate a complex expression pattern of prominin-1 molecules in developing adult brain. Given that prominin-1 is thought to act as an organizer of plasma membrane protrusions, they further suggest that a specific prominin-1 splice variant might play a role in morphogenesis and/or maintenance of the myelin sheath.

The extent of apoptosis of neural progenitors is known to influence the size of the cerebral cortex. Mouse embryos lacking Brca1, the ortholog of the human breast cancer susceptibility gene BRCA1, show apoptosis in the neural tube, but the consequences of this for brain development have not been studied. Here we investigated the role of Brca1 during mouse embryonic cortical development by deleting floxed Brca1 using Emx1-Cre, which leads to conditional gene ablation specifically in the dorsal telencephalon after embryonic day (E) 9.5. The postnatal Brca1-ablated cerebral cortex was substantially reduced in size with regard to both cortical thickness and surface area. Remarkably, although the thickness of the cortical layers (except for the upper-most layer) was decreased, cortical layering as such was essentially unperturbed. High levels of apoptosis were found at E11.5 and E13.5, but dropped to near-control levels by E16.5. The apoptosis at the early stage of neurogenesis occurred in both BrdU pulse-labeled neural progenitors and the neurons derived therefrom. No changes were observed in the mitotic index of apical (neuroepithelial, radial glial) progenitors and basal (intermediate) progenitors, indicating that Brca1 ablation did not affect cell cycle progression. Brca1 ablation did, however, result in the nuclear translocation of p53 in neural progenitors, suggesting that their apoptosis involved activation of the p53 pathway. Our results show that Brca1 is required for the cerebral cortex to develop to normal size by preventing the apoptosis of early cortical progenitors and their immediate progeny.

Although sufficient cholesterol supply is known to be crucial for neurons in the developing mammalian brain, the cholesterol requirement of neural stem and progenitor cells in the embryonic central nervous system has not been addressed. Here we have conditionally ablated the activity of squalene synthase (SQS), a key enzyme for endogenous cholesterol production, in the neural stem and progenitor cells of the ventricular zone (VZ) of the embryonic mouse brain. Mutant embryos exhibited a reduced brain size due to the atrophy of the neuronal layers, and died at birth. Analyses of the E11.5-E15.5 dorsal telencephalon and diencephalon revealed that this atrophy was due to massive apoptosis of newborn neurons, implying that this progeny of the SQS-ablated neural stem and progenitor cells was dependent on endogenous cholesterol biosynthesis for survival. Interestingly, the neural stem and progenitor cells of the VZ, the primary target of SQS inactivation, did not undergo significant apoptosis. Instead, vascular endothelial growth factor (VEGF) expression in these cells was strongly upregulated via a hypoxia-inducible factor-1-independent pathway, and angiogenesis in the VZ was increased. Consistent with an increased supply of lipoproteins to these cells, the level of lipid droplets containing triacylglycerides with unsaturated fatty acyl chains was found to be elevated. Our study establishes a direct link between intracellular cholesterol levels, VEGF expression, and angiogenesis. Moreover, our data reveal a hitherto unknown compensatory process by which the neural stem and progenitor cells of the developing mammalian brain evade the detrimental consequences of impaired endogenous cholesterol biosynthesis.

The basal process is an extension of certain types of neural progenitors during brain development; that is, the neuroepithelial and radial glial cells, which show radial orientation, emanating from their cell body. Originally, the basal process was considered to serve as a scaffold for the migration of newborn neurons, but recent observations obtained by advanced genetic manipulations and microscopic methods show that the basal process has additional roles. In this review, we first summarize the role of the radial glial basal process for neuronal migration and signaling and for the proper organization of the developing brain. We then focus on the emerging roles of the basal process during the division of neural progenitor cells, specifically the various modes of division of neuroepithelial and radial glial cells.

We previously reported on the occurrence of prominin-1-carrying membrane vesicles that are released into body fluids from microvilli of epithelial cells. This release has been implicated in cell differentiation. Here we have characterized these vesicles released from the differentiated Caco-2 cells. We find that in these vesicles, prominin-1 directly interacts with membrane cholesterol and is associated with a membrane microdomain. The cholesterol depletion using methyl-beta-cyclodextrin resulted in a marked increase in their release, and a dramatic change in the microvillar ultrastructure from a tubular shape to a "pearling" state, with multiple membrane constrictions, suggesting a role of membrane cholesterol in vesicle release from microvilli.

Prominin-1/CD133 (Prom-1) is a commonly used marker of neuronal, vascular, hematopoietic and other stem cells, yet little is known about its biological role and importance in vivo. Here, we show that loss of Prom-1 results in progressive degeneration of mature photoreceptors with complete loss of vision. Despite the expression of Prom-1 on endothelial progenitors, photoreceptor degeneration was not attributable to retinal vessel defects, but caused by intrinsic photoreceptor defects in disk formation, outer segment morphogenesis, and associated with visual pigment sorting and phototransduction abnormalities. These findings shed novel insight on how Prom-1 regulates neural retinal development and phototransduction in vertebrates.

IFT172, also known as Selective Lim-domain Binding protein (SLB), is a component of the intraflagellar transport (IFT) complex. In order to evaluate the biological role of the Ift172 gene, we generated a loss-of-function mutation in the mouse. The resulting Slb mutant embryos die between E12.5 and 13.0, and exhibit severe cranio-facial malformations, failure to close the cranial neural tube, holoprosencephaly, heart edema and extensive hemorrhages. Cilia outgrowth in cells of the neuroepithelium is initiated but the axonemes are severely truncated and do not contain visible microtubules. Morphological and molecular analyses revealed a global brain-patterning defect along the dorsal-ventral (DV) and anterior-posterior (AP) axes. We demonstrate that Ift172 gene function is required for early regulation of Fgf8 at the midbrain-hindbrain boundary and maintenance of the isthmic organizer. In addition, Ift172 is required for proper function of the embryonic node, the early embryonic organizer and for formation of the head organizing center (the anterior mesendoderm, or AME). We propose a model suggesting that forebrain and mid-hindbrain growth and AP patterning depends on the early function of Ift172 at gastrulation. Our data suggest that the formation and function of the node and AME in the mouse embryo relies on an indispensable role of Ift172 in cilia morphogenesis and cilia-mediated signaling.

RATIONALE: Hypertension is a complex trait with deranged autonomic control of the circulation. Chromogranin B (CHGB) is the most abundant core protein in human catecholamine secretory vesicles, playing an important role in their biogenesis. Does common inter-individual variation at the CHGB locus contribute to phenotypic variation in CHGB and catecholamine secretion, autonomic stability of the circulation, or blood pressure in the population? METHODS AND RESULTS: To probe inter-individual variability in CHGB, we systematically studied polymorphism across the locus by re-sequencing CHGB (~6 kbp footprint spanning the promoter, 5 exons, exon/intron borders, UTRs) in n=160 subjects (2n=320 chromosomes) of diverse biogeographic ancestries. We identified 53 SNPs, of which 22 were common. We then studied n=1182 subjects drawn from the most extreme BP values in the population (highest and lowest 5(th) %iles), typing 4 common polymorphisms spanning the ~14 kbp locus. Sliding-window haplotype analysis indicated BP associations peaking in the 5'/promoter region, and most prominent in men, and a peak effect in the proximal promoter at variant A-261T (A>T), accounting for ~8/~6 mmHg SBP/DBP in males. The promoter allele (A-261) that was a predictor of higher DBP and SBP was also associated with lower circulating/plasma CHGB concentration (CHGB(439-451) epitope) in twin pairs. In twins, the same CHGB variants that were predictors of lower basal CHGB secretion were also associated with exaggerated catecholamine secretion and BP response to environmental (cold) stress; likewise, women displayed increased plasma CHGB(439-451), but decreased catecholamine secretion as well as BP response to environmental stress. The effect of A-261T on CHGB expression was confirmed in chromaffin cells by site-directed mutagenesis on transfected CHGB promoter/luciferase reporter activity, and the allelic effects of A-261T on gene expression were directionally coordinate in cella and in vivo. To confirm these clinical associations experimentally, we undertook targeted homozygous (-/-) ablation of the mouse Chgb gene; knockout mice displayed substantially increased BP, by ~20/~18 mmHg SBP/DBP, confirming the mechanistic basis of our findings in humans. CONCLUSIONS: We conclude that common genetic variation at the CHGB locus, especially in the proximal promoter, influences CHGB expression, and later catecholamine secretion and the early heritable responses to environmental stress, eventuating in changes in resting/basal BP in the population. Both the early (gene expression) and late (population BP) consequences of CHGB variation are sex-dependent. The results point to new molecular strategies for probing autonomic control of the circulation, and ultimately the susceptibility to and pathogenesis of cardiovascular disease states such as hypertension.

The switch of neural stem and progenitor cells from proliferation to differentiation during development is a crucial determinant of brain size. This switch is intimately linked to the architecture of the two principal classes of neural stem and progenitor cells, the apical (neuroepithelial, radial glial) and basal (intermediate) progenitors, which in turn is crucial for their symmetric versus asymmetric divisions. Focusing on the developing rodent neocortex, we discuss here recent advances in understanding the cell biology of apical and basal progenitors, place key regulatory molecules into subcellular context, and highlight their roles in the control of proliferation versus differentiation.

Neurogenesis during the development of the mammalian cerebral cortex involves a switch of neural stem and progenitor cells from proliferation to differentiation. To explore the possible role of microRNAs (miRNAs) in this process, we conditionally ablated Dicer in the developing mouse neocortex using Emx1-Cre, which is specifically expressed in the dorsal telencephalon as early as embryonic day (E) 9.5. Dicer ablation in neuroepithelial cells, which are the primary neural stem and progenitor cells, and in the neurons derived from them, was evident from E10.5 onwards, as ascertained by the depletion of the normally abundant miRNAs miR-9 and miR-124. Dicer ablation resulted in massive hypotrophy of the postnatal cortex and death of the mice shortly after weaning. Analysis of the cytoarchitecture of the Dicer-ablated cortex revealed a marked reduction in radial thickness starting at E13.5, and defective cortical layering postnatally. Whereas the former was due to neuronal apoptosis starting at E12.5, which was the earliest detectable phenotype, the latter reflected dramatic impairment of neuronal differentiation. Remarkably, the primary target cells of Dicer ablation, the neuroepithelial cells, and the neurogenic progenitors derived from them, were unaffected by miRNA depletion with regard to cell cycle progression, cell division, differentiation and viability during the early stage of neurogenesis, and only underwent apoptosis starting at E14.5. Our results support the emerging concept that progenitors are less dependent on miRNAs than their differentiated progeny, and raise interesting perspectives as to the expansion of somatic stem cells.

Neuroepithelial (NE) cells, the primary stem and progenitor cells of the vertebrate central nervous system, are highly polarized and elongated. They retain a basal process extending to the basal lamina, while undergoing mitosis at the apical side of the ventricular zone. By studying NE cells in the embryonic mouse, chick and zebrafish central nervous system using confocal microscopy, electron microscopy and time-lapse imaging, we show here that the basal process of these cells can split during M phase. Splitting occurred in the basal-to-apical direction and was followed by inheritance of the processes by either one or both daughter cells. A cluster of anillin, an essential component of the cytokinesis machinery, appeared at the distal end of the basal process in prophase and was found to colocalize with F-actin at bifurcation sites, in both proliferative and neurogenic NE cells. GFP-anillin in the basal process moved apically to the cell body prior to anaphase onset, followed by basal-to-apical ingression of the cleavage furrow in telophase. The splitting of the basal process of M-phase NE cells has implications for cleavage plane orientation and the relationship between mitosis and cytokinesis.

Human prominin-1 (CD133) is expressed by various stem and progenitor cells originating from diverse sources. In addition to stem cells, its mouse ortholog is expressed in a broad range of adult epithelial cells, where it is selectively concentrated in their apical domain. The lack of detection of prominin-1 in adult human epithelia might be explained, at least in part, by the specificity of the widely used AC133 antibody, which recognizes an epitope that seems dependent on glycosylation. Here we decided to re-examine its expression in adult human tissues, particularly in glandular epithelia, using a novel monoclonal antibody (80B258) generated against the human prominin-1 polypeptide. In examined tissues, we observed 80B258 immunoreactivity at the apical or apicolateral membranes of polarized cells. For instance, we found expression in secretory serous and mucous cells as well as intercalated ducts of the large salivary and lacrimal glands. In sweat glands including the gland of Moll, 80B258 immunoreactivity was found in the secretory (eccrine and apocrine glands) and duct (eccrine glands) portion. In the liver, 80B258 immunoreactivity was identified in the canals of Hering, bile ductules, and small interlobular bile ducts. In the uterus, we detected 80B258 immunoreactivity in endometrial and cervical glands. Together these data show that the overall expression of human prominin-1 is beyond the rare primitive cells, and it seems to be a general marker of apical or apicolateral membrane of glandular epithelia. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Although the male reproductive system seems to be enriched in transcripts encoding for both Prominin genes, little is known about their spatial distribution in distinct segments of this organ system. This is especially true for the less-characterized second Prominin paralogue, Prominin-2. The present study, therefore, mainly examines the expression of Prominin-2 in male mice and reveals the existence of some crucial differences in the tissue compartmentalization of the two Prominin paralogues in the testis, epididymis, seminal vesicle, prostate and urinary bladder. Our in situ hybridization analysis demonstrates that the major domains of overlapping expression between the two Prominin genes are those compartments that are derived ontogenetically from the epigonadal mesonephric tubules, i.e. ductuli efferentes, or from the Wolffian-tube/ductus mesonephricus, for instance the corpus epididymidis and vesicula seminalis. In contrast, the sinus urogenitalis derivative urinary bladder epithelium expresses exclusively Prominin-2, but not Prominin-1 (CD133). The testis expresses only Prominin-1, not Prominin-2. In human prostate, we finally demonstrate that the expression of Prominin-2 (transcript and protein) is highly enriched in cells located in the basal compartment of the glandular epithelium where only a minute population was recently reported to be Prominin-1 positive. Taken together our data indicate that, except for the gonad, Prominin-2 is widely and abundantly expressed along the epithelia of various segments of the adult male genitourinary tract.

Basal (intermediate) progenitors are the major source of neurons in the mammalian neocortex. The molecular machinery governing basal progenitor biogenesis is unknown. Here, we show that the zinc-finger transcription factor Insm1 (insulinoma-associated 1) is expressed specifically in progenitors undergoing neurogenic divisions, has a panneurogenic role throughout the brain, and promotes basal progenitor formation in the neocortex. Mouse embryos lacking Insm1 contained half the number of basal progenitors and showed a marked reduction in cortical plate radial thickness. Forced premature expression of Insm1 in neuroepithelial cells resulted in their mitosis occurring at the basal (rather than apical) side of the ventricular zone and induced expression of the basal progenitor marker Tbr2. Remarkably, these cells remained negative for Tis21, a marker of neurogenic progenitors, and did not generate neurons but underwent self-amplification. Our data imply that Insm1 is involved in the generation and expansion of basal progenitors, a hallmark of neocortex evolution.

Relative brain size differs markedly between species. This variation might ultimately result from differences in the cell biology of neural progenitors, which might underlie their different proliferative potential. On the basis of the cell-biological properties of neural progenitors of animals of varying brain size and complexity (namely, Drosophila melanogaster, rodents and primates), we hypothesize that the evolution of four related cell-biological features has contributed to increases in neuron number. Three of these features-the pseudostratification of the progenitor layer, the loss of (Inscuteable-mediated) mitotic-spindle rotation and the evolution of proteins (such as Aspm) that maintain the precision of symmetric progenitor division-affect the mode of cell division in the apically dividing progenitors of the ventricular zone. The fourth feature, however, concerns the evolution of the basally dividing progenitors of the subventricular zone. In rodents, these basal (or intermediate) progenitors lack cell polarity, whereas in primates a subpopulation of radial, presumably polarized, progenitors has evolved (outer-subventricular-zone progenitors). These cells undergo basal mitoses and are thought to retain epithelial characteristics. We propose the epithelial-progenitor hypothesis, which argues that evolutionary changes that promote the maintenance of epithelial features in neural progenitors, including outer-subventricular-zone progenitors, have been instrumental in the expansion of the cerebral cortex in primates.

Familial macular degeneration is a clinically and genetically heterogeneous group of disorders characterized by progressive central vision loss. Here we show that an R373C missense mutation in the prominin 1 gene (PROM1) causes 3 forms of autosomal-dominant macular degeneration. In transgenic mice expressing R373C mutant human PROM1, both mutant and endogenous PROM1 were found throughout the layers of the photoreceptors, rather than at the base of the photoreceptor outer segments, where PROM1 is normally localized. Moreover, the outer segment disk membranes were greatly overgrown and misoriented, indicating defective disk morphogenesis. Immunoprecipitation studies showed that PROM1 interacted with protocadherin 21 (PCDH21), a photoreceptor-specific cadherin, and with actin filaments, both of which play critical roles in disk membrane morphogenesis. Collectively, our results identify what we believe to be a novel complex involved in photoreceptor disk morphogenesis and indicate a possible role for PROM1 and PCDH21 in macular degeneration.

The floor plate (FP), a signaling center and a structure rich in radial glia-like cells, has been traditionally thought to be devoid of neurons and neuronal progenitors. However, in the midbrain, the FP contains neurons of the dopaminergic (DA) lineage that require contact with radial glia-like cells for their induction. We, therefore, decided to explore the interaction relationship between radial glia and neurons during DA neurogenesis. Taking advantage of a novel FP radial glia-like cell culture system and retroviruses, DA neurons were lineage traced in vitro. In utero BrdU pulse-chases extensively labeled the midbrain FP and traced DA neurons both in vivo and in FP cultures. Moreover, from E9.5 to E13.5 the midbrain FP contained dividing cells only in the most apical part of the neuroepithelium, in cells identified as radial glia-like cells. We, therefore, hypothesized that midbrain FP radial glia-like cells could be DA progenitors and tested our hypothesis in vivo. Lineage tracing of DA progenitors with EGFP in Tis21-EGFP knock-in mice, and genetic fate mapping in GLAST::CreERT2/ZEG mice identified the neuroepithelium of the midbrain FP, and specifically, GLAST+ radial glia-like cells as DA progenitors. Combined, our experiments support the concept that the midbrain FP differs from other FP regions and demonstrate that FP radial glia-like cells in the midbrain are neurogenic and give rise to midbrain DA neurons.

Cerebrospinal fluid (CSF) is routinely used for diagnosing and monitoring neurological diseases. The CSF proteins used so far for diagnostic purposes (except for those associated with whole cells) are soluble. Here, we show that human CSF contains specific membrane particles that carry prominin-1/CD133, a neural stem cell marker implicated in brain tumors, notably glioblastoma. Differential and equilibrium centrifugation and detergent solubility analyses showed that these membrane particles were similar in physical properties and microdomain organization to small membrane vesicles previously shown to be released from neural stem cells in the mouse embryo. The levels of membrane particle-associated prominin-1/CD133 declined during childhood and remained constant thereafter, with a remarkably narrow range in healthy adults. Glioblastoma patients showed elevated levels of membrane particle-associated prominin-1/CD133, which decreased dramatically in the final stage of the disease. Hence, analysis of CSF for membrane particles carrying the somatic stem cell marker prominin-1/CD133 offers a novel approach for studying human central nervous system disease.

OBJECTIVES: Many efforts are spent in identifying stem cells in adult pancreas because these could provide a source of beta cells for cell-based therapy of type 1 diabetes. Prominin-1, particularly its specific glycosylation-dependent AC133 epitope, is expressed on stem/progenitor cells of various human tissues and can be used to isolate them. We, therefore, examined its expression in adult human pancreas. METHODS: To detect prominin-1 protein, monoclonal antibody CD133/1 (AC133 clone), which recognizes the AC133 epitope, and the alphahE2 antiserum, which is directed against the human prominin-1 polypeptide, were used. Prominin-1 RNA expression was analyzed by real-time polymerase chain reaction. RESULTS: We report that all duct-lining cells of the pancreas express prominin-1. Most notably, the cells that react with the alphahE2 antiserum also react with the AC133 antibody. After isolation and culture of human exocrine cells, we found a relative increase in prominin-1 expression both at protein and RNA expression level, which can be explained by an enrichment of cells with ductal phenotype in these cultures. CONCLUSIONS: Our data show that pancreatic duct cells express prominin-1 and surprisingly reveal that its particular AC133 epitope is not an exclusive stem and progenitor cell marker.

The neurons of the mammalian brain are generated by progenitors dividing either at the apical surface of the ventricular zone (neuroepithelial and radial glial cells, collectively referred to as apical progenitors) or at its basal side (basal progenitors, also called intermediate progenitors). For apical progenitors, the orientation of the cleavage plane relative to their apical-basal axis is thought to be of critical importance for the fate of the daughter cells. For basal progenitors, the relationship between cell polarity, cleavage plane orientation and the fate of daughter cells is unknown. Here, we have investigated these issues at the very onset of cortical neurogenesis. To directly observe the generation of neurons from apical and basal progenitors, we established a novel transgenic mouse line in which membrane GFP is expressed from the beta-III-tubulin promoter, an early pan-neuronal marker, and crossed this line with a previously described knock-in line in which nuclear GFP is expressed from the Tis21 promoter, a pan-neurogenic progenitor marker. Mitotic Tis21-positive basal progenitors nearly always divided symmetrically, generating two neurons, but, in contrast to symmetrically dividing apical progenitors, lacked apical-basal polarity and showed a nearly randomized cleavage plane orientation. Moreover, the appearance of beta-III-tubulin-driven GFP fluorescence in basal progenitor-derived neurons, in contrast to that in apical progenitor-derived neurons, was so rapid that it suggested the initiation of the neuronal phenotype already in the progenitor. Our observations imply that (i) the loss of apical-basal polarity restricts neuronal progenitors to the symmetric mode of cell division, and that (ii) basal progenitors initiate the expression of neuronal phenotype already before mitosis, in contrast to apical progenitors.

The major cephalic exocrine glands share many morphological and functional features and so can be simultaneously affected in certain autoimmune- and inherited disorders leading to glandular hypofunction. Phenotypic characterization of these exocrine glands is not only an interesting biological issue, but might also be of considerable clinical relevance. The major salivary and lacrimal glands might therefore be potential subjects of future cell-based regenerative/tissue engineering therapeutic approaches. In the present study, we described the expression of the stem and progenitor cell marker Prominin-1 and those of its paralogue, Prominin-2, in the three pairs of major salivary glands, i.e., submandibular-, major sublingual-, and parotid glands in adult mice. We have also documented their expression in the extraorbital lacrimal and meibomian glands (Glandulae tarsales) of the eyelid (Palpebra). Our analysis revealed that murine Prominin-1 and Prominin-2 were differentially expressed in these major cephalic exocrine organs. Expression of Prominin-1 was found to be associated with the duct system, while Prominin-2 expression was mostly, but not exclusively, found in the acinar compartment of these organs with marked differences among the various glands. Finally, we report that Prominin-2, like Prominin-1, is released into the human saliva associated with small membrane particles holding the potential for future diagnostic applications.

Prominin-1 (CD133), a pentaspan membrane glycoprotein that constitutes an important cell surface marker of various, either normal or cancerous, stem cell populations is widely used to isolate or characterize such cells in different systems. Occurring throughout the metazoan evolution with a remarkably conserved genomic organization, it may be expressed as different splice variants with distinctive characteristics. A rational nomenclature has been proposed earlier for their consistent designation across species. Although generally accepted, it seems to be misunderstood in view of the recent report of novel prominin-1 complementary DNAs in rhesus monkey and humans with improper naming. As this may lead to confusion, we have reexamined the genomic organization of prominin-1 in various primates to provide an update that should further clarify the rationale of the nomenclature for prominin-1 gene products. This report comprises (i) the determination of the genomic organization of prominin-1 gene in two non-human primates, i.e. Macaca mulatta and Pan troglodytes, commonly used in research, (ii) the mapping of a new exon that creates an alternative cytoplasmic C-terminal end of prominin-1, (iii) the identification of various potential PDZ-binding domains generated by alternative cytoplasmic C-terminal tails, suggesting that different prominin-1 splice variants might interact with distinct protein partners, and (iv) a summing up of the different prominin-1 splice variants.

Apical-basal polarity of epithelial cells is critical for their symmetric versus asymmetric division and commonly thought to be established in interphase. In a novel type of cell division termed "mirror-symmetric", apical cell constituents accumulate during M-phase at the cleavage furrow, resulting in epithelial daughter cells with opposite apical-basal polarity.

Prominin-2 is a pentaspan membrane glycoprotein structurally related to the cholesterol-binding protein prominin-1, which is expressed in epithelial and non-epithelial cells. Although prominin-1 expression is widespread throughout the organism, the loss of its function solely causes retinal degeneration. The finding that prominin-2 appears to be restricted to epithelial cells, such as those found in kidney tubules, raises the possibility that prominin-2 functionally substitutes prominin-1 in tissues other than the retina and provokes a search for a definition of its morphological and biochemical characteristics. Here, we have investigated, by using MDCK cells as an epithelial cell model, whether prominin-2 shares the biochemical and morphological properties of prominin-1. Interestingly, we have found that, whereas prominin-2 is not restricted to the apical domain like prominin-1 but is distributed in a non-polarized fashion between the apical and basolateral plasma membranes, it retains the main feature of prominin-1, i.e. its selective concentration in plasmalemmal protrusions; prominin-2 is confined to microvilli, cilia and other acetylated tubulin-positive protruding structures. Similar to prominin-1, prominin-2 is partly associated with detergent-resistant membranes in a cholesterol-dependent manner, suggesting its incorporation into membrane microdomains, and binds directly to plasma membrane cholesterol. Finally, prominin-2 is also associated with small membrane particles that are released into the culture media and found in a physiological fluid, i.e. urine. Together, these data show that all the characteristics of prominin-1 are shared by prominin-2, which is in agreement with a possible redundancy in their role as potential organizers of plasma membrane protrusions.

Expansion of the neocortex requires symmetric divisions of neuroepithelial cells, the primary progenitor cells of the developing mammalian central nervous system. Symmetrically dividing neuroepithelial cells are known to form a midbody at their apical (rather than lateral) surface. We show that apical midbodies of neuroepithelial cells concentrate prominin-1 (CD133), a somatic stem cell marker and defining constituent of a specific plasma membrane microdomain. Moreover, these apical midbodies are released, as a whole or in part, into the extracellular space, yielding the prominin-1-enriched membrane particles found in the neural tube fluid. The primary cilium of neuroepithelial cells also concentrates prominin-1 and appears to be a second source of the prominin-1-bearing extracellular membrane particles. Our data reveal novel origins of extracellular membrane traffic that enable neural stem and progenitor cells to avoid the asymmetric inheritance of the midbody observed for other cells and, by releasing a stem cell membrane microdomain, to potentially influence the balance of their proliferation versus differentiation.

Das zentrale Nervensystem von Wirbeltieren entsteht in der Embryonalentwicklung aus einem einschichtigen Epithel, dem Neuroepithel. Waehrend der Neurogenese bildet sich aus diesem einfachen Epithel das komplexe Netzwerk des vielschichtigen Gehirngewebes. Dabei produzieren neurale Stammzellen und die aus ihnen hervorgehenden neuronalen Vorlaeuferzellen (die wir der Einfachheit halber beide unter dem Oberbegriff progenitors zusammenfassen) alle Nervenzellen (Neurone) des Gehirns.

Neurons, astrocytes and oligodendrocytes of the central nervous system (CNS) arise from a common pool of multipotent neuroepithelial progenitor cells lining the walls of the neural tube. Initially, neuroepithelial cells undergo symmetric proliferative divisions, thereby expanding the progenitor pool and determining the size of brain compartments. At the onset of neurogenesis, a subset of progenitors switch to asymmetric or terminal symmetric neurogenic divisions. Maintenance of progenitor cell population throughout the period of neurogenesis is essential to generate the full diversity of neuronal cell types and proper histological pattern. However, the mechanisms responsible for the maintenance of progenitor cells proliferation are far from being fully understood. The family of Y-box proteins is involved in control of proliferation and transformation in various normal and pathological cellular systems, and therefore was considered as a candidate to exert such a function. Y-box proteins have a capacity to bind DNA and RNA, thereby controlling gene expression from transcription to translation. This study aimed to examine the expression of mouse Y-box protein 3 (MSY3) in the developing nervous system and elucidate its putative role in regulation of proliferation of progenitor cells. As presented in this work, the MSY3 protein in the embryonic CNS is expressed solely in progenitor cells and not neurons. Moreover, as shown by two independent approaches: morphologically, i.e. using immunofluorescence and confocal microscopy, and biochemically, MSY3 expression is downregulated concomitantly with the spatiotemporal progression of neurogenesis. Interestingly, in preliminary results it was shown that MSY3 is expressed in Dcx-positive transient amplifying precursors in germinal zones of the adult brain, and in EGF-dependent neurospheres. To evaluate whether MSY3 could regulate the neurogenesis, the levels of the MSY3 protein in the progenitors were acutely downregulated or elevated by electroporation of RNAi or MSY3 expression plasmids, respectively. Neither premature reduction of MSY3 in the neuroepithelium (E9.5-E11.5) nor prolonged expression at the developmental stage when this protein is endogenously downregulated (E10.5-14.5) did affect proliferation versus the cell cycle exit of progenitors. Moreover, in Notch1-deficient progenitors in the cerebellar anlage, which exhibit precocious differentiation, MSY3 was not prematurely downregulated, suggesting that MSY3 also is not an early marker of differentiation. Differential centrifugation, immunoprecipitation and polysomal analysis performed in this study revealed that the MSY3 protein in the developing embryo, as well as in Neuro-2A cells, is associated with RNA. On a sucrose density gradient MSY3 co-fractionates with ribosomes and actively translating polysomes, suggesting that it might have a role in regulation of translation. However, downregulation or overexpression of MSY3 in the Neuro-2A cell line did not affect global translation rates. Other researchers suggested that the MSY3 protein has the redundant function with Y-box protein 1 (YB-1). Accordingly, in our system the MSY3 protein could be co-immunoprecipitated with YB-1. Importantly, developmentally regulated expression of MSY3 is not a hallmark of general translation apparatus, as several other proteins involved in translation did not show similar downregulation. To summarise, this work showed that the MSY3 protein is a marker of proliferation of progenitor cells in the embryonic and adult brain, being absent from neurons. Discovery of the molecular mechanism by which MSY3 exerts its role in the cell could provide the link between the translational machinery and proliferation.

The detection of microRNAs (miRNAs) at single-cell resolution is important for studying the role of these posttranscriptional regulators. Here, we use a dual-fluorescent green fluorescent protein (GFP)-reporter/monomeric red fluorescent protein (mRFP)-sensor (DFRS) plasmid, injected into zebrafish blastomeres or electroporated into defined tissues of mouse embryos in utero or ex utero, to monitor the dynamics of specific miRNAs in individual live cells. This approach reveals, for example, that in the developing mouse central nervous system, miR-124a is expressed not only in postmitotic neurons but also in neuronal progenitor cells. Collectively, our results demonstrate that acute administration of DFRS plasmids offers an alternative to previous in situ hybridization and transgenic approaches and allows the monitoring of miRNA appearance and disappearance in defined cell lineages during vertebrate development.

Stem cell persistence into adulthood requires self-renewal from early developmental stages. In the developing mouse brain, only apical progenitors located at the ventricle are self-renewing, whereas basal progenitors gradually deplete. However, nothing is known about the mechanisms regulating the fundamental difference between these progenitors. Here we show that the conditional deletion of the small Rho-GTPase cdc42 at different stages of neurogenesis in mouse telencephalon results in an immediate increase in basal mitoses. Whereas cdc42-deficient progenitors have normal cell cycle length, orientation of cell division and basement membrane contact, the apical location of the Par complex and adherens junctions are gradually lost, leading to an increasing failure of apically directed interkinetic nuclear migration. These cells then undergo mitoses at basal positions and acquire the fate of basal progenitors. Thus, cdc42 has a crucial role at the apical pole of progenitors, thereby regulating the position of mitoses and cell fate.

The ASPM (abnormal spindle-like microcephaly-associated) protein has previously been implicated in the determination of human cerebral cortical size, but the cell biological basis of this regulation has not been studied. Here we investigate the role of Aspm in mouse embryonic neuroepithelial (NE) cells, the primary stem and progenitor cells of the mammalian brain. Aspm was found to be concentrated at mitotic spindle poles of NE cells and to be down-regulated with their switch from proliferative to neurogenic divisions. Upon RNA interference in telencephalic NE cells, Aspm mRNA is reduced, mitotic spindle poles lack Aspm protein, and the cleavage plane of NE cells is less frequently oriented perpendicular to the ventricular surface of the neuroepithelium. The alteration in the cleavage plane orientation of NE cells increases the probability that these highly polarized cells undergo asymmetric division, i.e., that apical plasma membrane is inherited by only one of the daughter cells. Concomitant with the resulting increase in abventricular cells in the ventricular zone, a larger proportion of NE cell progeny is found in the neuronal layer, implying a reduction in the number of NE progenitor cells upon Aspm knock-down relative to control. Our results demonstrate that Aspm is crucial for maintaining a cleavage plane orientation that allows symmetric, proliferative divisions of NE cells during brain development. These data provide a cell biological explanation of the primary microcephaly observed in humans with mutations in ASPM, which also has implications for the evolution of mammalian brains.

Drosophila Cornichon (Cni) is the founding member of a conserved protein family that also includes Erv14p, an integral component of the COPII-coated vesicles that mediate cargo export from the yeast endoplasmic reticulum (ER). During Drosophila oogenesis, Cni is required for transport of the TGFalpha growth factor Gurken (Grk) to the oocyte surface. Here, we show that Cni, but not the second Drosophila Cni homologue Cni-related (Cnir), binds to the extracellular domain of Grk, and propose that Cni acts as a cargo receptor recruiting Grk into COPII vesicles. Consequently, in the absence of Cni function, Grk fails to leave the oocyte ER. Proteolytic processing of Grk still occurs in cni mutant ovaries, demonstrating that release of the active growth factor from its transmembrane precursor occurs earlier during secretory transport than described for the other Drosophila TGFalpha homologues. Massive overexpression of Grk in a cni mutant background can overcome the requirement of Grk signalling for cni activity, confirming that cni is not essential for the production of the functional Grk ligand. However, the rescued egg chambers lack dorsoventral polarity. This demonstrates that the generation of temporally and spatially precisely coordinated Grk signals cannot be achieved by bulk flow secretion, but instead has to rely on fast and efficient ER export through cargo receptor-mediated recruitment of Grk into the secretory pathway.

The type and number of cell divisions of neuronal progenitors determine the number of neurons generated during the development of the vertebrate central nervous system. Over the past several years, there has been substantial progress in characterizing the various kinds of neuronal progenitors and the types of symmetric and asymmetric divisions they undergo. The understanding of the cell-biological basis of symmetric versus asymmetric progenitor cell division has been consolidated, and the molecular machinery controlling these divisions is beginning to be unravelled. Other recent advances include comparative studies of brain development in rodents and primates, as well as the identification of gene mutations in humans that affect the balance between the various types of cell division of neuronal progenitors.

During the development of the mammalian central nervous system, neural stem cells and their derivative progenitor cells generate neurons by asymmetric and symmetric divisions. The proliferation versus differentiation of these cells and the type of division are closely linked to their epithelial characteristics, notably, their apical-basal polarity and cell-cycle length. Here, we discuss how these features change during development from neuroepithelial to radial glial cells, and how this transition affects cell fate and neurogenesis.

During embryonic development of the mammalian brain, the average cell-cycle length of progenitor cells in the ventricular zone is known to increase. However, for any given region of the developing cortex and stage of neurogenesis, the length of the cell cycle is thought to be similar in the two coexisting subpopulations of progenitors [i.e., those undergoing (symmetric) proliferative divisions and those undergoing (either asymmetric or symmetric) neuron-generating divisions]. Using cumulative bromodeoxyuridine labeling of Tis21-green fluorescent protein knock-in mouse embryos, in which these two subpopulations of progenitors can be distinguished in vivo, we now show that at the onset as well as advanced stages of telencephalic neurogenesis, progenitors undergoing neuron-generating divisions are characterized by a significantly longer cell cycle than progenitors undergoing proliferative divisions. In addition, we find that the recently characterized neuronal progenitors dividing at the basal side of the ventricular zone and in the subventricular zone have a longer G(2) phase than those dividing at the ventricular surface. These findings are consistent with the hypothesis (Calegari and Huttner, 2003) that cell-cycle lengthening can causally contribute to neural progenitors switching from proliferative to neuron-generating divisions and may have important implications for the expansion of somatic stem cells in general.

Apical plasma membrane constituents of mammalian neural stem/progenitor cells have recently been implicated in maintaining their stem/progenitor cell state. Here, we report that in the developing embryonic mouse brain, the fluid in the lumen of the neural tube contains membrane particles carrying the stem cell marker prominin-1 (CD133), a pentaspan membrane protein found on membrane protrusions of the apical surface of neuroepithelial cells. Two size classes of prominin-1-containing membrane particles were observed in the ventricular fluid: approximately 600-nm particles, referred to as P2 particles, and 50-80-nm vesicles, referred to as P4 particles. The P2 and P4 particles appeared in the ventricular fluid at the very onset and during the early phase of neurogenesis, respectively. Concomitant with their appearance, the nature of the prominin-1-containing apical plasma membrane protrusions of neuroepithelial cells changed, in that microvilli were lost and large pleiomorphic protuberances appeared. P4 particles were found in various body fluids of adult humans, including saliva, seminal fluid and urine, and were released by the epithelial model cell line Caco-2 upon differentiation. Importantly, P4 particles were distinct from exosomes. Our results demonstrate the widespread occurrence of a novel class of extracellular membrane particles containing proteins characteristic of stem cells, and raise the possibility that the release of the corresponding membrane subdomains from the apical surface of neural progenitors and other epithelial cells may have a role in tissue development and maintenance. Moreover, the presence of prominin-1-containing membrane particles in human body fluids may provide the basis for a protein-based diagnosis of certain diseases.

The cerebellum is critical for motor coordination and cognitive function and is the target of transformation in medulloblastoma, the most common malignant brain tumor in children. Although the development of granule cells, the most abundant neurons in the cerebellum, has been studied in detail, the origins of other cerebellar neurons and glia remain poorly understood. Here we show that the murine postnatal cerebellum contains multipotent neural stem cells (NSCs). These cells can be prospectively isolated based on their expression of the NSC marker prominin-1 (CD133) and their lack of markers of neuronal and glial lineages (lin-). Purified prominin+ lin- cells form self-renewing neurospheres and can differentiate into astrocytes, oligodendrocytes and neurons in vitro. Moreover, they can generate each of these lineages after transplantation into the cerebellum. Identification of cerebellar stem cells has important implications for the understanding of cerebellar development and the origins of medulloblastoma.

Tyrosine O-sulfation of proteins is a widespread post-translational modification that occursin all animals. The sulfate transfer to tyrosine residues is catalyzed by one of two tyrosylprotein sulfotransferase (TPST) isoenzymes (EC 2.8.2.20; Fig. 14.7.1), which are integral membrane proteins of the trans-Golgi network (TGN), with their catalytic site facing the lumen. Consequently, all tyrosine-sulfated proteins must transit through the TGN. Moreover, the recognition by TPST of the tyrosine residue to be sulfated in a particular protein requires the presence of certain structural features (see Strategic Planning). Tyrosine sulfate in proteins can be identified by various methods, including metabolic labeling using inorganic [35S]-sulfate (see Basic Protocol 1) followed by tyrosine sulfate analysis (see Basic Protocol 2). The [35S]-labeling may be performed with whole animals, tissue explants, tissue slices, and cells in culture. This unit outlines procedures to determine whether a protein of interest contains a tyrosine-sulfated residue, using mammalian adherent cells.

Prominin-1/CD133 is a plasma membrane marker found in several types of somatic stem cells, including hematopoietic and neural stem cells. To study its role during development and with differentiation, we analyzed its temporal and spatial expression (mRNA and protein) in preimplantation embryos, undifferentiated mouse embryonic stem (ES) cells, and differentiated ES cell progeny. In early embryos, prominin-1 was expressed in trophoblast but not in cells of the inner cell mass; however, prominin-1 transcripts were detected in undifferentiated ES cells. Both ES-derived cells committed to differentiation and early progenitor cells coexpressed prominin-1 with early lineage markers, including the cytoskeletal markers (nestin, cytokeratin 18, desmin), fibulin-1, and valosin-containing protein. After spontaneous differentiation at terminal stages, prominin-1 expression was downregulated and no coexpression with markers characteristic for neuroectodermal, mesodermal, and endodermal cells was found. Upon induction of neuronal differentiation, some prominin-1-positive cells, which coexpressed nestin and showed the typical morphology of neural progenitor cells, persisted until terminal stages of differentiation. However, no coexpression of prominin-1 with markers of differentiated neural cells was detected. In conclusion, we present the somatic stem cell marker prominin-1 as a new parameter to define ES-derived committed and early progenitor cells.

Human prominin-1/CD133 has been reported to be expressed in neural and hematopoietic stem/progenitor cells and in embryonic, but not adult, epithelia. This lack of detection of human prominin-1, as defined by its glycosylation-dependent AC133 epitope, is surprising given the expression of the murine ortholog in adult epithelia. Here, we demonstrate, by using a novel prominin-1 antiserum (alphahE2), that the decrease of AC133 immunoreactivity observed during differentiation of the colonic adenocarcinoma-derived Caco-2 cells is not paralleled by a down-regulation of prominin-1. We have also shown that alphahE2 immunoreactivity, but not AC133 immunoreactivity, is present in several adult human tissues, such as kidney proximal tubules and the parietal layer of Bowman's capsule of juxtamedullary nephrons, and in lactiferous ducts of the mammary gland. These observations suggest that only the AC133 epitope is down-regulated upon cell differentiation. Furthermore, alphahE2 immunoreactivity has been detected in several kidney carcinomas derived from proximal tubules, independent of their grading. Interestingly, in one particular case, the AC133 epitope, which is restricted to stem cells in normal adult tissue, was up-regulated in the vicinity of the tumor. Our data thus show that (1) in adults, the expression of human prominin-1 is not limited to stem and progenitor cells, and (2) the epitopes of prominin-1 might be useful for investigating solid cancers.

Rab13 is recruited to tight junctions from a cytosolic pool after cell-cell contact formation. Tight junctions are intercellular junctions that separate apical from basolateral domains and are required for the establishment/maintenance of polarized transport in epithelial cells. They form selective barriers regulating the diffusion of ions and solutes between cells. They also maintain the cell surface asymmetry by forming a "fence" that prevents apical/basolateral diffusion of membrane proteins and lipids in the outer leaflet of the plasma membrane. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delays the formation of electrically tight epithelial monolayers, induces the leakage of small nonionic tracers from the apical domain, and disrupts the tight junction fence diffusion barrier. It also alters the tight junction strand structure and delays the localization of the tight junction transmembrane protein, claudin1. In contrast, the inactive Rab13T22N mutant does not disrupt tight junction functions, tight junction strand architecture, or claudin1 localization. Here we describe a set of assays that allows us to investigate the role of Rab13 in modulating tight junction structure and function.

During ontogenesis and the entire adult life hematopoietic stem and progenitor cells have the capability to migrate. In comparison to the process of peripheral leukocyte migration in inflammatory responses, the molecular and cellular mechanisms governing the migration of these cells remain poorly understood. A common feature of migrating cells is that they need to become polarized before they migrate. Here we have investigated the issue of cell polarity of hematopoietic stem/progenitor cells in detail. We found that human CD34(+) hematopoietic cells (1) acquire a polarized cell shape upon cultivation, with the formation of a leading edge at the front pole and a uropod at the rear pole; (2) exhibit an amoeboid movement, which is similar to the one described for migrating peripheral leukocytes; and (3) redistribute several lipid raft markers including cholesterol-binding protein prominin-1 (CD133) in specialized plasma membrane domains. Furthermore, polarization of CD34(+) cells is stimulated by early acting cytokines and requires the activity of phosphoinositol-3-kinase as previously reported for peripheral leukocyte polarization. Together, our data reveal a strong correlation between polarization and migration of peripheral leukocytes and hematopoietic stem/progenitor cells and suggest that they are governed by similar mechanisms.

Prominin-1/CD133 is a five-membrane-span glycoprotein that is thought to act as an organizer of plasma-membrane protrusions. Here, we report the molecular and cell-biological characterization of four novel prominin-1 splice variants isolated from a mouse testis cDNA library and referred to as prominin-1.s3 to prominin-1.s6. Compared with kidney-derived prominin-1.s1, the s3, s4 and s5 variants contain a distinct cytoplasmic C-terminal domain. The s4 and s5 variants bear, in addition, two and one inframe deletion(s), respectively, in the extracellular domains. The s6 variant displays a truncated C-terminal domain caused by a premature termination resulting from intron retention. Upon their ectopic expression in Chinese hamster ovary cells, the s3 and s6 variants were found to be concentrated in plasma-membrane protrusions, whereas the s4 and s5 variants did not reach the cell surface. Biochemical analyses suggest that most of the prominin-1 in the adult male reproductive system is expressed as the s6 variant. Immunohistological and electron microscopic analyses show that prominin-1 is: (1) confined to the apical surface of the epithelium all along the epididymal duct, with the exception of the initial segment; (2) concentrated in stereocilia of the epididymal duct epithelium; and (3) found on the tail of developing spermatozoa in seminiferous tubules. Our data suggest that prominin-1 is involved in the formation and/or stabilization of epididymal stereocilia and the tail of spermatozoa, and hence might play a dual role in the biogenesis of spermatozoa.

At the onset of neurogenesis in the mammalian central nervous system, neuroepithelial cells switch from symmetric, proliferative to asymmetric, neurogenic divisions. In analogy to the asymmetric division of Drosophila neuroblasts, this switch of mammalian neuroepithelial cells is thought to involve a change in cleavage plane orientation from perpendicular (vertical cleavage) to parallel (horizontal cleavage) relative to the apical surface of the neuroepithelium. Here, we report, using TIS21-GFP knock-in mouse embryos to identify neurogenic neuroepithelial cells, that at the onset as well as advanced stages of neurogenesis the vast majority of neurogenic divisions, like proliferative divisions, show vertical cleavage planes. Remarkably, however, neurogenic divisions of neuroepithelial cells, but not proliferative ones, involve an asymmetric distribution to the daughter cells of the apical plasma membrane, which constitutes only a minute fraction (1-2%) of the entire neuroepithelial cell plasma membrane. Our results support a novel concept for the cell biological basis of asymmetric, neurogenic divisions of neuroepithelial cells in the mammalian central nervous system.

We have expressed the neuroendocrine VGF protein in FRT rat thyroid cells to study the molecular mechanisms of its sorting to the regulated and polarized pathways of secretion. By immunoelectron microscopy, we have demonstrated that VGF localizes in dense-core granules. Rapid secretion of VGF is induced by PMA stimulation. Moreover, human chromogranin B, a protein of the regulated pathway, co-localizes in the same granules with VGF. In confluent, FRT monolayers on filters protein secretion occur from the apical cell domain. VGF deletion mutants have been generated. By confocal microscopy, we have found that in transient transfection, all mutant proteins are sorted into granules and co-localize with the full-length VGF. They all retain the apical polarity of secretion. We also found that intracellular VGF and its deletion mutants are largely in an aggregated form. We conclude that FRT thyroid cells correctly decode the sorting information of VGF. The signals present on the protein to enter the granules and to be secreted apically cannot be separated from each other and are not in just one discrete portion of the protein. We propose that selective aggregation might represent the signal for sorting VGF to the regulated, apical route.

In mammals, embryonic development is more difficult to analyze than in non-mammalian species because this development occurs in utero. Interestingly, whole embryo culture allows the normal development of mouse post-implantation embryos for up to 2 days in vitro. One limitation of this technology has been the difficulty of performing loss-of-gene function studies in this system. RNA interference (RNAi), whereby double-stranded RNA molecules suppress the expression of complementary genes, has rapidly become a widely used tool for gene function analyses. We have combined the technologies of mouse whole embryo culture and RNAi to allow the molecular dissection of developmental processes. Here, we review the manipulation by topical injection followed by directional electroporation of endoribonuclease-prepared siRNA to demonstrate that this technology may be useful to knock down genes in a tissue- and region-specific manner in several organs of the developing mouse embryo.

Neurons of the mammalian CNS are thought to originate from progenitors dividing at the apical surface of the neuroepithelium. Here we use mouse embryos expressing GFP from the Tis21 locus, a gene expressed throughout the neural tube in most, if not all, neuron-generating progenitors, to specifically reveal the cell divisions that produce CNS neurons. In addition to the apical, asymmetric divisions of neuroepithelial (NE) cells that generate another NE cell and a neuron, we find, from the onset of neurogenesis, a second population of progenitors that divide in the basal region of the neuroepithelium and generate two neurons. Basal progenitors are most frequent in the telencephalon, where they outnumber the apically dividing neuron-generating NE cells. Our observations reconcile previous data on the origin and lineage of CNS neurons and show that basal, rather than apical, progenitors are the major source of the neurons of the mammalian neocortex.

The O-sulfation of tyrosine residues of membrane and secretory proteins that transit through the secretory pathway of eukaryotic cells is a ubiquitous posttranslational modification conserved in all multicellular organisms. Tyrosine sulfation is catalyzed by tyrosylprotein sulfotransferase (TPST) isoenzymes, which are integral membrane proteins of the trans-Golgi network. Tyrosine sulfation has been shown to be important for protein–protein interactions occurring in diverse biological processes, ranging from the receptor binding of regulatory peptides to the interaction of viral envelope proteins with the cell surface.

The G1 phase of the cell cycle of neuroepithelial cells, the progenitors of all neurons of the mammalian central nervous system, has been known to lengthen concomitantly with the onset and progression of neurogenesis. We have investigated whether lengthening of the G1 phase of the neuroepithelial cell cycle is a cause, rather than a consequence, of neurogenesis. As an experimental system, we used whole mouse embryo culture, which was found to exactly reproduce the temporal and spatial gradients of the onset of neurogenesis occurring in utero. Olomoucine, a cell-permeable, highly specific inhibitor of cyclin-dependent kinases and G1 progression, was found to significantly lengthen, but not arrest, the cell cycle of neuroepithelial cells when used at 80 microM. This olomoucine treatment induced, in the telencephalic neuroepithelium of embryonic day 9.5 to 10.5 mouse embryos developing in whole embryo culture to embryonic day 10.5, (i) the premature up-regulation of TIS21, a marker identifying neuroepithelial cells that have switched from proliferative to neuron-generating divisions, and (ii) the premature generation of neurons. Our data indicate that lengthening G1 can alone be sufficient to induce neuroepithelial cell differentiation. We propose a model that links the effects of cell fate determinants and asymmetric cell division to the length of the cell cycle.

The majority of cells that build the nervous system of animals are generated early in embryonic development in a process called neurogenesis. Although the vertebrate nervous system is much more complex than that of insects, the underlying principles of neurogenesis are intriguingly similar. In both cases, neuronal cells are derived from polarized progenitor cells that divide asymmetrically. One daughter cell will continue to divide and the other daughter cell leaves the cell cycle and starts to differentiate as a neuron or a glia cell. In Drosophila, this process has been analyzed in great detail and several of the key players that control asymmetric cell division in the developing nervous system have been identified over the past years. Asymmetric cell division in vertebrate neurogenesis has been studied mostly at a descriptive level and so far little is known about the molecular mechanisms that control this process. In this review we will focus on recent findings dealing with asymmetric cell division during neurogenesis in Drosophila and vertebrates and will discuss common principles and apparent differences between both systems.

Prominin/CD133 is a 115/120-kDa integral membrane glycoprotein specifically associated with plasma membrane protrusions in epithelial and non-epithelial cells including neuroepithelial and hematopoietic stem cells. Here we report the identification as well as molecular and cell biological characterization of mouse, rat, and human prominin-2, a 112-kDa glycoprotein structurally related to prominin (referred to as prominin-1). Although the amino acid identity between prominin-2 and prominin-1 is low (<30%), their genomic organization is strikingly similar, suggesting an early gene duplication event. Like prominin-1, prominin-2 exhibits a characteristic membrane topology with five transmembrane segments and two large glycosylated extracellular loops. Upon its ectopic expression in Chinese hamster ovary cells as a green fluorescent protein fusion chimera, prominin-2 was also found to be associated with plasma membrane protrusions, as revealed by its co-localization with prominin-1, suggesting a related role. Consistent with this, prominin-2 shows a similar tissue distribution to prominin-1, being highly expressed in the adult kidney and detected all along the digestive tract as well as in various other epithelial tissues. However, in contrast to prominin-1, prominin-2 was not detected in the eye, which perhaps explains why a loss-of function mutation in the human prominin-1 gene causes retinal degeneration but no other obvious pathological signs. Finally, we present evidence for the existence of a family of pentaspan membrane proteins, the prominins, which are conserved in evolution.

We have characterized mammalian endophilin B1, a novel member of the endophilins and a representative of their B subgroup. The endophilins B show the same domain organization as the endophilins A, which contain an N-terminal domain responsible for lipid binding and lysophosphatidic acid acyl transferase activity, a central coiled-coil domain for oligomerization, a less conserved linker region, and a C-terminal Src homology 3 (SH3) domain. The endophilin B1 gene gives rise to at least three splice variants, endophilin B1a, which shows a widespread tissue distribution, and endophilins B1b and B1c, which appear to be brain-specific. Endophilin B1, like endophilins A, binds to palmitoyl-CoA, exhibits lysophosphatidic acid acyl transferase activity, and interacts with dynamin, amphiphysins 1 and 2, and huntingtin. However, in contrast to endophilins A, endophilin B1 does not bind to synaptojanin 1 and synapsin 1, and overexpression of its SH3 domain does not inhibit transferrin endocytosis. Consistent with this, immunofluorescence analysis of endophilin B1b transfected into fibroblasts shows an intracellular reticular staining, which in part overlaps with that of endogenous dynamin. Upon subcellular fractionation of brain and transfected fibroblasts, endophilin B1 is largely recovered in association with membranes. Together, our results suggest that the action of the endophilins is not confined to the formation of endocytic vesicles from the plasma membrane, with endophilin B1 being associated with, and presumably exerting a functional role at, intracellular membranes.

ATP is released from astrocytes and is involved in the propagation of calcium waves among them. Neuronal ATP secretion is quantal and calcium-dependent, but it has been suggested that ATP release from astrocytes may not be vesicular. Here we report that, besides the described basal ATP release facilitated by exposure to calcium-free medium, astrocytes release purine under conditions of elevated calcium. The evoked release was not affected by the gap-junction blockers anandamide and flufenamic acid, thus excluding purine efflux through connexin hemichannels. Sucrose-gradient analysis revealed that a fraction of ATP is stored in secretory granules, where it is accumulated down an electrochemical proton gradient sensitive to the v-ATPase inhibitor bafilomycin A(1). ATP release was partially sensitive to tetanus neurotoxin, whereas glutamate release from the same intoxicated astrocytes was almost completely impaired. Finally, the activation of metabotropic glutamate receptors, which strongly evokes glutamate release, was only slightly effective in promoting purine secretion. These data indicate that astrocytes concentrate ATP in granules and may release it via a regulated secretion pathway. They also suggest that ATP-storing vesicles may be distinct from glutamate-containing vesicles, thus opening up the possibility that their exocytosis is regulated differently.

Microtubules are essential for various cellular processes including cell division and intracellular organization. Their function depends on their ability to rearrange their distribution at different times and places. Microtubules are dynamic polymers and their behaviour is described as dynamic instability. Rearrangement of the microtubule cytoskeleton is made possible by proteins that modulate the parameters of dynamic instability. Studies using Xenopus egg extracts led to identification of a microtubule-associated protein called XMAP215 as a major regulator of physiological microtubule dynamics. XMAP215 belongs to an evolutionarily conserved protein family present in organisms ranging from yeast to mammals. Together with members of the Kin I family of kinesins, XMAP215 and its orthologues form an essential circuit for generating dynamic microtubules in vivo.

In epithelial cells, soluble cargo proteins destined for basolateral or apical secretion are packaged into distinct trans-Golgi network-derived transport carriers. Similar carriers, termed basolateral- and apical-like, have been observed in nonepithelial cells using ectopically expressed membrane marker proteins. Whether these cells are capable of selectively packaging secretory proteins into distinct carriers is still an open question. Here, we have addressed this issue by analyzing the packaging and transport of secretory human chromogranin B fusion proteins using a green fluorescent protein-based high-resolution, dual-color imaging technique. We were able to show that these secretory markers were selectively packaged at the Golgi into tubular/vesicular-like transport carriers containing basolateral membrane markers, resulting in extensive cotransport. In contrast, deletion mutants of the human chromogranin B fusion proteins lacking an N-terminal loop structure were efficiently transported in both basolateral- and apical-like carriers, the latter displaying a spherical morphology. Similarly, in polarized epithelial cells, the human chromogranin B fusion protein was secreted basolaterally and the loop-deleted analogue into both the basolateral and apical medium. These findings suggest that nonepithelial cells, like their epithelial counterparts, possess a sorting machinery capable of selective packaging of secretory cargo into distinct types of carriers.

Endophilin A1, a cytoplasmic protein essential for the budding and fission of synaptic vesicles from presynaptic plasma membranes, is implicated in the generation of membrane curvature. Endophilin A1 exhibits intrinsic lysophosphatidic acid acyl transferase activity, reflecting its interaction with both the hydrophobic portion and the headgroup of acidic phospholipids. A recent study demonstrates that endophilin A1 binds to liposomes and alone is sufficient to deform them into narrow tubules, thus generating positive bilayer curvature. The recently identified endophilins B, which associate with membranes of the early secretory pathway, also bind to acidic phospholipids and tubulate liposomes, as do the endophilin-interacting proteins amphiphysin and dynamin. Thus, a novel concept for tubulo-vesicular membrane dynamics emerges in which a team of proteins distinct from, but often operating in concert with, the classical coat proteins is pivotal in the generation of membrane curvature.

Microtubules are dynamically unstable polymers that interconvert stochastically between polymerization and depolymerization. Compared with microtubules assembled from purified tubulin, microtubules in a physiological environment polymerize faster and transit more frequently between polymerization and depolymerization. These dynamic properties are essential for the functions of the microtubule cytoskeleton during diverse cellular processes. Here, we have reconstituted the essential features of physiological microtubule dynamics by mixing three purified components: tubulin; a microtubule-stabilizing protein, XMAP215; and a microtubule-destabilizing kinesin, XKCM1. This represents an essential first step in the reconstitution of complex microtubule dynamics-dependent processes, such as chromosome segregation, from purified components.

At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids. Subsequently, spindles elongate, separating the sister chromosomes into two sets. Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication. We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation. Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation. Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1. Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1.

SNARE (Soluble N-ethyl-maleimide sensitive factor Attachment protein Receptor) proteins assemble in tight core complexes, which promote fusion of carrier vesicles with target compartments. Members of this class of proteins are expressed in all eukaryotic cells and are distributed in distinct subcellular compartments. The molecular mechanisms underlying sorting of SNAREs to their physiological sites of action are still poorly understood. Here have we analyzed the transport of syntaxin1A in epithelial cells. In line with previous data we found that syntaxin1A is not transported to the plasma membrane, but rather is retained intracellularly when overexpressed in MDCK and Caco-2 cells. Its delivery to the cell surface is recovered after munc-18-1 cotransfection. Furthermore, overexpression of the ubiquitous isoform of munc-18, munc-18-2, is also capable of rescuing the transport of the t-SNARE. The interaction between syntaxin 1A and munc-18 occurs in the biosynthetic pathway and is required to promote the exit of the t-SNARE from the Golgi complex. This enabled us to investigate the targeting of syntaxin1A in polarized cells. Confocal analysis of polarized monolayers demonstrates that syntaxin1A is delivered to both the apical and basolateral domains independently of the munc-18 proteins used in the cotranfection experiments. In search of the mechanisms underlying syntaxin 1A sorting to the cell surface, we found that a portion of the protein is included in non-ionic detergent insoluble complexes. Our results indicate that the munc-18 proteins represent limiting but essential factors in the transport of syntaxin1A from the Golgi complex to the epithelial cell surface. They also suggest the presence of codominant apical and basolateral sorting signals in the syntaxin1A sequence.

Molecular cell biology is now facing the challenges of the post-genomic era. In this regard, the recently established Max-Planck-Institute of Molecular Cell Biology and Genetics in Dresden, Germany, provides interesting perspectives. Its atypical structure and the unique mixture of research topics and model systems give this Max-Planck-Institute the necessary versatility and flexibility for this new phase of biology.

The >1 kb XL-exon of the rat XLalphas/Galphas gene encodes the 37 kDa XL-domain, the N-terminal half of the 78 kDa neuroendocrine-specific G-protein alpha-subunit XLalphas. Here, we describe a novel feature of the XL-exon, the presence of an alternative >1 kb open reading frame (ORF) that completely overlaps with the ORF encoding the XL-domain. The alternative ORF starts 32 nucleotides downstream of the start codon for the XL-domain and is terminated by a stop codon exactly at the end of the XL-exon. The alternative ORF encodes ALEX, a very basic (pI 11.8), proline-rich protein of 356 amino acids. Both XLalphas and ALEX are translated from the same mRNA. Like XLalphas, ALEX is expressed in neuroendocrine cells and tightly associated with the cytoplasmic leaflet of the plasma membrane. Remarkably, ALEX binds to the XL-domain of XLalphas. Our results reveal a mechanism of gene usage that is without precedent in mammalian genomes.

Mouse prominin is the first characterized member of a novel family of membrane glycoproteins. It displays a characteristic membrane topology with five transmembrane segments and two large glycosylated extracellular loops. Prominin orthologues and paralogues have been identified in human, fish, fly, and worm. Recently, a cDNA sequence encoding the rat homologue of mouse prominin has been reported [Zhu et al. (2001) Biochem. Biophys. Res. Commun. 281, 951-956]. Surprisingly, due to a single nucleotide deletion that shifts the reading frame and introduces a premature stop codon, the protein predicted from this cDNA would correspond to a C-terminally truncated form of prominin with only four transmembrane segments. Here we report evidence that is in contrast to the report of Zhu et al. (2001). We isolated a rat prominin cDNA devoid of any frameshift mutation, demonstrate that rat prominin, like the other mammalian prominins, is a full-length 120-kDa pentaspan membrane glycoprotein, and have not been able to detect any C-terminally truncated form of rat prominin.

During anaphase, mitotic spindles elongate up to five times their metaphase length. This process, known as anaphase B, is essential for correct segregation of chromosomes. Here, we examine the control of spindle length during anaphase in the budding yeast Saccharomyces cerevisiae. We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2. We further show that the activity of Stu2 is opposed by the activity of the kinesin-related protein Kip3. Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I). We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.

Prominin is the first identified member of a novel family of polytopic membrane proteins conserved throughout the animal kingdom. It has an unusual membrane topology, containing five transmembrane domains and two large glycosylated extracellular loops. In mammals, prominin is expressed in various embryonic and adult epithelial cells, as well as in nonepithelial cells, such as hematopoietic stem cells. At the subcellular level, prominin is selectively localized in microvilli and other plasma membrane protrusions, irrespective of cell type. At the molecular level, prominin specifically interacts with membrane cholesterol and is a marker of a novel type of cholesterol-based lipid 'raft'. A frameshift mutation in the human prominin gene, which results in a truncated protein that is no longer transported to the cell surface, is associated with retinal degeneration. Given that prominin is concentrated in the plasma membrane evaginations at the base of the outer segment of rod photoreceptor cells, which are essential precursor structures in the biogenesis of photoreceptive disks, it is proposed that prominin has a role in the generation of plasma membrane protrusions, their lipid composition and organization and their membrane-to-membrane interactions.

Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial polyposis, a form of inherited colon cancer. In addition to its role in mediating beta-catenin degradation in the Wnt signaling pathway, APC plays a role in regulating microtubules. This was suggested by its localization to the end of dynamic microtubules in actively migrating areas of cells and by the apparent correlation between the dissociation of APC from polymerizing microtubules and their subsequent depolymerization [1, 2]. The microtubule binding domain is deleted in the transforming mutations of APC [3, 4]; however, the direct effect of APC protein on microtubules has never been examined. Here we show that binding of APC to microtubules increases microtubule stability in vivo and in vitro. Deleting the previously identified microtubule binding site from the C-terminal domain of APC does not eliminate its binding to microtubules but decreases the ability of APC to stabilize them significantly. The interaction of APC with microtubules is decreased by phosphorylation of APC by GSK3 beta. These data confirm the hypothesis that APC is involved in stabilizing microtubule ends. They also suggest that binding of APC to microtubules is mediated by at least two distinct sites and is regulated by phosphorylation.

In the preceding paper (Pasolli, H. A., Klemke, M., Kehlenbach, R. H. , Wang, Y., and Huttner, W. B. (2000) J. Biol. Chem. 275, 33622-33632), we report on the tissue distribution and subcellular localization of XLalphas (extra large alphas), a neuroendocrine-specific, plasma membrane-associated protein consisting of a novel 37-kDa XL domain followed by a 41-kDa alphas domain encoded by exons 2-13 of the Galphas gene. Here, we have studied the signal transduction properties of XLalphas. Like Galphas, XLalphas undergoes a conformational change upon binding of GTPgammaS (guanosine 5'-O-(thio)triphosphate), as revealed by its partial resistance to tryptic digestion, which generated the same fragments as in the case of Galphas. Two approaches were used to analyze XLalphas-betagamma interactions: (i) ADP-ribosylation by cholera toxin to detect even weak or transient XLalphas-betagamma interactions and (ii) sucrose density gradient centrifugation to reveal stable heterotrimer formation. The addition of betagamma subunits resulted in an increased ADP-ribosylation of XLalphas as well as an increased sedimentation rate of XLalphas in sucrose density gradients, indicating that XLalphas interacts with the betagamma dimer. Surprisingly, however, XLalphas, in contrast to Galphas, was not activated by the beta2-adrenergic receptor upon reconstitution of S49cyc(-) membranes. Similarly, using photoaffinity labeling of pituitary membranes with azidoanilide-GTP, XLalphas was not activated upon stimulation of pituitary adenylyl cyclase-activating polypeptide (PACAP) receptors or other Galphas-coupled receptors known to be present in these membranes, whereas Galphas was. Despite the apparent inability of XLalphas to undergo receptor-mediated activation, XLalphas-GTPgammaS markedly stimulated adenylyl cyclase in S49cyc(-) membranes. Moreover, transfection of PC12 cells with a GTPase-deficient mutant of XLalphas, XLalphas-Q548L, resulted in a massive increase in adenylyl cyclase activity. Our results suggest that in neuroendocrine cells, the two related G proteins, Galphas and XLalphas, exhibit distinct properties with regard to receptor-mediated activation but converge onto the same effector system, adenylyl cyclase.

Membrane cholesterol-sphingolipid 'rafts', which are characterized by their insolubility in the non-ionic detergent Triton X-100 in the cold, have been implicated in the sorting of certain membrane proteins, such as placental alkaline phosphatase (PLAP), to the apical plasma membrane domain of epithelial cells. Here we show that prominin, an apically sorted pentaspan membrane protein, becomes associated in the trans-Golgi network with a lipid raft that is soluble in Triton X-100 but insoluble in another non-ionic detergent, Lubrol WX. At the cell surface, prominin remains insoluble in Lubrol WX and is selectively associated with microvilli, being largely segregated from the membrane subdomains containing PLAP. Cholesterol depletion results in the loss of prominin's microvillus-specific localization but does not lead to its complete intermixing with PLAP. We propose the coexistence within a membrane domain, such as the apical plasma membrane, of different cholesterol-based lipid rafts, which underlie the generation and maintenance of membrane subdomains.

The human AC133 antigen and mouse prominin are structurally related plasma membrane proteins. However, their tissue distribution is distinct, with the AC133 antigen being found on hematopoietic stem and progenitor cells and prominin on various epithelial cells. To determine whether the human AC133 antigen and mouse prominin are orthologues or distinct members of a protein family, we examined the human epithelial cell line Caco-2 for the possible expression of the AC133 antigen. By both immunofluorescence and immunoprecipitation, the AC133 antigen was found to be expressed on the surface of Caco-2 cells. Interestingly, immunoreactivity for the AC133 antigen, but not its mRNA level, was down-regulated upon differentiation of Caco-2 cells. The AC133 antigen was specifically located at the apical rather than basolateral plasma membrane. An apical localization of the AC133 antigen was also observed in various human embryonic epithelia including the neural tube, gut, and kidney. Electron microscopy revealed that, within the apical plasma membrane of Caco-2 cells, the AC133 antigen was confined to microvilli and absent from the planar, intermicrovillar regions. This specific subcellular localization did not depend on an epithelial phenotype, because the AC133 antigen on hematopoietic stem cells, as well as that ectopically expressed in fibroblasts, was selectively found in plasma membrane protrusions. Hence, the human AC133 antigen shows the features characteristic of mouse prominin in epithelial and transfected non-epithelial cells, i.e. a selective association with apical microvilli and plasma membrane protrusions, respectively. Conversely, flow cytometry of murine CD34(+) bone marrow progenitors revealed the cell surface expression of prominin. Taken together, the data strongly suggest that the AC133 antigen is the human orthologue of prominin.

The disks of vertebrate photoreceptors are produced by outgrowths of the plasma membrane. Hence genes that encode retinal proteins targeted to plasma membrane protrusions represent candidates for inherited retinal degenerations. One such candidate is the gene encoding human prominin (mouse)-like 1 (PROML1, previously known as AC133 antigen) which belongs to the prominin family of 5-transmembrane domain proteins. Murine prominin (prom) shows a strong preference for plasma membrane protrusions in a variety of epithelial cells whereas PROML1 is expressed in retinoblastoma cell lines and adult retina. In the present study, molecular genetic analyses of a pedigree segregating for autosomal recessive retinal degeneration indicated that the affected individuals were homozygous for a nucleotide 1878 deletion in PROML1. This alteration is predicted to result in a frameshift at codon 614 with premature termination of translation. Expression of a similar prom deletion mutant in CHO cells indicated that the truncated protein does not reach the cell surface. Immunocytochemistry revealed that prom is concentrated in the plasma membrane evaginations at the base of the outer segments of rod photoreceptors. These findings suggest that loss of prominin causes retinal degeneration, possibly because of impaired generation of the evaginations and/or impaired conversion of the evaginations to disks.

The small GTPase Rab5 regulates membrane docking and fusion in the early endocytic pathway. Here we reveal a new role for Rab5 in the regulation of endosome interactions with the microtubule network. Using Rab5 fused to green fluorescent protein we show that Rab5-positive endosomes move on microtubules in vivo. In vitro, Rab5 stimulates both association of early endosomes with microtubules and early-endosome motility towards the minus ends of microtubules. Moreover, similarly to endosome membrane docking and fusion, Rab5-dependent endosome movement depends on the phosphatidylinositol-3-OH kinase hVPS34. Thus, Rab5 functionally links regulation of membrane transport, motility and intracellular distribution of early endosomes.

Prominin is a recently identified polytopic membrane protein expressed in various epithelial cells, where it is selectively associated with microvilli. When expressed in non-epithelial cells, prominin is enriched in plasma membrane protrusions. This raises the question of whether the selective association of prominin with microvilli in epithelial cells is solely due to its preference for, and stabilization in, plasma membrane protrusions, or is due to both sorting to the apical plasma membrane domain and subsequent enrichment in plasma membrane protrusions. To investigate this question, we have generated stably transfected MDCK cells expressing either full-length or C-terminally truncated forms of mouse prominin. Confocal immunofluorescence and domain-selective cell surface biotinylation experiments on transfected MDCK cells grown on permeable supports demonstrated the virtually exclusive apical localization of prominin at steady state. Pulse-chase experiments in combination with domain-selective cell surface biotinylation showed that newly synthesized prominin was directly targeted to the apical plasma membrane domain. Immunoelectron microscopy revealed that prominin was confined to microvilli rather than the planar region of the apical plasma membrane. Truncation of the cytoplasmic C-terminal tail of prominin impaired neither its apical cell surface expression nor its selective retention in microvilli. Both the apical-specific localization of prominin and its selective retention in microvilli were maintained when MDCK cells were cultured in low-calcium medium, i.e. in the absence of tight junctions. Taken together, our results show that: (i) prominin contains dual targeting information, for direct delivery to the apical plasma membrane domain and for the enrichment in the microvillar subdomain; and (ii) this dual targeting does not require the cytoplasmic C-terminal tail of prominin and still occurs in the absence of tight junctions. The latter observation suggests that entry into, and retention in, plasma membrane protrusions may play an important role in the establishment and maintenance of the apical-basal polarity of epithelial cells.

Synaptic vesicles, which have been a paradigm for the fusion of a vesicle with its target membrane, also serve as a model for understanding the formation of a vesicle from its donor membrane. Synaptic vesicles, which are formed and recycled at the periphery of the neuron, contain a highly restricted set of neuronal proteins. Insight into the trafficking of synaptic vesicle proteins has come from studying not only neurons but also neuroendocrine cells, which form synaptic-like microvesicles (SLMVs). Formation and recycling of synaptic vesicles/SLMVs takes place from the early endosome and the plasma membrane. The cytoplasmic machinery of synaptic vesicle/SLMV formation and recycling has been studied by a variety of experimental approaches, in particular using cell-free systems. This has revealed distinct machineries for membrane budding and fission. Budding is mediated by clathrin and clathrin adaptors, whereas fission is mediated by dynamin and its interacting protein SH3p4, a lysophosphatidic acid acyl transferase.

Secretory granules are the cellular organelles mediating storage and regulated secretion of proteins. Their biogenesis involves sorting of secretory protein cargo and membrane constituents, which takes place at two distinct levels, the trans-Golgi network and the immature secretory granule. At both levels, sorting is accomplished by cargo aggregation and cargo-membrane recognition. Given not only the aggregative properties of the regulated secretory proteins but also the ability of lipids to form distinct membrane microdomains, self-organization of both lumenal and membrane constituents is proposed to play a crucial role in secretory granule biogenesis.

Chromogranins A and B, two widespread neuroendocrine secretory proteins, contain a homologous N-terminal disulfide-bonded loop that is required for sorting to secretory granules. Here we have investigated the role of this loop in the oligomerization of chromogranin A. Reduction of the disulfide bond or the addition of an excess of an N-terminal chromogranin A fragment containing the loop (CgA1-60) resulted in the dissociation into monomers of the chromogranin A dimer found at pH 7.4 and 6.4 and of the chromogranin tetramer found at pH 5.4. The addition of an excess of a synthetic peptide corresponding to the conserved C-terminal domain of chromogranin A (CgA406-431) had no effect on the chromogranin dimers at pH 7.4 and 6.4 and resulted in the dissociation of the chromogranin A tetramers at pH 5.4 into dimers. Fluorescence energy transfer experiments using fluorescently labeled CgA1-60 showed that the N-terminal disulfide-bonded loop has a high affinity for homodimerization (KD = 20 nM at pH 6.4), which was sufficient to mediate dimerization of full-length chromogranin A. Association and dissociation of loop-mediated chromogranin A dimerization approached completion within a few seconds. Our results imply that chromogranin A homodimerizes shortly after synthesis in the endoplasmic reticulum and that the loop-mediated homodimeric state is an essential prerequisite for its sorting, in the trans-Golgi-network, to secretory granules.