Genetic Targets of the Ligand-Ultraspiracle (RXR) Signaling Axis for Generation of the Epidermal Cuticular Barrier in Drosophila Melanogaster

Presentation Number: SUN 262
Date of Presentation: April 2nd, 2017

Anna Niewiadomska-Cimicka1, Agnes Sapa2, Rebecca Spokony3, Kamran Siddiqui4, Elizabeth Barajas5, Davy Jones6 and Grace Jones*6
1IGBMC, 2Wroclaw Medical University, Wroclaw, Poland, 3Baruch College, CUNY, New York, 4University of Kentucky, KY, 5University of Kentucky, 6University of Kentucky, Lexington, KY


Introduction: The apical surface barrier is essential for both vertebrates and invertebrates, protecting from dehydration, pathogens and external stresses.1,2 The acellular barrier has dynamic properties, such as being continuously shed or periodically molted. The barrier may form specialized and sclerotized structures such as human fingernails, or a puparium shell around the body in Drosophila. The ongoing restoration of the sloughed acellular barrier and formation of stage-specific specialized structures necessitates tight coordination of epidermal events. We report here advances in understanding a necessary natural ligand-RXR axis that regulates morphogenesis of the larval stage Drosophilaskin.

Methods: We used a Q288A L366A mutation to the ligand binding pocket of the Drosophila nuclear hormone receptor ultraspiracle (“USP,” an ortholog of vertebrate RXR) that strongly reduces binding affinity for the circulating terpenoid ligand methyl farnesoate. When this “QLUSP” is transgenically expressed from its natural promoter in –/-usp animals, the larval epidermis does not produce a puparium cuticle. 3,4 ChIP was performed using ABCAM anti-HA antibody. ChIP-qPCR measurement of USP occupancy of a potential direct target site was made relative to qPCR on the same immunoprecipitate using control ‘desert’ primers (Active Motif). Expression analysis of wandering stage integument was made using Affymetrix 3’ IVT Exon Chip (n=3 each).

Results: We performed ChIP-seq and ChIP-qPCR on +/+usp wandering stage animals that were transgenically expressing from the natural USP promoter either C-terminally HA-tagged wild type USP or QLUSP. ChIP-seq identified USP binding regions at the gene encoding the nuclear hormone receptor Dhr3 (= vertebrate ROR), including a region containing a consensus ultraspiracle-ecdysone receptor binding motif: AGGTCAATAAACT. ChIP-qPCR confirmed that occupancy of that region was significantly lower for the QLUSPHA (10.3+1.1, n=2) as compared to wild type USPHA (29.7+4.0, n=3) (t, p=0.03).

Epidermal expression analysis determined there was significant misexpression of several genes for end-pathway cuticle structural proteins in -/-usp animals transgenically expressing mutant QLUSP, as compared to normal siblings (FDR 0.25), including the gene obst-E (p = 0.007). Functional disruption of obst-Eexpression by either RNAi (VDRC UAS-RNAi 104901; “A58” GAL4 driver) or p-element insertion (BDSC stock 42089) yielded third instar animals with misformed puparium cuticle.

Conclusion: Our results support the model of a ligand/RXR (MF/USP) axis that mediates a transcriptional-to-structural regulatory network for generation of this model epidermal barrier.

 *First 3 authors: alphabetical co-leading authorship


Nothing to Disclose: AN, AS, RS, KS, EB, DJ, GJ