Genomic code for Sox10 activation reveals a key regulatory enhancer for cranial neural crest.
The neural crest is a multipotent, stem cell-like population that migrates extensively in the embryo and forms a wide array of derivatives, ranging from neurons to melanocytes and cartilage. Analyses of the gene regulatory network driving neural crest development revealed Sox10 as one of the earliest neural crest-specifying genes, cell-autonomously driving delamination and directly regulating numerous downstream effectors and differentiation gene batteries. In search of direct inputs to the neural crest specifier module, we dissected the chick Sox10 genomic region and isolated two downstream regulatory regions with distinct spatiotemporal activity. A unique element, Sox10E2 represents the earliest-acting neural crest cis-regulatory element, critical for initiating Sox10 expression in newly formed cranial, but not vagal and trunk neural crest. A second element, Sox10E1, acts in later migrating vagal and trunk crest cells. Deep characterization of Sox10E2 reveals Sox9, Ets1, and cMyb as direct inputs mediating enhancer activity. ChIP, DNA-pull down, and gel-shift assays demonstrate their direct binding to the Sox10E2 enhancer in vivo, whereas mutation of their corresponding binding sites, or inactivation of the three upstream regulators, abolishes both reporter and endogenous Sox10 expression. Using cis-regulatory analysis as a tool, our study makes critical connections within the neural crest gene regulatory network, thus being unique in establishing a direct link of upstream effectors to a key neural crest specifier.
PubMed ID: 20139305
PMC ID: PMC2840498
Article link: Proc Natl Acad Sci U S A.
Grant support: NS36585 NINDS NIH HHS , P01-HD037105 NICHD NIH HHS
Genes referenced: ets1 myb sox10 sox9
Article Images: [+] show captions
|Fig. 1. Sox10 cis-regulatory analysis. (A) Schematic diagram showing comparative genomic analysis using the ECR browser. Chicken, zebrafish, Xenopus, opossum, mouse, rat, and human genomic sequences were compared between Sox10 and neighboring genes, Slc16A8 and PolR2F: (red) highly conserved elements; (blue) coding exons; (green) transposable elements and simple repeats. Boxed Sox10 putative regulatory regions L8 (late) and E (early) show activity in neural crest. UTRs shaded in yellow. (B) At HH8+, GFP transcripts are detected by fluorescent in situ hybridization in CNC, similar to endogenous Sox10 expression (G). Distribution of EGFP transcripts (C–E) (HH9+, HH12, HH15) is similar to endogenous Sox10 in H to J, respectively. (D) EGFP expression at HH12 in rhombomere5 stream surrounding otic vesicle (OV) resembles endogenous Sox10 (I), but is missing in vagal neural crest (VNC). (F) Cross section of embryo in D shows specific Sox10E regulatory activity in CNC around optic vesicle (OpV). (G–J) Endogenous Sox10 expression at HH8 ± 15. OP, otic placode.|