An RNAi Screen for Skin Morphogenesis Unravels The Complexities of Rho GTPase Networks

During embryogenesis, antagonism and synergy between developmental signaling pathways dictate cell fate decisions. How these circuits orchestrate the complex cellular remodeling needed for tissue morphogenesis remains poorly understood. As effectors of cytoskeletal dynamics, Rho GTPases and their regulators are likely to be involved. However, their daunting complexity has hindered progress in dissecting their functions. Here we overcome this hurdle by developing an unbiased, high throughput RNAi-mediated screening approach to identify physiological regulators of morphogenesis. Exploiting in utero lentiviral delivery methods to transduce embryonic mouse skin progenitors, we screened for Rho GTPases and GTPase regulators that differentially function in epidermal versus hair follicle development. Our screen unveiled hitherto unrecognized roles for specific family members in cytoskeletal remodeling events that impact these diverse morphogenetic processes, including epidermal differentiation, follicle downgrowth and planar cell polarity. Our findings underscore the power of our screening strategy for unravel the physiological significance of complex gene families. Embryonic day 14.5 epidermal cell mRNA profiles from shScramble (control) and shRhou-505 transduced animals were generated by deep sequencing, in triplicate, using Illumina. Embryonic day 17.5 epidermal cell mRNA profiles from WNT intermediate epidermal, WNT high hair follicle and WNT low hair follicle cells were generated by deep sequencing, in duplicate, using Illumina. Differential expression was determined using a matrix of all samples (WNT Hi, Wnt Low, Epidermal).