Planar polarization in embryonic epidermis orchestrates global asymmetric morphogenesis of hair follicles

Abstract

Mammalian body hairs align along the anterior-posterior (A-P) axis and offer a striking but poorly understood example of global cell polarization, a phenomenon known as planar cell polarity (PCP). We have discovered that during embryogenesis, marked changes in cell shape and cytoskeletal polarization occur as nascent hair follicles become anteriorly angled, morphologically polarized and molecularly compartmentalized along the A-P axis. Hair follicle initiation coincides with asymmetric redistribution of Vangl2, Celsr1 and Fzd6 within the embryonic epidermal basal layer. Moreover, loss-of-function mutations in Vangl2 and Celsr1 show that they have an essential role in hair follicle polarization and orientation, which develop in part through non-autonomous mechanisms. Vangl2 and Celsr1 are both required for their planar localization in vivo, and physically associate in a complex in vitro. Finally, we provide in vitro evidence that homotypic intracellular interactions of Celsr1 are required to recruit Vangl2 and Fzd6 to sites of cell-cell contact.

Figure 1. HF angling is accompanied by polarized cell shape and cytoskeleton changes in anterior and posterior cells at the HF-epidermal boundary

(a–c) Sagittal sections through E16.5 HFs labelled with E-Cadherin to mark cell-cell borders. (a) Hair placodes (dashed line) are symmetric when they first invaginate into the dermis. (b–c) In early and late hair germs, anterior follicle cells next to the epidermis constrict basally (arrowheads) while posterior cells do not (arrows). (d–f) Confocal sections of E15.5 whole mount epidermis labelled with E-Cadherin. By the early hair germ stage anterior cells (expressing reduced levels of ECad) adopt shapes/orientations distinct from posterior cells. Asterisk denotes dermis (see schematic). Red dotted lines in schematic denote planes of view for images shown in (d-f). (g–i) Sagittal sections from E16.5 transgenic embryos expressing K14-GFPActin. GFPActin is enriched in apical (arrows) and basal (arrowheads) constrictions. (j–l) Protrusions enriched for keratin 5 (K5) emanate from basal epidermal cells on the anterior but not posterior side of hair germs and E18.5 hair pegs. bl= basal layer; sb=suprabasal layers; der= dermis; pl= placode; hg= hair germ. Scale bar 10um. (m) Proliferation in developing HFs. BrdU was administered to E16.5 embryos and chased for 1 hour. Bars represent the average number of BrdU positive cells/ anterior or posterior side of HF. Total #HFs counted: 152 placodes, 147 early hair germs, 122 late hair germs. n=4 embryos.

Figure 2. A-P-polarized gene expression in embryonic HFs

(a–d) Immunofluorescence images of E17.5 hair germs. Antibodies (Abs) colour coded according to the secondary Abs used. White dotted lines denote polarized zones for gene expression. Note upregulation of the adherens junction (AJ) protein P-cadherin (Pcad) and tight junction protein ZO-1 and downregulation of the AJ protein E-cadherin (Ecad) on the anterior side of cells in the lower portion of hair germs (arrowheads). Note upregulation of neural cell adhesion molecule (NCAM) on the posterior side of cells in the upper portion of the follicle (arrows). (e) Expression of the gene encoding signalling morphogen Shh by in situ hybridization. (f) Tracking early progeny of Shh-expressing cells through lineage tracing. ShhCreER^T2; R26R mice were treated with tamoxifen at E15.5. In P0 follicles LacZ-positive cells (blue, X-Gal staining) were only detected in the anterior and central portion of developing HFs. K5, keratin 5. Scale bars 10um.

Figure 3. Anterior-Posterior polarization of Vangl2 and Celsr1 in the hair germs and the epidermal basal layer of embryonic skin

Schematic indicates each of the planar views in optical confocal sections of E15.5 whole mount skins. Abs are indicated overhead. Nuclei are labelled with DAPI (blue) except in (c-c’) where transgenic expression of K14-H2B-GFP is pseudocoloured in blue. White dotted lines denote skin surface. Boxed areas are magnified as insets. (a–c) Expression and localization of Vangl2 and Celsr1 in hair germs. Note that Vangl2 and Celsr1 are polarized in the anterior-posterior lateral membranes of follicle cells, while E-cadherin is not. (d–g) Expression and localization of Vangl2 and Celsr1 within the basal layer of embryonic epidermis. Again, while E-cadherin is uniformly distributed at cell-cell borders, Celsr1 and Vangl2 are polarized along the A–P axis. sb=suprabasal layers; bl= basal layer; d= dermis; hg= hair germ. Scale bars 10um.

Figure 4. PCP establishment occurs early and requires signals from the embryo but develops independently of stratification and HF morphogenesis

(a–c) Confocal planar sections through the basal layer of whole mount E12.5–E14.5 skin labelled with Celsr1 antibodies and DAPI as indicated. Note that PCP begins to develop between E12.5 and E13.5, and is established by E14.5. (d–e) Skins were removed from E13.5 and E14.5 embryos and cultured ex vivo for two days under conditions permissive for HF morphogenesis. Afterwards, explants were labelled with Celsr1 (red), E-cadherin (green) and DAPI (blue) and imaged through the plane of the epidermis (d, e) or HF (d’,e’). Note that at E14.5 but not E13.5, epidermal PCP was maintained in vitro and nascent HFs still grew towards the anterior. (f–k) Sagittal and planar views of skins from embryos mutant for Shh, β-catenin or p63, labelled with Celsr1, E-cadherin and DAPI as indicated. Boxed areas are magnified, and co-labelling is shown to highlight that Celsr1 polarization in the basal layer is established independently of these early developmental regulatory pathways. hg= hair germ; d=dermis. Scale bars 10µm.

Figure 5. Genetic mutations in Vangl2 and Celsr1 result in loss of A–P alignment of hair follicles

(a–d) Saggital images of skin sections from WT, Lp/Lp, Crsh/Crsh, and Crc/Crc E18.5 embryos labelled with Abs against K5 and imaged at 10X magnification. (e) Schematics summarizing the quantifications of HF angles.×axis= angle between epidermis and HF on the anterior side. y-axis= number of follicles at angle x. The angles of ~10% of mutant follicles were oriented out of the plane of sectioning and hence were not measured. Note that Crsh (Celsr1) and Lp (Vangl2) mutants are defective in HF alignment, but Crc (Scribble), involved in some types of PCP, is not. (f–g) Projected Z-stacks of whole mount epidermis from Lp/+ and Lp/Lp embryos expressing K14-GFPactin at E18.5. Magnification 10X. Note that larger, more mature Lp/Lp follicles (arrowheads) orient randomly while early stage follicles (rest) point straight downward and appear uniformly circular by planar view. Scale bars 50µm.

Figure 6. Loss of hair follicle asymmetry in PCP mutants

(a–p). Immunofluorescence microscopy of planar (c,d) or sagittal (rest) confocal skin sections from WT or Lp/Lp mutant embryos labelled with Abs or epifluorescence as indicated. (a–b) Anterior cells near the top of Lp/Lp follicles (E16.5) no longer constrict basally (arrowheads). (c–d) Loss of anterior-posterior asymmetry in architecture of Lp/Lp hair germs (E15.5). (e–f) E16.5 WT and Lp/Lp embryos on transgenic background of K14-GFPactin. Note that the apical enrichment (arrows) and basal constrictions (arrowheads) of F-actin are lost at the anterior-posterior junctions between Lp/Lp hair germs and epidermis. (g–h) The asymmetric distribution of keratin 5 (K5) positive protrusions, typically on the anterior side of WT hair pegs at E18.5, is replaced by smaller K5-positive protrusions on both sides of Lp mutant hair pegs (arrowheads). (i–p) Intercellular adhesion proteins are no longer asymmetrically distributed within the anterior and posterior sides of hair germs in E17.5 Lp/Lp embryos. (q–q’) Chimeric embryos (E16.5) were generated from blastocysts composed of WT and Lp/Lp cells (see Experimental Procedures). WT cells are indicated by (+) symbols and are marked by the presence of Vangl2; Lp/Lp mutant cells are denoted by (−) symbols and marked by the absence of cell border Vangl2 staining (q). Note that despite consisting almost entirely of WT cells, HFs from chimeric mice fail to asymmetrically express NCAM (q’; green), or point towards the anterior when surrounded by Lp/Lp mutant interfollicular epidermal cells. Scale bars 10µm.

Figure 7. Evidence for interactions between Celsr1 and Vangl2 in vivo and in vitro

(a–f) In vivo studies. Planar confocal sections through the basal layer of whole mount skin from E15.5 WT, Lp/Lp, and Crsh/Crsh embryos, labelled with Celsr1 (green), Vangl2 (red) and DAPI (blue). Note that in Lp/Lp mutants, Celsr1’s A–P polarity is lost. In Crsh/Crsh mutants both Celsr1 and Vangl2 lose A–P polarity, and Vangl2 is punctate and discontinuous at cell borders. Boxed areas are magnified in insets. (g–t) In vitro studies. Cultured keratinocytes expressing fluorescently tagged Celsr1, Vangl2 or their mutant versions as indicated. Confluent monolayers were shifted to high Ca²⁺ medium to induce the formation of intercellular junctions, except in (i) where cells were kept in low Ca⁺². Each image shown is representative of ≥ 50 examples. Boxed areas are magnified, and red and green fluorescence is separated for additional clarity. Scale bars 10µm. Schematics depict domain structure of WT and mutant proteins. (g–l) Celsr1 data. Note that Celsr1^WTGFP shifts its localization to cell-cell borders in a calcium-dependent but α-catenin (adherens junction) independent fashion when two transfected cells come into direct contact. Note also that the Celsr1^Crsh mutant and Celsr1 lacking the C-terminal cytoplasmic tail (Δcyto) are compromised in this ability. (m–t) Vangl2 and co-expression data. Note that Vangl2 is unable to localize to cell-cell borders on its own. In the presence of Celsr1^WTGFP, Cherry-Vangl2 shifts its localization to cell-cell borders only when two Celsr1-expressing cells are in direct contact (p), and mutant versions of Celsr1 are compromised in this ability (q–r). Cherry-Vangl2 mutants lacking the C-terminal cytoplasmic tail or carrying the Lp point mutation fail to be recruited by Celsr1 to cell contacts. (u) Surface biotinylation assay. Biotinylated surface proteins from keratinocytes expressing Flag-Vangl2^WT or Flag-Vangl2^Lp were recovered with streptavidin-coated sepharose beads and analyzed by Western blot to show that the Lp Vangl2 mutation does not appear to compromise the relative stability and/or cell surface localization of Vangl2. (v) Protein extracts from keratinocytes expressing Flag-Vangl2 ± Celsr1-Myc as indicated were immunoprecipitated with anti-Myc Abs. Flag-Vangl2^WT but not Flag-Vangl2^Lp coimmunoprecipitated with Celsr1-Myc.

Figure 8. Fzd6 requires Vangl2 for its asymmetric localization in the epidermis and requires Celsr1 for it recruitment to cell contacts

(a–b’). Sagittal sections of WT E16.5 epidermis labelled with Abs as indicated. Like Celsr1 and Vangl2, Fzd6 is expressed in the basal layer (a) and early HFs (b), and is localized to lateral cell-cell borders. (c–e) Planar localization of Fzd6. Confocal sections through the basal layer of whole mount epidermis from WT, Lp/Lp, and Crsh/Crsh E15.5 embryos, labelled with Abs as indicated. Note enrichment of Fzd6 at A–P cell borders in WT, but not Lp/Lp and Crsh/Crsh epidermis. (f–h) Expression of Fzd6-Cherry and Celsr1-GFP in keratinocyte monolayers. Note that in the presence of Celsr1-GFP, Cherry-Fzd6 is recruited to cell-cell borders only when two Celsr1 expressing cells are in direct contact. Fzd6 recruitment by Celsr1 also occurs normally in Lp/Lp mutant keratinocytes, suggesting that Vangl2 is not required for Fzd6 cell contact localization. (i) Endogenous Fzd6 localization in keratinocyte monolayers. In the absence of Celsr1-GFP, Fzd6 is distributed throughout the cell (arrows), but is recruited to the interface between two Celsr1-expressing cells (arrowheads).