Neural crest survival and differentiation in zebrafish depends on mont blanc/tfap2a gene function

Neural crest progenitor cells are the main contributors to craniofacial cartilage and connective tissue of the vertebrate head. These progenitor cells also give rise to the pigment, neuronal and glial cell lineages. To study the molecular basis of neural crest differentiation, we have cloned the gene disrupted in the mont blanc (mob(m610)) mutation, which affects all neural crest derivatives. Using a positional candidate cloning approach we identified an A to G transition within the 3' splice site of the sixth intron of the tfap2a gene that abolishes the last exon encoding the crucial protein dimerization and DNA-binding domains. Neural crest induction and specification are not hindered in mob(m610) mutant embryos, as revealed by normal expression of early neural crest specific genes such as snail2, foxd3 and sox10. In addition, the initial stages of cranial neural crest migration appear undisturbed, while at a later phase the craniofacial primordia in pharyngeal arches two to seven fail to express their typical set of genes (sox9a, wnt5a, dlx2, hoxa2/b2). In mob(m610) mutant embryos, the cell number of neuronal and glial derivatives of neural crest is greatly reduced, suggesting that tfap2a is required for their normal development. By tracing the fate of neural crest progenitors in live mont blanc (mob(m610)) embryos, we found that at 24 hpf neural crest cells migrate normally in the first pharyngeal arch while the preotic and postotic neural crest cells begin migration but fail to descend to the pharyngeal region of the head. TUNEL assay and Acridine Orange staining revealed that in the absence of tfap2a a subset of neural crest cells are unable to undergo terminal differentiation and die by apoptosis. Furthermore, surviving neural crest cells in tfap2a/mob(m610) mutant embryos proliferate normally and later differentiate to individual derivatives. Our results indicate that tfap2a is essential to turn on the normal developmental program in arches 2-7 and in trunk neural crest. Thus, tfap2a does not appear to be involved in early specification and cell proliferation of neural crest, but it is a key regulator of an early differentiation phase and is required for cell survival in neural crest derived cell lineages.     

Deletion of Shp2 in the brain leads to defective proliferation and differentiation in neural stem cells and early postnatal lethality

The intracellular signaling controlling neural stem/progenitor cell (NSC) self-renewal and neuronal/glial differentiation is not fully understood. We show here that Shp2, an introcellular tyrosine phosphatase with two SH2 domains, plays a critical role in NSC activities. Conditional deletion of Shp2 in neural progenitor cells mediated by Nestin-Cre resulted in early postnatal lethality, impaired corticogenesis, and reduced proliferation of progenitor cells in the ventricular zone. In vitro analyses suggest that Shp2 mediates basic fibroblast growth factor signals in stimulating self-renewing proliferation of NSCs, partly through control of Bmi-1 expression. Furthermore, Shp2 regulates cell fate decisions, by promoting neurogenesis while suppressing astrogliogenesis, through reciprocal regulation of the Erk and Stat3 signaling pathways. Together, these results identify Shp2 as a critical signaling molecule in coordinated regulation of progenitor cell proliferation and neuronal/astroglial cell differentiation.     

Regulation of neural progenitor cell state by ephrin-B

Maintaining a balance between self-renewal and differentiation in neural progenitor cells during development is important to ensure that correct numbers of neural cells are generated. We report that the ephrin-B-PDZ-RGS3 signaling pathway functions to regulate this balance in the developing mammalian cerebral cortex. During cortical neurogenesis, expression of ephrin-B1 and PDZ-RGS3 is specifically seen in progenitor cells and is turned off at the onset of neuronal differentiation. Persistent expression of ephrin-B1 and PDZ-RGS3 prevents differentiation of neural progenitor cells. Blocking RGS-mediated ephrin-B1 signaling in progenitor cells through RNA interference or expression of dominant-negative mutants results in differentiation. Genetic knockout of ephrin-B1 causes early cell cycle exit and leads to a concomitant loss of neural progenitor cells. Our results indicate that ephrin-B function is critical for the maintenance of the neural progenitor cell state and that this role of ephrin-B is mediated by PDZ-RGS3, likely via interacting with the noncanonical G protein signaling pathway, which is essential in neural progenitor asymmetrical cell division.     

Microarray analysis of selected genes in neural stem and progenitor cells

To access and compare gene expression in fetal neuroepithelial cells (NEPs) and progenitor cells, we have used microarrays containing approximately 500 known genes related to cell cycle regulation, apoptosis, growth and differentiation. We have identified 152 genes that are expressed in NEPs and 209 genes expressed by progenitor cells. The majority of genes (141) detected in NEPs are also present in progenitor populations. There are 68 genes specifically expressed in progenitors with little or no expression in NEPs, and a few genes that appear to be present exclusively in NEPs. Using cell sorting, RT-PCR, in situ hybridization or immunocytochemistry, we have examined the segregation of expression to neuronal and glial progenitors, and identified several that appeared to be enriched in neuronal (e.g. CDK5, neuropilin, EphrinB2, FGF11) or glial (e.g. CXCR4, RhoC, CD44, tenascin C) precursors. Our data provide a first report of gene expression profiles of neural stem and progenitor cells at early stages of development, and provide evidence for the potential roles of specific cell cycle regulators, chemokines, cytokines and extracellular matrix molecules in neural development and lineage segregation.     

Wnt2b inhibits differentiation of retinal progenitor cells in the absence of Notch activity by downregulating the expression of proneural genes

During the development of the central nervous system, cell proliferation and differentiation are precisely regulated. In the vertebrate eye, progenitor cells located in the marginal-most region of the neural retina continue to proliferate for a much longer period compared to the ones in the central retina, thus showing stem-cell-like properties. Wnt2b is expressed in the anterior rim of the optic vesicles, and has been shown to control differentiation of the progenitor cells in the marginal retina. In this paper, we show that stable overexpression of Wnt2b in retinal explants inhibited cellular differentiation and induced continuous growth of the tissue. Notably, Wnt2b maintained the undifferentiated progenitor cells in the explants even under the conditions where Notch signaling was blocked. Wnt2b downregulated the expression of multiple proneural bHLH genes as well as Notch. In addition, expression of Cath5 under the control of an exogenous promoter suppressed the negative effect of Wnt2b on neuronal differentiation. Importantly, Wnt2b inhibited neuronal differentiation independently of cell cycle progression. We propose that Wnt2b maintains the naive state of marginal progenitor cells by attenuating the expression of both proneural and neurogenic genes, thus preventing those cells from launching out into the differentiation cascade regulated by proneural genes and Notch.     

The on/off of Pax6 controls the tempo of neuronal differentiation in the developing spinal cord

During neurogenesis, complex networks of genes act sequentially to control neuronal differentiation. In the neural tube, the expression of Pax6, a paired-box-containing gene, just precedes the appearance of the first post-mitotic neurons. So far, its only reported function in the spinal cord is in specifying subsets of neurons. Here we address its possible function in controlling the balance between proliferation and commitment of neural progenitors. We report that increasing Pax6 level is sufficient to push neural progenitors toward cell cycle exit and neuronal commitment via Neurogenin 2 (Ngn2) upregulation. However, neuronal precursors maintaining Pax6(On) fail to perform neuronal differentiation. Conversely, turning off Pax6 function in these precursors is sufficient to provoke premature differentiation and the number of differentiated neurons depends of the amount of Pax6 protein. Moreover, we found that Pax6 expression involves negative feedback regulation by Ngn2 and this repression is critical for the proneural activity of Ngn2. We present a model in which the level of Pax6 activity first conditions the moment when a given progenitor will leave the cell cycle and second, the moment when a selected neuronal precursor will irreversibly differentiate.     

BM88 is a dual function molecule inducing cell cycle exit and neuronal differentiation of neuroblastoma cells via cyclin D1 down-regulation and retinoblastoma protein hypophosphorylation

Control of cell cycle progression/exit and differentiation of neuronal precursors is of paramount importance during brain development. BM88 is a neuronal protein associated with terminal neuron-generating divisions in vivo and is implicated in mechanisms underlying neuronal differentiation. Here we have used mouse neuroblastoma Neuro 2a cells as an in vitro model of neuronal differentiation to dissect the functional properties of BM88 by implementing gain- and loss-of-function approaches. We demonstrate that stably transfected cells overexpressing BM88 acquire a neuronal phenotype in the absence of external stimuli, as judged by enhanced expression of neuronal markers and neurite outgrowth-inducing signaling molecules. In addition, cell cycle measurements involving cell growth assays, BrdUrd incorporation, and fluorescence-activated cell sorting analysis revealed that the BM88-transfected cells have a prolonged G(1) phase, most probably corresponding to cell cycle exit at the G(0) restriction point, as compared with controls. BM88 overexpression also results in increased levels of the cell cycle regulatory protein p53, and accumulation of the hypophosphorylated form of the retinoblastoma protein leading to cell cycle arrest, with concomitant decreased levels and, in many cells, cytoplasmic localization of cyclin D1. Conversely, BM88 gene silencing using RNA interference experiments resulted in acceleration of cell proliferation accompanied by impairment of retinoic acid-induced neuronal differentiation of Neuro 2a cells. Taken together, our results suggest that BM88 plays an essential role in regulating cell cycle exit and differentiation of Neuro 2a cells toward a neuronal phenotype and further support its involvement in the proliferation/differentiation transition of neural stem/progenitor cells during embryonic development.     

Decrease in expression of alpha 5 beta 1 integrin during neuronal differentiation of cortical progenitor cells

Neuronal differentiation of embryonic neural progenitor cells is regulated by both intrinsic and extrinsic signals. Since dynamic changes in cell shape typify neuronal differentiation, cell adhesion molecules could be relevant to this process. Although it has been reported that fibronectin-integrin interactions are important for the proliferation of neural progenitor cells, little is known about the contribution of integrins to neuronal differentiation. In order to address this shortfall, we examined integrin expression on cortical progenitor cells by using immunohistochemistry and FACS analysis of cells in which GFP expression was driven by regulatory (promoter) regions of the nestin gene (nestin-GFP(+)). We here report that high levels of nestin promoter activity correlated with high expression levels of alpha(5)beta(1) integrin (alpha(5)beta(1)(high) cells). FACS analysis of nestin-GFP(+) cortical cells revealed an additional subpopulation with reduced expression of alpha(5)beta(1) integrin (alpha(5)beta(1)(low) cells). The size of the alpha(5)beta(1)(low) subpopulation increased during cortical development. To investigate the correlation between integrin and neuronal differentiation, nestin-GFP(+) cortical progenitor cells were sorted into alpha(5)beta(1)(high) or alpha(5)beta(1)(low) populations, and each potential to differentiate was analyzed. We show that the nestin-GFP(+) alpha(5)beta(1)(high) population corresponded to broadly multipotential neural progenitor cells, whereas nestin-GFP(+) alpha(5)beta(1)(low) cells appeared to be committed to a neuronal fate. These findings suggest that alpha(5)beta(1) expression on cortical progenitor cells is developmentally regulated and its downregulation is involved in the process of neuronal differentiation.     

Sox11 modulates neocortical development by regulating the proliferation and neuronal differentiation of cortical intermediate precursors

Neural precursor cells play important roles in the neocortical development, but the mechanisms of neural progenitor proliferation, neuronal differentiation, and migration, as well as patterning are still unclear. Sox11, one of SoxC family members, has been reported to be essential for embryonic and adult neurogenesis. But there is no report about the roles of Sox11 in corticogenesis. In order to investigate Sox11 function during cortical development, loss of function experiment was performed in this study. Knockdown of Sox11 by Sox11 siRNA constructs resulted in a diminished neuronal differentiation, but enhanced proliferation of intermediate progenitors. Accompanied with the high expression of Sox11 in the postmitotic neurons, but low expression of Sox11 in the dividing neural progenitors, all the observations indicate that Sox11 induces neuronal differentiation during the neocortical development.     

Wnt7a Regulates Multiple Steps of Neurogenesis

Although Wnt7a has been implicated in axon guidance and synapse formation, investigations of its role in the early steps of neurogenesis have just begun. We show here that Wnt7a is essential for neural stem cell self-renewal and neural progenitor cell cycle progression in adult mouse brains. Loss of Wnt7a expression dramatically reduced the neural stem cell population and increased the rate of cell cycle exit in neural progenitors in the hippocampal dentate gyrus of adult mice. Furthermore, Wnt7a is important for neuronal differentiation and maturation. Loss of Wnt7a expression led to a substantial decrease in the number of newborn neurons in the hippocampal dentate gyrus. Wnt7a^−/− dentate granule neurons exhibited dramatically impaired dendritic development. Moreover, Wnt7a activated β-catenin and its downstream target genes to regulate neural stem cell proliferation and differentiation. Wnt7a stimulated neural stem cell proliferation by activating the β-catenin–cyclin D1 pathway and promoted neuronal differentiation and maturation by inducing the β-catenin–neurogenin 2 pathway. Thus, Wnt7a exercised critical control over multiple steps of neurogenesis by regulating genes involved in both cell cycle control and neuronal differentiation.     

Mammalian neurogenesis requires Treacle-Plk1 for precise control of spindle orientation, mitotic progression, and maintenance of neural progenitor cells

The cerebral cortex is a specialized region of the brain that processes cognitive, motor, somatosensory, auditory, and visual functions. Its characteristic architecture and size is dependent upon the number of neurons generated during embryogenesis and has been postulated to be governed by symmetric versus asymmetric cell divisions, which mediate the balance between progenitor cell maintenance and neuron differentiation, respectively. The mechanistic importance of spindle orientation remains controversial, hence there is considerable interest in understanding how neural progenitor cell mitosis is controlled during neurogenesis. We discovered that Treacle, which is encoded by the Tcof1 gene, is a novel centrosome- and kinetochore-associated protein that is critical for spindle fidelity and mitotic progression. Tcof1/Treacle loss-of-function disrupts spindle orientation and cell cycle progression, which perturbs the maintenance, proliferation, and localization of neural progenitors during cortical neurogenesis. Consistent with this, Tcof1(+/-) mice exhibit reduced brain size as a consequence of defects in neural progenitor maintenance. We determined that Treacle elicits its effect via a direct interaction with Polo-like kinase1 (Plk1), and furthermore we discovered novel in vivo roles for Plk1 in governing mitotic progression and spindle orientation in the developing mammalian cortex. Increased asymmetric cell division, however, did not promote increased neuronal differentiation. Collectively our research has therefore identified Treacle and Plk1 as novel in vivo regulators of spindle fidelity, mitotic progression, and proliferation in the maintenance and localization of neural progenitor cells. Together, Treacle and Plk1 are critically required for proper cortical neurogenesis, which has important implications in the regulation of mammalian brain size and the pathogenesis of congenital neurodevelopmental disorders such as microcephaly.     

Atg5 and Ambra1 differentially modulate neurogenesis in neural stem cells

Neuroepithelial cells undergoing differentiation efficiently remodel their cytoskeleton and shape in an energy-consuming process. The capacity of autophagy to recycle cellular components and provide energy could fulfill these requirements, thus supporting differentiation. However, little is known regarding the role of basal autophagy in neural differentiation. Here we report an increase in the expression of the autophagy genes Atg7, Becn1, Ambra1 and LC3 in vivo in the mouse embryonic olfactory bulb (OB) during the initial period of neuronal differentiation at E15.5, along with a parallel increase in neuronal markers. In addition, we observed an increase in LC3 lipidation and autophagic flux during neuronal differentiation in cultured OB-derived stem/progenitor cells. Pharmacological inhibition of autophagy with 3-MA or wortmannin markedly decreased neurogenesis. These observations were supported by similar findings in two autophagy-deficient genetic models. In Ambra1 loss-of-function homozygous mice (gt/gt) the expression of several neural markers was decreased in the OB at E13.5 in vivo. In vitro, Ambra1 haploinsufficient cells developed as small neurospheres with an impaired capacity for neuronal generation. The addition of methylpyruvate during stem/progenitor cell differentiation in culture largely reversed the inhibition of neurogenesis induced by either 3-MA or Ambra1 haploinsufficiency, suggesting that neural stem/progenitor cells activate autophagy to fulfill their high energy demands. Further supporting the role of autophagy for neuronal differentiation Atg5-null OB cells differentiating in culture displayed decreased TuJ1 levels and lower number of cells with neurites. These results reveal new roles for autophagy-related molecules Atg5 and Ambra1 during early neuronal differentiation of stem/progenitor cells.