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 p27Kip1 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. p27Kip1 promotes neuronal migration by blocking RhoA signaling, an activity that resides in its C-terminal half. Thus, p27Kip1 plays a key role in cortical development, acting as a modular protein that independently regulates and couples multiple cellular pathways contributing to neurogenesis.[Keywords: Neurogenesis; neurogenin; radial migration; RhoA; electroporation; RNA interference] Supplemental material is available at http://www.genesdev.org.

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 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 of Mash1 and Ngn2 were swapped. Using this approach, we demonstrate that Mash1 has the capacity to respecify the identity of neuronal populations normally derived from Ngn2-expressing progenitors in the dorsal telencephalon and ventral spinal cord. In contrast, misexpression of Ngn2 in Mash1-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 Ngn2 functions 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 of Mash1 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. The mechanisms underlying the generation by multipotent stem cells of the vast array of neuronal and glial subtypes that constitute the nervous system remain poorly characterized. Recently, evidence have been obtained that genes of the bHLH class are important regulators of several steps in neural lineage development (Brunet and Ghysen 1999;Cepko 1999;Guillemot 1999;Morrison 2001). In particular, a subset of neural bHLH genes, the proneural genes, has been shown to play a central role in the selection of neural progenitor cells. Two families of proneural genes have been identified in Drosophila, the achaete-scute genes (ac-sc) which control the generation of progenitors for the central nervous system (CNS) and external sense organs in the peripheral nervous system (PNS), and genes of the atonal family (ato) which control the generation of progenitors for photoreceptors, chordotonal organs, and olfactory receptors (Modolell 1997;Campos-Ortega 1998). In vertebrates, a large number of bHLH genes are expressed in the developing nervous system, but only a fraction of them appear to have a proneural function. Loss-of-function (LOF) analyses in mouse have demonstrated that the ac-sc homolog Mash1 and the ato-related genes Neurogenin (Ngn) 1 and Ngn2 are required for the generation by neural stem cells of various populations of progenitor cell populations in the PNS and CNS, including progenitors in the ventral telencephalon and olfactory epithelium for Mash1, and in the cranial and dorsal root ganglia, the dorsal telencephalon and the ventral spinal cord for Ngn1 and Ngn2 (Cau et al. 1997;Fode et al. 1998Fode et al. , 2...

The molecular mechanisms specifying the dendritic morphology of different neuronal subtypes are poorly understood. Here we demonstrate that the bHLH transcription factor Neurogenin2 (Ngn2) is both necessary and sufficient for specifying the dendritic morphology of pyramidal neurons in vivo by specifying the polarity of its leading process during the initiation of radial migration. The ability of Ngn2 to promote a polarized leading process outgrowth requires the phosphorylation of a single tyrosine residue at position 241, an event that is neither involved in Ngn2 direct transactivation properties nor its proneural function. Interestingly, the migration defect observed in the Ngn2 knockout mouse and in progenitors expressing the Ngn2(Y241F) mutation can be rescued by inhibiting the activity of the small-GTPase RhoA in cortical progenitors. Our results demonstrate that Ngn2 coordinates the acquisition of the radial migration properties and the unipolar dendritic morphology characterizing pyramidal neurons through molecular mechanisms distinct from those mediating its proneural activity.