Using Genomic Transgenes and the CRISPR/Cas9 Gene Editing System to Understand How Hedgehog Signaling Regulates Costal2 and Cubitus Interruptus in Drosophila melanogaster

Abstract

The Hedgehog protein (Hh) is a morphogen that is necessary for cell survival, growth and patterning in flies and mammals. In germline cells, alterations in the Hh signaling pathway can result in developmental disorders; in somatic cells, misregulation of the Hh signaling pathway can result in cancer. Most components of the signaling pathway were identified by genetic screens in Drosophila that were later found to be conserved in mammals. In the presence of the Hh signal, multiple Hh signaling components interact to mediate the induction of Hh target genes. In flies, Cubitus Interruptus (Ci) is the singular transcription factor of the pathway that is regulated by multiple upstream components of the pathway including Costal2 (Cos2). Cos2 is a scaffold protein that can both positively and negatively regulate Hh signaling by binding to Ci and various kinases such as Fused (Fu). We disrupted the binding of Cos2 to Fu using a physiological expressed genomic Costal2 transgene (gCosΔFu) and found that Fu must bind to Cos2 to promote efficient processing and activation of full-length Ci (Ci-155). Fu was thought to activate Ci-155 by phosphorylating Cos2 at sites S931 and S572, but we found that gCosS931A and gCosS572A did not reduce Ci activity in the fly wing disc. Instead, we hypothesize that Fu could directly phosphorylate Ci-155 or another unknown protein. To investigate if another protein was involved we developed a Hh sensitized genetic screen. We obtained multiple “hits” from the genetic screen but we did not find an obvious candidate that could be a substrate for Fu. Instead, we identified Mago Nashi and Srp54 which we found to be involved with the post-transcriptional regulation of ci RNA. We confirmed the existence of ci isoforms A and B and found that knockdown of Mago Nashi, resulted in an altered splicing pattern while knockdown of Srp54 reduced ci RNA levels. Mago Nashi inhibition and intronless Ci reduced Ci-155 protein levels, which suggests efficient splicing is necessary for normal Ci-155 levels. Furthermore, we found that reduced Ci-155 levels only affected Ci activity in sub-optimal Hh signaling conditions. In order to further dissect the mechanism Ci processing, activation and stabilization, we used physiologically expressed genomic Ci (gCi) and CRISPR Ci variants (crCi). First we examined Ci-S849A, which prevents Ci processing and we found that in the absence of processing, Ci-155 levels are uniformly high throughout the wing disc. Cos2 and PKA are necessary for Ci processing but we wanted to know if they had an additional role in Ci silencing We found that Cos2 but not PKA can silence and stabilize Ci-155 in the absence of processing. Activated Fu in the Ci-S849A wing disc highly activated and destabilized Ci-155, which was similar to Hh signaling at the AP Border. To test if Ci is the direct target of Fu, we are testing physiologically expressed Ci with point mutations and deletions that are near the Suppressor of Fused (Su(fu)) binding site to examine whether they are unresponsive to activated Fu. Su(fu) binds to Ci-155 to stabilize Ci- 155 levels and inhibit Ci activity, but the mechanism is not well understood. We developed Ci transgenes that have altered Su(fu) binding to determine if Su(fu) inhibits Ci by cytoplasmic anchoring, co-repressor recruitment, or by blocking a co-activator.