Forward and reverse genetic approaches to identify genes involved in leg morphogenesis in Drosophila

The body plan of metazoan animals is ultimately defined by a series of coordinated morphogenetic events, some of which are controlled by hormone or endocrine signals. Development of Drosophila leg imaginal discs provides an ideal model to study hormoneregulated morphogenesis. During the onset of metamorphosis, a pulse of steroid hormone ecdysone triggers the rapid transformation of flat leg imaginal discs into rudimentary adult fly legs, largely through coordination of cell shape changes and cell rearrangements. The ecdysoneinducible early gene broad (br), is required for this morphogenesis. Leg development in amorphic br 5 mutant animals is arrested at a stage similar to that of a wild-type disc at puparium formation. Hypomorphic alleles of br, including br1, demonstrate a low penetrance of malformed adult legs. To better understand how ecdysone regulates tissue morphogenesis, I used both forward and reverse genetic approaches to study leg morphogenesis in my graduate studies.;Previously, the Ward lab conducted two large-scale genetic screens and determined that the Rho1 signaling pathway interacts with br and plays a central role in imaginal discs morphogenesis. From these screens several unknown dominant Enhancer of br or E( br) alleles that showed interactions with both br and Rho1 pathway genes were isolated. My first project was to characterize E(br)165, which I determined was a mutation in Sec61alpha and renamed it Sec61alpha1. Sec61alpha encodes the main subunit of the translocon complex for co-translational import of proteins into the endoplasmic reticulum (ER). Since Sec61alpha 1 (an EMS strong loss of function allele) is embryonic lethal, I concentrated on studying its role in embryogenesis. Sec61alpha 1 specifically perturbs dorsal closure during embryogenesis. During dorsal closure in Drosophila, signaling events in the dorsalmost row of epidermal cells (DME cells) direct the migration of lateral epidermal sheets towards the dorsal midline where they fuse to enclose the embryo. A Jun amino-terminal kinase (JNK) cascade in the DME cells induces the expression of Decapentaplegic (Dpp). Dpp signaling then regulates the cytoskeleton in the DME cells and amnioserosa to affect the cell shape changes necessary to complete dorsal closure. JNK signaling is normal in Sec61alpha mutant embryos, but Dpp signaling is attenuated, and the DME cells fail to establish an actinomyosin cable and epithelial migration fails. Consistent with this model, dorsal closure is rescued in Sec61alpha mutant embryos by an activated form of the Dpp receptor Thick veins. With regards to leg morphogenesis, these observations suggest that normal secretion levels have to be maintained during this process, and one or more dose-sensitive secreted proteins may be essential for hormone-regulated imaginal disc morphogenesis.;My second project was to identify and characterize genes that are regulated by ecdysone and the transcription factor br at the onset of metamorphosis in leg imaginal discs. To accomplish this, I conducted a series of microarray experiments to identify genes that are differently regulated by br in leg discs at the onset of metamorphosis. I also used microarrays to identify genes differentially expressed between mid third instar larva and 0 hr prepupa in wild type animals. Interestingly there is only a small subset of genes that are induced or repressed by both ecdysone and br. Northern blot analysis showed that, instead of br expression being induced by the late larval ecdysone pulse, br transcripts are already present in late third instar larvae. This suggests that br may function in concert with ecdysone to regulate leg morphogenesis. Consistent with this idea, early activation of br or early supply of ecdysone does not induce premature leg imaginal disc elongation, indicating that neither br nor ecdysone alone is sufficient to induce a leg development program. To examine the function of these br- and ecdysone-induced genes in more details, I conducted functional analyses by RNA interference knockdown in distal leg segments. Ten of the 27 ecdysone- and br-induced genes thus far examined produce animals showing at least 20% malformed legs.;Some of the genes that were identified from the forward and reverse genetic screens overlap, or function in the similar pathways, indicating their important roles in ecdysone-regulated leg morphogenesis. These findings may thus help us to better understand hormone-regulated morphogenetic events in Drosophila and other organisms.