Knockout of either of two Drosophila Troponin C genes that are expressed in either the flight muscle or the jump muscle resulted in expansion of transcription of its paralogue into the affected muscle. Although either isoform can support normal jumping, only the flight isoform can support flight.
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.
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.
The muscles of Drosophila show similar structure and pathway of development to the muscles of humans, and allows Drosophila to model human muscle disorders. However, little is known about many of the genes and their potential role in muscle development. Thus, the focus of this project is to efficiently screen for potential transcriptional regulators of muscle development and identify associated disorder phenotypes. Genes were selected based on having a potential role in transcription, which were silenced using RNA interference (RNAi). The 1151‐gal4 driver was utilized to induce RNAi in developing adult muscles. Combined with the driver, the flies harbored enhancers of either Flightin‐LacZ or TpnC41C‐LacZ. The resulting transgenic flies were collected and assayed to measure the effects of this genetic loss. All 101 genes in the latest set have been assayed for a change of enhancer activity of less than 70% or greater than 130%, in Flightin and TpnC41C; the genes selected for immunostaining exhibited semi‐lethality or lethality, and indicated a loss or gain of LacZ. The immunostaining would reveal several factors: muscle morphology, and expansion or loss of enhancer activity associated with specific flight and jump muscle genes. The resulting data was classified into three categories, severe loss, moderate loss, or no change in muscle development. Genes with a severe loss indicates a potential issue with myoblasts fusion, and genes with a moderate loss indicates issues with adult muscle formation and maturation. Therefore, the results demonstrate that the screen is efficient in identifying potential transcriptional regulators and muscle disorder phenotypes.Support or Funding InformationNational Institute of Health ROI GM061738This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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