Region-specific reversal of epidermal planar polarity in the rosette fancy mouse

Abstract

The planar cell polarity (PCP) pathway collectively orients cells with respect to a body axis. Hair follicles of the murine epidermis provide a striking readout of PCP activity in their uniform alignment across the skin. Here, we characterize, from the molecular to tissue-scale, PCP establishment in the rosette fancy mouse, a natural variant with posterior-specific whorls in its fur, to understand how epidermal polarity is coordinated across the tissue. We find that rosette hair follicles emerge with reversed orientations specifically in the posterior region, creating a mirror image of epidermal polarity. The rosette trait is associated with a missense mutation in the core PCP gene Fzd6, which alters a consensus site for N-linked glycosylation, inhibiting its membrane localization. Unexpectedly, the Fzd6 trafficking defect does not block asymmetric localization of the other PCP proteins. Rather, the normally uniform axis of PCP asymmetry rotates where the PCP-directed cell movements that orient follicles are reversed, suggesting the PCP axis rotates 180°. Collectively, our multiscale analysis of epidermal polarity reveals PCP patterning can be regionally decoupled to produce posterior whorls in the rosette fancy mouse.

Keywords: Epidermis; Frizzled 6; Hair follicle; Morphogenesis; Mouse; Planar cell polarity.

Fig. 1.

Posterior hair follicles emerge with reversed orientations in rosette mutants. (A) Hair patterns of C57BL/6 controls (left) and the rosette fancy mouse (right). (B-D) Orientation of emerging hair follicles at E15.5. (B) Representative scanning confocal projections of whole-mount dorsal skins labeled with the asymmetrically distributed follicle markers P-Cad (green) and Sox9 (magenta). Scale bar: 100 μm. Zoomed in view of individual follicles are shown below. A1, A2, etc. indicate anterior follicles; P1, P2, etc. indicate posterior follicles. Scale bar: 10 μm. (C, top) Voronoi diagrams of follicle orientation represented by color (cool colors, anteriorly oriented; warm colors, posteriorly oriented; black, unpolarized). A↔P, anterior-posterior; M↔L, medial-lateral. (C, bottom) Graph showing individual follicle orientation along the AP axis. The three shades indicate three embryos per genotype. (D) Voronoi diagrams representing local order of follicle orientations (high coordination, white; low coordination, red; unpolarized, black).

Fig. 2.

Coordinated hair follicle reversal persists into postnatal stages. (A) Representative images of cleared, flat-mounted dorsal skins at P4 from rosette/+ controls (left), Fzd6^−/− (center) and rosette (right). Hair follicle orientation is indicated by color (cool colors, anteriorly oriented; warm colors, posteriorly oriented). Scale bars: 1 mm. (B) Local order of follicle orientation (high order, white; low order, red). A↔P, anterior-posterior. M↔L, medial-lateral, with M indicating the midline. (C) Rear paws from indicated genotypes.

Fig. 3.

The rosette phenotype correlates with a missense mutation in Fzd6. (A) PCA of the filtered SNP dataset for all mice analyzed. Circle shading indicates C57BL/6 ancestry. Percent variation for the first two axes are included. (B) Candidate region associated with the whorled phenotype on chromosome 15 (dashed line) identified by outlier loci from the likelihood ratio test statistic (top panel) and outlier β values (lower panel, gray shading). (C) A novel missense mutation in the rosette mutant compared with the C57BL/6 reference sequence. G1061>A results in an amino acid change in the Fzd6 protein, S354I (yellow). (D) Sanger sequencing of Fzd6 position 1061 (asterisk) from rosette and non-whorled fancy mice outside our colony. (E,F) (Top) Voronoi diagrams of hair follicle orientations at E15.5 (cool colors, anteriorly oriented; warm colors, posteriorly oriented; black, unpolarized). A↔P, anterior-posterior; M↔L, medial-lateral. (Bottom) Graphs showing individual follicle orientations with respect to AP position. Three different shades indicate three embryos per genotype.

Fig. 4.

Membrane localization of Fz6 is lost in rosette mutants. (A) Scanning confocal images of whole-mount epidermis labeled with E-cadherin and Fzd6 antibodies and Hoechst in control (top) and rosette (bottom) embryos at E15.5. (B) Colocalization of E-cadherin and Fzd6 represented as Pearson's correlation coefficient. Three animals were analyzed per genotype, represented by unique colors. Circles indicate a single analyzed image; the triangle is the average/animal. Data are mean±s.e.m. across all animals per group. Two-tailed t-test: P<0.00001, anterior; P<0.00005, posterior. (C) Schematic of tagged Fzd6 constructs. (D) Keratinocytes transfected with HA-Fzd6-tdTomato constructs (wild type, S354I and N352A) and labeled using anti-HA antibodies without permeabilization. Maximum projections of wide-field stacks are shown. (E) Quantification of surface HA levels relative to the tdTomato signal. Wild type, n=36; N354I, n=34; N352A, n=35 cells from three experiments. Each experiment is shown in a different color. Replicates are indicated by circles; triangles indicate the average of the experiment. Data are mean±s.e.m. Two-tailed t-test: P=0.00059 (wild type versus S354I), P=0.00099 (wild type versus N352A). Scale bars: 10 μm.

Fig. 5.

The axis of PCP asymmetry rotates in the rosette epidermis. (A) Schematic of PCP asymmetry in wild-type basal epidermal cells. (B-E) Celsr1 polarity in the basal layer of E15.5 epidermis. The areas shown are in the transition zone of rosette mutants and the corresponding area in Fzd6^−/− and controls. (B) Circular histograms of Celsr1 polarity angles weighted by magnitude. Control, n=46,256 cells; Fzd6^−/−, n=45,854 cells; rosette, n=48,031 cells from three embryos/genotype. (C, top) Scanning confocal images of whole-mount epidermis labeled for Celsr1. Scale bar: 10 μm. (C, bottom) Celsr1 polarity nematic calculated by PCA with QuantifyPolarity. Lines indicate the sides of the cell with the highest levels of Celsr1 and are color coded by angle (green, AP; magenta, ML). Magnitude is indicated by the length of the line, with longer lines indicating higher magnitudes. (D) Extended AP region showing the average polarity nematic for a zone of ∼10 cells. Blank spaces are newly formed follicles. Scale bar: 100 μm. (E) Spatial map of local Celsr1 polarity angle (color) and magnitude (saturation). Each cell is color-coded to represent the average angle and magnitude of a local area with a radius of 40 pixels (center cells and approximately two radii of neighbors). Black circular regions correspond to emerging hair follicles and arrows depict their orientations. White circles indicate unpolarized follicles that point straight down. Blank spaces represent newly formed follicles that are too early to categorize or areas that could not be segmented. Scale bar: 100 μm.

Fig. 6.

PCP-directed collective cell movements are reversed in posterior rosette hair placodes. (A,B) Spinning disk confocal images from a time series of rosette placode cells expressing mTomato (top). Cells were segmented and false colored in a rainbow pattern of vertical lines at t=0. Cell tracks show the movement of cells during the designated time window, with circles indicating the last position (bottom). Smoothed tracks with arrow overlays show overall movements through the course of the time series (upper right). (A) Anterior placode; see Movie 3 and additional example from another explant in Movie 4; n=7. (B) Posterior placode from the same explant as in A; see Movie 5 and additional example from another explant in Movie 6; n=9. Scale bars: 10 µm. Anterior is towards the left.