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First published online 14 February 2007
doi: 10.1242/dev.02798

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A homeo-paired domain-binding motif directs Myf5 expression in progenitor cells of limb muscle

Department of Cell and Molecular Biology, Technical University of Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany.

View larger version (76K): [in this window] [in a new window]   Fig. 1. The -58/-56 kb Myf5 enhancer controls gene activity in muscle progenitor cells in limbs and myotomal cells in somites. Whole-mount ß-gal staining of embryos from stable mouse lines that harbor the enhancer containing transgene Myf5-IV. The transgene is strongly expressed in limb buds and myotomes between E10.5 and 11.5 (A-C) and persists in fetal muscles of an E13.0 embryo (D). Strictly myotomal expression is illustrated on the transverse section of an E10.5 embryo (inset in A). Two embryos carrying the control transgene Myf5-XX that lacks the enhancer show no expression in limb buds and significantly reduced expression in somites (E,F). Schematic drawings below the images illustrate the transgene constructs.

View larger version (50K): [in this window] [in a new window]   Fig. 2. Putative transcription factor binding sites in highly conserved homology regions H1 and H2 that are part of the -58/-56 kb enhancer. (A,B) Only those homology sequences were analyzed that contributed to enhancer activity in a previous study (Buchberger et al., 2003). The consensus sequences marked by gray shaded boxes were designated according to the prediction obtained by Transfac database. Homeo (TAATT) and paired (GTAACT) domain motifs within the putative Xvent2 site were also predicted by the Transfac program. Binding sites highlighted in dark gray were functionally tested by site-directed mutagenesis of the underlined sequences in the complete -58/-56 kb enhancer. Individual nucleotide exchanges are indicated.

View larger version (94K): [in this window] [in a new window]   Fig. 3. Specific mutations in the -58/-56 kb distal Myf5 enhancer disturb expression patterns in somites. Mouse embryos carrying transgenes with mutations in the putative Mef3 (A,B) and Smad (C,D) binding sites show abnormal expression in somites but not in limb buds. Myf5-IV transgenic control embryos at corresponding stages are shown for comparison (E,F). Both mutated enhancers cause a shift of the expression domain from anterior to posterior halves of somites (A,C). Sagittal (A') and transverse sections (B',C',D') of E10.5 embryos (levels of sections indicated in A and C by lines) illustrate that both transgenes are predominantly expressed in the dermomyotome. The Smad mutation also leads to massive ectopic transgene activation in dispersed and presumably migrating cells at limb level (C',D') and in addition may cause a slight delay of activation in myogenic cells in limb buds (compare D with F). Note, however, that both mutations do not prevent expression in myogenic progenitor cells in limbs.

View larger version (76K): [in this window] [in a new window]   Fig. 4. Mutation of the Xvent (homeo/paired) site in the -58/-56 kb distal Myf5 enhancer abolishes expression of the transgene in limb muscle progenitor cells, whereas the mutated Tcf/Lef site shows little change of transgene expression. Embryos containing the transgene that carries the mutation in the homeobox consensus sequence (Xvent) show no expression in limbs at E10.5 (A) or E13.5 (B). It also appears that expression in somites at E10.5 and particularly in trunk muscles at E13.5 is reduced with this mutant enhancer. Transgenic embryos containing the Tcf/Lef mutation (C,D) exhibit a fairly normal expression pattern in somites and muscle precursors of limbs (D). The mutation may possibly cause a minor reduction of transgene expression in somites (C) and trunk muscles at later stages (D). Transgenic control embryos (Myf5-IV) at similar developmental stages are shown for comparison (E,F).

View larger version (59K): [in this window] [in a new window]   Fig. 5. An oligonucleotide (30mer) encompassing the intact binding motifs for homeo- and paired domains confers expression in myogenic progenitor cells in limb buds and enhances expression in somites. Oligonucleotides with wild-type sequence (A-D), mutated homeobox binding site (E,F) and truncated sequence lacking the potential Pax3-binding sites (G,H) were used to generate transgene constructs as illustrated schematically. ß-Gal staining in whole mount of transgenic embryos demonstrates that dimerized wild-type oligonucleotide directs robust transgene expression in limbs and somites from E10.5 to 13.5 (A-D). By contrast, embryos containing the transgene in which the homeobox consensus sequence TAATT has been mutated (highlighted by blue letters) do not show expression in limb buds and exhibit drastically reduced expression in somites at E11.5 (E) and E13.5 (F). Likewise, a truncation of the oligonucleotide sequence that retains the homeobox but removes the potential Pax3 and paired domain binding sequences results in embryos that fail to express the transgene in limb buds and only weakly express it in somites (G,H).

View larger version (49K): [in this window] [in a new window]   Fig. 6. Pax3 can bind to but fails to transactivate the minimal limb enhancer. Electrophoretic mobility shift assays, with in vitro translated Pax3 (A), nuclear extract from limb buds and somites of mouse embryos (B) and in vitro synthesized Meox2 (C). All shifts were performed on the indicated wild-type oligonucleotide (wt). Specificity of complex formation was assessed by competition with wild type or various mutant oligonucleotides (mt1 to mt5) as outlined in A. Wild-type consensus motifs for protein binding are highlighted by light gray and mutations by dark gray boxes. Mutant mt5 has been chosen according to the Pax3-binding site proposed in Bajard et al. (Bajard et al., 2006). Protein complexes supershifted with specific antibodies are indicated by arrows. (D) Transactivation of luciferase reporter controlled by Myf5-XXI oligonucleotide sequence (WT, light columns) in comparison to the mutant1 oligonucleotide (Mut, dark columns). 10T1/2 cells were transfected with 200 ng of indicated luciferase reporter plasmids (wild type or homeobox mutant), 200 ng expression vector encoding the indicated transcription factors and 100 ng Renilla luciferase vector for transfection control. The results are expressed as fold activation over transfection with empty vector (pcDNA3.1) that had essentially no effect on either reporter. Note the strong transcriptional activation that is dependent on the wild-type homeobox motif by Pax3-VP16, while Pax3, Meox2 and both in combination are only marginally active. Mean values of three independent transfections for each construct are shown.

View larger version (95K): [in this window] [in a new window]   Fig. 7. The homeodomain transcription factor Meox2 does not control Myf5 expression in myogenic progenitor cells in the limb. Wild type (A-C,G-I) and Meox2-null mutant (D-F,J-L) embryos carrying the transgenes Myf4-IV (A-F) or BAC195APZ (G-L) display very similar expression of either transgene in fore- and hind limbs between E11.5 and 13.5. Note, however, that distinct muscles in limbs of homozygous Meox2 mutants are missing.