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Planar Cell Polarity Cadherin Celsr1 Regulates Skin Hair Patterning in the Mouse

2009; Elsevier BV; Volume: 129; Issue: 10 Linguagem: Inglês

10.1038/jid.2009.84

1523-1747

Aurélia Ravni, Yibo Qu, André M. Goffinet, Fadel Tissir,

Hair Growth and Disorders

cadherin, EGF LAG seven-pass G-type receptor Flamingo/Starry night planar cell polarity Skin hairs develop in a highly coordinated manner, and a complex genetic program controls their size, texture, density, and patterns (Schmidt-Ullrich and Paus, 2005Schmidt-Ullrich R. Paus R. Molecular principles of hair follicle induction and morphogenesis.Bioessays. 2005; 27: 247-261Crossref PubMed Scopus (357) Google Scholar; Sick et al., 2006Sick S. Reinker S. Timmer J. Schlake T. WNT and DKK determine hair follicle spacing through a reaction-diffusion mechanism.Science. 2006; 314: 1447-1450Crossref PubMed Scopus (394) Google Scholar; Schlake, 2007Schlake T. Determination of hair structure and shape.Semin Cell Dev Biol. 2007; 18: 267-273Crossref PubMed Scopus (67) Google Scholar). In addition to their physical features, hairs are distributed in macroscopic patterns. In rodents, all body hairs are oriented caudally and those on the feet are pointed distally. Here, we report that hair patterning on the body and legs in mice are disturbed by targeted inactivation of Celsr1, a seven-pass cadherin (Celsr) that is homologous to the Drosophila planar cell polarity (PCP) protein Flamingo/Starry night (Fmi/Stan; Strutt and Strutt, 2007Strutt D. Strutt H. Differential activities of the core planar polarity proteins during Drosophila wing patterning.Dev Biol. 2007; 302: 181-194Crossref PubMed Scopus (85) Google Scholar). Celsr1 conditional mutant mice were produced by introduction of loxP sites in introns 25 and 29 of the Celsr1 gene, which contains 35 exons. They were crossed with PGK-Cre mice to generate a mutant allele with deletion of exons 26–29 (Figure 1a). Analysis of mutant RNA by reverse transcriptase-PCR and sequencing confirmed that exons 26–29 (nucleotides 7812–8257, GI 115648152), which encode transmembrane segments 5–7, were deleted. Translation of the mutant mRNA is predicted to produce a truncated protein with a scrambled sequence of 15 amino acids and a premature stop downstream of T2603 (GI:115648153). Quantitative RT-PCR showed that the concentration of the Celsr1 transcript was fivefold lower in the mutant than in the wild type (Figure 1b), pointing to mRNA instability and suggesting that our allele (Celsr1ko) is a null. Homozygous animals had a kinky or looping tail, and around 20% died in utero with various degrees of neural tube opening (Figure S1). Some animals displayed a striking hair-patterning defect that developed independently of the looptail trait. In wildtype and heterozygous Celsr1ko mice, guard and pelage hairs were normally oriented, pointing towards the caudal part of the body and the distal extremity of limbs. In homozygous Celsr1ko/ko mice, with the exception of vibrissae, hairs formed abnormal, whorl-like patterns that spiraled around a few centers on the body and the head (Figure 2a, arrowheads). On the limbs, hairs did not point distally like in control animals, but were oriented in a rosette-like manner (Figure 2a, arrowhead). In Celsr1/Emx1 mice, where Celsr1 was inactivated following Cre expression in the embryonic limb ectoderm from E11.5 (see Figure S1b in Zhou et al., 2008Zhou L. Bar I. Achouri Y. Campbell K. De Backer O. Hebert J.M. et al.Early forebrain wiring: genetic dissection using conditional Celsr3 mutant mice.Science. 2008; 320: 946-949Crossref PubMed Scopus (128) Google Scholar), the rosette-like pattern was seen on limbs, but no whorls developed on the body. Inasmuch as Emx1 is not expressed in the mesoderm and neural crest, this observation indicates that Celsr1 acts autonomously in the epidermis and not in other lineages. Download .jpg (.07 MB) Help with files Figures S1Celsr1ko/Celsr1ko mutant mice have a looping tail (arrowhead) and open neural tube. E15.5 (left) and E18.5 (right) embryos with cranioschisis (upper panel) and with fully open neural tube (lower panel). Mutant hairs were straight and appeared to have a normal physical structure (not shown), indicating that abnormal patterns did not result from hair curvature or twisting, but rather from aberrant disposition and orientation. Examination of histological sections in the dorsal skin of 10- and 21-days-old mice confirmed that hair follicles were regularly orientated in wildtype animals (Figure 2b and c left, respectively). In contrast, in regions of abnormal patterning in mutants, follicles and hairs in areas located within a few millimeters from each other assumed very different orientations (Figure 2b and c right, respectively). Flat-mount preparations from the dorsal skin of wildtype and Celsr1 mutant mice at postnatal day 1 disclosed the abnormal angular distribution of growing hairs, which was confirmed using circular statistics. The circular standard deviation was 22.924° for wildtype and 84.99° for Celsr1ko/ko (P<0.01; Figure 2d and e). Mutant hair bulbs were histologically unremarkable, and both the density and distribution of mitotic figures, detected using phosphohistone-H3 immunohistochemistry, were normal (Figure S2). Download .jpg (.1 MB) Help with files Figures S2Immunocytochemical staining of skin cryostat sections using phosphohistone-H3 antibody. The Celsr1 mutation does not modify the proportion of cells in mitosis. Scale bar = 50μm. A recent study of the embryonic epidermis of a mouse with mutations in Celsr1 (Celsr1Crsh allele) showed that mutant hair bulbs fail to acquire a normal oblique orientation between the hair placode and the late germ (peg) stage, but instead remain oriented radially to the skin at all stages (Devenport and Fuchs, 2008Devenport D. Fuchs E. Planar polarization in embryonic epidermis orchestrates global asymmetric morphogenesis of hair follicles.Nat Cell Biol. 2008; 10: 1257-1268Crossref PubMed Scopus (230) Google Scholar). Our observation that Celsr1 mutant follicles are obliquely orientated suggests that a secondary orientation occurs during the early postnatal period. Body and leg hair-patterning defects similar to those described here are mimicked by inactivation of frizzled6, an ortholog of Drosophila Frizzled gene (Guo et al., 2004Guo N. Hawkins C. Nathans J. Frizzled6 controls hair patterning in mice.Proc Natl Acad Sci USA. 2004; 101: 9277-9281Crossref PubMed Scopus (222) Google Scholar; Wang et al., 2006Wang Y. Badea T. Nathans J. Order from disorder: Self-organization in mammalian hair patterning.Proc Natl Acad Sci USA. 2006; 103: 19800-19805Crossref PubMed Scopus (67) Google Scholar). In addition, mice deficient in Celsr3 and Frizzled3 have similar brain-wiring anomalies. This suggests that Celsr1-Frizzled6 and Celsr3-Frizzled3 act in similar pathways (Tissir et al., 2005Tissir F. Bar I. Jossin Y. De Backer O. Goffinet A.M. Protocadherin Celsr3 is crucial in axonal tract development.Nat Neurosci. 2005; 8: 451-457Crossref PubMed Scopus (190) Google Scholar). In flies, Frizzled controls patterning of skin appendages, together with Fmi/Stan and other core PCP genes such as Van Gogh, Disheveled, and Prickle (Wang and Nathans, 2007Wang Y. Nathans J. Tissue/planar cell polarity in vertebrates: new insights and new questions.Development. 2007; 134: 647-658Crossref PubMed Scopus (334) Google Scholar; Simons and Mlodzik, 2008Simons M. Mlodzik M. Planar cell polarity signaling: from fly development to human disease.Annu Rev Genet. 2008; 42: 517-540Crossref PubMed Scopus (403) Google Scholar). The hair-patterning phenotypes in Celsr1 and Frizzled6 mutant mice resemble the defects seen in Fmi/Stan and Frizzled Drosophila mutants, thus providing further evidence that Frizzled and Celsr (Fmi/Stan) are key elements of PCP-related pathways that pattern ectodermal derivatives and are evolutionary conserved. In the Drosophila wing, Frizzled localizes to the distal side of cells, Van Gogh to the proximal side, and Fmi/Stan to both. This polarized distribution is thought to affect the hair orientation by controlling the cytoskeletal machinery responsible for hair assembly. Similarly, in the embryonic skin, polarization of the Celsr1, van gogh-like 2, and Frizzled6 is found in wildtype, but not in corresponding mutant mice (Devenport and Fuchs, 2008Devenport D. Fuchs E. Planar polarization in embryonic epidermis orchestrates global asymmetric morphogenesis of hair follicles.Nat Cell Biol. 2008; 10: 1257-1268Crossref PubMed Scopus (230) Google Scholar). It will be interesting to study the protein–protein interactions that generate this polarized distribution. The authors state no conflict of interest. This work was supported by the following grants: Actions de recherches concertées (ARC-186), FRFC 2.4504.01, FRSM 3.4501.07, Interuniversity Poles of Attraction (SSTC, PAI p6/20), Fondation médicale Reine Elisabeth, DIANE program from Région Wallonne, all from Belgium. AR and FT are, respectively, postdoctoral researcher and research associate of the National Funds for Scientific Research (FNRS). Figure S1. Celsr1ko/Celsr1ko mutant mice have a looping tail (arrowhead) and open neural tube. Figure S2. Immunocytochemical staining of skin cryostat sections using phosphohistone-H3 antibody.