Morphogenesis is a fundamental developmental process that encompasses myriad cellular processes, including cell shape changes that determine an organism's form and function. The RhoA signal transduction pathway is highly conserved at cellular and molecular levels and significantly influences morphogenetic processes through regulation of cell shape changes. RhoA accomplishes this, in part, by regulating contraction of nonmuscle myosin and the subsequent sliding of the apical actin cytoskeleton to change cell shape. Since these processes are so highly conserved, Drosophila melanogaster serves as an effective model organism for these studies. Drosophila requires RhoA function during embryonic and pupal morphogenesis. RhoA mutants are recessive embryonic lethals that disrupt head involution. However, later developmental stages also require RhoA. In order to study the RhoA pathway during post-embryonic phases, a heat-inducible wild-type RhoA transgene (hRh) was constructed using PCR amplification and standard subcloning techniques. The hRh transgene was inserted into the Drosophila genome by injection into pre-cellularized Drosophila embryos by Rainbow Transgenic Flies, Inc. Seven transgenic lines were generated, and insertion locations were chromosomally mapped by a series of segregation analysis matings. Four of the seven lines had single transgene inserts; one of them mapped to the X-chromosome and the other three mapped to the Second chromosome. The remaining three lines exhibited two or three insertions within the genome, mapping to the Second, Third and/or Fourth chromosomes. A series of crosses were performed to accurately map these insertions while simultaneously generating sublines from each of them that carry only a single transgene insertion. Finally, the X-linked hRh transgene insertion was successfully introduced into a RhoA loss-of-function mutant background. In the future, these stocks will be analyzed for heat-induced rescue of the RhoA embryonic lethality.
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Vol. 85 • No. 1