Fig. 6

(A,B) ChIP-seq on wild-type embryos at 75-85% epiboly reveals endogenous Tbx16 and Tbxta binding events within 120 kb flanking the myf5 transcriptional start site (TSS). H3K4me3 indicates TSSs. H3K4me1 indicates putative enhancers. H3K27ac indicates active enhancers. Known transcripts with exons (black) and introns (arrowheads) are indicated. Purple and cyan boxes indicate validated and other mentioned Tbx16 binding sites, respectively. (C) ChIP-qPCR validation of Tbx16 peaks on myf5 distal element (5DE) and proximal element 2 (5PE1). Error bars indicate s.e.m. for biological triplicate experiments. (D) Schematic of Tbx16 direct-target assay. (E) Wild-type embryos injected with tbx16-GR mRNA treated with CHD±DEX. CHD alone permits wild-type myf5 expression at 75-80% epiboly, whereas CHD+DEX induced mosaic ectopic myf5 expression with strong (white arrowheads, comparable to wild-type pre-adaxial level) and weak (black arrowheads, comparable to wild-type paraxial level) staining. Numbers indicate the fraction of embryos with the expression pattern(s) shown. Inset shows an unusual induction of myf5 in anterior regions that was not observed with myod. (F) Injection of tbx16 mRNA (200 pg) into embryos from a myf5hu2022/+ heterozygote incross led to ectopic upregulation of myod mRNA in the dorsal germ ring (arrows) irrespective of genotype. Numbers indicate fraction of embryos showing ectopic mRNA/total analysed. Scale bars: 100 µm.