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Figures
- Fig. 1.
ChIP-seq from isolated ZPA and AER cells identifies candidate regulatory elements. (A) Schematic showing cells from the ZPA and AER isolated by FACS, followed by ChIP-seq with an antibody against H3K27ac. (B) Different genomic categories are associated with H3K27ac ZPA and AER peaks. (C) ZPA (blue) and AER (red) ChIP-seq peaks overlap enhancers from the VISTA Enhancer Browser (Visel et al., 2007). Both ZPA and AER peaks show significant overlap with enhancers in the whole limb, but have little overlap with forebrain enhancers (*P<0.0001; Fisher's exact test, one-tailed). ZPA and AER enhancers also overlap more with their respective tissue compared with the forebrain enhancers. (D) GREAT (McLean et al., 2010) shows significantly enriched terms related to ZPA and AER biological function and phenotypes.
- Fig. 2.
Genomic regions around limb patterning genes show epigenetic active signatures only in the appropriate tissue. All coordinates are mm9. (A) The Shh gene and its enhancer ZRS are active in the ZPA and overlap H3K27ac. The AER has no peak at this locus. (B) En1, an AER- and ventral ectoderm-expressed gene, has an H3K27ac peak in the AER, but not in the ZPA. (C) The locus around Alx4, a gene expressed in the anterior limb mesenchyme, does not have an H3K27ac peak in either signaling center.
- Fig. 3.
ZPA and AER specific peak analyses. (A) By overlapping ZPA and AER H3K27ac peaks to whole-limb H3K27ac peaks, we obtained 1233 ZPA and 715 AER-specific ChIP-seq peaks. (B) ZPA peak 44 shows weak lacZ expression in the ZPA in a mouse transgenic enhancer assay. (C-E) AER peaks 2292, 3723 and 4460 show AER limb expression in a mouse transgenic assay. Arrowheads highlight β-galactosidase-stained regions.
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