The polarity of mouse hair follicles is controlled by the Frizzled (Fzd) receptors and other membrane planar cell polarity (PCP) proteins. Whether Wnt proteins can act as PCP ligands in the skin remains unknown. Here, we show that overexpression of Wnt5a in the posterior part of mouse embryos causes a local disruption of hair follicle orientation. The misoriented hair follicle phenotype in Wnt5a overexpressing mice can be rescued by a heterozygous loss of Fzd6, suggesting Wnt5a is likely to signal through Fzd6. Although the membrane distribution of PCP proteins seems unaffected by Wnt5a overexpression, transcriptional profiling analyses identify a set of genes as potential targets of the skin polarization program controlled by Wnt5a/Fzd6 signaling. Surprisingly, deletion of Wnt5a globally or in the posterior part of the mouse embryos does not affect hair follicle orientation. We show that many other Wnts are highly expressed in the developing skin. They can activate the Fzd6 signaling pathway in vitro and may act together with Wnt5a to regulate the Fzd6-mediated skin polarization. Our experiments demonstrate for the first time that Wnt5a can function as an orienting cue for mouse skin PCP.

During embryogenesis, many different cell types are generated and organized into specific patterns to give rise to intricate tissues and organs. For each individual cell, the spatial information might include front-rear polarity of migrating mesenchymal cells, dendrite-axon polarity of neurons and apical-basal polarity of epithelial cells. For a group of cells, they need to coordinate with each other and establish their orientation with respect to the tissue and body axes. The coordination of orientation across a two-dimensional sheet, orthogonal to the axis of apical-basal polarity, is referred to as planar cell polarity (PCP) or tissue polarity (Goodrich and Strutt, 2011). PCP was initially studied in Drosophila wing epithelia. Genetic experiments have identified that a core set of three membrane family proteins are essential for the correct alignment of cuticles to the body axes: frizzled (Fz), flamingo (Celsr in vertebrates) and Van Gogh (Vangl in vertebrates). In mice, skin has been used to study mammalian PCP since the discovery that frizzled (Fzd) 6, the ortholog of Drosophila Fz, is required for proper hair follicle alignment (Guo et al., 2004). In Fzd6–/– mice, hair follicles develop normally with respect to their intrinsic structure, but they exhibit random orientations relative to the body axes. The polarity of other hair-associated structures, such as sebaceous glands, arrector pili muscles, sensory nerve endings and Merkel cell clusters are also affected in the Fzd6–/– mice (Chang and Nathans, 2013). Mutations in the other two PCP family genes, Vangl2 and Celsr1, have also been reported to cause hair orientation phenotypes (Devenport and Fuchs, 2008; Ravni et al., 2009), suggesting that the PCP pathway is a conserved mechanism that regulates the orientation of cuticles in Drosophila and hair follicles in mice.

Wnts are the extracellular ligands of frizzled receptors (Bhanot et al., 1996). There are 19 Wnts in mammals and recent studies have suggested multiple roles of Wnts in skin development (Lim and Nusse, 2013; Nusse and Clevers, 2017). For example, Wnt5a has been identified as a direct target of Notch signaling in dermal papilla cells (Hu et al., 2010). Although Wnt5a–/– skin shows no detectable defects in hair follicle morphogenesis and differentiation at embryonic day (E) 18.5, defective dermal papilla functions, such as impaired hair follicle-inductive properties, downregulated of hair follicle differentiation markers and inappropriate cytokeratin 1 expression are found in E17.5 embryonic skin grafted onto nude mice (Hu et al., 2010). WNT10A mutations have been found in individuals with odonto-onycho-dermal dysplasia (OODD), a rare autosomal recessive syndrome that presents with dry hair, severe hypodontia, smooth tongue, onychodysplasia, keratoderma and hyperhidrosis of palms and soles, and hyperkeratosis of the skin (Adaimy et al., 2007). Wnt10a–/– mice show various phenotypes of organogenetic failure and defects in skin wound healing (Wang et al., 2018). Wnt4–/– mice die within 24 h of birth due to the lack of kidney function (Stark et al., 1994) and newborn Wnt4–/– mice show an increased accumulation of type I collagen fibrils in the dermis (Saitoh et al., 1998). Although significant progress has been made regarding the role of Wnts in developing skin and homeostasis, no involvement of any Wnts in skin PCP has been reported.

Wnt5a can signal through both the canonical and non-canonical pathways. Depending on the cell types and the availability of receptors and co-receptors, distinct signaling outputs can be generated (Mikels and Nusse, 2006; van Amerongen et al., 2012). Several studies support a role of Wnt5a as a PCP Wnt. For example, Wnt5a controls convergent extension movement during gastrulation in zebrafish (Wallingford et al., 2001). In mice, the Wnt5a gradient in the limb bud patterns developing chondrocytes along the proximal-distal axis (Gao et al., 2011). Wnt5a also genetically interacts with Vangl2 to regulate hair cell orientation in the cochlea and neural tube closure (Qian et al., 2007). A recent study in Xenopus early ectoderm shows that Wnt5a is capable of orienting Prickle3/Vangl2 complexes away from the Wnt5a source across many cell diameters (Chu and Sokol, 2016). In this study, we use both gain- and loss-of-function approaches to study the possible role of Wnt5a in skin PCP. We have found that overexpression of Wnt5a in the posterior part of the mouse body causes a misorientation of hair follicles. The disruption of hair follicle polarity by Wnt5a overexpression can be rescued by a heterozygous loss of Fzd6. Transcriptional profiling analyses identify a set of genes as mediators of the skin polarization program controlled by Wnt5a/Fzd6 signaling. In addition, we have found that posterior or global knockout of Wnt5a does not affect hair follicle orientation. Our in vitro screening reveals that multiple Wnts might act redundantly with Wnt5a in controlling skin polarization. This study demonstrates for the first time that Wnt is capable of regulating skin PCP and supports the idea of Wnt5a being a PCP ligand.

Wnt5a overexpression in the posterior part of mouse embryos disrupts hair follicle orientation

To determine the potential role of Wnt5a in skin PCP, we used an inducible transgenic mouse model that allows a spatiotemporal control of ectopic Wnt5a expression. In the Z/Wnt5a transgenic line, a Cre-activated Wnt5a cassette was targeted to the ubiquitin B (Ubb) locus, the site of transgene insertion in the Z/AP mouse line (Lobe et al., 1999). Excision of the transcription stop sequences by Cre allows the expression of Wnt5a (Fig. 1A). We first tested the functionality of the Z/Wnt5a mice using the Sox2-Cre, in which Cre is ubiquitously expressed in all epiblast cells by E6.5 (Hayashi et al., 2002). Conditional overexpression of Wnt5a by Sox2-Cre caused a severe disruption in embryogenesis. All mutant embryos died and were resorbed at E13.5. Mutant embryos were alive at E10.5, but they all had open neural tube defects and their tails failed to turn (Fig. S1A). We also crossed the Z/Wnt5a allele to Foxg1-Cre mice, in which Cre is expressed widely in the head, including in the developing otic placode (Hébert and McConnell, 2000). Z/Wnt5a;Foxg1-Cre mice showed a thin cochlea sensory epithelium and an extra row of outer hair cells in some regions of the cochlea (Fig. S1B). The hair cell patterning phenotype caused by Wnt5a overexpression in the cochlea is similar to that of Wnt5a−/− mice, as they also show an extra row of outer hair cells (Qian et al., 2007).

Fig. 1.

Wnt5a overexpression in the posterior part of the mouse body disrupts hair follicle orientation. (A) Diagram showing Wnt5a manipulation in mouse embryos using the Z/Wnt5a allele, which has a Cre-activated Wnt5a expression cassette inserted upstream of the Ubb gene. Cre-mediated recombination, such as the Cdx2-Cre, eliminates a loxP-flanked β-geo and three transcription termination sites, permitting transcription of Wnt5a. (B) Overexpression of Wnt5a in the posterior back skin, assessed by qRT-PCR on E15.5 embryos. All qRT-PCR experiments were repeated three times, and three biological replicates were included each time. Gapdh was used as a control. The expression levels of Wnt5a are presented as mean±s.e.m. and compared using ANOVA. WT-A and WT-P, anterior and posterior skin from wild-type embryos; Mut-A and Mut-P, anterior and posterior skin from the Z/Wnt5a;Cdx2-Cre embryos. **P<0.01; ns, not significant. (C) Z/Wnt5a;Cdx2-Cre mice had a tail bent towards the right side of their body. (D) Flat mounts of anterior and posterior back skin from P3 wild-type and Z/Wnt5a;Cdx2-Cre mice. Z/Wnt5a;Cdx2-Cre mice showed a misoriented hair phenotype in the lower back, where Wnt5a was overexpressed. The anterior region had normal hair follicle orientations. A→P, anterior to posterior. Scale bar: 0.5 mm. (E) Quantification of the hair follicle angle relative to the A-P body axis. Rectangles in A and C show where the anterior and posterior back skins were harvested in embryonic and early postnatal mice.

Fig. 1.

Wnt5a overexpression in the posterior part of the mouse body disrupts hair follicle orientation. (A) Diagram showing Wnt5a manipulation in mouse embryos using the Z/Wnt5a allele, which has a Cre-activated Wnt5a expression cassette inserted upstream of the Ubb gene. Cre-mediated recombination, such as the Cdx2-Cre, eliminates a loxP-flanked β-geo and three transcription termination sites, permitting transcription of Wnt5a. (B) Overexpression of Wnt5a in the posterior back skin, assessed by qRT-PCR on E15.5 embryos. All qRT-PCR experiments were repeated three times, and three biological replicates were included each time. Gapdh was used as a control. The expression levels of Wnt5a are presented as mean±s.e.m. and compared using ANOVA. WT-A and WT-P, anterior and posterior skin from wild-type embryos; Mut-A and Mut-P, anterior and posterior skin from the Z/Wnt5a;Cdx2-Cre embryos. **P<0.01; ns, not significant. (C) Z/Wnt5a;Cdx2-Cre mice had a tail bent towards the right side of their body. (D) Flat mounts of anterior and posterior back skin from P3 wild-type and Z/Wnt5a;Cdx2-Cre mice. Z/Wnt5a;Cdx2-Cre mice showed a misoriented hair phenotype in the lower back, where Wnt5a was overexpressed. The anterior region had normal hair follicle orientations. A→P, anterior to posterior. Scale bar: 0.5 mm. (E) Quantification of the hair follicle angle relative to the A-P body axis. Rectangles in A and C show where the anterior and posterior back skins were harvested in embryonic and early postnatal mice.

To determine the effects of Wnt5a overexpression in the skin, we crossed the Z/Wnt5a mice with the Cdx2-Cre (Hinoi et al., 2007), in which Cre is expressed in nearly all embryonic tissues, including the ectoderm, in the posterior part of the body (Fig. 1A). Cdx2-Cre expression begins before E11.5, a time before the initiation of skin and hair follicle patterning (Chang et al., 2016). We collected skin from the anterior (in the neck region) and posterior (in the lower back at the hindlimb level) parts of the E15.5 embryos and performed qRT-PCR to determine the Wnt5a expression levels. In wild-type embryos, Wnt5a was expressed in both the anterior and posterior skin, with a level ∼40% lower in the posterior skin compared with the anterior. As expected, in Z/Wnt5a;Cdx2-Cre embryos, the Wnt5a expression level in the anterior skin was similar to wild type, but its level in the posterior skin was ∼3.3-fold higher than the wild type (Fig. 1B). Z/Wnt5a;Cdx2-Cre mice were born at a normal Mendelian ratio with similar body weight to wild-type littermates. However, all mutant mice had a tail that bent towards the right side of the body (Fig. 1C). We harvested the back skins from postnatal day (P) 3 mice and found that all mutant mice had misoriented hair follicles in the posterior regions where Wnt5a was overexpressed (Fig. 1D,E).

Hair follicles are polarized in random orientations or lose anterior-posterior polarity upon Wnt5a overexpression

To determine whether hair follicles lose their anteroposterior polarity in the Z/Wnt5a;Cdx2-Cre mice, we collected back skins from E15.5 embryos, the time when the 1st wave of hair follicles acquires an anterior-to-posterior polarity. We performed whole-mount immunostaining with E-cadherin antibodies to examine the polarity of hair follicles. Consistent with the literature (Devenport and Fuchs, 2008), anterior cells in wild-type hair follicles adopted elongated cell shapes and expressed reduced levels of E-cadherin. In addition, we found that ZO-1, an anterior marker previously used for labeling hair germs of older embryos, was highly expressed in the E-cadherin weak cells of E15.5 hair follicles (Fig. 2A,B). The complementary staining pattern of E-cadherin and ZO-1 permits a clear determination of which directions the developing hair follicles are tilted, and the orientations of hair follicles already correlate robustly with the anterior-posterior body axis at E15.5. In Z/Wnt5a;Cdx2-Cre embryos, hair follicle orientations in the anterior regions matched to the body axis (Fig. 2C). However, broad distributions of follicle orientations were observed in the posterior part of the Z/Wnt5a;Cdx2-Cre embryos, where Wnt5a was overexpressed (Fig. 2D,E). In hair follicles with a tilted direction, either from anterior to posterior (same orientation as the body axis), from posterior to anterior (a reversed orientation to the body axis) or any other direction, hair follicles showed an asymmetry and the distribution of E-cadherin-weak/ZO-1-strong cells correlated with the orientation of hair follicles. In hair follicles that pointed straight down in the skin, this anteroposterior asymmetry in architecture was lost (Fig. 2F). We quantified the hair follicle orientations (Fig. 2G,H) and noticed that the phenotype was similar to the Fzd6 knockout mice, but less severe than the dominant-negative Vangl2lp and Celsr1Crsh mutants or Fzd3/6 double knockout mice, suggesting a partial loss of PCP in the Z/Wnt5a;Cdx2-Cre posterior skin (Cetera et al., 2017; Devenport and Fuchs, 2008; Dong et al., 2018). We also stained sagittal sections of back skin at E17.5 with makers for polarization and found that the polarization of hair follicles still existed in E17.5 Z/Wnt5a;Cdx2-Cre embryos, suggesting that the asymmetry of hair follicles is maintained as hair follicles grow (data not shown). Together, these data suggest hair follicles in Z/Wnt5a;Cdx2-Cre mice partially lose their anterior-posterior polarity.

Fig. 2.

Hair follicles are polarized in random orientations or lose the anterior-posterior polarity upon Wnt5a overexpression. (A-F″) Whole-mount immunostaining of E15.5 back skins using E-cadherin and ZO-1 antibodies. Schematic on the left indicates the locations of the samples. (A-B″) Hair follicles in wild-type anterior and posterior back skins showed an asymmetry and their orientation aligned with the A-P body axis. Anterior cells in the hair follicles adopted elongated cell shapes and expressed low levels of E-cadherin (red) and high levels of ZO-1 (green). (C-F″) Hair follicles in the anterior region (Cre-negative) of Z/Wnt5a;Cdx2-Cre back skin had a normal orientation (C-C″). However, hair follicles in the posterior back skin showed different orientations (D-E″). In hair follicles pointing straight down in the skin, this anteroposterior asymmetry in architecture was lost (F-F″). Arrowheads indicate the asymmetric distribution of E-cadherin and ZO-1 in the hair follicles. Dotted lines in A″-F″ outline the hair follicles. (G) Quantification of the hair follicle angle relative to the A-P body axis. (H) Frequency of angled versus vertical hair follicles in the anterior and posterior wild-type and Z/Wnt5a;Cdx2-Cre back skins. A→P, anterior to posterior. Scale bar: 20 µm. WT-A and WT-P, anterior and posterior skin from wild-type embryos; Mut-A and Mut-P, anterior and posterior skin from the Z/Wnt5a;Cdx2-Cre embryos.

Fig. 2.

Hair follicles are polarized in random orientations or lose the anterior-posterior polarity upon Wnt5a overexpression. (A-F″) Whole-mount immunostaining of E15.5 back skins using E-cadherin and ZO-1 antibodies. Schematic on the left indicates the locations of the samples. (A-B″) Hair follicles in wild-type anterior and posterior back skins showed an asymmetry and their orientation aligned with the A-P body axis. Anterior cells in the hair follicles adopted elongated cell shapes and expressed low levels of E-cadherin (red) and high levels of ZO-1 (green). (C-F″) Hair follicles in the anterior region (Cre-negative) of Z/Wnt5a;Cdx2-Cre back skin had a normal orientation (C-C″). However, hair follicles in the posterior back skin showed different orientations (D-E″). In hair follicles pointing straight down in the skin, this anteroposterior asymmetry in architecture was lost (F-F″). Arrowheads indicate the asymmetric distribution of E-cadherin and ZO-1 in the hair follicles. Dotted lines in A″-F″ outline the hair follicles. (G) Quantification of the hair follicle angle relative to the A-P body axis. (H) Frequency of angled versus vertical hair follicles in the anterior and posterior wild-type and Z/Wnt5a;Cdx2-Cre back skins. A→P, anterior to posterior. Scale bar: 20 µm. WT-A and WT-P, anterior and posterior skin from wild-type embryos; Mut-A and Mut-P, anterior and posterior skin from the Z/Wnt5a;Cdx2-Cre embryos.

Distribution of PCP proteins Fzd6 and Vangl2 remains unchanged in skin epithelial cells upon Wnt5a overexpression

To determine whether Wnt5a overexpression affects membrane PCP protein distribution in the skin, we collected posterior back skins from E15.5 Z/Wnt5a;Cdx2-Cre embryos and stained them using Fzd6 and E-cadherin antibodies (E-cadherin was used to outline the plasma membrane). Similar to previous reports, we observed Fzd6 proteins on all sides of the epithelial cells in the wild-type skin (Fig. 3A) (Dong et al., 2018). We quantified the fluorescence intensity on the anterior, posterior and mediolateral borders, and identified no significant difference in Fzd6 immunostaining intensity among all sides of the wild-type epithelial cells (Fig. 3B). Wnt5a overexpression did not change the distribution pattern of Fzd6 in the skin epithelial cells (Fig. 3A,B). We also stained the skin with Vangl2 and β-catenin antibodies, and observed similar results (Fig. 3C,D). β-Catenin antibody was used to outline the plasma membrane because Vangl2 antibody was raised in the same host species as the E-cadherin antibody. These data suggest that Wnt5a overexpression does not significantly change the membrane distribution pattern of Fzd6 and Vangl2 in the skin. We note that asymmetric distribution of Fzd6 protein (fused with 3xGFP) has been recently shown in mouse skin (70% versus 30%, enriched at the posterior side) using a high-resolution imaging method (Basta et al., 2021). It is possible that Wnt5a overexpression initiates only a slight asymmetry that is too subtle to detect by our immunostaining method. Feedback mechanisms then amplify the asymmetrical signal, resulting in a misoriented hair follicle.

Fig. 3.

Localization of Fzd6 and Vangl2 in skin epithelial cells remains unchanged upon Wnt5a overexpression. (A) Whole-mount immunostaining of E15.5 wild-type and Z/Wnt5a;Cdx2-Cre posterior back skins with Fzd6 and E-cadherin antibodies. E-cadherin was used to outline the plasma membrane. (B) Quantification of fluorescence intensity of Fzd6 and E-cadherin on the anterior (A), posterior (P) and mediolateral sides (M-L) of the epithelial cells. The fluorescence intensity on each side was normalized by the average intensity of all sides for any given cells. (C) Whole-mount immunostaining of E15.5 wild-type and Z/Wnt5a;Cdx2-Cre posterior back skins with Vangl2 and β-catenin antibodies. β-Catenin was used to replace E-cadherin for outlining the plasma membrane because Vangl2 antibody was raised in the same host species as E-cadherin. (D) Quantification of fluorescence intensity of Vangl2 and β-catenin on the anterior, posterior and mediolateral sides of the epithelial cells. A→P, anterior to posterior. Scale bars: 20 µm.

Fig. 3.

Localization of Fzd6 and Vangl2 in skin epithelial cells remains unchanged upon Wnt5a overexpression. (A) Whole-mount immunostaining of E15.5 wild-type and Z/Wnt5a;Cdx2-Cre posterior back skins with Fzd6 and E-cadherin antibodies. E-cadherin was used to outline the plasma membrane. (B) Quantification of fluorescence intensity of Fzd6 and E-cadherin on the anterior (A), posterior (P) and mediolateral sides (M-L) of the epithelial cells. The fluorescence intensity on each side was normalized by the average intensity of all sides for any given cells. (C) Whole-mount immunostaining of E15.5 wild-type and Z/Wnt5a;Cdx2-Cre posterior back skins with Vangl2 and β-catenin antibodies. β-Catenin was used to replace E-cadherin for outlining the plasma membrane because Vangl2 antibody was raised in the same host species as E-cadherin. (D) Quantification of fluorescence intensity of Vangl2 and β-catenin on the anterior, posterior and mediolateral sides of the epithelial cells. A→P, anterior to posterior. Scale bars: 20 µm.

Posterior Wnt5a overexpression disrupts vertebrae column formation

Global overexpression of Wnt5a has been reported to cause multiple skeletal defects, affecting both endochondral and intramembranous ossification (van Amerongen et al., 2012). To examine the bent tail phenotype in detail and take a closer look at bone formation in the Z/Wnt5a;Cdx2-Cre mice, we performed skeletal staining on P3 pups using Alizarin Red and Alcian Blue (Fig. S2). Surprisingly, all long bones in hindlimbs (femur, tibia and fibula), pelvic bones and foot bones appeared to be fully developed and ossified, as in wild-type littermates. The most prominent defects in Z/Wnt5a;Cdx2-Cre mice were mis-shapen lumbar and sacral vertebrae. Although mutant mice had the normal number of six lumbar and four sacral vertebrae, these bones were wider and the space between adjacent vertebrae was narrower. As a result, the lumbar vertebrae columns in Z/Wnt5a;Cdx2-Cre mice were ∼10% shorter but ∼20% wider than in wild-type mice. In addition, the bending in the lower sacral vertebrae (S3 and S4) and upper caudal vertebrae in the Z/Wnt5a;Cdx2-Cre mice caused the tail to turn towards the right side of the body. All bones in the upper body (above the lumbar vertebra L1) of the Z/Wnt5a;Cdx2-Cre mice appeared normal, as those regions did not have the ectopic expression of Wnt5a. The mechanism causing the tail to turn towards the right side of the body in the Z/Wnt5a;Cdx2-Cre mice remains unknown.

One allele loss of Fzd6 rescues the Wnt5a overexpression phenotypes

Overexpression of Wnt5a in the posterior part of the mouse body caused a local disruption of hair follicle orientations. To determine whether the phenotype is due to hyper-activated Fzd6 signaling or to an artifact of forced Wnt5a expression, we introduced one allele loss of Fzd6 to see whether heterozygous loss of Fzd6 could attenuate the hyper-activated signal, thus resulting in a genetic rescue of the polarity phenotype. We crossed the Z/Wnt5a;Cdx2-Cre to the Fzd6+/− background. Although all Z/Wnt5a;Cdx2-Cre mice showed misoriented hair follicles in the posterior skin, 10 out of the 13 Z/Wnt5a;Cdx2-Cre;Fzd6+/− mice that we collected at P3 had a complete rescue of the hair follicle misorientation phenotype, and the normal anterior-to-posterior follicle orientation in the lower back was restored. The other three Z/Wnt5a;Cdx2-Cre;Fzd6+/− mice had a partial or no rescue (Fig. 4A-H). We also stained the posterior skin from three E15.5 embryos of the rescue genotype with ZO1 and E-cadherin antibodies, and the asymmetry of ZO1/E-cadherin was completely restored (Fig. S3). The vertebrae column showed a limited degree of rescue, as only one mouse (out of 13) showed a partial rescue, in which the space between adjacent vertebrae was restored to normal, but some vertebrae remained wider than in wild type (Fig. 4I-M). The bending tail phenotype was rescued in seven out of 13 mice (Fig. 4N-Q). Detailed information about the rescue experiments is summarized in Table S1. The rescue of Wnt5a overexpression phenotype in the skin by heterozygous loss of Fzd6 suggests that Wnt5a is likely to signal through Fzd6. The incomplete or failed rescue in the sacral and caudal vertebrae suggests either that these tissues are more sensitive to the Wnt5a/Fzd6 signaling disturbance or that another receptor transduces the Wnt5a signaling in these tissues.

Fig. 4.

One allele loss of Fzd6 rescues the Wnt5a overexpression skin and bone phenotypes to various degrees. (A-D) Flat mounts of posterior back skin from P3 wild-type, Z/Wnt5a;Cdx2-Cre and Z/Wnt5a;Cdx2-Cre;Fzd6+/− mice. Wild-type mice showed normal hair follicle orientations (A). Z/Wnt5a;Cdx2-Cre mice showed a misoriented hair phenotype (B), which can be fully (C) or partially (D) rescued by one allele loss of Fzd6. The detailed information of the rescue experiments is summarized in Table S1. A→P, anterior-to-posterior. Scale bar: 0.5 mm. (E-H) The corresponding vector maps of A-D; anterior is towards the left and posterior is towards the right. Vector maps were constructed by sampling hair follicle orientations at each point on the vector grid using a similar method to that of Chang et al. (2015). Quantifications of hair follicle angles to the A-P body axis are shown as bar graphs. (I-Q) Alizarin Red and Alcian Blue stained bones from P3 wild-type (I,N), Z/Wnt5a;Cdx2-Cre (J,O) and Z/Wnt5a;Cdx2-Cre;Fzd6+/− (K,L,P,Q) mice. Wild-type mice showed normal lumbar vertebral morphology (I) and a straight tail (N). Z/Wnt5a;Cdx2-Cre mice showed wider lumbar vertebrae with narrower space between them (J) and a bent tail (O), which can be partially rescued by one allele loss of Fzd6 (K,P). (M) Quantifications of the lumbar vertebrae length and maximum width of wild-type (I), overexpressing (J) and rescue (K,L) mice. *P<0.05; **P<0.01. WT, wild type; OE, Z/Wnt5a;Cdx2-Cre; Res, Z/Wnt5a;Cdx2-Cre;Fzd6+/−. T, thoracic vertebrae; L, lumbar vertebrae; S, sacral vertebrae. Scale bars: 2 mm.

Fig. 4.

One allele loss of Fzd6 rescues the Wnt5a overexpression skin and bone phenotypes to various degrees. (A-D) Flat mounts of posterior back skin from P3 wild-type, Z/Wnt5a;Cdx2-Cre and Z/Wnt5a;Cdx2-Cre;Fzd6+/− mice. Wild-type mice showed normal hair follicle orientations (A). Z/Wnt5a;Cdx2-Cre mice showed a misoriented hair phenotype (B), which can be fully (C) or partially (D) rescued by one allele loss of Fzd6. The detailed information of the rescue experiments is summarized in Table S1. A→P, anterior-to-posterior. Scale bar: 0.5 mm. (E-H) The corresponding vector maps of A-D; anterior is towards the left and posterior is towards the right. Vector maps were constructed by sampling hair follicle orientations at each point on the vector grid using a similar method to that of Chang et al. (2015). Quantifications of hair follicle angles to the A-P body axis are shown as bar graphs. (I-Q) Alizarin Red and Alcian Blue stained bones from P3 wild-type (I,N), Z/Wnt5a;Cdx2-Cre (J,O) and Z/Wnt5a;Cdx2-Cre;Fzd6+/− (K,L,P,Q) mice. Wild-type mice showed normal lumbar vertebral morphology (I) and a straight tail (N). Z/Wnt5a;Cdx2-Cre mice showed wider lumbar vertebrae with narrower space between them (J) and a bent tail (O), which can be partially rescued by one allele loss of Fzd6 (K,P). (M) Quantifications of the lumbar vertebrae length and maximum width of wild-type (I), overexpressing (J) and rescue (K,L) mice. *P<0.05; **P<0.01. WT, wild type; OE, Z/Wnt5a;Cdx2-Cre; Res, Z/Wnt5a;Cdx2-Cre;Fzd6+/−. T, thoracic vertebrae; L, lumbar vertebrae; S, sacral vertebrae. Scale bars: 2 mm.

Wnt5a regulates multiple downstream targets in developing skin

Wnt5a has been reported to signal through both the canonical and PCP pathways (van Amerongen et al., 2012). As we did not observe any changes in the distribution of membrane PCP proteins Fzd6 and Vangl2 in the skin epithelial cells upon Wnt5a overexpression, we examined the transcriptional profile changes of Wnt target genes as an alternative mechanism for disrupted hair follicle polarity. We employed a Qiagen RT² Profiler PCR Array to profile the transcriptional changes of 84 Wnt signaling target genes in the posterior skin from E15.5 wild-type and Z/Wnt5a;Cdx2-Cre embryos (Fig. 5A). This PCR array contains three Wnt genes: Wnt3a, Wnt5a and Wnt9a. As expected, Wnt5a showed a much higher expression level (3.57-fold) in Z/Wnt5a;Cdx2-Cre skin compared with wild type. Our analysis also identified 11 other differentially expressed target genes (with a fold change >1.2 and P<0.05). These include three upregulated (Fst, Fn1 and Gdnf) and eight downregulated [Cdkn2a, Fgf4, Gdf5, Nanog, Mmp7, Wisp2 (Ccn5), Pou5f1 and Tle1] genes. Interestingly, several canonical Wnt signaling targets, such as Axin2, Ccnd1, Lef1, Tcf4 and Tcf7, showed no changes upon Wnt5a overexpression. We also performed similar experiments using the anterior skin from the E15.5 wild-type and Z/Wnt5a;Cdx2-Cre embryos, and did not see any difference in the expression of Wnt target genes (Fig. S4). This is not surprising because no genetic manipulation happened in the Cre-negative anterior regions.

Fig. 5.

Wnt5a regulates multiple downstream targets in developing mouse skin. (A) Effects of Wnt5a overexpression (OE) on the 84 Wnt signaling target genes in the posterior back skins, using the Qiagen RT² Profiler PCR Array (PAMM-243ZA). Ct values for the genes were uploaded to the Qiagen GeneGlobe Data Analysis Center and analyzed using Actb, B2m, Gapdh, Gusb and Hsp90ab1 as reference genes. The data analysis web portal calculated the fold change of three independent experiments using the ΔΔCT method. (Left) Heat map showing upregulated genes in red and downregulated genes in blue. (Right) Volcano plot showing the log2 of the average of Z/Wnt5a;Cdx2-Cre divided by wild-type transcript abundances on the x-axis and the log10 P-value on the y-axis. (B) Effects of heterozygous loss of Fzd6 on the transcriptional changes of 84 Wnt signaling target genes caused by Wnt5a overexpression, using wild-type posterior back skins as controls. (Left) Heat map showing upregulated genes in red and downregulated genes in blue. (Right) Volcano plot showing the log2 of the average of Z/Wnt5a;Cdx2-Cre;Fzd6+/− divided by wild-type transcript abundances on the x-axis and the log10 P-value on the y-axis. (C) Lists and a Venn diagram showing differentially expressed target genes (with a fold change >1.2 and P<0.05) in the Z/Wnt5a;Cdx2-Cre versus wild-type comparison and Z/Wnt5a;Cdx2-Cre;Fzd6+/− versus wild-type comparison. (D) Effects of heterozygous loss of Fzd6 on the transcriptional changes of 84 Wnt signaling target genes caused by Wnt5a overexpression, using Fzd6+/− posterior back skins as control. (Left) Heat map showing upregulated genes in red and downregulated genes in blue. (Right) Volcano plot showing the log2 of the average of Z/Wnt5a;Cdx2-Cre;Fzd6+/− divided by Fzd6+/− transcript abundances on the x-axis and the log10 P-value on the y-axis. (E) Lists and a Venn diagram showing differentially expressed target genes (with a fold change >1.2 and P<0.05) in the Z/Wnt5a;Cdx2-Cre versus wild-type comparison and Z/Wnt5a;Cdx2-Cre;Fzd6+/− versus Fzd6+/− comparison.

Fig. 5.

Wnt5a regulates multiple downstream targets in developing mouse skin. (A) Effects of Wnt5a overexpression (OE) on the 84 Wnt signaling target genes in the posterior back skins, using the Qiagen RT² Profiler PCR Array (PAMM-243ZA). Ct values for the genes were uploaded to the Qiagen GeneGlobe Data Analysis Center and analyzed using Actb, B2m, Gapdh, Gusb and Hsp90ab1 as reference genes. The data analysis web portal calculated the fold change of three independent experiments using the ΔΔCT method. (Left) Heat map showing upregulated genes in red and downregulated genes in blue. (Right) Volcano plot showing the log2 of the average of Z/Wnt5a;Cdx2-Cre divided by wild-type transcript abundances on the x-axis and the log10 P-value on the y-axis. (B) Effects of heterozygous loss of Fzd6 on the transcriptional changes of 84 Wnt signaling target genes caused by Wnt5a overexpression, using wild-type posterior back skins as controls. (Left) Heat map showing upregulated genes in red and downregulated genes in blue. (Right) Volcano plot showing the log2 of the average of Z/Wnt5a;Cdx2-Cre;Fzd6+/− divided by wild-type transcript abundances on the x-axis and the log10 P-value on the y-axis. (C) Lists and a Venn diagram showing differentially expressed target genes (with a fold change >1.2 and P<0.05) in the Z/Wnt5a;Cdx2-Cre versus wild-type comparison and Z/Wnt5a;Cdx2-Cre;Fzd6+/− versus wild-type comparison. (D) Effects of heterozygous loss of Fzd6 on the transcriptional changes of 84 Wnt signaling target genes caused by Wnt5a overexpression, using Fzd6+/− posterior back skins as control. (Left) Heat map showing upregulated genes in red and downregulated genes in blue. (Right) Volcano plot showing the log2 of the average of Z/Wnt5a;Cdx2-Cre;Fzd6+/− divided by Fzd6+/− transcript abundances on the x-axis and the log10 P-value on the y-axis. (E) Lists and a Venn diagram showing differentially expressed target genes (with a fold change >1.2 and P<0.05) in the Z/Wnt5a;Cdx2-Cre versus wild-type comparison and Z/Wnt5a;Cdx2-Cre;Fzd6+/− versus Fzd6+/− comparison.

Next, we performed the PCR array experiments on the posterior skin from E15.5 Z/Wnt5a;Cdx2-Cre;Fzd6+/− embryos (Fig. 5B). Transcriptional profiles revealed that Wnt5a expression remained very high (3.86-fold) in Z/Wnt5a;Cdx2-Cre;Fzd6+/− skin compared with wild type, but the expression levels of three upregulated genes induced by Wnt5a overexpression (Fst, Fn1 and Gdnf) were rescued to normal levels by the heterozygous loss of Fzd6. Tle1 is one of the eight genes downregulated by Wnt5a overexpression, and its level was also restored in the Z/Wnt5a;Cdx2-Cre;Fzd6+/− skin. The expression of the other seven genes (Cdkn2a, Fgf4, Gdf5, Nanog, Mmp7, Wisp2 and Pou5f1) were not rescued by the heterozygous loss of Fzd6. In addition, Z/Wnt5a;Cdx2-Cre;Fzd6+/− and wild type comparison showed three downregulated genes that were not shared by the Z/Wnt5a;Cdx2-Cre and wild type comparison: Fosl1, Cebpd and Wnt9a (Fig. 5C). We also compared the transcriptional profiles of Z/Wnt5a;Cdx2-Cre;Fzd6+/− posterior skin to Fzd6+/−. Very strikingly, only four genes (including Wnt5a) were found to be differentially expressed, suggesting a nearly complete rescue of the target gene expression by one allele loss of Fzd6 in the Wnt5a overexpressing skin (Fig. 5D,E). The rescue of polarity phenotype in most of the Z/Wnt5a;Cdx2-Cre;Fzd6+/− mice, together with the transcription profiling data, suggest that Fst, Fn1, Gdnf and Tle1 might be mediators of the skin polarization program controlled by Wnt5a/Fzd6 signaling.

Posterior or global knockout of Wnt5a does not affect hair follicle polarity

To further extend our study beyond gain of function and determine whether Wnt5a is required for controlling hair follicle orientation, we used the existing Wnt5a conditional knockout allele in which the critical exon 2 of the Wnt5a gene was flanked by loxP sites (Ryu et al., 2013). We crossed the Wnt5a conditional allele to Cdx2-Cre to generate mice with a posterior deletion of Wnt5a. Wnt5afx/−;Cdx2-Cre mutant mice survive postnatally but showed a severe truncation in the hindlimbs and the tail (Fig. 6A), consistent with the known function of Wnt5a in regulating distal extension of multiple structures from the primary body axis (Yamaguchi et al., 1999). The anterior part of the body is normal, as Cre is not expressed in these regions. We harvested the back skin from P3 mutant mice and found that hair follicles in the posterior part of the body were normally oriented (Fig. 6B). We also collected back skin from E17.5 embryos and performed whole-mount staining using E-cadherin antibodies. No defects in hair follicle orientation were found in the posterior part of Wnt5afx/−;Cdx2-Cre embryos, as hair follicles showed a narrow distribution centered on the anterior-posterior body axis, identical to wild type (Fig. 6C). To exclude the possibility that the posterior skin receives the Wnt5a from the anterior sources (via diffusion) or cell non-autonomous effects interfere with the phenotype, we examined the hair follicle orientation in the Wnt5a straight knockout mice. For this experiment, we used a keratin 17 (K17)-GFP transgene, which is expressed specifically in hair follicles, to visualize and quantify follicle orientations (Bianchi et al., 2005). At E18.5, no defects in hair follicle orientation were found in any parts of Wnt5a−/− embryos (Fig. 6D). We also performed whole-mount immunostaining on E15.5 back skins from Wnt5afx/−;Cdx2-Cre and Wnt5a−/− embryos with E-cadherin and ZO-1 antibodies, and found no abnormalities in hair follicle polarity (Fig. 6E). The observation that posterior or global deletion of Wnt5a does not affect hair follicle orientation strongly suggests that Wnt5a is not the sole orienting ligand for skin polarization. We examined the expression levels of all 19 Wnts in the Wnt5a−/− skin by RT-PCR and observed no significant changes other than in Wnt5a, suggesting that the already existing endogenous level of other Wnt(s) might be sufficient to take over Wnt5a function. We also examined the mRNA levels of Fn1, Fst, Gdnf and Tle1 in the Wnt5a−/− skin by RT-PCR and did not see any significant changes compared with the wild type (Fig. S5).

Fig. 6.

Posterior or global knockout of Wnt5a does not affect hair follicle polarity. (A) At P3, Wnt5afx/−;Cdx2-Cre mice show a severe truncation in the hindlimbs and the tail, indicating a specific deletion of Wnt5a in the posterior part of the body. Wild type on the left; mutant on the right. (B) Flat mounts of posterior back skin from P3 wild-type and Wnt5afx/−;Cdx2-Cre mice, showing normal hair follicle orientations in the Wnt5afx/−;Cdx2-Cre mice. A→P, anterior to posterior. Scale bar: 0.5 mm. Quantification of hair follicle angles to the A-P body axis is shown as bar graphs. (C) (Left) Whole-mount immunostaining of E17.5 back skins from wild-type and Wnt5afx/−;Cdx2-Cre embryos with E-cadherin antibodies. Hair follicles in Wnt5afx/−;Cdx2-Cre skin showed a normal anterior-to-posterior orientation. Scale bar: 20 µm. (Right) Hair follicle angles to the plane of the skin were compared using a two-tailed unpaired Student's t-test. Wild type, n=265 hair follicles; Wnt5afx/−;Cdx2-Cre, n=272 hair follicles. ns, not significant. (D) (Left) Back skin flat mounts showing normal hair follicle orientations in the E18.5 Wnt5a−/− embryos. Hair follicles are visualized with a K17-GFP reporter. Scale bar: 1 mm. (Right) Hair follicle angles to the plane of the skin were compared using a two-tailed unpaired Student's t-test. Wild type, n=321 hair follicles; Wnt5a−/−, n=334 hair follicles. (E) Whole-mount immunostaining of E-cadherin and ZO-1 on posterior back skins from E15.5 wild-type (a-a″), Wnt5afx/−;Cdx2-Cre (b-b″) and Wnt5a−/− mice (c-c″). Quantification of hair follicle angles to the A-P body axis are shown as bar graphs. A→P, anterior to posterior. Scale bar: 20 µm.

Fig. 6.

Posterior or global knockout of Wnt5a does not affect hair follicle polarity. (A) At P3, Wnt5afx/−;Cdx2-Cre mice show a severe truncation in the hindlimbs and the tail, indicating a specific deletion of Wnt5a in the posterior part of the body. Wild type on the left; mutant on the right. (B) Flat mounts of posterior back skin from P3 wild-type and Wnt5afx/−;Cdx2-Cre mice, showing normal hair follicle orientations in the Wnt5afx/−;Cdx2-Cre mice. A→P, anterior to posterior. Scale bar: 0.5 mm. Quantification of hair follicle angles to the A-P body axis is shown as bar graphs. (C) (Left) Whole-mount immunostaining of E17.5 back skins from wild-type and Wnt5afx/−;Cdx2-Cre embryos with E-cadherin antibodies. Hair follicles in Wnt5afx/−;Cdx2-Cre skin showed a normal anterior-to-posterior orientation. Scale bar: 20 µm. (Right) Hair follicle angles to the plane of the skin were compared using a two-tailed unpaired Student's t-test. Wild type, n=265 hair follicles; Wnt5afx/−;Cdx2-Cre, n=272 hair follicles. ns, not significant. (D) (Left) Back skin flat mounts showing normal hair follicle orientations in the E18.5 Wnt5a−/− embryos. Hair follicles are visualized with a K17-GFP reporter. Scale bar: 1 mm. (Right) Hair follicle angles to the plane of the skin were compared using a two-tailed unpaired Student's t-test. Wild type, n=321 hair follicles; Wnt5a−/−, n=334 hair follicles. (E) Whole-mount immunostaining of E-cadherin and ZO-1 on posterior back skins from E15.5 wild-type (a-a″), Wnt5afx/−;Cdx2-Cre (b-b″) and Wnt5a−/− mice (c-c″). Quantification of hair follicle angles to the A-P body axis are shown as bar graphs. A→P, anterior to posterior. Scale bar: 20 µm.

Potential functional redundancy of Wnt genes in controlling hair follicle polarity

Developing mouse skin expresses many Wnts, including Wnt5a (Reddy et al., 2001). To determine which Wnt ligands might also be involved in controlling hair follicle polarity, we examined the expression of all 19 mammalian Wnt genes in E15.5 back skin by RT-PCR (Fig. 7A). We found that the 19 Wnt genes could be categorized into three groups based on differential expression levels: (1) Wnt3, Wnt4, Wnt5a, Wnt6, Wnt9a, Wnt10a, Wnt10b, Wnt11 and Wnt16 were highly expressed; (2) Wnt2, Wnt5b, Wnt7a and Wnt7b were expressed at a lower level; and (3) the rest of six Wnts (Wnt1, Wnt2b, Wnt3a, Wnt8a, Wnt8b and Wnt9b) were either not expressed or expressed at a very low level. To determine which other Wnts can contribute to skin PCP (like Wnt5a), we tried to establish an in vitro screening assay using HEK293T cells. We took advantage of our discovery that Fst, Fn1, Gdnf and Tle1 might be specific targets involved in skin polarization and tested whether these targets could be used for monitoring PCP activity in vitro. We chose HEK293T cells because no detectable level of FZD6 or FZD3 was found in these cells, whereas mouse keratinocytes or skin express high levels of Fzd6, Fzd3 and Lrp5, making them unsuitable for monitoring the activation of Fzd6 signaling (Dong et al., 2018). We transiently transfected HEK293T cells with Fzd6, the co-receptor Lrp5, or with Fzd6+Lrp5 expression plasmids. C59 was used to block the endogenous production of Wnt proteins (Koo et al., 2015; Proffitt et al., 2013; Teh et al., 2015). Cells were then treated with Wnt5a conditional media for 6 h, and the expression levels of target genes were analyzed by qRT-PCR (Fig. 7B). Wnt5a conditional media treatment significantly increased the expression of FN1 in HEK293T cells expressing Fzd6 and Lrp5, whereas it had no effects on control HEK293T cells transfected with the empty pRK5 vector plasmids. These data suggest that activation of Wnt5a/Fzd6 signaling is sufficient to drive the expression of FN1 in HEK293T cells. We noticed that the transfection of Fzd6 and Lrp5 alone can increase the expression of FN1 by more than twofold, suggesting that other endogenous ligands (most likely non-Wnt ligands) exist to activate Fzd6/Lrp5. The expression levels of GDNF, FST and TLE1 did not change significantly upon Wnt5a treatment (Fig. S6), highlighting the cell type specificity of downstream targets of Fzd6 in mouse skin epithelial cells and human embryonic kidney cells.

Fig. 7.

Multiple Wnts can regulate the Fzd6-mediated signaling pathway in vitro. (A) Endogenous expression of Wnt genes in E15.5 mouse back skin, as assessed by RT-PCR. Among 19 Wnt genes, Wnt3, Wnt4, Wnt5a, Wnt6, Wnt9a, Wnt10a, Wnt10b, Wnt11 and Wnt16 are highly expressed. Numbers refer to the expected size (bp) of PCR products. (B) Wnt5a is sufficient to drive FN1 expression in HEK293T cells. qRT-PCR shows a significant change in FN1 mRNA levels after 6 h of Wnt5a treatment in HEK293T cells transiently transfected with Fzd6 and the co-receptor Lrp5 (group g versus h). Transfection of Fzd6 and Lrp5 alone can increase the expression of FN1 by twofold (group g versus a), suggesting that other endogenous ligands exist to activate Fzd6/Lrp5. (C) Effects of eight other highly expressed Wnts on Fzd6-induced FN1 expression. Like Wnt5a, Wnt3, Wnt10b, Wnt11 and Wnt16, conditional media treatment can significantly upregulate FN1 expression (group g versus h). Lrp5 is essential for activating the Wnt/Fzd6 signaling pathway (comparing group c with d). (Right) Heat plot showing the fold change of FN1 expression levels upon various combinations of Wnt, Fzd6 and Lrp5. The normalized fold change was calculated by dividing the (Fzd6+Lrp5+Wnt) group by the average of (Lrp5+Wnt) and (Fzd6+Lrp5) groups to reveal the fold increase referable specifically to the interaction between Fzd6 and Wnts. All qRT-PCR data are mean±s.e.m. of three biological replicates. GAPDH was used as a control. Quantification of data was compared using ANOVA followed by Dunnett's test. *P<0.05; **P<0.01; ns, not significant.

Fig. 7.

Multiple Wnts can regulate the Fzd6-mediated signaling pathway in vitro. (A) Endogenous expression of Wnt genes in E15.5 mouse back skin, as assessed by RT-PCR. Among 19 Wnt genes, Wnt3, Wnt4, Wnt5a, Wnt6, Wnt9a, Wnt10a, Wnt10b, Wnt11 and Wnt16 are highly expressed. Numbers refer to the expected size (bp) of PCR products. (B) Wnt5a is sufficient to drive FN1 expression in HEK293T cells. qRT-PCR shows a significant change in FN1 mRNA levels after 6 h of Wnt5a treatment in HEK293T cells transiently transfected with Fzd6 and the co-receptor Lrp5 (group g versus h). Transfection of Fzd6 and Lrp5 alone can increase the expression of FN1 by twofold (group g versus a), suggesting that other endogenous ligands exist to activate Fzd6/Lrp5. (C) Effects of eight other highly expressed Wnts on Fzd6-induced FN1 expression. Like Wnt5a, Wnt3, Wnt10b, Wnt11 and Wnt16, conditional media treatment can significantly upregulate FN1 expression (group g versus h). Lrp5 is essential for activating the Wnt/Fzd6 signaling pathway (comparing group c with d). (Right) Heat plot showing the fold change of FN1 expression levels upon various combinations of Wnt, Fzd6 and Lrp5. The normalized fold change was calculated by dividing the (Fzd6+Lrp5+Wnt) group by the average of (Lrp5+Wnt) and (Fzd6+Lrp5) groups to reveal the fold increase referable specifically to the interaction between Fzd6 and Wnts. All qRT-PCR data are mean±s.e.m. of three biological replicates. GAPDH was used as a control. Quantification of data was compared using ANOVA followed by Dunnett's test. *P<0.05; **P<0.01; ns, not significant.

Next, we tested the combination of Fzd6 with various other Wnt ligands in regulating FN1 expression in HEK293T cells. We focused on the eight Wnts that are highly expressed in E15.5 skin: Wnt3, Wnt4, Wnt6, Wnt9a, Wnt10a, Wnt10b, Wnt11 and Wnt16. When analyzing the FN1 expression levels in Fzd6, Lrp5 and Fzd6+Lrp5 transfected cells with or without treatment with Wnts, several patterns emerged. First, adding Wnts to Fzd6-expressing cells without Lrp5 does not change the FN1 expression level (comparing group c with d in Fig. 7B,C, except for Wnt9a), suggesting that Lrp5 is required for the Fzd6 pathway activation. Second, when Fzd6/Lrp5 is not present, different Wnts can still have either a positive or negative effect on FN1 expression, suggesting that FN1 transcription is not only controlled by Wnt/Fzd6 (comparing group a with b). The up- or downregulation of FN1 might depend on whether Wnts can activate other non-Fzd6 receptors or compete with endogenous ligands of parallel mechanisms. Third, the most informative comparison is between Fzd6+Lrp5 and Fzd6+Lrp5+Wnt groups (group g versus h). Similar to Wnt5a, Wnt3, Wnt10b, Wnt11 and Wnt16 can activate Fzd6 and promote the expression of FN1 (Fig. 7C). We also normalized the FN1 expression levels of the Fzd6+Lrp5+Wnt groups to Lrp5+Wnt and Fzd6+Lrp5 to reveal the fold change that refers specifically to the interaction between Fzd6 and Wnts. After normalization, the top three Wnts are Wnt11, Wnt10b and Wnt16, which can increase the expression level of FN1 by 1.96, 1.89 and 1.81-fold, respectively. These results suggest that multiple Wnts can regulate FN1 expression and might play a role in skin PCP.

The experiments described here show that Wnt5a can regulate mouse skin PCP. In particular, we report that (1) overexpression of Wnt5a in the posterior part of the mouse body causes misorientation of hair follicles and malformation of the vertebrae column; (2) the misoriented hair follicle phenotype in Wnt5a overexpressing mice can be rescued by a heterozygous loss of Fzd6; (3) Fst, Fn1, Gdnf and Tle1 are possible targets of the skin polarization program controlled by the Wnt5a/Fzd6 signaling; (4) posterior or global deletion of Wnt5a does not affect hair follicle orientation; and (5) multiple other Wnts are highly expressed in the mouse skin and are capable of activating the Fzd6-induced signaling pathway.

The function of Wnts in the skin

Wnt signaling is involved in almost all aspects of development (Nusse and Clevers, 2017). Its role in the skin has also been extensively studied since the initial observation that Lef1 knockout mice lack whiskers and hairs (Lim and Nusse, 2013; van Genderen et al., 1994). The lack of hair formation in Lef1 knockout mice is due to the failure of stem cells to differentiate into follicular keratinocytes, instead adopting an epidermal fate when the canonical Wnt signaling is suppressed (Huelsken et al., 2001). In addition to the lineage specification of follicular keratinocytes, Wnt signaling is also required for the proper specification of the sebocyte (Niemann and Horsley, 2012) and melanocyte (Dunn et al., 2000). A recent addition to the list is that Wnt signaling controls the lineage specification of stem cells located at the junctional zone of mouse upper hair follicles, and abnormal Wnt signaling causes pathological changes that resemble human acne (Shang et al., 2021). Persistent activation of Wnt signaling leads to junctional zone cyst formation and sebaceous gland atrophy, whereas loss of Wnt signaling leads to the enlargement of the junctional zone, infundibulum and sebaceous gland.

Here for the first time, we show that Wnt can regulate skin PCP in mice. We determined the role of Wnt5a in skin polarization using the Wnt5a conditional knockout and overexpression alleles in combination with an anatomically-localized Cre driver, which allows Wnt5a activity to be selectively eliminated or overactivated in the posterior part of the embryos. Our data demonstrate that: (1) overexpression of Wnt5a in the posterior part of the body causes misorientation of hair follicles; and (2) deletion of Wnt5a in the posterior part of the body does not affect hair orientation. Given that the Wnt5a expression level is higher in the anterior part than the posterior, these results lead us to hypothesize that an anterior-high and posterior-low gradient of Wnt5a is essential for controlling hair follicle orientation during development. The normal hair follicle orientation in the Wnt5a−/− mice suggests that other Wnt(s) can recapitulate the Wnt5a function in its absence. At present, there are no Cre lines that are expressed in the anterior half of the body that is complementary to Cdx2-Cre. If the anterior Cre lines existed, we would predict that overexpressing Wnt5a in the anterior part of the body would result in no disruption of hair follicle orientations as the anterior to posterior gradient of signaling intensity is not disrupted. Our findings contrast with two recent studies in Drosophila wing epithelium showing that Wnts are dispensable for the establishment of wing hair polarity (Ewen-Campen et al., 2020; Yu et al., 2020), highlighting a fundamental difference between the vertebrate and invertebrate systems or maybe even among different tissue types of the same species. It has been reported that ectopic Fz expression is sufficient to produce the asymmetric localization of Dsh and alter the polarity pattern on the fly wing (Axelrod, 2001). However, overexpression of Fzd6 from either the Rosa26 or Ubb locus in a wild-type background produces no effect on skin polarity (Hua et al., 2014). One wild but simple explanation for this discrepancy is that fly wing PCP relies more on the frizzled gradient, but mouse skin PCP is more dependent on the ligand gradient and Fzd6 needs to be present to passively transduce the signal.

Canonical and non-canonical targets of Wnt5a

Wnt5a can bind to both frizzled receptors and receptor tyrosine kinases, such as orphan receptor 2 (Ror2), and cause distinct signaling outputs. For example, cell culture studies have shown that Wnt5a can inhibit canonical Wnt signaling when Ror2 is expressed but activate the canonical Wnt signaling with the presence of Fzd5 or Fzd4 and Lrp5 co-receptor (He et al., 1997; Mikels and Nusse, 2006). It has also been reported that Wnt5a can activate and inhibit canonical Wnt signaling in the skull and skin, respectively (van Amerongen et al., 2012). Increasing evidence suggests that the inhibition of the canonical Wnt signaling by Wnt5a is independent of Ror2 and possibly due to competition with other canonical Wnts for binding to receptors (Grumolato et al., 2010; Ho et al., 2012). These studies highlight the complexity of the Wnt signaling pathway and the importance of receptor/co-receptor availability in determining the signaling outcomes of Wnt5a.

Interestingly, Wnt5a overexpression in mouse embryos using a tet-ON system has been reported to cause severe defects in hair follicle formation due to a suppression of the canonical Wnt signaling in the skin (van Amerongen et al., 2012). Here, we have adopted a genetic knock-in approach and overexpressed Wnt5a at a level about three times that of the endogenous gene. We observed no defects in hair follicle formation; instead, we observed misoriented hair follicles in the region where Wnt5a was overexpressed. Our qRT-PCR array analysis on the skin revealed that canonical Wnt signaling is also not severely suppressed upon Wnt5a overexpression. Many canonical Wnt target genes (e.g. Axin2, Ccnd1, Lef1, Tcf4 and Tcf7) showed no expression changes in the Z/Wnt5a;Cdx2-Cre skin. The disparities between the previous report and our observations are likely due to the Wnt5a transgene expression level. We observed significant changes in the expression of Fst, Fn1, Gdnf and Tle1 upon Wnt5a overexpression, which can be rescued by a heterozygous loss of Fzd6. Fst encodes glycoprotein follistatin that is believed to function as a TGFβ antagonist by binding to activin (Seachrist and Keri, 2019). Fn1 encodes an extracellular matrix protein, fibronectin, which plays a major role in cell adhesion, migration and differentiation (Pankov and Yamada, 2002). Gdnf encodes a neuron survival protein glial cell line-derived neurotrophic factor (Cintrón-Colón et al., 2020), and it is expressed at a lower level in the mouse skin (Fig. S5). Tle1 encodes a transcriptional co-repressor that can regulate transcription in several signaling pathways, including Notch and Wnt (Chodaparambil et al., 2014). Our qRT-PCR array data comparing the Wnt5a overexpressing skin with disrupted polarity and Fzd6+/− rescue skin with restored polarity suggest that these four genes are possible targets of the Wnt5a-Fzd6 signaling pathway. Given that the establishment of polarity involves dramatic cell structure rearrangements in the skin (Cetera et al., 2018), that fibronectin plays a general role in cell adhesion and migration, and that our in vitro data demonstrate that FN1 expression is significantly upregulated by Wnt/Fzd6 activation, we predict that Fn1 might play a key role in regulating skin polarization.

Monitoring the Fzd6 signaling pathway and PCP activation in vitro

Many in vitro and in vivo tools have been generated to monitor the activation of canonical Wnt signaling. HEK293 cells carrying a luciferase reporter under the control of seven LEF/TCF-binding sites (Super TOP-FLASH, STF cells) are widely used to quantitatively monitor the activation of the canonical Wnt signaling in vitro (Xu et al., 2004). Transgenic mice carrying a LacZ or EGFP reporter under various promoters and different numbers of TCF/LEF-binding sites enable us to visualize the activation of canonical Wnt signaling by X-gal staining or fluorescence microscopy (DasGupta and Fuchs, 1999; Jho et al., 2002; Lustig et al., 2002). Recently, a cell membrane-targeted EGFP reporter driven by Axin2 was generated to provide a better tool to track cell movements and shape changes in vivo (Wang et al., 2021). In addition to these reporters, the immunostaining method to detect nuclear localized β-catenin or non-phosphorylated (activated) β-catenin can also be used to monitor the activation of the canonical Wnt signaling.

Monitoring the non-canonical PCP signaling is difficult because the downstream targets remain mysterious. Detecting the colocalization of Fzds with Celsr1 on the membrane of keratinocytes or MDCK cells has provided some insights into whether a particular Fzd can function as a PCP receptor (Devenport and Fuchs, 2008; Yu et al., 2012), but this is limited to the proteins that can physically interact with Celsr1; how the binding is physiologically relevant to the polarization of cell or tissue is unknown. Here, we have identified that activation of Fst, Fn1 and Gdnf, and inhibition of Tle1 are potential downstream targets of the Wnt5a-Fzd6 signaling pathway in skin polarization. We show that the transcriptional levels of these genes, especially FN1, can be used to identify the ligand/activator of Fzd6 in HEK293T cells. In our screening of the eight Wnts that are highly expressed in the developing skin, Wnt3, Wnt10b, Wnt11 and Wnt16 showed a similar capability to upregulate FN1 expression when Fzd6 and Lrp5 were present. It will be interesting to determine whether these Wnts are essential for polarizing the skin in vivo and how much functional redundancy they share with Wnt5a.

Mouse lines and husbandry

The following mouse alleles were used: Z/Wnt5a (JAX #018141), Cdx2-Cre (Hinoi et al., 2007), Fzd6−/− (Guo et al., 2004), Wnt5afx/fx (Ryu et al., 2013), Sox2-Cre (Hayashi et al., 2002), Foxg1-Cre (Hébert and McConnell, 2000) and K17-GFP (Bianchi et al., 2005). Mice were handled and housed according to the approved Institutional Animal Care and Use Committee (IACUC) protocol M005675 of the University of Wisconsin-Madison.

Immunostaining

For whole-mount immunostaining of embryonic skins, embryos were fixed for 1 h in 4% formaldehyde. Back skins were dissected, rinsed with PBS, washed with 0.3% PBST for 30 min and incubated with primary antibodies in 0.3% PBST containing 5% normal donkey serum overnight at 4°C. The primary antibodies used were rat anti-E-cadherin (ab11512-100; Abcam; 1:400), rabbit anti-ZO-1 (61-7300; Life Technologies; 1:200), goat anti-Fzd6 (AF1526; R&D Systems; 1:400), rat anti-Vangl2 (MABN750; Millipore; 1:100) and rabbit anti-β-catenin (9587; Cell Signaling; 1:800). Skins were then washed in 0.3% PBST three times for 30 min each, incubated in secondary antibodies in 0.3% PBST at room temperature for 2 h, washed in 0.3% PBST and flat-mounted in Fluoromount G (Southern Biotech). The secondary antibodies used were Alexa Fluor 488- or 594-conjugated donkey anti-rat, -rabbit and -goat IgG antibodies (Invitrogen). Immunostained samples were imaged using the Nikon A1R-Si+ confocal microscope with NIS-Elements software.

Skin whole mounts

The procedures for preparation and processing of skin whole mounts for imaging of hair follicles based on melanin content were similar to previously described methods (Chang and Nathans, 2013; Chang et al., 2014). Dorsal back skins were dissected and flattened by pinning the edges to a flat Sylguard surface, fixed overnight in 4% paraformaldehyde in PBS, dehydrated through a graded alcohol series and then clarified with benzyl benzoate: benzyl alcohol (BBBA) in a glass dish. Images were collected with a Zeiss Stemi 508 microscope with a color Axiocam 105 in combination with Zen software.

Skeleton preparations

Alcian Blue/Alizarin Red staining on P3 skeletons was performed similarly to the methods of McPherron et al. (1999). P3 pups were euthanized, skinned and eviscerated. Samples were fixed in 80% ethanol, dehydrated in 95% ethanol for 1 day and acetone for 3 days. Skeletons were then stained for 2 days with 10% acetic acid in ethanol containing 0.003% Alizarin Red and 0.0045% Alcian Blue. After staining, skeletons were cleared in 1% potassium hydroxide and transferred to 20%, 50%, 80% and 100% glycerol over several days. Images were collected with a Zeiss Stemi 508 microscope with a color Axiocam 105 in combination with Zen software.

Cell culture

1D4-tagged mouse Fzd6- and Wnt-expressing plasmids have been described previously (Yu et al., 2012). Lrp5 full-length cDNA was purchased from Open Biosystems (MMM1013-202761137, clone ID 3154246). Standard procedures were used to transfect plasmid DNA into HEK293T grown in DMEM/F12 medium with 10% fetal bovine serum and penicillin/streptomycin. For each well of cells to be transfected in a 12-well plate, 0.5 µg of each plasmid DNA was diluted in 50 µl of serum-free media. 1.8 µl of 1 mg/ml polyethylenimine (PEI) was added into the diluted DNA solution, mixed gently and incubated for 10 min at room temperature before adding to the cells. To test the activation of Fzd6 signaling, HEK293T cells were transfected with control pRK5 plasmids or Fzd6 and Lrp5 plasmids on day 1, treated with 5 nM C59 (Abcam, ab142216) on day 2, and changed into various Wnt-containing conditional media or control media on day 3. After 6 h of Wnt treatment at 37°C in the cell culture chamber, cells were harvested for RNA collection.

Semi-quantitative RT-PCR and quantitative real-time PCR (qRT-PCR)

RNA was extracted using Trizol (Invitrogen) and RNeasy (Qiagen) kits. cDNA synthesis was performed with the SuperScript IV First-Strand Synthesis System (18091150, Invitrogen) following the manufacturer's instructions. Quantitative real-time RT-PCR was performed in triplicate in 20 μl reactions with SYBR Premix Ex Taq II ROX plus (RR82LR, Takara) with 40 ng first-strand cDNA and 0.2 μg each forward and reverse primers. Samples were cycled once at 95°C for 2 min, followed by 40 cycles of 95°C, 58°C and 72°C for 30 s each. Relative mRNA levels were calculated using the ΔΔCT method with Gapdh/GAPDH as an endogenous control. Primers used for semi-quantitative RT-PCR and qRT-PCR were designed or selected from the PrimerBank database (listed in Table S2). The effects of Wnt5a overexpression on the Wnt signaling pathway were assessed using the Qiagen RT² Profiler PCR Array Mouse WNT Signaling Pathway (PAMM-243ZA) following the manufacturer's instructions. Ct values for the genes were uploaded to the Qiagen GeneGlobe Data Analysis Center and analyzed using Actb, B2m, Gapdh, Gusb and Hsp90ab1 as reference genes. The data analysis web portal (https://geneglobe.qiagen.com/us/analyze) calculated fold change using the ΔΔCT method.

Statistical analysis

Statistical analyses were performed with two-tailed unpaired Student's t-test between two experimental groups and one-way analysis of variance (ANOVA) for more than two experimental groups followed by Dunnett's or Tukey's test for multiple comparisons using the GraphPad Prism 9 software. P<0.05 was considered significant.

The work was supported by grants from the National Institutes of Health (the Skin Diseases Research Center Core grant P30 AR066524) and the University of Wisconsin Carbone Cancer Center (support grant P30 CA014520).

Author contributions

Conceptualization: H.C.; Methodology: L.S., H.C.; Formal analysis: L.S., H.C.; Investigation: L.S., E.O., H.C.; Data curation: L.S., E.O., H.C.; Writing - original draft: H.C.; Writing - review & editing: L.S., E.O., H.C.; Supervision: H.C.; Project administration: H.C.; Funding acquisition: H.C.

The work was supported by the National Institutes of Health (R01GM129259 to H.C.). H.C. was also supported in part by a Gary S. Wood Dermatology Research Bascom Endowed Professorship. Deposited in PMC for release after 12 months.

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Competing interests

The authors declare no competing or financial interests.

Supplementary information