Notch signaling is required for the maintenance of enteric neural crest progenitors

Notch signaling is involved in neurogenesis, including that of the peripheral nervous system as derived from neural crest cells (NCCs). However, it remains unclear which step is regulated by this signaling. To address this question, we took advantage of the Cre-loxP system to specifically eliminate the protein O-fucosyltransferase 1 (Pofut1) gene, which is a core component of Notch signaling, in NCCs. NCC-specific Pofut1-knockout mice died within 1 day of birth, accompanied by a defect of enteric nervous system (ENS) development. These embryos showed a reduction in enteric neural crest cells (ENCCs) resulting from premature neurogenesis. We found that Sox10 expression, which is normally maintained in ENCC progenitors, was decreased in Pofut1-null ENCCs. By contrast, the number of ENCCs that expressed Mash1, a potent repressor of Sox10, was increased in the Pofut1-null mouse. Given that Mash1 is suppressed via the Notch signaling pathway, we propose a model in which ENCCs have a cell-autonomous differentiating program for neurons as reflected in the expression of Mash1, and in which Notch signaling is required for the maintenance of ENS progenitors by attenuating this cell-autonomous program via the suppression of Mash1.     

Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.     

In vivo fate analysis reveals the multipotent and self-renewal capacities of Sox2+ neural stem cells in the adult hippocampus

To characterize the properties of adult neural stem cells (NSCs), we generated and analyzed Sox2-GFP transgenic mice. Sox2-GFP cells in the subgranular zone (SGZ) express markers specific for progenitors, but they represent two morphologically distinct populations that differ in proliferation levels. Lentivirus- and retrovirus-mediated fate-tracing studies showed that Sox2+ cells in the SGZ have potential to give rise to neurons and astrocytes, revealing their multipotency at the population as well as at a single-cell level. A subpopulation of Sox2+ cells gives rise to cells that retain Sox2, highlighting Sox2+ cells as a primary source for adult NSCs. In response to mitotic signals, increased proliferation of Sox2+ cells is coupled with the generation of Sox2+ NSCs as well as neuronal precursors. An asymmetric contribution of Sox2+ NSCs may play an important role in maintaining the constant size of the NSC pool and producing newly born neurons during adult neurogenesis.     

Proliferating neural progenitors in the developing CNS of zebrafish require Jagged2 and Jagged1b

In the central nervous system (CNS), giving rise to the diversity and the complexity of neurons is the spatial and temporal differentiation of neural stem cells and/or neural precursors. Here, we investigated the role of Jagged-mediated Notch signaling in the maintenance and differentiation of progenitor cells during late neurogenesis by analyzing the expression patterns of zebrafish jagged homologues, and by injecting their morpholinos. Expression of both jagged2 and jagged1b mRNA in the CNS suggested that they might be involved in control of differentiating neural progenitors in which they are involved later in development. In Jagged2 and Jagged1b knock-down embryos, the overall rate of cell division dramatically decreased, and the ectopic VeMe neurons were generated. The results suggest that Jagged-Notch signaling plays a critical role in the maintenance of proliferating neural precursors, and that the generation of late-born neurons, especially VeMe neurons, is regulated by the interplay between Jagged2 and Jagged1b.     

Synaptically-competent neurons derived from canine embryonic stem cells by lineage selection with EGF and Noggin

Pluripotent stem cell lines have been generated in several domestic animal species; however, these lines traditionally show poor self-renewal and differentiation. Using canine embryonic stem cell (cESC) lines previously shown to have sufficient self-renewal capacity and potency, we generated and compared canine neural stem cell (cNSC) lines derived by lineage selection with epidermal growth factor (EGF) or Noggin along the neural default differentiation pathway, or by directed differentiation with retinoic acid (RA)-induced floating sphere assay. Lineage selection produced large populations of SOX2+ neural stem/progenitor cell populations and neuronal derivatives while directed differentiation produced few and improper neuronal derivatives. Primary canine neural lines were generated from fetal tissue and used as a positive control for differentiation and electrophysiology. Differentiation of EGF- and Noggin-directed cNSC lines in N2B27 with low-dose growth factors (BDNF/NT-3 or PDGFαα) produced phenotypes equivalent to primary canine neural cells including 3CB2+ radial progenitors, MOSP+ glia restricted precursors, VIM+/GFAP+ astrocytes, and TUBB3+/MAP2+/NFH+/SYN+ neurons. Conversely, induction with RA and neuronal differentiation produced inadequate putative neurons for further study, even though appropriate neuronal gene expression profiles were observed by RT-PCR (including Nestin, TUBB3, PSD95, STX1A, SYNPR, MAP2). Co-culture of cESC-derived neurons with primary canine fetal cells on canine astrocytes was used to test functional maturity of putative neurons. Canine ESC-derived neurons received functional GABA(A)- and AMPA-receptor mediated synaptic input, but only when co-cultured with primary neurons. This study presents established neural stem/progenitor cell populations and functional neural derivatives in the dog, providing the proof-of-concept required to translate stem cell transplantation strategies into a clinically relevant animal model.     

Inhibition of leukotriene receptors boosts neural progenitor proliferation

Neural stem and progenitor cells serve as a reservoir for new neurons in the adult brain throughout lifetime. One of the critical steps determining the net production of new neurons is neural progenitor proliferation, which needs to be tightly controlled. Since inflammation has detrimental effects on neurogenesis and the 5-lipoxygenase/leukotriene pathway is involved in inflammatory processes, we investigated the effects of leukotrienes and montelukast, a small molecule inhibitor of the leukotriene receptors CysLT(1)R and GPR17, on neural stem and progenitor cell proliferation. We demonstrate expression of the leukotriene receptor GPR17 by neural progenitors and by neural stem cells. Stimulation with excess amounts of leukotrienes did not affect progenitor proliferation, whereas blockade of GPR17 with montelukast strongly elevated neural stem and progenitor proliferation, while maintaining their differentiation fate and potential. This effect was associated with increased ERK1/2 phosphorylation suggesting an involvement of the EGF signaling cascade. Based on our results, montelukast and the inhibition of the 5-LOX pathway might be potent candidates for future therapies employing neurogenesis to promote structural and functional improvement in neurodegeneration, neuropsychiatric disease and ageing.     

The early postnatal nonhuman primate neocortex contains self-renewing multipotent neural progenitor cells

The postnatal neocortex has traditionally been considered a non-neurogenic region, under non-pathological conditions. A few studies suggest, however, that a small subpopulation of neural cells born during postnatal life can differentiate into neurons that take up residence within the neocortex, implying that postnatal neurogenesis could occur in this region, albeit at a low level. Evidence to support this hypothesis remains controversial while the source of putative neural progenitors responsible for generating new neurons in the postnatal neocortex is unknown. Here we report the identification of self-renewing multipotent neural progenitor cells (NPCs) derived from the postnatal day 14 (PD14) marmoset monkey primary visual cortex (V1, striate cortex). While neuronal maturation within V1 is well advanced by PD14, we observed cells throughout this region that co-expressed Sox2 and Ki67, defining a population of resident proliferating progenitor cells. When cultured at low density in the presence of epidermal growth factor (EGF) and/or fibroblast growth factor 2 (FGF-2), dissociated V1 tissue gave rise to multipotent neurospheres that exhibited the ability to differentiate into neurons, oligodendrocytes and astrocytes. While the capacity to generate neurones and oligodendrocytes was not observed beyond the third passage, astrocyte-restricted neurospheres could be maintained for up to 6 passages. This study provides the first direct evidence for the existence of multipotent NPCs within the postnatal neocortex of the nonhuman primate. The potential contribution of neocortical NPCs to neural repair following injury raises exciting new possibilities for the field of regenerative medicine.     

Olig bHLH proteins interact with homeodomain proteins to regulate cell fate acquisition in progenitors of the ventral neural tube

BACKGROUND: Organizing signals such as Sonic hedgehog are thought to specify neuronal subtype identity by regulating the expression of homeodomain proteins in progenitors of the embryonic neural tube. One of these, Nkx2.2, is necessary and sufficient for the development of V3 interneurons. RESULTS: We report that Olig genes, encoding basic helix-loop-helix (bHLH) proteins, are expressed in a subset of Nkx2.2 progenitors before the establishment of interneurons and oligodendroglial precursors. Gain-of-function analysis in transgenic mouse embryos indicates that Olig genes specifically inhibit the establishment of Sim1-expressing V3 interneurons. Moreover, coexpression of Olig2 with Nkx2.2 in the chick neural tube generated cells expressing Sox10, a marker of oligodendroglial precursors. Colocalization of Olig and Nkx2.2 proteins at the dorsal extent of the Nkx2.2 expression domain is consistent with regulatory interactions that define the potential of progenitor cells in the border region. CONCLUSIONS: Interactions between homeodomain and Olig bHLH proteins evidently regulate neural cell fate acquisition and diversification in the ventral neural tube. In particular, interactions between Olig and Nkx2.2 proteins inhibit V3 interneuron development and promote the formation of alternate cell types, including those expressing Sox10.     

Physiological Notch signaling promotes gliogenesis in the developing peripheral and central nervous systems

Constitutive activation of the Notch pathway can promote gliogenesis by peripheral (PNS) and central (CNS) nervous system progenitors. This raises the question of whether physiological Notch signaling regulates gliogenesis in vivo. To test this, we conditionally deleted Rbpsuh (Rbpj) from mouse PNS or CNS progenitors using Wnt1-Cre or Nestin-Cre. Rbpsuh encodes a DNA-binding protein (RBP/J) that is required for canonical signaling by all Notch receptors. In most regions of the developing PNS and spinal cord, Rbpsuh deletion caused only mild defects in neurogenesis, but severe defects in gliogenesis. These resulted from defects in glial specification or differentiation, not premature depletion of neural progenitors, because we were able to culture undifferentiated progenitors from the PNS and spinal cord despite their failure to form glia in vivo. In spinal cord progenitors, Rbpsuh was required to maintain Sox9 expression during gliogenesis, demonstrating that Notch signaling promotes the expression of a glial-specification gene. These results demonstrate that physiological Notch signaling is required for gliogenesis in vivo, independent of the role of Notch in the maintenance of undifferentiated neural progenitors.     

MYC proteins promote neuronal differentiation by controlling the mode of progenitor cell division

The role of MYC proteins in somatic stem and progenitor cells during development is poorly understood. We have taken advantage of a chick in vivo model to examine their role in progenitor cells of the developing neural tube. Our results show that depletion of endogenous MYC in radial glial precursors (RGPs) is incompatible with differentiation and conversely, that overexpression of MYC induces neurogenesis independently of premature or upregulated expression of proneural gene programs. Unexpectedly, the neurogenic function of MYC depends on the integrity of the polarized neural tissue, in contrast to the situation in dissociated RGPs where MYC is mitogenic. Within the polarized RGPs of the neural tube, MYC drives differentiation by inhibiting Notch signaling and by increasing neurogenic cell division, eventually resulting in a depletion of progenitor cells. These results reveal an unexpected role of MYC in the control of stemness versus differentiation of neural stem cells in vivo.     

Reelin sets the pace of neocortical neurogenesis

Migration of neurons during cortical development is often assumed to rely on purely post-proliferative reelin signaling. However, Notch signaling, long known to regulate neural precursor formation and maintenance, is required for the effects of reelin on neuronal migration. Here, we show that reelin gain-of-function causes a higher expression of Notch target genes in radial glia and accelerates the production of both neurons and intermediate progenitor cells. Converse alterations correlate with reelin loss-of-function, consistent with reelin controlling Notch signaling during neurogenesis. Ectopic expression of reelin in isolated clones of progenitors causes a severe reduction in neuronal differentiation. In mosaic cell cultures, reelin-primed progenitor cells respond to wild-type cells by further decreasing neuronal differentiation, consistent with an increased sensitivity to lateral inhibition. These results indicate that reelin and Notch signaling cooperate to set the pace of neocortical neurogenesis, a prerequisite for proper neuronal migration and cortical layering.     

Six1 is indispensable for production of functional progenitor cells during olfactory epithelial development

The rodent olfactory epithelium (OE) is a good model system for studying the principles of stem and progenitor cell biology, because of its capacity for continuous neurogenesis throughout life and relatively well-characterized neuronal lineage. The development of mouse OE is divided into two stages, early and established neurogenesis. In established neurogenesis, which starts at embryonic day (E) 12.5, sustentacular cells and olfactory receptor neurons (ORNs) are produced from apical and basal progenitors, respectively. We previously reported that Six1(-/-) shows a lack of mature ORNs throughout development and disorganization of OE after E12.5. However, the molecular bases for these defects have not been addressed. Here, we show that Six1 is expressed in both apical and basal progenitors. In Six1(-/-) mice, apical proliferating cells were absent and no morphologically identifiable sustentacular cells were observed. Consistently, the expression of Notch2 and Jagged1 in the apical layer was absent in Six1(-/-) mice. On the other hand, basal proliferating cells were observed in Six1(-/-) animals, but the expression of Ngn1, NeuroD, Notch1, and Jagged2 in the basal layer was absent. The expression of Mash1, the determination gene for ORNs, and Hes genes was enhanced in Six1(-/-) mice. The present findings suggest that Six1 regulates production of functional apical and basal progenitors during OE development, through the regulation of various genes, such as neuronal basic helix-loop-helix (bHLH), neuronal repressor bHLH, and genes involved in the Notch signaling pathway.