An RNAi screen unravels the complexities of Rho GTPase networks in skin morphogenesis
- The Rockefeller University, United States
Abstract
During mammalian embryogenesis, extensive cellular remodeling is needed for tissue morphogenesis. As effectors of cytoskeletal dynamics, Rho GTPases and their regulators are likely involved, but their daunting complexity has hindered progress in dissecting their functions. We overcome this hurdle by employing high throughput in utero RNAi-mediated screening to identify key Rho regulators of skin morphogenesis. Our screen unveiled hitherto unrecognized roles for Rho-mediated cytoskeletal remodeling events that impact hair follicle specification, differentiation, downgrowth and planar cell polarity. Coupling our top hit with gain/loss-of-function genetics, interactome proteomics and tissue imaging, we show that RHOU, an atypical Rho, governs the cytoskeletal-junction dynamics that establish columnar shape and planar cell polarity in epidermal progenitors. Conversely, RHOU downregulation is required to remodel to a conical cellular shape that enables hair bud invagination and downgrowth. Our findings underscore the power of coupling screens with proteomics to unravel the physiological significance of complex gene families.
Data availability
Sequencing data have been deposited in NCBI GEO under accession number GSE123047. All data generated or analysed during this study are included in the manuscript and supporting files
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RNA-sequencing from E14.5 epidermal cells from shScr and shRhou transduced miceNCBI Gene Expression Omnibus, GSE123047.
Article and author information
Author details
Funding
Howard Hughes Medical Institute
- Elaine Fuchs
Canadian Institutes of Health Research
- Melanie Laurin
Burroughs Wellcome Fund (Postdoctoral Enrichment Program Award)
- Nicholas C Gomez
National Institutes of Health (Ruth L. Kirschstein National Research Service Award F32CA221353)
- Nicholas C Gomez
Human Frontier Science Program
- Ataman Sendoel
Marie Curie Foundation
- Ataman Sendoel
Cancer Research Society
- Melanie Laurin
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Maddy Parsons, King's College London, United Kingdom
Ethics
Animal experimentation: All mouse strains were housed in an AAALAC-accredited facility and experiments were conducted according to the Rockefeller University's Institutional Animal Care and Use Committee, and NIH guidelines for Animal Care and Use.All animal procedures used in this study are described in our #17020-H protocol named Lentiviral RNAi and Skin, which had been previously reviewed and approved by the Rockefeller University Institutional Animal Care and Use Committee (IACUC)
Version history
- Received: July 16, 2019
- Accepted: September 24, 2019
- Accepted Manuscript published: September 25, 2019 (version 1)
- Version of Record published: September 30, 2019 (version 2)
Copyright
© 2019, Laurin et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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An RNAi screen unravels the complexities of Rho GTPase networks in skin morphogenesis
eLife 8:e50226.
https://doi.org/10.7554/eLife.50226
Further reading
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Large-scale cell flow characterizes gastrulation in animal development. In amniote gastrulation, particularly in avian gastrula, a bilateral vortex-like counter-rotating cell flow, called ‘polonaise movements’, appears along the midline. Here, through experimental manipulations, we addressed relationships between the polonaise movements and morphogenesis of the primitive streak, the earliest midline structure in amniotes. Suppression of the Wnt/planar cell polarity (PCP) signaling pathway maintains the polonaise movements along a deformed primitive streak. Mitotic arrest leads to diminished extension and development of the primitive streak and maintains the early phase of the polonaise movements. Ectopically induced Vg1, an axis-inducing morphogen, generates the polonaise movements, aligned to the induced midline, but disturbs the stereotypical cell flow pattern at the authentic midline. Despite the altered cell flow, induction and extension of the primitive streak are preserved along both authentic and induced midlines. Finally, we show that ectopic axis-inducing morphogen, Vg1, is capable of initiating the polonaise movements without concomitant PS extension under mitotic arrest conditions. These results are consistent with a model wherein primitive streak morphogenesis is required for the maintenance of the polonaise movements, but the polonaise movements are not necessarily responsible for primitive streak morphogenesis. Our data describe a previously undefined relationship between the large-scale cell flow and midline morphogenesis in gastrulation.
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Geometric criteria can be used to assess whether cell intercalation is active or passive during the convergent extension of tissue.
