Muscle LIM proteins are associated with muscle sarcomeres and require dMEF2 for their expression during Drosophila myogenesis

Abstract

A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Previously, we characterized two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, which show restricted expression in differentiating muscle lineages. To extend our analysis of Drosophila Mlps, we characterized the expression of Mlps in mutant backgrounds that disrupt specific aspects of muscle development. We show a genetic requirement for the transcription factor dMEF2 in regulating Mlp expression and an ability of dMEF2 to bind, in vitro, to consensus MEF2 sites derived from those present in Mlp genomic sequences. These data suggest that the Mlp genes may be direct targets of dMEF2 within the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation, which are dedicated primarily to assembly of the contractile apparatus, we analyzed the subcellular distribution of Mlp84B in detail. Immunofluorescent studies revealed the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and alpha-actinin, key components of these structures, also failed to perturb Mlp84B distribution. In conclusion, we have used molecular epistasis analysis to position Mlp function downstream of events involving mesoderm specification and patterning and concomitant with terminal muscle differentiation. Furthermore, our results are consistent with a structural role for Mlps as components of muscle cytoarchitecture.

Figure 1

Schematic representation of the CRP family of LIM domain proteins. In vertebrates, there are three conserved isoforms, CRP1, CRP2, and CRP3/MLP, encoded by unique genes. All share the same molecular architecture with two LIM domains, each followed by short glycine-rich regions (black box). In Drosophila, there are two proteins related to the vertebrate CRPs. Mlp60A exhibits a single LIM-glycine motif. Mlp84B comprises five tandem LIM-glycine modules.

Figure 2

The transcription factor dMEF2 is essential for Mlp expression. dMEF2 heterozygous embryos express Mlp60A (A) and Mlp84B (B) in somatic, visceral, and pharyngeal muscles. These embryos also carry the balancer chromosome from which the lacZ gene is expressed. Thus, by indirect immunofluorescence, we detect not only Mlp expression in these embryos but expression of β-galactosidase in single cells dispersed throughout the embryo (A and B, arrowheads). Mlp60A (C) and Mlp84B (D) fail to be expressed in the dMEF2 null mutant embryos. Null embryos display defective midgut morphology and no β-galactosidase–positive cells. All panels show lateral views of stage 16 (13- to 16-h) embryos oriented with dorsal up and anterior to the left. Bar, 50 μm.

Figure 3

Ectopic dMEF2 expression stimulates expression of myosin and Mlp84B but not Mlp60A. In wild-type embryos, myosin (A), Mlp84B (B), and Mlp60A (C) are detected by indirect immunofluorescence in embryonic somatic muscles (sm) but not epidermal cells (ep). Myosin (D) and Mlp84B (E) expression can be induced in the epidermis by ectopic expression of dMEF2 under the control of the 69B GAL4 enhancer. Unlike myosin and Mlp84B, Mlp60A is not up-regulated by ectopic expression of epidermal dMEF2 (F). All panels display lateral views of 12- to 14-h embryos oriented with dorsal up and anterior to the left. Bar, 20 μm.

Figure 4

dMEF2 protein binds to consensus MEF2 sites found in the genomic regions of the Mlp genes. (A) Diagram of the genomic organization of the Mlp60A and Mlp84B genes. The boxed areas indicate the regions that correspond to cDNA sequences. The sticks indicate positions of 10-bp elements that exactly match the MEF2 consensus binding site. The two sites indicated by filled circles are those tested in the in vitro dMEF2 binding assay shown in C. The exact nucleotide range of the Mlp84B sites is noted in GenBank accession number AF090832. (B) The potential MEF2 binding elements found in the putative regulatory regions of Mlp60A and Mlp84B are shown aligned with the MEF2 consensus, the MEF2 regulatory site from the MCK enhancer, and the Mutant 6 form of the MCK site, which does not support MEF2 binding. Letters to the left correspond to the sites diagrammed in A, and the filled letters indicate those sites tested in C. (C) Mobility shift assays with in vitro–translated dMEF2 protein were performed with oligonucleotides corresponding to the MCK MEF2 site and one MEF2 element from each of the Mlp genes. The MEF2 elements used as probes were MCK MEF2 (lanes 1–6), Mlp60A-B (lanes 7–11), and Mlp84B-D (lanes 12–16). For each probe a control lysate lacking translated dMEF2 was included in parallel (lanes 1, 7, and 12). Competitor oligonucleotides were used at 100-fold molar excess of each probe. dMEF2 binds to each of the probes specifically (lanes 2, 8, and 13). Each of the Mlp MEF2 sites binds dMEF2 and competes for binding to the MCK element and their cognate site. A mutant form of the MCK element (Mutant 6) fails to compete for binding with any of the sites tested (lanes 4, 11, and 16).

Figure 5

Myoblast fusion is not required for muscle LIM protein expression. Immunofluorescent detection of Mlp60A (A and C) or Mlp84B (B and D) in embryos mutant for the rollingstone gene is shown. These embryos display severe defects in myoblast fusion. Both Mlps are expressed in single unfused myoblasts (mb) as well as in myotubes (mt) that have formed (see higher magnification; C and D). All panels display ventral-lateral views of late stage embryos with anterior to the left. Bars: A and B, 50 μm; C and D, 20 μm.

Figure 6

Mlp84B localizes to discrete sites within sarcomeres of larval midgut visceral muscles. The mesoderm surrounding a third instar larval midgut has been double labeled for α-actinin (red), to visualize Z-bands, and Mlp84B (green). Most of the visceral muscles are positioned horizontally, but muscle cells can be seen on top of these positioned vertically in the figure. Mlp84B localizes as doublets within the muscles (B). In the merged image (C), Mlp84B is observed to flank α-actinin in a region adjacent to the Z-bands (see diagram of two adjacent sarcomeres with bands indicated in D). Bar, 10 μm.

Figure 7

Loss of α-actinin does not affect Mlp84B localization within visceral muscles. α-Actinin null mutant l(1)HC288/Y and wild-type midgut muscles have been double labeled for α-actinin (A and C) and Mlp84B (B and D). The null mutant lacks staining for α-actinin protein (C), and wild type is shown for comparison (A). Note that Mlp84B distribution is similar in the wild-type (B) and mutant (D) visceral muscles showing doublets flanking the Z-bands. In the mutant, Mlp84B still localizes to discrete sites within sarcomeres. Bar, 10 μm.

Figure 8

Mlp84B colocalizes with β_PS integrin at the MASs. Ventral-lateral longitudinal muscles of a stage 16 embryo are double labeled for Mlp84B (green) and the β_PS subunit of integrin (red). The merged image (C) reveals significant colocalization of the two proteins (yellow) at the MASs. The arrow in C indicates β_PS complexes in the tendon cells. Bar, 20 μm.

Figure 9

Mlp84B is capable of associating with MASs in the absence of integrin–ligand interactions. Immunofluorescent detection of Mlp84B in somatic muscles of stage 16 wild-type (A and B), mys (C and D), or if (E and F) embryos is shown. Note the characteristic rounded muscles observed in embryos mutant for either integrin subunit. Arrows indicate Mlp84B enrichment at the MASs of wild-type embryos (A and B) or at remnant junctions in mutant embryos (C-F). Bar, 20 μm.