Identification of novel genes involved in planar cell polarity establishment in the Drosophila eye

Abstract

In multicellular organisms proper functioning of tissues requires precise patterning of cells, which is acquired by apical-basal and planar cell polarization. To establish planar cell polarity (PCP), cells have to organize themselves along the plane of the epithelium. The mechanisms leading to this organization include several signalling pathways and cytoskeletal arrangements. Although general aspects of PCP have been elucidated, a complete view has not been established yet. It is known that genetic control of planar cell polarization is highly conserved among vertebrates and invertebrates. The Drosophila eye is a remarkable model system to study PCP, which is evident in organization of photoreceptors (PRs) into trapezoidal structures pointing to opposite directions in different halves of the eye. In the process of polarization, PR clusters require correct specification of R3/R4 cells, followed by chirality establishment and ommatidial rotation. We aimed to identify novel genes involved in PCP establishment by following two approaches. In the first approach, effects of six putative R3/R4 specific genes (CG33259, cropped, faint sausage, polychaetoid, Stubble, taranis) were analyzed by RNAi down-regulation, and faint sausage (fas) was determined as a promising candidate. Then, we generated recombinant fas-mutant flies to use in further mutant analyses. In the second approach, R3/R4 cells were sorted by FACS and analyzed by RNA-Seq in order to identify differentially expressed (DE) genes that might have role in PCP establishment. From this analysis, ets domain lacking (edl) appeared to be an interesting candidate. Additionally, we used this dataset to predict putative transcription factors that might be regulating differentially expressed genes. These analyses yielded six putative regulators of the DE genes in R3/R4: Trithorax-like, Grainy head, Jim, DNA replication-related element factor, Cropped, and CG7928. Furthermore, gene regulatory network data were used to dissect the Svp targetome, hypothesizing that this targetome might be containing genes contributing to PCP in the eye. Out of four putative targets that were selected for validation (couch potato, bruchpilot, futsch, pebbled), only one, pebbled, was verified as being regulated by Svp.