Differential requirements for Myocyte Enhancer Factor-2 during adult myogenesis in Drosophila.

Identifying the genetic program that leads to formation of functionally and morphologically distinct muscle fibers is one of the major challenges in developmental biology. In Drosophila, the Myocyte Enhancer Factor-2 (MEF2) transcription factor is important for all types of embryonic muscle differentiation. In this study we investigated the role of MEF2 at different stages of adult skeletal muscle formation, where a diverse group of specialized muscles arises. Through stage- and tissue-specific expression of Mef2 RNAi constructs, we demonstrate that MEF2 is critical at the early stages of adult myoblast fusion: mutant myoblasts are attracted normally to their founder cell targets, but are unable to fuse to form myotubes. Interestingly, ablation of Mef2 expression at later stages of development showed MEF2 to be more dispensable for structural gene expression: after myoblast fusion, Mef2 knockdown did not interrupt expression of major structural gene transcripts, and myofibrils were formed. However, the MEF2-depleted fibers showed impaired integrity and a lack of fibrillar organization. When Mef2 RNAi was induced in muscles following eclosion, we found no adverse effects of attenuating Mef2 function. We conclude that in the context of adult myogenesis, MEF2 remains an essential factor, participating in control of myoblast fusion, and myofibrillogenesis in developing myotubes. However, MEF2 does not show a major requirement in the maintenance of muscle structural gene expression. Our findings point to the importance of a diversity of regulatory factors that are required for the formation and function of the distinct muscle fibers found in animals.

A molecular mechanism of temperature sensitivity for mutations affecting the Drosophila muscle regulator Myocyte enhancer factor-2.

Temperature-sensitive (TS) mutations are a useful tool for elucidating gene function where a gene of interest is essential at multiple stages of development. However, the molecular mechanisms behind TS alleles vary. TS mutations of the myogenic regulator Myocyte enhancer factor-2 (MEF2) in Drosophila arise in the heteroallelic combination Mef2(30-5)/Mef2(44-5). We show that the 30-5 mutation affects the N-terminal MADS domain, causing impaired DNA binding ability and failure of homozygous mutants to survive to adulthood. The 44-5 mutation deletes a downstream splice acceptor site and results in a truncated protein that is unable to activate MEF2 targets. 44-5 homozygotes consequently show severely impaired myogenesis and die as embryos. We propose that in heteroallelic mutants at the permissive temperature, 30-5/44-5 heterodimers form and have a sufficiently stable interaction with DNA to activate myogenic gene expression; at the restrictive temperature, 44-5 homodimers displace 30-5/44-5 heterodimers from target genes, thus acting in a dominant-negative manner. To test this, we show that 30-5/44-5 heterodimers can form, and we study additional Mef2 alleles for complementation with the 30-5 allele. An allele affecting the DNA binding domain fails to complement 30-5, whereas two alleles affecting downstream residues show temperature-dependent complementation. Thus, by combining one MEF2 isoform having weakened DNA binding ability with a second truncated MEF2 mutant that has lost its activation ability, a TS form of intragenic complementation can be generated. These findings will provide new insight and guidance into the functions of dimeric proteins and how they might be engineered to generate TS combinations.

Adult myogenesis in Drosophila melanogaster can proceed independently of myocyte enhancer factor-2.

Myocyte enhancer factor-2 (MEF2) is a transcription factor that is necessary for embryonic muscle development in Drosophila and vertebrates; however, whether this factor is required during later muscle development remains largely unknown. Using heteroallelic combinations of different Mef2 mutant alleles, we isolated and characterized a temperature-sensitive combination. Through temperature-shift experiments, we obtained adult animals that were lacking proper MEF2 function. Many of these individuals died as mature pupae, and those that eclosed showed poor locomotion and an inability to fly. Histological analysis of these animals revealed a requirement for MEF2 in skeletal muscle patterning, although these animals had strikingly normal amounts of muscle tissue. Using quantitative polymerase chain reaction, we determined that expression of the MEF2-regulated actin gene Act57B was severely reduced in these animals. By contrast myofibrillar actin genes unique to the adult stage were only mildly affected. Since MEF2 mutant adults were still capable of forming muscle tissue, we conclude that MEF2 is required for the expression of only a subset of muscle structural genes in the adult. These results indicate that additional muscle-specific factors function to control the myogenesis of complex and diverse muscle in the adult.