Figure 1.
Human, mouse and fugu sequence conservation surrounding the Bmp4 gene.
Homologous genomic pair-wise alignments between human and mouse (Human-Mouse) and Fugu and mouse (Fugu-Mouse) were generated using BLASTZ. Genomic sequence of 159 kb surrounding the mouse Bmp4 gene (76 kb 5′ of the Bmp4 transcriptional start site and 83 kb 3′ of it) was used as reference. The Bmp4 gene (green) consists of 4 exons as (triangles denote IA, IB, II, III and IV) with the 2.4 kb proximal Bmp4IA promoter region (brown) upstream of exon IA. Transcriptional direction is designated by the exon triangles and genomic positions upstream and downstream of the transcriptional start site (position 0) are denoted in (−) and (+) numbers, respectively. Human-Mouse and Fugu-Mouse homologies of >75% identity over 50 bp regions are shown as blocks of sequence conservation. CONS1, CONS2, CONS3 and CONS4 (pink) encompass clusters of conservation blocks. Note: Two blocks of Fugu-Mouse sequence conservation are located 46 kb upstream and 80 kb downstream of the Bmp4 transcription start site, respectively. The 46 kb upstream Fugu-Mouse conservation block is embedded within the CONS3 region.
Figure 2.
Conserved region driven β-galactosidase activity in the orofacial region and limb.
(A) Schematic of the transgenic reporter construct used in transient transgenic analyses. The black rectangle denotes the location of CONS region insertions upstream of the human beta globin promoter in pGLKS (see Materials and Methods). (B) Schematic of the Bmp4lacZneo allele [45] (white boxes, exons; gray boxes, coding regions). (C) Number of transient transgenic embryos with various CONS derivative constructs that supported β-galactosidase expression in limb or orofacial tissue.
Figure 3.
Bmp4lacZneo and TgCONS3 β-galactosidase activity in the limb and orofacial region.
(A) Lateral views of developing fore- or hindlimbs of Bmp4lacZneo control mouse embryos (upper row). TgCONS3 transgenic embryos from permanent transgenic line TL3459 (lower row). The CONS3 transgene expression largely recapitulates endogenous Bmp4 expression in the AER from E9.5–13.5. (B) Frontal views of the developing head of Bmp4lacZneo and TgCONS3 transgenic embryos from permanent line TL3459. From E9.5 to E11.0, Bmp4 is expressed in the epithelium overlying the distal first branchial arch, maxillary and mandibular processes and medial and lateral nasal processes. At these stages, the CONS3 enhancer recapitulates endogenous expression in the ectoderm overlying the distal part of the first branchial arch at E9.5 and the mandibular process at E10.5. At E12.5, endogenous Bmp4 expression shifts to the mesenchyme, while CONS3 expression persists in the epithelium overlying the mandibular arch and premaxilla, including incisor tooth germs. At E12.5 and 13.5, endogenous Bmp4 is concentrated in the mesenchyme of the midface including the whisker follicles. CONS3 transgene expression persists in the epithelium that overlies the mandible, pre-maxilla and nasal pits, and fails to shift to the underlying mesenchyme. Abbreviations: MxP, maxillary process; MdP, mandibular process; MNP, medial nasal process; LNP, lateral nasal process; NP, nasal pit; Mx, maxilla; Md, mandible.
Figure 4.
Deletion analysis of the Bmp4 enhancer CONS3.
(A) Schematic of the four regions (CONS1-4; black boxes), each containing multiple human-mouse sequence conservation blocks, tested in a transient transgenic mouse reporter assay in the same reporter shown in Figure 2. The CONS3 enhancer was systemically narrowed down to 758 bp region, CONS3.5, which contains the Fugu-Mouse upstream conservation block, and then to a minimal 396 bp minimal enhancer. (B) The number of E11.5 transgenic mouse embryos showing endogenous Bmp4 orofacial and limb expression (in the AER and mesenchyme region of fore- and hindlimbs) is reported over the total number of transgenic embryos analyzed (No. Stained/No. Tg).
Figure 5.
Pitx binds to the minimal Bmp4 enhancer CRM in vitro and the Pitx binding site is necessary for enhancer activity.
(A) Electrophoretic Mobility Shift Assay (EMSA) exhibits robust binding of Pitx1 protein to both a positive control bicoid DNA sequence and to the consensus Pitx1/2 binding site in with a 25 bp probe sequence in the CONS3.8 sequence. Competition with specific or non-specific unlabelled probes indicates sequence-specific binding of GST:Pitx1 fusion protein to the consensus Pitx1/2 DNA binding motif. (B) Number of transient transgenic embryos that supported β-galactosidase expression in forelimbs, hindlimbs, or orofacial tissue. Asterisk indicates statistically significant differences (p<0.05) compared to wild-type CONS3 by Fisher’s exact test.
Figure 6.
Pitx binds the minimal Bmp4 enhancer in vivo.
(A) Schematic representation of the ChIP assay design. The 396-bp Bmp4 CRM containing the Pitx1/2 binding site is shown along with two control regions, C1 and C2. C1 and C2 are located upstream of the Bmp4 transcription start site and do not contain Pitx1/2 binding sites (see Materials and Methods). The transcriptional start site (TSS) is marked as +1 and Pr is the Bmp4 proximal promoter. Chromatin Immunoprecipitation (ChIP) assays were performed in LS-8 cells. Lane 1 is a no template control using the test primers, lane 2 is the Pitx2 antibody immunoprecipitated (IP-Pitx2) chromatin, lane 3 is the normal rabbit immunoglobulin G (IgG) immunoprecipitated chromatin, and lane 4 is the input chromatin amplified using the test primers. The top gel (Bmp4 enhancer) represents the test primers encompassing Pitx1/2 motif in the 396-bp Bmp4 CRM. Lane 2 exhibits a 338 amplicon of the expected size. The lower two gels reveal the absence of amplicon products from the two control regions C1 and C2. An amplicon from these regions are only detected in the input control groups. (B) Quantitative real-time PCR was performed using the same conditions for the ChIP assays as described in (A). The 4.7-fold enrichment of the region containing the 396-bp Bmp4 CRM in the Pitx2 antibody immunoprecipitated (Pitx2-IP) chromatin is shown in the bar graph. Significance is denoted by p = 0.0018 for the enrichment of amplified product in Pitx2-IP versus an IgG control set at 1.0. (C) Sequencing analysis demonstrates the presence of a Pitx1/2 binding site in the product amplified using the Bmp4 minimal CRM region-specific primers and Pitx2-IP chromatin as template.