5 Ascl1flox/flox mice resulted in a significant reduction of the radial migration of electroporated cells at E17.5 when compared with electroporation of only GFP ( Figure 1A), demonstrating that Ascl1 is required for proper neuronal migration in the embryonic cortex. We next asked whether Rnd2, which mediates the promigratory activity of Neurog2, is also regulating cortical neuron migration downstream of Ascl1. We found that Rnd2 transcripts are normally present in the telencephalon of Ascl1 mutant embryos,
whereas they are clearly depleted in Neurog2 mutants ( Heng et al., 2008; Figure S1D), suggesting that Ascl1 does not regulate Rnd2 expression. To identify alternative mechanisms through which Ascl1 promotes migration, Venetoclax chemical structure we searched for candidate target genes of Ascl1 that might be involved http://www.selleck.co.jp/products/Bleomycin-sulfate.html in regulating cell migration ( Gohlke et al., 2008; Figure S1E). By using gene expression microarrays, we found that Rnd3/RhoE, a member of the Rnd family of small GTP-binding proteins that also includes Rnd2 ( Chardin, 2006), was significantly downregulated in the embryonic cortex of Ascl1 null mutant embryos and upregulated in the ventral telencephalon of embryos electroporated with an Ascl1 expression
construct ( Figure S1E). Rnd3 transcripts are found throughout embryonic development in the VZ and the CP of the cerebral cortex ( Figures 1B–1E), as well as in the VZ and SVZ of the ventral telencephalon ( Figures 1C–1E). Rnd3 transcript levels were markedly reduced in embryos mutant for Ascl1, while they were unaffected in Neurog2 mutant embryos ( Figures 1F–1H and Figure S1D). To determine whether Rnd3 is a direct transcriptional target of Ascl1, we performed an in silico search for putative Ascl1-regulated elements within the Rnd3 gene locus and identified 21 distinct evolutionarily conserved regions which contained a consensus Ascl1 binding motif (CAGSTG) ( Figure S1F). CYTH4 To evaluate Ascl1 occupancy within these putative regulatory regions, we carried out chromatin immunoprecipitation (ChIP) with an antibody against Ascl1 and chromatin prepared from embryonic telencephalon and found that Ascl1 was bound in vivo to two of these conserved elements
(Rnd3 E1, located 59 kb 3′ of the gene and Rnd3 E5, located 110 kb 3′ of Rnd3; Figures 1I and 1J and Figure S1F). We examined the gene regulatory activity of these regions by using a transgenic mouse enhancer assay and we established that one element, Rnd3 E1, had enhancer activity in the embryonic cortex (n = 6; Figure 1K and data not shown). We also used a luciferase reporter assay in the embryonal carcinoma cell line P19 to show that Ascl1 activates transcription from the E1 element and to a lesser extent from the E5 element and that intact Ascl1 binding motifs are required for this activity ( Figures 1L and 1M). Together, these results indicate that Ascl1 regulates Rnd3 expression in the embryonic cortex by direct regulation of the E1 enhancer and possibly other elements in the Rnd3 locus.