Comparison of the generic neuronal differentiation and neuron subtype specification functions of mammalian achaete-scute and atonal homologs in cultured neural progenitor cells

In the vertebrate peripheral nervous system, the proneural genes neurogenin 1 and neurogenin 2 (Ngn1 and Ngn2), and Mash1 are required for sensory and autonomic neurogenesis, respectively. In cultures of neural tube-derived, primitive PNS progenitors NGNs promote expression of sensory markers and MASH1 that of autonomic markers. These effects do not simply reflect enhanced neuronal differentiation, suggesting that both bHLH factors also specify neuronal identity like their Drosophila counterparts. At high concentrations of BMP2 or in neural crest stem cells (NCSCs), however, NGNs like MASH1 promote only autonomic marker expression. These data suggest that that the identity specification function of NGNs is more sensitive to context than is that of MASH1. In NCSCs, MASH1 is more sensitive to Notch-mediated inhibition of neurogenesis and cell cycle arrest, than are the NGNs. Thus, the two proneural genes differ in other functional properties besides the neuron subtype identities they can promote. These properties may explain cellular differences between MASH1- and NGN-dependent lineages in the timing of neuronal differentiation and cell cycle exit.     

Crossregulation between Neurogenin2 and pathways specifying neuronal identity in the spinal cord

We have examined how genetic pathways that specify neuronal identity and regulate neurogenesis interface in the vertebrate neural tube. Here, we demonstrate that expression of the proneural gene Neurogenin2 (Ngn2) in the ventral spinal cord results from the modular activity of three enhancers active in distinct progenitor domains, suggesting that Ngn2 expression is controlled by dorsoventral patterning signals. Consistent with this hypothesis, Ngn2 enhancer activity is dependent on the function of Pax6, a homeodomain factor involved in specifying the identity of ventral spinal cord progenitors. Moreover, we show that Ngn2 is required for the correct expression of Pax6 and several homeodomain proteins expressed in defined neuronal populations. Thus, neuronal differentiation involves crossregulatory interactions between a bHLH-driven program of neurogenesis and genetic pathways specifying progenitor and neuronal identity in the spinal cord.     

Contrasting and brain region-specific roles of neurogenin2 and mash1 in GABAergic neuron differentiation in vitro

We have cultivated highly uniform populations of neural precursor cells, which retain their region-specific identities, from various rat embryonic brain regions. The roles of the proneural basic-helix-loop-helix (bHLH) factors neurogenin2 (Ngn2) and Mash1 in gamma-aminobutyric acid (GABA) neuron differentiation were explored in the region-specific cultures. Consistent with previous in vivo studies, forced expression of Mash1 promoted GABA neuron formation from the precursors derived from the developing forebrains, whereas Ngn2 displayed an inhibitory role in forebrain GABA neuron differentiation. Functional analyses of mutant bHLH proteins indicated that the helix-loop-helix domains of Mash1 and Ngn2, known as the structures for protein-protein interactions, impart the distinct activities. Intriguingly, the regulatory activities of Mash1 and Ngn2 in GABA neuron differentiation from the hindbrain- and spinal cord-derived precursor cells were completely opposite of those observed in the forebrain-derived cultures: increased GABA neuron yield by Ngn2 and decreased yield by Mash1 were shown in the precursors of those posterior brain regions. No clear difference that depended on dorsal-ventral brain regions was observed in the bHLH-mediated activities. Finally, we demonstrated that Otx2, the expression of which is developmentally confined to the regions anterior to the isthmus, is a factor responsible for the anterior-posterior region-dependent opposite effects of the bHLH proteins.     

Crossinhibitory activities of Ngn1 and Math1 allow specification of distinct dorsal interneurons

Distinct classes of neurons are generated from progenitor cells distributed in characteristic dorsoventral patterns in the developing spinal neural tube. We define restricted neural progenitor populations by the discrete, nonoverlapping expression of Ngn1, Math1, and Mash1. Crossinhibition between these bHLH factors is demonstrated and provides a mechanism for the generation of discrete bHLH expression domains. This precise control of bHLH factor expression is essential for proper neural development since as demonstrated in both loss- and gain-of-function experiments, expression of Math1 or Ngn1 in dorsal progenitor cells determines whether LH2A/B- or dorsal Lim1/2-expressing interneurons will develop. Together, the data suggest that although Math1 and Ngn1 appear to be redundant with respect to neurogenesis, they have distinct functions in specifying neuronal subtype in the dorsal neural tube.