INTRODUCTION The process of neurogenesis in the nervous system of vertebrates produces a large variety of neuronal cell types. Neurons are generated from populations of neural progenitor cells, which differ in their proliferative characteristics and differentiation potentials (McConnell, 1995). The genes that control the com- mitment of ectodermal cells to a neural fate, the proliferation of neural progenitor cells and their differentiation into post-mitotic neurons, are still poorly characterized in vertebrates. In contrast, studies in invertebrates, in particular in Drosophila, have led to the identification of a number of genes, functioning at success- ive stages in the developmental pathways, leading to differenti- ated neurons. Proneural genes control the generation of neural precursors from uncommitted ectodermal cells. The main Drosophila proneural genes are the genes of the achaete-scute complex (asc) and atonal (ato), which encode transcription factors of the basic helix-loop-helix (bHLH) class (Campuzano and Modolell, 1992; Jarman et al., 1993). Loss-of-function mutations in proneural genes prevent the generation of neural precursors (Ghysen and Dambly-Chaudière, 1988; Jarman et al., 1993) whereas gain-of-function mutations result in the transfor- mation of ectodermal cells into neural precursors at ectopic positions (Rodriguez et al., 1990; Ghysen et al., 1993). Genes homologous to Drosophila asc have been identified in vertebrate species. ash1 is a vertebrate asc homolog expressed in subsets of neural progenitors in the central nervous system (CNS) and the peripheral nervous system (PNS), and it has been identified in all species examined (Johnson et al., 1990; Guillemot and Joyner, 1993; reviewed in Guillemot, 1995). A targeted mutation of Mash1 in the mouse results in the elimination of most olfactory and autonomic neurons, demonstrating a role for Mash1 in the development of particular neural lineages (Guillemot et al., 1993; Sommer et al., 1995; Blaugrund et al., 1996). A number of bHLH genes with similarities to Drosophila ato have also been isolated in vertebrates. Math1, the closest murine relative of ato, is expressed in the dorsal neural tube and the developing cerebellum (Akazawa et al., 1995). NeuroD/Beta2 (Lee et al., 1995; Naya et al., 1995), neuroD2/NDRF/kw8 (Kawakami et al., 1996; Kume et al., 1996; McCormick et al., 1996; Yasunami et al., 1996), Nex1/Math2 (Bartholomä and Nave, 1994; Shimizu et al., 1995) and Math3 (Takebayashi et al., 1997) constitute a subfamily of genes distantly related to ato, and with very similar bHLH domains. These genes are expressed either tran- siently in differentiating neurons or permanently in terminally differentiated cells. In contrast, neurogenin1/neuroD3 and 1611 Development 124, 1611-1621 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 DEV2133 The lineage of olfactory neurons has been relatively well characterized at the cellular level, but the genes that regulate the proliferation and differentiation of their pro- genitors are currently unknown. In this study, we report the isolation of a novel murine gene, Math4C/neurogenin1, which is distantly related to the Drosophila proneural gene atonal. We show that Math4C/neurogenin1 and the basic helix-loop-helix gene Mash1 are expressed in the olfactory epithelium by different dividing progenitor populations, while another basic helix-loop-helix gene, NeuroD, is expressed at the onset of neuronal differentiation. These expression patterns suggest that each gene marks a distinct stage of olfactory neuron progenitor development, in the following sequence: Mash1>Math4C/neurogenin1>NeuroD. We have previously reported that inactivation of Mash1 function leads to a severe reduction in the number of olfactory neurons. We show here that most cells in the olfactory epithelium of Mash1 mutant embryos fail to express Math4C/neurogenin1 or NeuroD. Strikingly, a subset of progenitor cells in a ventrocaudal domain of Mash1 mutant olfactory epithelium still express Math4C/neurogenin1 and NeuroD and differentiate into neurons. Cells in this domain also express Math4A/neuro- genin2, another member of the Math4/neurogenin gene family, and not Mash1. Our results demonstrate that Mash1 is required at an early stage in the olfactory neuron lineage to initiate a differentiation program involving Math4C/neurogenin1 and NeuroD. Another gene activates a similar program in a separate population of olfactory neuron progenitors. Key words: neurogenesis, neural progenitors, olfactory epithelium, olfactory placodes, olfactory receptor neurons, bHLH proteins, Mash1, NeuroD, achaete-scute homolog, atonal homolog, mouse SUMMARY Mash1 activates a cascade of bHLH regulators in olfactory neuron progenitors Elise Cau, Gérard Gradwohl, Carol Fode and François Guillemot* Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur, BP 163, 67404 Illkirch Cédex, CU de Strasbourg, France *Author for correspondence (e-mail: francois@igbmc.u-strasbg.fr)