Abstract
During mouse development, core planar cell polarity (PCP) proteins become polarized in the epidermal plane to guide angling/morphogenesis of hair follicles. How PCP is established is poorly understood. Here, we identify a key role for Wdr1 (also known as Aip1), an F-actin-binding protein that enhances cofilin/destrin-mediated F-actin disassembly. We show that cofilin and destrin function redundantly in developing epidermis, but their combined depletion perturbs cell adhesion, cytokinesis, apicobasal polarity and PCP. Although Wdr1 depletion accentuates single-loss-of-cofilin/destrin phenotypes, alone it resembles core PCP mutations. Seeking a mechanism, we find that Wdr1 and cofilin/destrin-mediated actomyosin remodelling are essential for generating or maintaining cortical tension within the developing epidermal sheet and driving the cell shape and planar orientation changes that accompany establishment of PCP in mammalian epidermis. Our findings suggest intriguing evolutionary parallels but mechanistic modifications to the distal wing hinge-mediated mechanical forces that drive cell shape change and orient PCP in the Drosophila wing disc.
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Acknowledgements
We thank D. Devenport, S. Williams, S. Beronja, A. R. Folgueras, D. Schramek, I. Matos and E. Ezratty for intellectual input; D. Oristian and A. Aldeguer as mouse specialists; Comparative Bioscience Center (AAALAC accredited) for care of mice in accordance with National Institutes of Health (NIH) guidelines; Bioimaging Center (A. North, director) for advice; Flow Cytometry facility (S. Mazel, director) for FACS sorting. Cfl–GFP was a generous gift from J. Condeelis (Albert Einstein college of Medicine, New York, USA); E.F. is an Investigator of the Howard Hughes Medical Institute. This research was supported by a grant from the NIH (R37-AR27883, E.F.), a Starr Stem Cell Postdoctoral Fellowship (C.L.) and a Genetics Training Grant by the NIH (E.H.).
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C.L. and E.F. conceived the study. C.L., E.H. and E.F. designed the experiments. C.L. and E.H. carried out the experiments and analysed the data. H.A.P. performed the ultrastructural analyses (Supplementary Fig. 1S). S.C. made the Wdr1-rescue construct, C.L. and N.S. performed the in utero injections. C.L. and M.N. prepared high-titre viruses. C.L., E.H. and E.F. wrote the paper. All authors provided intellectual input, vetted and approved the final manuscript.
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Supplementary Figure 2 Wdr1 knockdown epidermis display normal intercellular adhesion and attachment to the basement membrane.
Correlative light and electron microscopy of E16.5 headskin in WT and Wdr1-368. Whole mount skin was immunolabeled for GFP (and subsequently embedded for electron microscopy. (a) and (b) Semithin sections (1 μm) were stained with toluidine blue and examined for the presence of GFP positive cells (brown nuclei). Dotted line indicated dermo-epidermal boundary. (b and b′) consecutive ultrathin sections were examined at the electron microscope. For Wdr1-368, the same exact cells that were GFP+ by immunohistochemistry were identified (pseudocolored in green) and photographed at high resolution. Red box enlarged in (c) and yellow box enlarged in (e). (c) Epidermal cells in Wdr1-368 display normal adhesion, with intact desmosomes (de) shown in boxed area enlarged in (d) No gaps were observed between the cells. Some inevitable cytoplasmic damage (asterisks) is due to detergent permeabilization necessary for immunolabelling. (e) Electron microscopic analysis showed intact basal lamina (bl) and hemidesmosomes (hd). Bars in (a) and (c), 2 μm, also valid for a′, b and b′. Bars in (d) and (e), 500 nm.
Supplementary Figure 3 Normal adhesion and apicobasal polarity in Wdr1 knockdown epidermis.
10 μm sagittal sections of E16.5 backskins were labelled for: (a) E-cadherin (E-cad) or ZO1 and co-labelled for integrin β4 (β4) and Laminin 5 (Lam5) (b) ABP markers Par3 and pericentrin. DAPI was used to label chromatin. Dotted line denotes the dermal-epidermal border. Inserts denote infected cells (H2B-GFP+ nuclei). Scale bar, 10 μm.
Supplementary Figure 4 Wdr1 knockdown by a second hairpin (Wdr1-1622) results in PCP defects analogous to Wdr1-368 and validation of Vangl2 shRNAs.
(a) 10 μm sagittal sections of E15.5 backskins were labelled for Par3 or Pericentrin (PC) or co-labelled for Keratin 5 (K5) and Keratin 10 (K10). (b) Whole-mount immunofluorescence of E15.5 backskins co-labelled for E-cadherin (E-cad) and Celsr1 and imaged in the mid-plane of the basal layer. (c) Whole-mount immunofluorescence of E18.5 HFs labelled for E-cadherin. (d) Quantifications of data shown in (c), n = 36 hair follicles from 3 embryos. Note similarities to data compiled on similarly aged Wdr1-368 knockdown embryonic epidermis (see Figs 1 and 2). Green arrows denote normal HF orientation, red arrows denote abnormal HF orientation. Red circles denote perpendicularly oriented HFs. Insets denote transduced cells (H2B-RFP+ nuclei). (e) In vitro Knockdown efficiencies of shRNAs against Vangl2 to achieve specific loss of PCP in the skin, n = 3 independent measurements/hairpin. Error bars indicate mean ± s.e.m. Scale bars, 10 μm.
Supplementary Figure 5 Generation and expression of a hairpin-resistant Wdr1-GFP lentiviral transgene.
(a) Wdr1 coding sequence recognized by Wdr1-368 hairpin and a mutant, shown in red, that renders it refactory to the hairpin. (b,c) Western blots of protein extracts from WT cells or cells transduced with lentiviruses that contain either Wdr1-368 shRNA, Scrambled control shRNA or the Wdr1-GFP hairpin-resistant mutant cDNA expression vector. Blots were probed with WDR1 antibodies (Ab) (b) or GFP Ab (c). Note the ∼93 kDa WDR1-GFP protein (endogenous WDR1 = 66 kDa, GFP = 27 kDa) detected with both Abs. Glyceraldehyde dehydrogenase (GAPDH) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) Abs were used as loading controls.
Supplementary Figure 6 Normal localization of Cofilin and Destrin in Wdr1-368 KD epidermis.
(a) 10 μm sagittal sections of E15.5 control and WDR1-368 KD backskins were labelled for Cofilin (Cfl) or Destrin (Dstn). Inserts denote transduced cells (H2B-GFP+ nuclei). DAPI in blue. (c) Western blot analyses of protein extracts from control (Scr) and Wdr1-368 KD 10MK were probed with antibodies against: WDR1, Cofilin (Cfl) phospho-serine 3-cofilin (p-Cfl) and α-tubulin (αTub, loading control). (d) qPCR of mRNAs were from control and Cfl1KD/Dstn-null (KD/KO) 10MK. n = 3 samples/condition Cfl1 levels in Ctrl versus KD/KO p = 0.0026; Dstn levels in Ctrl. versus KD/KO p = 0.009. Dotted line denotes the dermal-epidermal border. Insets show transduced cells (H2B-GFP+ nuclei). Scale bars, 10 μm.
Supplementary Figure 7 Quantification of F-actin content, actin severing, and endogeneous levels of cofilin in Wdr1-KD and cofilin-overexpressing keratinocytes.
(a) Uncropped Western blots used to evaluate Wdr1 knockdown and endogeneous levels of actin in Fig. 1a. (b) Uncropped Western blots used to evalulate endogeneous cofilin and phosph-cofilin levels in transduced keratinocytes in Fig. 5a, b. Immunoblot for GFP indicates overexpression of histone 2B (H2B)-GFP (∼51 kDa), used as a marker of lentiviral transduction in scramble- and Wdr1-KD keratinocytes, and overexpression of GFP-CflS3A (∼52 kDa) in the others. Relative levels were quantified by fluorescence intensity on a LiCor Odyssey CLx imager and normalized to GAPDH levels. (c) Uncropped Western blots used to quantify F/G actin ratios. G-actin standards were loaded at the indicated quantities and used to construct a calibration curve. Actin levels were normalized to the cytoplasmic HPRT signal in each lysate.
Supplementary Figure 8 Defects in mitotic rounding in Wdr1-368 KD epidermis.
(a) Whole-mount immunofluorescence of E15.5 backskin labelled for Ecadherin (E-cad) and imaged at the basal layer plane. (b) Quantifications of data from (a). Control: 1.14 ± 0.088; Wdr1 KD: 1.39 ± 0.27, p = 5.27 × 10−5 (unpaired t-test), n = 23 cells (WT); n = 29 cells (Wdr1 KD) from 3 embryos per condition. Box and whiskers plot: line = median, box = 50% range, whiskers = 100% range. Arrow denotes early mitotic cells. Inserts denote infected cells (H2B-GFP+ nuclei). Scale bar, 10 μm.
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Luxenburg, C., Heller, E., Pasolli, H. et al. Wdr1-mediated cell shape dynamics and cortical tension are essential for epidermal planar cell polarity. Nat Cell Biol 17, 592–604 (2015). https://doi.org/10.1038/ncb3146
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DOI: https://doi.org/10.1038/ncb3146
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