The molecular mechanisms mediating the retinogenic potential of multipotent retinal progenitor cells (RPCs) are poorly defined. Prior to initiating retinogenesis, RPCs express a limited set of transcription factors implicated in the evolutionary ancient genetic network that initiates eye development. We elucidated the function of one of these factors, Pax6, in the RPCs of the intact developing eye by conditional gene targeting. Upon Pax6 inactivation, the potential of RPCs becomes entirely restricted to only one of the cell fates normally available to RPCs, resulting in the exclusive generation of amacrine interneurons. Our findings demonstrate furthermore that Pax6 directly controls the transcriptional activation of retinogenic bHLH factors that bias subsets of RPCs toward the different retinal cell fates, thereby mediating the full retinogenic potential of RPCs.
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...
Expression of the proneural gene Neurogenin2 is controlled by several enhancer elements, with the E1 element active in restricted progenitor domains in the embryonic spinal cord and telencephalon that express the homeodomain protein Pax6. We show that Pax6 function is both required and sufficient to activate this enhancer, and we identify one evolutionary conserved sequence in the E1 element with high similarity to a consensus Pax6 binding site. This conserved sequence binds Pax6 protein with low affinity both in vitro and in vivo, and its disruption results in a severe decrease in E1 activity in the spinal cord and in its abolition in the cerebral cortex. The regulation of Neurogenin2 by Pax6 is thus direct. Pax6 is expressed in concentration gradients in both spinal cord and telencephalon. We demonstrate that the E1 element is only activated by high concentrations of Pax6 protein, and that this requirement explains the restriction of E1 enhancer activity to domains of high Pax6 expression levels in the medioventral spinal cord and lateral cortex. By modifying the E1 enhancer sequence, we also show that the spatial pattern of enhancer activity is determined by the affinity of its binding site for Pax6. Together, these data demonstrate that direct transcriptional regulation accounts for the coordination between mechanisms of patterning and neurogenesis. They also provide evidence that Pax6 expression gradients are involved in establishing borders of gene expression domains in different regions of the nervous system.
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