Embryonic Neuroepithelial Cells Research Articles

Single-cell Profiling of Reprogrammed Human Neural Stem Cells Unveils High Similarity to Neural Progenitors in the Developing Central Nervous System.

Similar to induced pluripotent cells (iPSCs), induced neural stem cells (iNSCs) can be directly converted from human somatic cells such as dermal fibroblasts and peripheral blood monocytes. While previous studies have demonstrated the resemblance of iNSCs to neural stem cells derived from primary sources and embryonic stem cells, respectively, a comprehensive analysis of the correlation between iNSCs and their physiological counterparts remained to be investigated. Nowadays, single-cell sequencing technologies provide unique opportunities for in-depth cellular benchmarking of complex cell populations. Our study involves the comprehensive profiling of converted human iNSCs at a single-cell transcriptomic level, alongside conventional methods, like flow cytometry and immunofluorescence stainings. Our results show that the iNSC conversion yields a homogeneous cell population expressing bona fide neural stem cell markers. Extracting transcriptomic signatures from published single cell transcriptomic atlas data and comparison to the iNSC transcriptome reveals resemblance to embryonic neuroepithelial cells of early neurodevelopmental stages observed in vivo at 5 weeks of development. Our data underscore the physiological relevance of directly converted iNSCs, making them a valuable in vitro system for modeling human central nervous system development and establishing translational applications in cell therapy and compound screening.

Transcriptomic analysis of spinal cord regeneration after injury in Cynops orientalis.

Cynops orientalis (C. orientalis) has a pronounced ability to regenerate its spinal cord after injury. Thus, exploring the molecular mechanism of this process could provide new approaches for promoting mammalian spinal cord regeneration. In this study, we established a model of spinal cord thoracic transection injury in C. orientalis, which is an endemic species in China. We performed RNA sequencing of the contused axolotl spinal cord at two early time points after spinal cord injury - during the very acute stage (4 days) and the subacute stage (7 days) - and identified differentially expressed genes; additionally, we performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses, at each time point. Transcriptome sequencing showed that 13,059 genes were differentially expressed during C. orientalis spinal cord regeneration compared with uninjured animals, among which 4273 were continuously down-regulated and 1564 were continuously up-regulated. Down-regulated genes were most enriched in the Gene Ontology term "multicellular organismal process" and in the ribosome pathway at 10 days following spinal cord injury. We found that multiple genes associated with energy metabolism were down-regulated and multiple genes associated with the lysosome were up-regulated after spinal cord injury, indicating the importance of low metabolic activity during wound healing. Immune response-associated pathways were activated during the early acute phase (4 days), while the expression of extracellular matrix proteins such as glycosaminoglycan and collagen, as well as tight junction proteins, was lower at 10 days post-spinal cord injury than 4 days post-spinal cord injury. However, compared with 4 days post-injury, at 10 days post-injury neuroactive ligand-receptor interactions were no longer down-regulated, up-regulated differentially expressed genes were enriched in pathways associated with cancer and the cell cycle, and SHH, VIM, and Sox2 were prominently up-regulated. Immunofluorescence staining showed that glial fibrillary acidic protein was up-regulated in axolotl ependymoglial cells after injury, similar to what is observed in mammalian astrocytes after spinal cord injury, even though axolotls do not form a glial scar during regeneration. We suggest that low intracellular energy production could slow the rapid amplification of ependymoglial cells, thereby inhibiting reactive gliosis, at early stages after spinal cord injury. Extracellular matrix degradation slows cellular responses, represses the expression of neurogenic genes, and reactivates a transcriptional program similar to that of embryonic neuroepithelial cells. These ependymoglial cells act as neural stem cells: they migrate and proliferate to repair the lesion and then differentiate to replace lost glial cells and neurons. This provides the regenerative microenvironment that allows axon growth after injury.

Impaired neurogenesis alters brain biomechanics in a neuroprogenitor-based genetic subtype of congenital hydrocephalus.

Hydrocephalus, characterized by cerebral ventricular dilatation, is routinely attributed to primary defects in cerebrospinal fluid (CSF) homeostasis. This fosters CSF shunting as the leading reason for brain surgery in children despite considerable disease heterogeneity. In this study, by integrating human brain transcriptomics with whole-exome sequencing of 483 patients with congenital hydrocephalus (CH), we found convergence of CH risk genes in embryonic neuroepithelial stem cells. Of all CH risk genes, TRIM71/lin-41 harbors the most de novo mutations and is most specifically expressed in neuroepithelial cells. Mice harboring neuroepithelial cell-specific Trim71 deletion or CH-specific Trim71 mutation exhibit prenatal hydrocephalus. CH mutations disrupt TRIM71 binding to its RNA targets, causing premature neuroepithelial cell differentiation and reduced neurogenesis. Cortical hypoplasia leads to a hypercompliant cortex and secondary ventricular enlargement without primary defects in CSF circulation. These data highlight the importance of precisely regulated neuroepithelial cell fate for normal brain-CSF biomechanics and support a clinically relevant neuroprogenitor-based paradigm of CH.

Viscoelastic properties of embryonic neuroepithelial cells in neural tube defects mice

Objective To explore the role of mechanical properties of embryonic neuroepithelial cells in the process of neural tube closure. Methods Neural tube defects (NTDs) mouse model was established by intragastric administration with all-trans retinoic acid at embryo 7.25 day. All the pregnant mice were sacrificed at embryo 9.5 day and 11.5 day, respectively, and the primary neuroepithelial cells were isolated from neural tube tissue of normal and NTDs mice, respectively. The mechanical characteristics of neuroepithelial cells were analyzed using micropipette aspiration technique combined with a standard solid viscoelastic mechanical model. Results The mechanical characteristics of mouse embryonic neuroepithelial cells showed typical viscoelastic solid characteristics. Compared with the control group, the three viscoelastic parameters, i.e. equilibrium modulus, transient modulus and apparent viscosity coefficient, of the neuroepithelial cells in the NTDs group were significantly increased, and the differences were statistically significant (all P 0.05). Conclusion The decrease in the deformability of embryonic neuroepithelial cells may be one of the factors responsible for neural tube closure disorders. Key words: Feural tube defects; Embryonic neuroepithelial cells; Micropipette aspiration; Viscoelasticity; Cell deformability

Accelerated cell divisions drive the outgrowth of the regenerating spinal cord in axolotls.

Axolotls are unique in their ability to regenerate the spinal cord. However, the mechanisms that underlie this phenomenon remain poorly understood. Previously, we showed that regenerating stem cells in the axolotl spinal cord revert to a molecular state resembling embryonic neuroepithelial cells and functionally acquire rapid proliferative divisions (Rodrigo Albors et al., 2015). Here, we refine the analysis of cell proliferation in space and time and identify a high-proliferation zone in the regenerating spinal cord that shifts posteriorly over time. By tracking sparsely-labeled cells, we also quantify cell influx into the regenerate. Taking a mathematical modeling approach, we integrate these quantitative datasets of cell proliferation, neural stem cell activation and cell influx, to predict regenerative tissue outgrowth. Our model shows that while cell influx and neural stem cell activation play a minor role, the acceleration of the cell cycle is the major driver of regenerative spinal cord outgrowth in axolotls.

Planar cell polarity-mediated induction of neural stem cell expansion during axolotl spinal cord regeneration

Axolotls are uniquely able to mobilize neural stem cells to regenerate all missing regions of the spinal cord. How a neural stem cell under homeostasis converts after injury to a highly regenerative cell remains unknown. Here, we show that during regeneration, axolotl neural stem cells repress neurogenic genes and reactivate a transcriptional program similar to embryonic neuroepithelial cells. This dedifferentiation includes the acquisition of rapid cell cycles, the switch from neurogenic to proliferative divisions, and the re-expression of planar cell polarity (PCP) pathway components. We show that PCP induction is essential to reorient mitotic spindles along the anterior-posterior axis of elongation, and orthogonal to the cell apical-basal axis. Disruption of this property results in premature neurogenesis and halts regeneration. Our findings reveal a key role for PCP in coordinating the morphogenesis of spinal cord outgrowth with the switch from a homeostatic to a regenerative stem cell that restores missing tissue.

Erythropoietin production in neuroepithelial and neural crest cells during primitive erythropoiesis

Erythropoietin (Epo) supports both primitive erythropoiesis in the yolk sac and definitive erythropoiesis in the fetal liver and bone marrow. Although definitive erythropoiesis requires kidney- and liver-secreted Epo, it is unclear which cells produce Epo for primitive erythropoiesis. Here we find neural Epo-producing (NEP) cells in mid-gestational stage embryos using mouse lines that express green fluorescent protein (GFP) under the Epo gene regulation. In these mice, GFP is expressed exclusively in a subpopulation of neural and neural crest cells at embryonic day 9.0 when Epo-deficient embryos exhibit abnormalities in primitive erythropoiesis. The GFP-positive NEP cells express Epo mRNA and the ex vivo culture of embryonic day 8.5 neural tubes results in the secretion of Epo, which is able to induce the proliferation and differentiation of yolk sac-derived erythroid cells. These results thus suggest that NEP cells secrete Epo and might support the development of primitive erythropoiesis.

C-Jun NH2-Terminal Kinase 1/2 and Endoplasmic Reticulum Stress as Interdependent and Reciprocal Causation in Diabetic Embryopathy

Embryos exposed to high glucose exhibit aberrant maturational and cytoarchitectural cellular changes, implicating cellular organelle stress in diabetic embryopathy. c-Jun-N-terminal kinase 1/2 (JNK1/2) activation is a causal event in maternal diabetes–induced neural tube defects (NTD). However, the relationship between JNK1/2 activation and endoplasmic reticulum (ER) stress in diabetic embryopathy has never been explored. We found that maternal diabetes significantly increased ER stress markers and induced swollen/enlarged ER lumens in embryonic neuroepithelial cells during neurulation. Deletion of either jnk1 or jnk2 gene diminished hyperglycemia-increased ER stress markers and ER chaperone gene expression. In embryos cultured under high-glucose conditions (20 mmol/L), the use of 4-phenylbutyric acid (4-PBA), an ER chemical chaperone, diminished ER stress markers and abolished the activation of JNK1/2 and its downstream transcription factors, caspase 3 and caspase 8, and Sox1 neural progenitor apoptosis. Consequently, both 1 and 2 mmol/L 4-PBA significantly ameliorated high glucose–induced NTD. We conclude that hyperglycemia induces ER stress, which is responsible for the proapoptotic JNK1/2 pathway activation, apoptosis, and NTD induction. Suppressing JNK1/2 activation by either jnk1 or jnk2 gene deletion prevents ER stress. Thus, our study reveals a reciprocal causation of ER stress and JNK1/2 in mediating the teratogenicity of maternal diabetes.

Spinal cord neuroepithelial progenitor cells display developmental plasticity when co-cultured with embryonic spinal cord slices at different stages of development

All neurons and glial cells of the vertebrate CNS are derived from embryonic neuroepithelial progenitor cells (NEP). Distinct modes of radial neuronal migration, locomotion, and somal translocation have been described in the cerebral cortex, but less is known about the migratory behavior of neuroepithelial cells and their neuronal and glial descendants in the developing spinal cord. Here a novel spinal cord slice co-culture was developed to investigate the migration and differentiation potential of NEPs in the developing spinal cord. E12 NEPs from eGFP transgenic mouse cells were co-cultured with E12, E14, E16, and E18 organotypic spinal cord slices. Time-lapse confocal microscopy and quantitative 3D image analysis revealed that the co-cultured E12 eGFP NEP cells differentiated at a faster rate with increasing age of embryonic spinal cord slice but migrated further in younger slices. Furthermore, it revealed fast tangentially migrating cells and slower radially migrating cells undergoing locomotion and somal translocation. The ability of NEP cells to alter their migration and differentiation within embryonic microenvironments of different ages highlights their developmental plasticity and ability to respond to temporally expressed extrinsic signals.

Plxdc2 Is a Mitogen for Neural Progenitors

The development of different brain regions involves the coordinated control of proliferation and cell fate specification along and across the neuraxis. Here, we identify Plxdc2 as a novel regulator of these processes, using in ovo electroporation and in vitro cultures of mammalian cells. Plxdc2 is a type I transmembrane protein with some homology to nidogen and to plexins. It is expressed in a highly discrete and dynamic pattern in the developing nervous system, with prominent expression in various patterning centres. In the chick neural tube, where Plxdc2 expression parallels that seen in the mouse, misexpression of Plxdc2 increases proliferation and alters patterns of neurogenesis, resulting in neural tube thickening at early stages. Expression of the Plxdc2 extracellular domain alone, which can be cleaved and shed in vivo, is sufficient for this activity, demonstrating a cell non-autonomous function. Induction of proliferation is also observed in cultured embryonic neuroepithelial cells (ENCs) derived from E9.5 mouse neural tube, which express a Plxdc2-binding activity. These experiments uncover a direct molecular activity of Plxdc2 in the control of proliferation, of relevance in understanding the role of this protein in various cancers, where its expression has been shown to be altered. They also implicate Plxdc2 as a novel component of the network of signalling molecules known to coordinate proliferation and differentiation in the developing nervous system.

Developmentally regulated expression of Sox9 and microRNAs 124,128 and 23 in neuroepithelial stem cells in the developing spinal cord

Central nervous system development is a complex process involving many interacting factors. MicroRNAs have recently been identified as playing key intrinsic roles in development however few of their specific targets have been identified in vivo. The transcription factor Sox9 has recently been identified as a target of miR-124 in the adult mouse sub-ventricular zone. Here we investigate the expression of the microRNAs miR-124, miR-128 and miR-23 and that of transcription factor Sox9, in neuroepithelial stem cells in the developing spinal cord. Furthermore we investigate if neurogenesis in embryonic neuroepithelial cells in the spinal cord might also be regulated by the interaction of Sox9 and miR-124. We provide evidence of the spatial and temporal regulation of miR-124, miR-128, miR-23 and Sox9, and taken together with recent findings we provide evidence that Sox9 may also be target of miR-124 in developing spinal cord neuroepithelial cells.

Involvement of β1-Integrin Up-regulation in Basic Fibroblast Growth Factor- and Epidermal Growth Factor-induced Proliferation of Mouse Neuroepithelial Cells

In neural stem cells, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promote cell proliferation and self-renewal. In the bFGF- and EGF-responsive neural stem cells, beta1-integrin also plays important roles in crucial cellular processes, including proliferation, migration, and apoptosis. The cross-talk of the signaling pathways mediated by these growth factors and beta1-integrin, however, has not been fully elucidated. Here we report a novel molecular mechanism through which bFGF or EGF promotes the proliferation of mouse neuroepithelial cells (NECs). In the NECs, total beta1-integrin expression levels and proliferation were dose-dependently increased by bFGF but not by EGF. EGF rather than bFGF strongly induced the increase of beta1-integrin localization on the NEC surface. bFGF- and EGF-induced beta1-integrin up-regulation and proliferation were inhibited after treatment with a mitogen-activated protein kinase kinase inhibitor, U0126, which indicates the dependence on the mitogen-activated protein kinase pathway. Involvement of beta1-integrin in bFGF- and EGF-induced proliferation was confirmed by the finding that NEC proliferation and adhesion to fibronectin-coated dishes were inhibited by knockdown of beta1-integrin using small interfering RNA. On the other hand, apoptosis was induced in NECs treated with RGD peptide, a small beta1-integrin inhibitor peptide with the Arg-Gly-Asp motif, but it was independent of beta1-integrin expression levels. Those results suggest that regulation of beta1-integrin expression/localization is involved in cellular processes, such as proliferation, induced by bFGF and EGF in NECs. The mechanism underlying the proliferation through beta1-integrin would not be expected to be completely identical, however, for bFGF and EGF.

Transgenic mice overproducing human thioredoxin-1, an antioxidative and anti-apoptotic protein, prevents diabetic embryopathy

Experimental studies have suggested that apoptosis is involved in diabetic embryopathy through oxidative stress. However, the precise mechanism of diabetic embryopathy is not yet clear. Thioredoxin (TRX) is a small, ubiquitous, multifunctional protein, which has recently been shown to protect cells from oxidative stress and apoptosis. Using transgenic mice that overproduce human TRX-1 (TRX-Tg mice), we examined whether oxidative stress is involved in fetal dysmorphogenesis in diabetic pregnancies. Non-diabetic and streptozotocin-induced diabetic (DM) female mice were mated with male TRX-Tg mice. Pregnant mice were killed either at day 10 or day 17 of gestation, and viable fetuses and their placentas were recovered, weighed and assessed for gross and histological morphology, biochemical markers and gene expression. In both wild-type (WT) and transgenic (Tg) groups, fetal and placental weights in the diabetic group were significantly decreased compared with the non-diabetic group. The incidence of malformation was higher in the diabetic group, and was significantly decreased in the TRX-Tg group (DM-WT vs DM-Tg; 28.6% vs 10.4%). Oxidative stress markers such as thiobarbituric acid reactive substances and 8-hydroxy-2'-deoxyguanosine were increased in DM-WT group fetuses but were decreased in fetuses from the DM-Tg group. Furthermore, immunohistochemically assayed apoptosis and cleaved caspase-3 production in embryonic neuroepithelial cells was significantly increased in the DM-WT group, and was significantly decreased in the DM-Tg group. These results indicate that oxidative stress is involved in diabetic embryopathy, and that the antioxidative protein TRX at least partially prevents diabetic embryopathy via suppression of apoptosis.

O‐linked β‐N‐acetylglucosaminylation in mouse embryonic neural precursor cells

In neural stem cells (NSCs), glycoconjugates and carbohydrate antigens are known not only to serve as excellent cell surface biomarkers for cellular differentiation and development but also to play important functional roles in determining cell fate. O-linked beta-N-acetylglucosamine (O-GlcNAc), which modifies nuclear and cytoplasmic proteins on the serine and threonine residues, is also expected to play an important regulatory role. It is not known, however, whether O-GlcNAc is expressed in NSCs or what the function of this expression is. In this study, we evaluated the patterns and possible functions of O-GlcNAcylation in mouse embryonic neuroepithelial cells (NECs), which are known to be rich in NSCs. We confirmed the expression of O-GlcNAc transferase, O-GlcNAcase, and several O-GlcNAcylated proteins in NECs. Treatment of NECs with O-GlcNAcase inhibitors, PUGNAc and streptozotocin, induced robust accumulation of O-GlcNAc in NECs and reduction of number of NECs. In O-GlcNAcase inhibitor-treated NECs, the Ras-mitogen-activated protein kinase pathway and the phosphatidylinositol 3-kinase-Akt pathway, important for proliferation and survival, respectively, were intact, but caspase-3, an executioner for cell death, was activated. These results suggest the possibility that O-GlcNAc is involved in cell death signaling in NECs. Furthermore, in NECs, we identified an O-GlcNAc-modified protein, Sp1 transcription factor. Our study is the first to evaluate expression and functions of O-GlcNAc in NECs.

O‐GlcNAc modification of proteins in mouse embryonic neural stem cells

In neural stem cells (NSCs), glycoconjugates and carbohydrate antigens are known to serve as excellent biomarkers for cellular differentiation and to play functional roles in determining cell fate. O‐Linked β‐N‐acetylglucosamine (O‐GlcNAc) modifying nuclear and cytoplasmic proteins on the serine/threonine residues is also expected to play the regulatory roles. It is not known, however, if O‐GlcNAc modification is expressed in NSCs and what the function of this expression is. In this study, we evaluated the patterns and possible functions O‐GlcNAcylation in mouse embryonic neuroepithelial cells (NECs) which are rich in NSCs. We found the expression of O‐GlcNAc transferase, O‐GlcNAcase, and a number of O‐GlcNAcylated proteins in NECs. Treatment of NECs with O‐GlcNAcase inhibitors induced robust O‐GlcNAc accumulation in NECs and reduction of the cell number. In O‐GlcNAcase inhibitor‐treated NECs, the Ras‐MAPK pathway and the PI3K‐Akt pathway, important for proliferation and survival, respectively, were intact, but caspase‐3, an executioner of cell death, was activated. These results suggest that O‐GlcNAc is involved in cell death signaling in NECs. Furthermore, we identified an O‐GlcNAc‐modified protein, Sp1 transcription factor. This work was supported by USPHS grants NS11853, AG027199 and a grant from the Childrens' Medical Research Foundation, Chicago, IL.

Reversing to the Nucleus

Membrane-bound ephrinB proteins bind to and activate EphB receptor tyrosine kinases on adjacent cells after cell-to-cell contact and regulate many processes, including cell adhesion. The activation of EphB receptors is considered a "forward" signal of ephrinB. In the cell expressing ephrinB, "reverse signaling" occurs and involves phosphorylation of the C terminus of ephrinB leading to the recruitment of the adaptor protein Grb4 (growth factor receptor-bound protein 4) and the activation of focal adhesion kinase (FAK). Sequence analysis of the ephrinB C terminus revealed the presence of the conserved YXXQ motif that is important for the binding and activation of signal transducer and activator of transcription 3 (STAT3), which led Bong et al . to investigate whether ephrinB might also activate STAT3. The authors performed a series of immunoprecipitation and Western blotting experiments in Xenopus oocytes (and mammalian cell lines) transfected with wild-type and mutant forms of ephrinB and STAT3. ephrinB was activated either by the addition of the EphB2 ectodomain fused to the Fc portion of human immunoglobulin or in response to fibroblast growth factor (FGF) receptor activation, which is known to lead to ephrinB phosphorylation. ephrinB that was phosphorylated at two specific tyrosine residues recruited STAT3, which resulted in STAT3 phosphorylation that was Janus kinase 2 (Jak2)-dependent. Reporter assays and gel-shift experiments showed that ephrinB activation also increased both the DNA-binding and transcriptional activity of STAT3. Immunofluorescence microscopy in mouse embryonic neuroepithelial cells showed that ephrinB activation resulted in the phosphorylation and nuclear localization of STAT3. Together, these data suggest that STAT3-mediated regulation of gene transcription may represent another "reverse" signaling pathway for ephrinB. Y.-S. Bong, H.-S. Lee, L. Carim-Todd, K. Mood, T. G. Nishanian, L. Tessarollo, I. O. Daar, ephrinB1 signals from the cell surface to the nucleus by recruitment of STAT3. Proc. Natl. Acad. Sci. U.S.A. 104 , 17305-17310 (2007). [Abstract] [Full Text]

Letter to the Glyco-Forum: Cholera toxin B subunit binding does not correlate with GM1 expression: a study using mouse embryonic neural precursor cells

Gangliosides, sialic acid-containing glycosphingolipids, are ubiquitously expressed in all eukaryotic cells and are primarily localized in the plasma membrane. Cholera toxin B subunit (Ctxb), a component of a heat-labile enterotoxin produced by Vibrio cholerae, has been frequently used as a probe to detect GM1 ganglioside because of its high affinity for this glycolipid. In this study, we evaluated the reactivity of Ctxb and the expression of GM1 in mouse embryonic neuroepithelial cells (NECs). Analysis of Ctxb reactivity of NECs based on flow cytometry revealed that about 80% of the cells are Ctxb positive. A detailed biochemical analysis, however, indicated that GM1 was expressed in NECs in barely detectable quantities. Thus, it was thought that reactivity of Ctxb in the NECs could arise from high-affinity interaction with GM1. Because Ctxb is commonly used as a reagent for flow cytometry and GM1 cell staining, we recommend that using this reagent alone would be inconclusive and that biochemical analysis of GM1 should also be performed to avoid overestimation of GM1 expression and/or mischaracterization of the ganglioside species.

P.338 Two-step reconstruction of the mandible body after wide tumour resections

We examined the myelin repair potential of transplanted neural precursor cells derived from the adult human brain from tissue removed during surgery. Sections of removed brain indicated that nestin-positive cells were found predominately in the subventricular zone around the anterior horns of the lateral ventricle and in the dentate nucleus. Neurospheres were established and the nestin-positive cells were clonally expanded in EGF and bFGF. Upon mitogen withdrawal in vitro, the cells differentiated into neuron- and glia-like cells as distinguished by antigenic profiles; the majority of cells in culture showed neuronal and astrocytic properties with a small number of cells showing properties of oligodendrocytes and Schwann cells. When transplanted into the demyelinated adult rat spinal cord immediately upon mitogen withdrawal, the cells elicited extensive remyelination with a peripheral myelin pattern similar to Schwann cell myelination characterized by large cytoplasmic and nuclear regions, a basement membrane, and P0 immunoreactivity. The remyelinated axons conducted impulses at near normal conduction velocities. This suggests that a common neural progenitor cell for CNS and PNS previously described for embryonic neuroepithelial cells may be present in the adult human brain and that transplantation of these cells into the demyelinated spinal cord results in functional remyelination.

Aspm specifically maintains symmetric proliferative divisions of neuroepithelial cells.

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.

The GATA2 transcription factor negatively regulates the proliferation of neuronal progenitors

Postmitotic neurons are produced from a pool of cycling progenitors in an orderly fashion that requires proper spatial and temporal coordination of proliferation, fate determination, differentiation and morphogenesis. This probably relies on complex interplay between mechanisms that control cell cycle, specification and differentiation. In this respect, we have studied the possible implication of GATA2, a transcription factor that is involved in several neuronal specification pathways, in the control of the proliferation of neural progenitors in the embryonic spinal cord. Using gain- and loss-of-function manipulations, we have shown that Gata2 can drive neural progenitors out of the cycle and, to some extent, into differentiation. This correlates with the control of cyclin D1 transcription and of the expression of the p27/Kip1 protein. Interestingly, this functional aspect is not only associated with silencing of the Notch pathway but also appears to be independent of proneural function. Consistently, GATA2 also controls the proliferation capacity of mouse embryonic neuroepithelial cells in culture. Indeed, Gata2 inactivation enhances the proliferation rate in these cells. By contrast, GATA2 overexpression is sufficient to force such cells and neuroblastoma cells to stop dividing but not to drive either type of cell into differentiation. Furthermore, a non-cell autonomous effect of Gata2 expression was observed in vivo as well as in vitro. Hence, our data have provided evidence for the ability of Gata2 to inhibit the proliferation of neural progenitors, and they further suggest that, in this regard, Gata2 can operate independently of neuronal differentiation.