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In Xenopus neuroectoderm, posterior cells start differentiating at the end of gastrulation, while anterior cells display an extended proliferative period and undergo neurogenesis only at tailbud stage. Recent studies have identified several important components of the molecular pathways controlling posterior neurogenesis, but little is known about those controlling the timing and positioning of anterior neurogenesis. We investigate the role of Xrx1, a homeobox gene required for eye and anterior brain development, in the control of proliferation and neurogenesis of the anterior neural plate. Xrx1 is expressed in the entire proliferative region of the anterior neural plate delimited by cells expressing the neuronal determination gene X-ngnr-1, the neurogenic gene X-Delta-1, and the cell cycle inhibitor p27Xic1. Positive and negative signals position Xrx1 expression to this region. Xrx1 is activated by chordin and Hedgehog gene signaling, which induce anterior and proliferative fate, and is repressed by the differentiation-promoting activity of neurogenin and retinoic acid. Xrx1 is required for anterior neural plate proliferation and, when overexpressed, induces proliferation, inhibits X-ngnr-1, X-Delta-1 and N-tubulin and counteracts X-ngnr-1- and retinoic acid-mediated differentiation. We find that Xrx1 does not act by increasing lateral inhibition but by inducing the antineurogenic transcriptional repressors Xhairy2 and Zic2, and by repressing p27Xic1. The effects of Xrx1 on proliferation, neurogenesis and gene expression are restricted to the most rostral region of the embryo, implicating this gene as an anterior regulator of neurogenesis.

bHLH transcription factors are expressed sequentially during the development of neural lineages, suggesting that they operate in genetic cascades. In the olfactory epithelium, the proneural genes Mash1 and neurogenin1 are expressed at distinct steps in the same olfactory sensory neuron lineage. Here, we show by loss-of-function analysis that both genes are required for the generation of olfactory sensory neurons. However, their mutant phenotypes are strikingly different, indicating that they have divergent functions. In Mash1 null mutant mice, olfactory progenitors are not produced and the Notch signalling pathway is not activated, establishing Mash1 as a determination gene for olfactory sensory neurons. In neurogenin1 null mutant mice, olfactory progenitors are generated but they express only a subset of their normal repertoire of regulatory molecules and their differentiation is blocked. Thus neurogenin1 is required for the activation of one of several parallel genetic programs functioning downstream of Mash1 in the differentiation of olfactory sensory neurons. These results illustrate the versatility of neural bHLH genes which adopt either a determination or a differentiation function, depending primarily on the timing of their expression in neural progenitors.

We have characterised the functions of the bHLH transcriptional repressors HES1 and HES5 in neurogenesis, using the development of the olfactory placodes in mouse embryos as a model. Hes1 and Hes5 are expressed with distinct patterns in the olfactory placodes and are subject to different regulatory mechanisms. Hes1 is expressed in a broad placodal domain, which is maintained in absence of the neural determination gene Mash1. In contrast, expression of Hes5 is restricted to clusters of neural progenitor cells and requires Mash1 function. Mutations in Hes1 and Hes5 also have distinct consequences on olfactory placode neurogenesis. Loss of Hes1 function leads both to expression of Mash1 outside of the normal domain of neurogenesis and to increased density of MASH1-positive progenitors within this domain, and results in an excess of neurons after a delay. A mutation in Hes5 does not produce any apparent defect. However, olfactory placodes that are double mutant for Hes1 and Hes5 upregulate Ngn1, a neural bHLH gene activated downstream of Mash1, and show a strong and rapid increase in neuronal density. Together, our results suggest that Hes1 regulates Mash1 transcription in the olfactory placode in two different contexts, initially as a prepattern gene defining the placodal domain undergoing neurogenesis and, subsequently, as a neurogenic gene controlling the density of neural progenitors in this domain. Hes5 synergizes with Hes1 and regulates neurogenesis at the level of Ngn1 expression. Therefore, the olfactory sensory neuron lineage is regulated at several steps by negative signals acting through different Hes genes and targeting the expression of different proneural gene homologs.

Previous studies have shown that mice mutant for the gene Mash1 display severe neuronal losses in the olfactory epithelium and ganglia of the autonomic nervous system, demonstrating a role for Mash1 in development of neuronal lineages in the peripheral nervous system. Here, we have begun to analyse Mash1 function in the central nervous system, focusing our studies on the ventral telencephalon where it is expressed at high levels during neurogenesis. Mash1 mutant mice present a severe loss of progenitors, particularly of neuronal precursors in the subventricular zone of the medial ganglionic eminence. Discrete neuronal populations of the basal ganglia and cerebral cortex are subsequently missing. An analysis of candidate effectors of Mash1 function revealed that the Notch ligands Dll1 and Dll3, and the target of Notch signaling Hes5, fail to be expressed in Mash1 mutant ventral telencephalon. In the lateral ganglionic eminence, loss of Notch signaling activity correlates with premature expression of a number of subventricular zone markers by ventricular zone cells. Therefore, Mash1 is an important regulator of neurogenesis in the ventral telencephalon, where it is required both to specify neuronal precursors and to control the timing of their production.

Like other tissues and organs in vertebrates, multipotential stem cells serve as the origin of diverse cell types during genesis of the mammalian central nervous system (CNS). During early development, stem cells self-renew and increase their total cell numbers without overt differentiation. At later stages, the cells withdraw from this self-renewal mode, and are fated to differentiate into neurons and glia in a spatially and temporally regulated manner. However, the molecular mechanisms underlying this important step in cell differentiation remain poorly understood. In this study, we present evidence that the expression and function of the neural-specific transcription factors Mash-1 and Prox-1 are involved in this process. In vivo, Mash-1- and Prox-1-expressing cells were defined as a transient proliferating population that was molecularly distinct from self-renewing stem cells. By taking advantage of in vitro culture systems, we showed that induction of Mash-1 and Prox-1 coincided with an initial step of differentiation of stem cells. Furthermore, forced expression of Mash-1 led to the down-regulation of nestin, a marker for undifferentiated neuroepithelial cells, and up-regulation of Prox-1, suggesting that Mash-1 positively regulates cell differentiation. In support of these observations in vitro, we found specific defects in cellular differentiation and loss of expression of Prox-1 in the developing brain of Mash-1 mutant mice in vivo. Thus, we propose that induction of Mash-1 and Prox-1 is one of the critical molecular events that control early development of the CNS.