Muscles organise pseudo-crystalline arrays of actin, myosin and titin filaments to build force-producing sarcomeres. To study sarcomerogenesis, we have generated a transcriptomics resource of developing Drosophila flight muscles and identified 40 distinct expression profile clusters. Strikingly, most sarcomeric components group in two clusters, which are strongly induced after all myofibrils have been assembled, indicating a transcriptional transition during myofibrillogenesis. Following myofibril assembly, many short sarcomeres are added to each myofibril. Subsequently, all sarcomeres mature, reaching 1.5 µm diameter and 3.2 µm length and acquiring stretch-sensitivity. The efficient induction of the transcriptional transition during myofibrillogenesis, including the transcriptional boost of sarcomeric components, requires in part the transcriptional regulator Spalt major. As a consequence of Spalt knock-down, sarcomere maturation is defective and fibers fail to gain stretch-sensitivity. Together, this defines an ordered sarcomere morphogenesis process under precise transcriptional control – a concept that may also apply to vertebrate muscle or heart development.
The development of clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) technologies promises a quantum leap in genome engineering of model organisms. However, CRISPR-mediated gene targeting reports in Drosophila melanogaster are still restricted to a few genes, use variable experimental conditions, and vary in efficiency, questioning the universal applicability of the method. Here, we developed an efficient two-step strategy to flexibly engineer the fly genome by combining CRISPR with recombinase-mediated cassette exchange (RMCE). In the first step, two sgRNAs, whose activity had been tested in cell culture, were co-injected together with a donor plasmid into transgenic Act5C-Cas9, Ligase4 mutant embryos and the homologous integration events were identified by eye fluorescence. In the second step, the eye marker was replaced with DNA sequences of choice using RMCE enabling flexible gene modification. We applied this strategy to engineer four different locations in the genome, including a gene on the fourth chromosome, at comparably high efficiencies. Our data suggest that any fly laboratory can engineer their favorite gene for a broad range of applications within approximately 3 months.
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