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

A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elabo rate and maintain the differentiated muscle phenotype through transcription al regulation of muscle-specific target genes. Much work suggests that memb ers of the cysteine-rich protein (CRP) family of LIM domain proteins also p lay a role in muscle differentiation; however, the specific functions of CR Ps in this process remain undefined. Previously, we characterized two membe rs of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, which show restricted expression in differentiating muscle lineages. To ex tend our analysis of Drosophila Mlps, we characterized the expression of Ml ps in mutant backgrounds that disrupt specific aspects of muscle developmen t. We show a genetic requirement for the transcription factor dMEF2 in regu lating Mlp expression and an ability of dMEF2 to bind, in vitro, to consens us MEF2 sites derived from those present in Mlp genomic sequences. These da ta suggest that the Mlp genes may be direct targets of dMEF2 within the gen etic hierarchy controlling muscle differentiation. Mutations that disrupt m yoblast fusion fail to affect Mlp expression. Ln later stages of myogenic d ifferentiation, which are dedicated primarily to assembly of the contractil e apparatus, we analyzed the subcellular distribution of Mlp84B in detail. Immunofluorescent studies revealed the localization of Mlp84B to muscle att achment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and alpha-actinin, key compon ents of these structures, also failed to perturb Mlp84B distribution. Ln co nclusion, we have used molecular epistasis analysis to position Mlp functio n 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 cyto architecture.