During development and differentiation, structural tissue-specific proteins undergo isoform switching. Although instances of multiple isoform expression have been described for all multicellular organisms, particular mechanisms of isoform switching in many cases still remain unclear. Among the five Drosophila Troponin C isoforms, TpnC4 is predominantly expressed in the fibrillar-type fibers of the indirect flight muscle, whereas TpnC41C is the main isoform of the tubular-type fibers of jump muscle. In our study, we show that these two isoforms are essential for the functioning of thoracic muscles, and can mutually replace each other when the expression of each is down-regulated.
To analyze functional importance of the two TpnC isoforms, we created genetic lines with single gene deletions for either TpnC4 or TpnC41C, and a line with a double deletion for both genes. We found that the removal of one of the isoforms resulted in reciprocal activation of expression of the other isoform in both fiber types. Moreover, minimal enhancers of TpnC4 and TpnC41C can be activated in both fiber types, suggesting that normal isoform expression pattern is rather a result of transcriptional repression in non-native muscles. However, functionally these two isoforms were not equivalent. Although TpnC4 expressed in jump muscles was able to support effective jumping, TpnC41C expressed in flight muscles could not sustain flight. Flies with the TpnC4/TpnC41C double deletion showed flightless and jumpless phenotypes. Similar data were obtained in experiments with RNAi knockdown of these genes. Analysis of the ultrastructural muscle organization in the flies with the double deletion revealed defects in myofibril alignment and Z-line integrity.
Our study provides insight into isoform switching mechanisms for muscle genes, and defines the importance of TpnC for normal muscle assembly.