Identification and characterization of genes that modify tubulogenesis defects caused by over-expression of Imaginal Disc Growth Factor-3 using the Drosophila dorsal appendage model, a partial genomic expedition

Abstract

Biological tube formation underlies organ development, and when disrupted, can cause severe birth defects. To investigate the genetic basis of tubulogenesis, I study the formation of Drosophila melanogaster eggshell structures, called dorsal appendages, which are produced by epithelial tubes. Previously in my lab we found that precise levels of Drosophila Chitinase-like proteins (CLPs), encoded by the Imaginal disc growth factor (Idgf) gene family, are needed to regulate dorsal-appendage tube closure and tube migration. To identify factors that act in the Idgf pathway, I developed a genetic modifier screen based on the finding that overexpressing Idgf3 causes dorsal appendage defects with ~50% frequency. Using a library of partially overlapping heterozygous deficiencies, I scanned chromosome 3L and found regions that enhanced or suppressed the Idgf3-overexpression phenotype. Using smaller deletions, RNAi, and mutant alleles, I further mapped five regions and refined the interactions to 58 candidate genes. Importantly, mutant alleles identified combover (cmb), a substrate of Rho-kinase (Rok) and a component of the Planar Cell Polarity (PCP) pathway, as an Idgf3-interacting gene: loss of function enhanced while gain of function suppressed the dorsal appendage defects. Since PCP drives cell intercalation in other systems, I asked if cmb/+ affected cell intercalation in my model, but I found no evidence of its involvement in this step. Instead, I found that loss of cmb dominantly enhanced tube defects associated with Idgf3-overexpression by expanding the apical area of dorsal appendage cells. Apical surface area determines tube volume and shape; in this way, Idgf3 and cmb regulate tube morphology. Additionally, I narrowed down the strongest enhancer region to eIF3e as a gene candidate. Validation of an interaction between Idgf3 and eIF3e remains to be tested, as a null allele for eIF3e is not available. Also, I found that by silencing eIF3e, eclosion and egg production fail, preventing me from testing this interaction with the genetic tools available. By testing genes known to interact with eIF3e, I looked for indirect evidence that Idgf3 interacts with eIF3e for dorsal appendage formation. eIF3e is part of a translation regulatory complex, which plays a role in regulating Mical translation. Mical interacts with PlexA for neuronal dendrite pruning. I found evidence that suggest overexpression of Mical might suppress the DA phenotypes that result from overexpressing Idgf3. Additionally, I found a role for PlexA in DA formation, and that lowering expression of PlexA enhances the Idgf3-overexpression phenotype. I suggest different experiments that can allow me to verify this interaction, and I hypothesize models that explain how Idgf3 might interact with the PlexA-Mical pathway, only if PlexA and Mical interact in the dorsal-appendage making cells during DA formation as they interact in neurons during dendrite pruning. Finally, I quickly tested other genes that, in my lab, we have found to play a role in DA formation. In this way I identified other possible Idgf3-interacting genes.