Regulation of fiber-specific actin expression by the Drosophila SRF ortholog Blistered.

Serum response factor (SRF) has an established role in controlling actin homeostasis in mammalian cells, yet its role in non-vertebrate muscle development has remained enigmatic. Here, we demonstrate that the single Drosophila SRF ortholog, termed Blistered (Bs), is expressed in all adult muscles, but Bs is required for muscle organization only in the adult indirect flight muscles. Bs is a direct activator of the flight muscle actin gene Act88F, via a conserved promoter-proximal binding site. However, Bs only activates Act88F expression in the context of the flight muscle regulatory program provided by the Pbx and Meis orthologs Extradenticle and Homothorax, and appears to function in a similar manner to mammalian SRF in muscle maturation. These studies place Bs in a regulatory framework where it functions to sustain the flight muscle phenotype in Drosophila Our studies uncover an evolutionarily ancient role for SRF in regulating muscle actin expression, and provide a model for how SRF might function to sustain muscle fate downstream of pioneer factors.

Myogenesis in Drosophila melanogaster: Dissection of Distinct Muscle Types for Molecular Analysis.

Drosophila is a useful model organism for studying the molecular signatures that define specific muscle types during myogenesis. It possesses significant genetic conservation with humans for muscle disease causing genes and a lack of redundancy that simplifies functional analysis. Traditional molecular methods can be utilized to understand muscle developmental processes such as Western blots, in situ hybridizations, RT-PCR and RNAseq, to name a few. However, one challenge for these molecular methods is the ability to dissect different muscle types. In this protocol we describe some useful techniques for extracting muscles from the pupal and adult stages of development using flight and jump muscles as an example.

Functional redundancy and nonredundancy between two Troponin C isoforms in Drosophila adult muscles.

We investigated the functional overlap of two muscle Troponin C (TpnC) genes that are expressed in the adult fruit fly, Drosophila melanogaster: TpnC4 is predominantly expressed in the indirect flight muscles (IFMs), whereas TpnC41C is the main isoform in the tergal depressor of the trochanter muscle (TDT; jump muscle). Using CRISPR/Cas9, we created a transgenic line with a homozygous deletion of TpnC41C and compared its phenotype to a line lacking functional TpnC4 We found that the removal of either of these genes leads to expression of the other isoform in both muscle types. The switching between isoforms occurs at the transcriptional level and involves minimal enhancers located upstream of the transcription start points of each gene. Functionally, the two TpnC isoforms were not equal. Although ectopic TpnC4 in TDT muscles was able to maintain jumping ability, TpnC41C in IFMs could not effectively support flying. Simultaneous functional disruption of both TpnC genes resulted in jump-defective and flightless phenotypes of the survivors, as well as abnormal sarcomere organization. These results indicated that TpnC is required for myofibril assembly, and that there is functional specialization among TpnC isoforms in Drosophila.

Arrest is a regulator of fiber-specific alternative splicing in the indirect flight muscles of Drosophila.

Drosophila melanogaster flight muscles are distinct from other skeletal muscles, such as jump muscles, and express several uniquely spliced muscle-associated transcripts. We sought to identify factors mediating splicing differences between the flight and jump muscle fiber types. We found that the ribonucleic acid-binding protein Arrest (Aret) is expressed in flight muscles: in founder cells, Aret accumulates in a novel intranuclear compartment that we termed the Bruno body, and after the onset of muscle differentiation, Aret disperses in the nucleus. Down-regulation of the aret gene led to ultrastructural changes and functional impairment of flight muscles, and transcripts of structural genes expressed in the flight muscles became spliced in a manner characteristic of jump muscles. Aret also potently promoted flight muscle splicing patterns when ectopically expressed in jump muscles or tissue culture cells. Genetically, aret is located downstream of exd (extradenticle), hth (homothorax), and salm (spalt major), transcription factors that control fiber identity. Our observations provide insight into a transcriptional and splicing regulatory network for muscle fiber specification.