Fig. 4

Fig. 4 Identification of vertebrate msx CRMs

(A) Representation of the zebrafish msxB and mouse Msx1 loci depicting the location of the CRMs and vertebrate sequence conservation (Cons). For zebrafish, histone 3 lysine 4 single and triple methylation patterns indicative of open chromatin are also indicated. Block conservation tracks for select species are represented for both loci. (B) In situ expression driven by msx CRMs. Dorsal (anterior to the top) and lateral (anterior to the left) views of transgenic zebrafish embryos at the open neural plate stages (3–6 somites). Embryos were injected with either msxB-CRM or Msx1-CRM constructs driving gfp and stable transgenic lines were subsequently bred. Stable transgenic embryos were stained for msxB and gfp expression, which was detected by in situ hybridization. Both CRMs drive patterns resembling the endogenous msxB pattern. The zebrafish DNA isolated contains sufficient information to drive a pattern resembling the endogenous msxB expression pattern. The cloned mouse CRM also is capable of responding to regulatory cues in the zebrafish embryo to drive expression resembling that of the zebrafish msxB gene (as well as the endogenous Msx1 gene in mice [62]). Note that this embryo is tilted in a slightly more rostral direction than the other embryos shown from the dorsal perspective, which results in bands of anterior expression coming into view. (C) EMSA experiment in which a radiolabeled oligonucleotide probe carrying the zAE element (Msx11F in Fig. S3) was incubated with extracts from S2 cells over-expressing activated Tkv (to induce BMP signaling), Med and Mad. When the GC-rich region of the mad1 binding site is mutated (GCR1), pMM biding is abolished (the same loss of binding was also observed for the mutation in the mad2 site, GCR2 - see Fig. S3C). (D) Mutation of the pMM zAE site greatly reduces specific expression driven by the 671 bp msxB CRM. Dorsal (anterior to the top) and lateral (anterior to the left) views of injected zebrafish embryos (6–8 somites). Embryos were injected with GFP-reporter constructs under the control of the intact 671 bp msxB-CRM, a 30 bp mutant deleting the zAE (msxB-CRM^DEL), or a point mutant version of the zAE that abolishes pMM binding – see panel C (msxB-CRM^GCR1). Both zAE mutant constructs show greatly reduced reporter expression. Transient gfp mRNA expression was detected by in situ hybridization.