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Drosophila Lame duck, a novel member of the Gli superfamily, acts as a key regulator of myogenesis by controlling fusion-competent myoblast development

¹ Department of Medicine (Division of Cardiology) and
² Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
³ Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA

View larger version (120K): [in a new window]   Fig. 1. lame duck (lmd) mutant embryos exhibit severe defects within the somatic muscle lineage. Wild-type and mutant embryos (trans-heterozygous for lmd1 and Df(3R)M95A)were stained with an antibody against MEF2 (A-D) or MHC (E-H). (A,B) Lateral views of stage 13 embryos. When compared with control embryo (A), mutant embryo shows a dramatic reduction in MEF2 expression in somatic mesodermal cells. (C,D) Dorsal views of stage 16 embryos with normal MEF2 expression in cardioblasts (cb; arrowheads) but a reduced number of MEF2-positive somatic muscle nuclei (arrows in D). (E,F) Multinucleate MHC-positive fibers are present in late stage 15 control embryo (E) while only elongated, mononucleate MHC-expressing muscle cells are detected in mutant embryo (arrows in F). (G,H) MHC expression in cardioblasts and gut muscles is normal.

View larger version (90K): [in a new window]   Fig. 2. lmd mutant phenotype involves loss of Mef2 and sns expression specifically in fusion-competent myoblasts. Late stage 13 wild-type and lmd1 mutant embryos, both of which also carry a twist-dependent Mef2-lacZ insertion (A,B) (Nguyen and Xu, 1998) or the enhancer trap insertion rP298-lacZ (C,D) (Nose et al., 1998) were double-stained with antibodies against MEF2 and lacZ, and analyzed by confocal microscopy. Control embryo (A) exhibits coincident nuclear MEF2 (red) and cytoplasmic ß-gal (green) expression in somatic myoblasts and cardioblasts at the dorsal margin while mutant embryo (B) shows a significant number of lacZ-positive somatic myoblasts that do not exhibit nuclear MEF2 expression. Control embryo (C) shows founders that are rP298-lacZ positive/MEF2 positive (yellow signals) and fusion-competent myoblasts that are only MEF2-positive (red), while mutant embryo (D) exhibits MEF2 expression only in lacZ-positive founders and cardioblasts. (E,F) Embryos were stained with an anti-Kr antibody. Kr-positive multinucleate muscle precursors in control embryo (E) are equivalent in position and number to Kr-positive mononucleate muscle precursors in lmd1 mutant embryo (F). (G,H) Embryos were hybridized with a digoxigenin-labeled sns RNA probe. In lmd mutant embryo, sns expression in fusion-competent myoblasts is completely abolished. Residual sns expression is in presumed garland cells (arrowheads).

View larger version (68K): [in a new window]   Fig. 3. Lmd is a C2H2-type of Zn-finger protein with homology to Gli family members. (A) Diagram of the predicted Lmd protein. Checked and black boxes denote the conserved Zn-finger domain and putative phosphorylation sites, respectively. Position of the mutation in lmd1 (nucleotide change from C to T, converting the Gln residue at position 127 to a nonsense residue) is indicated. (B) Sequence comparison of the Zn-finger domain of Lmd and representatives of the Gli superfamily was done using the Clustal W program: Lmd (Accession Number, AY032609); mouse Gli3 (Accession Number, Q61602; mouse Gli1 (Accession Number, BAA85004); human Gli2 (Accession Number, P10070); Drosophila Ci (Accession Number, A38926); C. elegans Tra-1 (Accession Number, P34708); mouse Zic4 (Accession Number, Q61467); ascidian Macho-1 (Accession Number, BAB19958). The Cys and His residues of each finger are highlighted in black. Amino acid residues that are identical or similar in at least 50% of the aligned sequences at a particular position are boxed in dark or light gray, respectively. (C) Phylogenetic tree of Lmd and representatives of the Gli superfamily, based upon Zn-finger domains shown in B. Programs Clustal X and TREEVIEW (Page, 1996) were used to generate the tree that displays the possible way in which the protein sequences may have evolved.

View larger version (99K): [in a new window]   Fig. 4. lmd gene expression in somatic mesodermal cells is severely affected in wg and abolished in N mutant embryos. Embryos were hybridized with a lmd RNA probe (A-D). (A) Dorsal view of late stage 11 embryo with expression in patches of visceral mesoderm. (B) Lateral view of late stage 12 embryos with expression in both somatic and visceral mesodermal layers. (C,D) Stage 13 embryos show decreasing expression in somatic mesodermal cells. Expression is no longer detectable in visceral mesodermal cells. (E-G) Embryos were hybridized to a lmd RNA probe (green) and stained with anti-Eve antibody (red), followed by confocal microscopy. When compared with control embryo (E), wgcx4 mutant embryo (F) shows a dramatic decrease in lmd expression in somatic mesodermal cells in dorsolateral and lateral regions while ventrally located cells (sm; arrow) are not strongly affected. Visceral mesoderm (vm; arrow) is expanded in mutant embryo. In N5419 mutant embryo (G), lmd expression is abolished in all somatic mesodermal cells. Visceral mesoderm is reduced in mutant embryo.

View larger version (70K): [in a new window]   Fig. 5. Lmd protein expression is high in fusion-competent myoblasts and not exclusively nuclear. (A-D) Two segments of a representative rP298-lacZ embryo that was triple-stained with antibodies against Lmd, MEF2 and ß-gal, and analyzed by confocal microscopy. Different channel combinations of the same scan are shown. Co-expression of Lmd (green) and MEF2 (blue) is observed in lacZ-negative fusion-competent myoblasts (green/turquoise signals in A,B). Hollow arrowheads identify representative lacZ-positive (red) founders that express MEF2 (red/pink signals in C,D). A very low level of Lmd expression is observed in these founders (hollow arrowheads in A). (E-H) Three segments of a representative late stage 12 embryo that was triple-stained with antibodies against Lmd, MEF2 and nuclear lamin. Different channel combinations of the same scan are shown. Cy3-labeled lamin (red) demarcates the nuclear envelope. Lmd expression (green) is observed in both the cytoplasm and nucleus (arrowheads in E,G), whereas MEF2 expression (blue) is strictly nuclear (arrowheads in H); see also high magnification views in E1-H1. Co-expression of nuclear Lmd and MEF2 is observed in fusion-competent myoblasts (green/turquoise signals in F). Arrows identify representative myoblasts with exclusively cytoplasmic Lmd and no MEF2 expression; see also high magnification views in E2-H2. Hollow arrowheads identify MEF2-positive founder cells that lack Lmd expression.

View larger version (83K): [in a new window]   Fig. 6. Somatic myoblast enhancer I-E is not activated in lmd mutant embryos. Early stage 13 wild-type and lmd1 mutant embryos, both of which also carry the construct I-E were double-labeled for MEF2 and ß-gal, and analyzed by confocal microscopy. (A,B) Control embryo shows MEF2 expression (A) in somatic myoblasts and cardioblasts at the dorsal margin and lacZ expression (B), directed by enhancer I-E, in somatic (fusion-competent) myoblasts. (C,D) lmd1 mutant embryo shows reduced MEF2 expression (C) in the somatic mesoderm and a complete absence of lacZ expression (D) in somatic myoblasts. Arrow denotes ectopic lacZ expression in non-mesodermal cells.

View larger version (60K): [in a new window]   Fig. 7. Molecular screen with minimal sequences from myoblast enhancer I-ED5 identifies Lmd as the DNA-binding factor. (A) Diagram of deletion constructs, each of which has an internal deletion (thin line) within the 170 bp myoblast enhancer I-ED5. Vertical lines bracket the endpoints of the essential [C/D]* region. (B) Sequence of enhancer I-ED5. Arrows demarcate the [C/D]* region and mutated sequences in constructs I-ED5-mt1, I-ED5-mt2, I-ED5-mt3 and I-ED5-mt4 are underlined. (C) Diagram of the 12 cDNA clones encoding partial Lmd proteins from the one-hybrid screen with [C/D]* as a specific target. Unrelated target T2 was used to check for specificity. The number of clones recovered for each type is in parentheses. Lmd derivatives used to localize the DNA binding domain are also shown. The checked box denotes the Zn-finger domain. Activation of His and lacZ expression was monitored; N.D. denotes not determined. (D) DNA binding assays with in vitro translated Lmd protein and 32P-labeled I-ED5 probe, in the absence or presence of 50x molar excess of cold specific competitor DNA. Free probe is marked as (U). Lane 1, probe alone; Lane 2, lysate without DNA template; Lane 3, Lmd protein lysate; Lane 4, Lmd + cold I-ED5 DNA; Lane 5, Lmd + cold unrelated III-F7 DNA. (E-J) Transgenic embryos were stained with an anti-ß-gal antibody. Robust levels of lacZ expression are seen in somatic myoblasts of embryo with I-ED5 construct (E), whereas embryo with I-ED5-DelD shows a nearly complete loss of expression (F). Construct 5x[C/D]* drives expression in myoblasts similarly to parental I-ED5 (compare E with G). Embryo with I-ED5-mt1 (H) or I-ED5-mt2 (I) exhibits dramatic loss of reporter gene expression in somatic myoblasts, while embryo with I-ED5-mt3 (data not shown) or I-ED5-mt4 (J) shows normal levels of expression. Ectopic expression in I-ED5-mt2 embryo is not in somatic myoblasts.