EMSA experiments with combinations of proteins to test co-binding or competition on the HBox2 probe. The labelled HBox2 probe, which contains Pax3 and Six binding sites was used in limiting molar amounts, compared to the proteins added. (A) Pax3 and Six4 proteins can bind together to the same DNA sequence. Pax3 binding (lane 1) is supershifted with anti-Pax3 antibodies (lane 2, asterisk). Six4 binding (lane 3) is supershifted when anti-Six4 antibodies are present (lane 4, asterisk). When both proteins are present, an additional slower migrating band is detected (lane 5, arrow) and the intensity of the band corresponding to Six4 binding alone is reduced, compared to lane 3. When anti-Pax3 (lane 6) or anti-Six4 (lane7) antibodies are added, this band, which is therefore due to co-binding of Pax3 and Six4 on the same oligo, is disrupted and the bands due to Pax3 or Six4 binding alone are supershifted. Similar results were obtained with Pax3 and Six1, with disruption of the Pax3/Six1 complex with anti-Pax3 antibodies (results not shown). Lane C is the control with crude lysate. (B) Meox2 and Six1/4 co-binding is not detectable. EMSA was performed with constant amounts of Meox2 and increasing amounts of either Six1 (left side of panel) or Six4 (right side of panel). Presence of anti-Meox2 (lanes 5) or anti-Six1 (left, lanes 6,8) or Six4 (right, lanes 6,8) antibodies are indicated above the panel. No additional slower migrating band, suggesting co-binding of Meox2 and Six proteins, is detected. A weak band (indicated by an arrow) was detected in most samples, including the control with crude lysate (lane C). Asterisks indicate the position of bands supershifted by antibodies. (C) Meox2 and Pax3 co-binding is not detectable. In the presence of constant amounts of Meox2 (left side of panel), no Pax3 (lane 1) or increasing amounts of Pax3 were added (lanes 2-4). No supplementary band is detected when both proteins are present. Controls are shown with Pax3 alone (lane 7) or with Pax3 and anti-Pax3 antibody (lane 8) or lysate alone (C). In the presence of constant amounts of Pax3 (right side of panel), no Meox2 (lane 1) or increasing amounts of Meox2 were added (lanes 2-4). No additional slower migrating band is detected when both proteins are present in the binding reaction. A weak band (arrow) was detected in most samples, including those without Meox2 and appears to represent a Pax3 complex with this lysate. Addition of anti-Meox2 (lanes 5, 8) or anti-Pax3 (lane 6) antibodies disrupt the bandshifts and, in the case of Pax3, generate a supershift (asterisk). Controls are shown with Meox2 alone (lane 7) or with Meox2 and anti-Meox2 antibody (lane 8). (D) Msx1 and Six4 co-binding is not detectable. Increasing amounts of Msx1 alone (lanes 1-3) or with constant amounts of Six4 added at the same time (lanes 4-6) do not produce an additional slower migrating band when both proteins are present. Six4 binding is reduced when higher amounts of Msx1 are added (lanes 4-6). This displacement occurs even if Six4 protein is added before Msx1 (lanes 7-9). (E) Msx1 binding is displaced by increasing amounts of Pax3. When constant amounts of Msx1 without (lane 1) or with 2.5, 5, or 10 fold amounts of Pax3 (lanes 1-4) are added, reduction of Msx1 binding is seen as Pax3 levels increase, under gel conditions which facilitate distinction of Pax3 and Msx1 binding with lower levels of Pax3. This is confirmed by the histogram showing a scan of this autoradiograph with ImageJ 1.47v. (F) Msx1 and Pax3 can bind together when the spacing between the sites is increased. One copy of a mutated HBox2* site, which does not bind Msx1 (see Fig. 4), was intercalated on either side of the bona fide HBox2 site, thus increasing the distance between Pax3 and Six binding sites. In the presence of constant amounts of Msx1 (lanes 2-6) and increasing amounts of Pax3 (lanes 3-5), a supplementary slower migrating complex appears (arrow). When anti-Pax3 antibody is added, this band disappears and Pax3 complexes are supershifted (asterisk, lane 6). This band does not appear if Pax3 is added alone (lane 7) but is detected when Pax3 and Msx1-HA proteins are added together or sequentially (lane 8) and disappears if anti-HA antibodies are added (lane 9). Note that bands corresponding to Pax3 or Msx1-HA complexes alone migrate at almost the same positions as shown in Fig. 1 (G) The drawing resumes the competition between homeoproteins such as Msx1 and Meox2 and Pax3/Six proteins for in vitro binding to the oligonucleotide probe in the EMSA experiments presented. The binding of a homeoprotein at the HBox2 site (red) prevents the binding of Pax3 or Six1/4.

Msx1 and Meox2 proteins interact in vivo with the 145-bp Myf5 regulatory sequence. ChIP experiments were carried out with two different anti-HA antibodies (aHA1-2), to immunoprecipitate chromatin prepared from the thoracic region of the trunk including forelimb buds (forelimb region), of E10-E10.5 Msx1^Tag/Tag embryos, or with anti-Meox2 antibodies to immunoprecipitate chromatin prepared from the equivalent region of wild-type embryos at E10.5. Histograms represent the fold change in occupancy of the 145-bp Myf5 element, versus two different negative control regions located respectively at −257.5 kb (black) and −55.2 kb (grey) upstream of the Myf5 gene transcription start site (Bajard et al., 2006; Giordani et al., 2007). A control using IgGs from non-immune serum is shown for experiments with wild-type chromatin. Results on the right part of the figure were obtained with ChIP on extracts from interlimb regions, excluding limb buds, of Msx1^Tag/Tag embryos at E10.5, using anti-HA antibodies or control non immune IgGs. Biological replicates of these ChIP experiments were carried out with three different preparations of chromatin. Error bars indicate s.e.m.

Mutation of HBox2, without affecting Pax3 and Six1/4 binding, results in premature activation of the −58/−57 kb Myf5 limb enhancer. (A). Single mutation scanning in the HBox2 binding site (red) region by successive replacements of nucleotides by a cytosine (C) (Mut1 to 6). Pax3 and Six1/4 binding sites are shown as shadowed boxes. (B) EMSA experiments were performed with the wild-type (WT) sequence or the mutated probe corresponding to Mut6 in order to test the binding of Pax3, Six1/4, Msx1 and Meox2 in vitro synthesized proteins. Compared with the wild-type sequence (WT), mutation 6 (Mut6-HBox2*) does not affect the binding of Pax3 (lanes 2), Six 1 (lanes 3) and Six4 (lanes 4), but compromises the binding of Msx1 (lanes 5) and Meox2 (lanes 6) (arrows). Controls with crude lysate are shown in lanes 1. (C-L) Examples of X-gal stained transient transgenic embryos at E10.5 (35/36 somites), obtained with a −58/−57baMyf5nLacZ transgene in which the 145-bp sequence within the −58/−57 region contains a mutated HBox2* sequence (C-G) or a wild-type HBox2 (H-L). Whole mount X-Gal stained embryos are shown in C and H, with close-ups of the forelimb region in D and I, where the arrow points to X-gal staining. FL, forelimb bud, asterisk shows branchial arch expression – this and variable neural tube expression are due to sequences in the Myf5 proximal promoter region used (baMyf5). (E-G,J-L) Serial cryostat sections at the forelimb level where the plane of section is shown by a line in C for E,F, and in H for J,K. Sections including the hypaxial somite and proximal forelimb are shown in G and L. These X-Gal stained sections (Myf5-β-Gal) were treated with anti-Pax3 antibodies, revealed by horse radish peroxidase (PAX3-HRP) to label Pax3-positive myogenic progenitors (HDM, hypaxial dermomyotome).

Msx1 Cre-mediated inactivation in Pax3-positive cells leads to premature expression of Myf5 in the hypaxial somite. (A) Immunochemistry on transverse cryosections at forelimb level of Pax3^Cre/+;Msx1^fl/+;Msx2^fl/+(control, left), Pax3^Cre/+;Msx1^fl/fl;Msx2^fl/+ (M1, middle) and Pax3^Cre/+;Msx1^fl/fl;Msx2^fl/fl (M1M2, right) embryos at E9.75 (28-30 somites), showing merged images after immunostaining with anti-Pax3 (green) and anti-Myf5 antibodies (red). Contrary to control embryos, double positive Myf5+/Pax3+ cells are found in the region of the hypaxial dermomyotome (HDM), indicated by white arrows in the higher magnification shown for the M1M2 embryo. White lines in merged images represent the upper limit (corresponding to the ventral part of the neighbouring neural tube) below which Myf5+ cells in the Pax3+ population were counted. The contour of the embryos is marked by a white line. FL, forelimb; DM, dermomyotome; M, myotome. (B) Histogram representing the percentage of Myf5+ cells among the Pax3+ cells counted in cryosections equivalent to those shown in (A), with 870-960 Pax3+ cells counted from three embryos for each genotype. Error bars indicate s.e.m. 0.01<*P<0.05; 0.001<**P<0.01; versus control. (C) Comparison of wild-type Msx1^+/+ and Msx1^nLacZ/nLacZ (Msx1^−/−) mutant embryos hybridized with an anti-sense Myf5 riboprobe. Whole mount lateral enlarged views at the forelimb level of embryos at E11.5 are shown.

Meox2 inactivation induces a delay in Myf5 expression in forelimb buds, which operates via HBox sequences in the 145-bp Myf5 element. (A-F) Lateral views showing X-Gal staining at 36 (A,D), 37 (B,E) and 39 (C,F) somite stages of Meox2^+/−Myf5^+/nLacZ (Myf5^+/−A-C) or Meox2^−/−Myf5^+/nLacZ (Myf5^+/− D-F) embryos. Activation of the Myf5nLacZ allele in myogenic progenitor cells in the forelimb bud is delayed in the absence of Meox2 (arrows in B,E). (G-L) X-Gal stained Meox2^+/− (G-I) and Meox2^−/− (J-L) embryos (E10.5) obtained after crossing with a transgenic 145-baMyf5nlacZ line, with magnifications of the dorsal aspect of the forelimbs shown in H,I,K,L. There is a striking reduction in β-galactosidase positive cells in the mutant at this stage (red arrows). (M-O) X-Gal stained transient transgenic embryos at E10.5, with a −58/−57baMyf5nlacZ transgene (−58/−57) (M) or with the same transgene in which HBoxes 1-3 sequences in the 145-bp sequence have been mutated (−58/−57 Mut HBox1/2*/3) (N,O). An enlargement at the forelimb level is shown in O. When all 3 HBox sequences are mutated transgene expression in the forelimb (red arrow) is absent at E10.5. Expression in the branchial arches and neural tube is due to sequences in the proximal promoter region of Myf5 (baMyf5), present in the transgene.