Figure 1.
Mutation analysis of the Tbx5 forelimb regulatory element.
A. Schematic representation of the mouse Tbx5 forelimb regulatory element. This 361 bp sequence contains six Hox binding sites (Hbs; blue boxes). B–I. E9.5 trangenic embryos (B–H) and E9.0 embryo (I) stained for β-galactosidase. Control (B and H) and mutated constructs (C–G and I) of the Tbx5 int2(361) reporter. C–G and I Results following mutation of one of the six Hox binding sites; mutation of Hbs1 (C), Hbs3 (D), Hbs4 (E), Hbs5 (F), Hbs6 (G) or Hbs2 and an additional 6 bp sequence located 3′ of Hbs2 (6bp3′) (I) J. Schematic representation of the 565 bp fragment of the Tbx5 regulatory element. The additional 204 bp sequence contains three putative Hox binding sites (blue/white hatched boxes). K–N. Representative β-galactosidase stainings for WT Tbx5 int2(565) reporter construct (K), construct with mutation(s) on both Hbs2 and 6bp3′ (L), 6bp3′ (M) or Hbs2 (N) alone. The red arrows indicate the caudal extent of staining. Forelimb bud (white dashed line) and presumptive hindlimb region (black dashed line) were marked. O. Tabulation of the number of embryos showing lacZ expression.
Figure 2.
Comparison of Tbx5 and HoxC gene expression domains.
A–G. In situ hybridization for Tbx5 (A) or HoxC genes (B–G) on 20–22 somites stages chick embryos. Tbx5 is expressed lateral to somites 13–20. The forelimb-forming region is indicated by the vertical bar. The red arrowheads indicate the rostral extent of expression. H–N. In situ hybridization for Tbx5 (H) or HoxC (I–N) on 11–13 somite stages mouse embryos. Tbx5 is expressed lateral to somites 4–11. The forelimb-forming region is indicated by the vertical bar. The red arrowheads indicate the rostral extent of expression.
Figure 3.
Hoxc9 can repress activity of the Tbx5 forelimb regulatory element via Hbs2.
A–D. The Tbx5 int2(361) LacZ reporter construct (A) was electroporated with pCAβ-dsRed-Express in the presumptive forelimb region of HH14-15 chick embryos. After 24 hours, electroporation efficiency was assessed by dsRed expression (B–D) and embryos were stained for LacZ to analyze the enhancer activity (B′–D′). Co-electroporation of pcDNA-mHoxc9 (C–C′) but not pcDNA-mHoxc5 (D–D′) reduced LacZ expression. E. Tabulation of the numbers of embryos showing β-galactosidase staining for the constructs described in B–D. F–I. Equivalent series with the Tbx5 int2(361) reporter plasmid with mutations on Hbs2 (F). The reporter plasmid was electroporated with pCAβ-dsRed-Express and assessed for dsRed and LacZ activity (G–G′). With the Hbs2 mutant reporter, co-electroporation of Hoxc9 (H–H′) did not repress LacZ activity. As in the control, Hoxc5 (I–I′) did not affect enhancer activity. J. Tabulation of the numbers of embryos showing β-galactosidase staining for the constructs described in G–I.
Figure 4.
Hoxc9 can repress endogeneous Tbx5 expression.
A–A″. pCAGGS-mHoxc9 was electroporated and Tbx5 expression was examined by whole mount in situ hybridization. dsRed was used to assay electroporation efficiency(A). Tbx5 was repressed in the electroporated right forelimb LPM (A′). Section of the embryo shown in panel A and A′ (A″). The region affected is bracketed. B–B″. Pitx1, a hindlimb-restricted gene, was induced ectopically in forelimb LPM after Hoxc9 ectopic expression (B′). The right panel shows a higher-magnification image. Section of the embryo shown in panel B and B′ (B″). The region affected is bracketed.
Figure 5.
Hox proteins can bind the Hbs2 site.
A. Binding of in vitro translated Hoxc5 flag-tagged proteins to the Hbs2 site. Hoxc5 forms a complex with an oligonucleotide probe containing Hbs2 (lane2) and this can be specifically competed with unlabelled oligo (lane 3–4). Unlabelled oligo containing mutated Hbs2 (mut Hbs2) does not compete with labelled probe (lane 5–6). B. Hoxc9 also makes a complex with a probe containing Hbs2 (lane 2) and can be competed with unlabelled probe but not by mutated Hbs2 (mut Hbs2) probe (lane 3–6). C. The Hoxc5-Hbs2 complex (lane 2–3) and the Hoxc9-Hbs2 complex (lane 4–5) can be super-shifted by addition of α-flag antibody (lanes 3 and 5). D. EMSA using nuclear extracts obtained from forelimb-forming rostral LPM (lane 2–6) or caudal LPM (lane 7–11) of E9.0 mouse embryos. 2 specific bands are produced (arrowed) from both rostral and caudal extracts (lane 2 and 7). Competition assay is performed using no mutation oligo (lane 3–4 and 8–9) or mut Hbs2 oligo (lane 5–6 and 10–11). E. Sequence of the oligonucleotide probe used containing Hbs2 (blue).
Figure 6.
Functional mapping of the Hoxc9 repressor domains.
A. Schematic representation of Hoxc5, Hoxc9 and chimeric proteins. Domains from Hoxc5 and Hoxc9 are shown in yellow and green, respectively. The specificity module is comprised of a domain including the hexapeptide motif (W) and N-terminal residues of the homeodomain (NHD). B–I. The Tbx5 int2(361) LacZ reporter construct was electroporated together with constructs encoding chimeric proteins. dsRed expression (from the pCAβ-dsRed-Express reporter) indicating successful targeting of the forelimb 24 hours after electroporation (B–I). LacZ staining of the same embryos shown in B–I (B′–I′). Co-electroporation of a Hoxc5C expression construct has no effect on the activation of the reporter in the forelimb (B′). Reporter expression is repressed by Hoxc9N5C (C′) but not by Hoxc9N15C (D′) or Hoxc9N25C (E′). Reporter expression is repressed by Hoxc5N9C (F′), Hoxc5(9WNH) (G′) and Hoxc5(9WN) (H′) but not by Hoxc5(9HC) (I′).
Figure 7.
Model for the combinatorial regulation of forelimb-restricted Tbx5 expression by distinct paralogous Hox gene inputs.
A. Hox genes expressed in the rostral forelimb-forming LPM induce Tbx5 expression (yellow arrows). In the caudal flank there is a latent capacity to activate Tbx5 expression (grey arrows) that is normally masked by the presence of caudally-expressed Hox genes (Hoxc8, Hoxc9 and Hoxc10) that repress expression of Tbx5 (green arrows). Thus, a combination of Hox colinear expression and the specific activator or repressor activities of distinct Hox protein paralogs dictates positioning of forelimb-forming region. B. The transcriptional repression of Tbx5 is controlled by caudally-expressed Hox genes, such as Hoxc9, bound on Hbs2. This site is occupied both in forelimb-forming region (Hox PG4/5) and in caudal LPM (Hoxc9). However, only Hoxc9 forms a repressive complex by recruiting co-repressor(s). HD, homeodomain; SM, specificity module; N, N-terminus.