fushi tarazu and fushi tarazu factor 1, novel re-wiring in the Tribolium castaneum pair-rule gene network
Authors: Ximena Gutierrez Ramos; Patricia L. Graham; Leslie Pick
Affiliation: University of Maryland, College Park
Keywords: l. evo-devo; f. pattern formation
Segmentation is a fixed feature of the insect body plan, yet surprisingly, the genetic network controlling the formation of segments has changed over the course of insect evolution. In Drosophila, pair-rule genes are responsible for allocating groups of cells to specific body regions that develop into separate, morphologically distinct body segments. Transcription factor (TF)-encoding genes regulate sets of downstream genes that include those encoding other TFs, signaling proteins, and products directly involved in morphogenesis. The evolutionary variation seen for pair-rule genes contrast with the conservation of expression of downstream target genes. How does the regulatory network re-wire to maintain downstream gene expression while upstream regulators are lost, gained, or changed in function? We are exploring this question by examining the expression, function, and interactions of the pair-rule gene fushi tarazu (ftz) in the flour beetle, Tribolium castaneum (Tc). Tc-FTZ contains an LXXLL motif that is necessary for the functional interaction of FTZ and its obligatory partner FTZ-F1 in Drosophila. Despite this, a large genomic deletion (Stuart et al., 1991) and RNAi experiments (Choe et al., 2006 and data not shown) indicate that Tc-ftz plays no role in segment formation in Tribolium. In contrast, FTZ-F1 is expressed in pair-rule stripes in Tribolium and RNAi knockdown resulted in pair-rule defects (Heffer et al., 2013). To definitively determine whether Tc-ftz plays a role in segmentation, we are using CRISPR/Cas9 to generate a genomic deletion of this gene. Further, to understand how FTZ-F1 regulates pair-rule patterning in Tribolium without the input of FTZ, we are examining cis-regulatory elements of the conserved target gene engrailed (en). We are testing approaches to using CRISPR-based and piggybac-based enhancer reporter genes to identify en regulatory elements and assess their dependency on Tc-FTZ-F1, using dsRNA to knockdown Tc-ftz-f1 expression. Other TFs binding to identified cis-regulatory elements will be identified and characterized. Simultaneously, we have generated a FLAG-tagged version of Tc-FTZ-F1 to identify other genomic targets, determine whether or not Tc-FTZ plays a role in their regulation and to identify the DNA binding partners using co-immunoprecipitation. This will allow us to understand how a rearrangement of upstream regulators occurs to maintain a conserved downstream gene expression pattern, and provide information on the transcriptional regulation of the downstream genes within the Tribolium pair-rule gene network.