Neocortical projection neurons, which segregate into six cortical layers according to their birthdate, have diverse morphologies, axonal projections and molecular profiles, yet they share a common cortical regional identity and glutamatergic neurotransmission phenotype. Here we demonstrate that distinct genetic programs operate at different stages of corticogenesis to specify the properties shared by all neocortical neurons. Ngn1 and Ngn2 are required to specify the cortical (regional), glutamatergic (neurotransmitter) and laminar (temporal) characters of early‐born (lower‐layer) neurons, while simultaneously repressing an alternative subcortical, GABAergic neuronal phenotype. Subsequently, later‐born (upper‐layer) cortical neurons are specified in an Ngn ‐independent manner, requiring instead the synergistic activities of Pax6 and Tlx , which also control a binary choice between cortical/glutamatergic and subcortical/GABAergic fates. Our study thus reveals an unanticipated heterogeneity in the genetic mechanisms specifying the identity of neocortical projection neurons.

The generation of neurons by progenitor cells involves the tight coordination of multiple cellular activities, including cell cycle exit, initiation of neuronal differentiation, and cell migration. The mechanisms that integrate these different events into a coherent developmental program are not well understood. Here we show that the cyclin-dependent kinase inhibitor p27^Kip1 plays an important role in neurogenesis in the mouse cerebral cortex by promoting the differentiation and radial migration of cortical projection neurons. Importantly, these two functions of p27^Kip1 involve distinct activities, which are independent of its role in cell cycle regulation. p27^Kip1 promotes neuronal differentiation by stabilizing Neurogenin2 protein, an activity carried by the N-terminal half of the protein. p27^Kip1 promotes neuronal migration by blocking RhoA signaling, an activity that resides in its C-terminal half. Thus, p27^Kip1 plays a key role in cortical development, acting as a modular protein that independently regulates and couples multiple cellular pathways contributing to neurogenesis.

The neural bHLH genes Mash1 and Ngn2 are expressed in complementary populations of neural progenitors in the central and peripheral nervous systems. Here, we have systematically compared the activities of the two genes during neural development by generating replacement mutations in mice in which the coding sequences ofMash1 and Ngn2 were swapped. Using this approach, we demonstrate that Mash1 has the capacity to respecify the identity of neuronal populations normally derived fromNgn2-expressing progenitors in the dorsal telencephalon and ventral spinal cord. In contrast, misexpression of Ngn2 inMash1-expressing progenitors does not result in any overt change in neuronal phenotype. Taken together, these results demonstrate that Mash1 and Ngn2 have divergent functions in specification of neuronal subtype identity, with Mash1 having the characteristics of an instructive determinant whereas Ngn2functions as a permissive factor that must act in combination with other factors to specify neuronal phenotypes. Moreover, the ectopic expression of Ngn2 can rescue the neurogenesis defects ofMash1 null mutants in the ventral telencephalon and sympathetic ganglia but not in the ventral spinal cord and the locus coeruleus, indicating that Mash1 contribution to the specification of neuronal fates varies greatly in different lineages, presumably depending on the presence of other determinants of neuronal identity.