In this study we used Ptch2 ^−/− mutant mice (Lee et al., 2006). All experimental procedures involving mice were approved by the Ethical Committees on the Use and Care of Animals and the Animal Facility at the University of Helsinki.

All animals (3 per genotype) were anesthetized prior the procedure. Analgesics were administered prior to wounding and for 3 consecutive days after wounding. Two to four full-thickness wounds were made on the dorso-rostral skin using a 4-mm biopsy punch as previously described (Aragona and Blanpain, 2017). Mice were sacrificed 7 days post-wounding and tissue was processed for histological and immunostaining analyses.

Mouse mandibles and neurovascular bundles (NVBs) were isolated from 3-4-week-old mice following an established protocol (Balic, 2019). Dorsal skins were collected from postnatal animals at days 1, 14, and 50. All samples were fixed with 10% formalin overnight at 4°C. Mandibles and skins were dehydrated, embedded in paraffin wax, and sectioned into 7–12 μm sections. NVBs were processed for whole-mount immunofluorescent staining. Long bones were collected from 4-5-week-old mice, cleaned from the surrounding tissue, and kept in 100% ethanol until µCT scanning.

MEFs obtained from E14.5 wild-type and Ptch2 ^−/− (Lee et al., 2006), and E10.5, Ptch1 ^−/− embryos (gift from Dr. Ben Allen), were plated on chamber slides with removable wells at a density of 10,000 cells/0.1 cm². Culture media were used as previously described (Binder et al., 2019). The following day, cultures were washed and processed for immunofluorescent staining.

Bone marrow stromal cells were isolated from the metaphyses of cleaned tibiae and cultured as previously described (Balic et al., 2010). CFU assay was performed as previously described (Yang et al., 2013). ImageJ was used to quantify the number of colonies formed after 7 days of culture. Data are represented as relative values normalized against wild-type control in each experimental replicate.

Mineralization and adipogenesis were induced in confluent cultures at day 7 or 10, respectively, as previously described (Balic et al., 2010). Alizarin Red staining (AR-S) was used to assay the extent of mineralization in terminated cultures. Adipocytes were visualized by Nile Red staining of live cultures. Briefly, cultures were incubated with Nile Red (1:1000 dilution of stock solution) for 1 h at 37°C, washed, and imaged under a fluorescent microscope. ImageJ was used to quantify the surface of AR-S and Nile Red staining. Data are represented as relative values normalized against wild-type control in each experimental replicate.

H&E stained skin sections were visualized using high-throughput brightfield scanning microscope equipped with 4 MP CMOS camera with 130 fps speed. Slides were scanned with extended focus (Z-stack) with 0.24 μm/pixel resolution with the combined 20X/0.8 NA objective (equivalent to ×40 magnification). Fixed-size images (2.5 × 1 mm) of random sections were collected for each sample (minimum 5 sections) using Case viewer software (3DHISTECH). Skin area in each image was calculated using ImageJ.

Cultures of wild-type, Ptch1 ^−/− , and Ptch2 ^−/− MEFs were immunostained against Gli1, Gli2, or Gli3 protein. DAPI staining was used to label the nucleus. Obtained images were generated by Zeiss LSM 700 confocal microscope. ImageJ was used to quantify the intensity of the immunofluorescence staining in the area defined by DAPI staining as previously described (Jensen, 2013).

Nuclear and cytoplasmic protein fractions were isolated from confluent cultures using NE-PER kit (Nuclear and Cytoplasmic Extraction Reagents, #78833, Thermo Fisher Scientific) according to the manufacturer’s instructions. Obtained extracts were separated by SDS-PAGE and transferred to nitrocellulose membranes (Trans-BlotTurboTransfer pack, Bio-Rad). After blocking in 5% milk in 1% Tween-20-tris-buffered saline, the membranes were incubated with the primary antibodies at 4°C overnight. Membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000) for 2 h at room temperature. Blots were developed using enhanced chemiluminescence substrate (Thermo Fischer Scientific).

Limb bone µCT data was acquired using a µCT (SkyScan 1272, Bruker) with the following data acquisition parameters: 60–70 kV, 142–166 μA, isotropic voxel size 4.5–5.0 µm. Three-dimensional (3D) reconstructions and length and volume measurements were performed with the software Avizo 9.0 (Thermo Fisher Scientific), using the 3D length and material statistics tools (the latter after segmentation with the magic wand tool).

One-way ANOVA tests were used for the analyses of limb bones. Student’s t-test was used for skin analyses. Two-way ANOVA tests were used for the analysis of the intensity of Gli protein immunostainings.

In the adult mouse incisor Ptch2 is expressed exclusively in the epithelium. Loss of Ptch2 increases the differentiation rate of epithelial SCs into ameloblasts and increases deposition of mineralized matrices (Binder et al., 2019). Incisor growth, however, also depends on MSC populations, both resident and those recruited via neurovascular bundle (NVB) (Figure 1A) (Feng et al., 2011; Kaukua et al., 2014; Zhao et al., 2014). 3-4-week-old Ptch2 ^−/− NVBs were grossly comparable to the NVBs of the wild-type littermates, as shown by the expression of Myelin Basic Protein (MBP) (Figures 1B,C). However, Ptch2 ^−/− NVBs at this stage show increased vascularization identified by the expression of endothelial marker CD31 (Figures 1B,C). Similarly, at this stage, Ptch2 ^−/− incisors showed increased number of blood vessels in the dental pulps, compared to their wild-type littermates (Figures 1D–K). The average number of blood vessels found in Ptch2 ^−/− dental pulp (n = 3) was 13.4 ± 2.07/section in comparison to 9 ± 1/section observed in the wild-type (n = 3). Increased vascularization implies higher numbers of recruited, blood-vessel-associated MSCs, like perivascular cells or pericytes identified by the expression of CD146, Gli1, and αSMA, among others (Feng et al., 2011; Zhao et al., 2014; Vidovic et al., 2017). We confirmed increased numbers of CD146⁺ and Gli1⁺ cells by immunostaining of the proximal end of the incisor (compare Figures 1F,G to Figures 1J,K, respectively).

By 4 weeks of age, Ptch2 ^−/− mice exhibited a trend of increased body weight, albeit not statistically significant (Figure 2A). μCT analyses revealed size-related changes of skeletal tissue, including longer Ptch2 ^−/− femora (Figures 2B,C) and tibiae (Figures 2B,E) and increased total bone volume (Figures 2D,F). In the long bones both Hh receptors are partially co-expressed at the mRNA and protein levels (Figures 2G–J). In comparison to broadly expressed Ptch1 (Figure 2H), Ptch2 is expressed in a narrow domain, in the resting zone of the growth plate (Figures 2I,J), periosteum layer (arrow in Figure 2J), and cells lining the endosteal side of the bone (arrowheads in Figure 2J). These locations are considered putative niches for the MSCs in the bone (Ambrosi et al., 2019), which suggests that the observed phenotype could relate to the effect of Ptch2 on MSCs. Correspondingly, bone marrow stromal cells isolated from the 4-week-old Ptch2 ^−/− long bones exhibited increased colony forming potential (CFU), mineralization, and adipogenic potential (Figures 2K–O). These analyses suggest that Ptch2 regulates skeletal MSC populations in the long bones, and, as shown in the incisor tooth, acts as an inhibitor of their differentiation activity.

Another organ affected by the absence of Ptch2 is skin. Previous studies have shown that most of the Ptch2 hypomorph mutants display a normal skin phenotype, although some males can develop alopecia, ulcerations, or epidermal hyperplasia later in life (Nieuwenhuis et al., 2006). During early skin morphogenesis, Ptch2 is broadly expressed in the skin (Figure 3A), but at later stages, Ptch2 expression is limited to fewer cells both in the epithelium and the mesenchyme (Figure 3B) (Motoyama et al., 1998; Adolphe et al., 2014; Joost et al., 2020). The scattered Ptch2⁺ cells found within the mesenchyme (dermis) during late morphogenesis are likely fibroblast and fibroblast-like cells (Joost et al., 2020), while in the epithelium Ptch2 expression is restricted to the hair follicle bulge, adjacent to the secondary hair germ, where it partially co-localizes with hair bulge SC marker Lhx2 (Figure 3B) (Rhee et al., 2006; Mardaryev et al., 2011). Loss of Ptch2, did not affect the epithelial compartment of the skin, as we observed no change in the morphology of the hair follicles, or the expression of hair follicle SC markers, like Lhx2, in the adult Ptch2 ^−/− skin (Figure 3C). However, we observed thicker skin in Ptch2 ^−/− mice throughout early postnatal morphogenesis (Figures 3D,F) and until the first resting phase (P50), at which Ptch2 ^−/− skin was thinner (Figures 3E,F). Interestingly, decreased thickness of adult Ptch2 ^−/− skin is entirely due to decreased thickness of the dermis (Figure 3G).

We performed a skin wounding experiment to test if abrogation of Ptch2 affects skin reparative potential (Figures 3H–M). Within 7 days, wounds on 8-week-old wild-type animals (N = 3) completely epithelialized (Figures 3H,M) (Biggs et al., 2015; Aragona et al., 2017), This contrasted with Ptch2 ^−/− wounds (N = 3), which showed distinct epithelial fronts (arrows in Figures 3I,K) separated by inflammatory cells and a thin strip of mesenchymal cells. Analysis of cell proliferation showed that the majority of the proliferating cells localized to the leading edge of wound closure in both epithelium and the mesenchyme of Ptch2 ^−/− skins (arrows in Figure 3M). To determine if delayed wound closure was due to altered migration of highly proliferative Ptch2 ^−/− skin mesenchyme, we performed in vitro scratch assay on fibroblasts isolated from adult Ptch2 ^−/− and wild-type skins. The motility of the mutant fibroblasts was comparable to wild-type (15.12 ± 0.4 μm/h vs. 15.23 ± 2.2 μm/h, respectively), and mutant scratch wounds were closing in a similar temporal manner (not shown).

Observed changes in the Ptch2 ^−/− mice indicate an independent role for Ptch2 in Hh signaling, which is supported by data published by us and others, that show independent Ptch2 regulation of genes involved in the cellular hierarchy of the incisor epithelial stem cell niche (Binder et al., 2019), and presence of a non-canonical Hh signaling cascade mediated by Ptch2 (Kim et al., 2020). To further unravel the mechanism through which Ptch2 exerts the independent role, we analyzed the expression and distribution of Gli proteins in Hh responsive cells, namely mouse embryonic fibroblasts (MEFs) that are frequently used for functional in vitro studies of Hh pathway (Figure 4). In mice, three Gli proteins (Gli1, 2, and 3) establish a canonical Hh signaling cascade (Hui and Angers, 2011). These proteins share an overlapping activator function, while only Gli3, and to lesser extent Gli2, contribute to repressor function (Hui and Angers, 2011). We analyzed the expression of Gli3, the principal repressor of Hh pathway, in untreated, proliferating cultures of wild-type, Ptch1 ^−/− , and Ptch2 ^−/− MEFs. Compared to wild-type MEFs, Gli3 was significantly reduced in both mutants (Figures 4A,B), which suggests activated Hh signaling in both mutant MEFs. Comparable levels of Gli3 nuclear intensity in the mutant MEFs (Figure 4B) indicate that Ptch receptors inhibit Hh signaling at similar levels. Examination of the expression levels and localization of the Gli1 protein showed significantly increased levels of nuclear Gli1 only in Ptch2 ^−/− MEFs (Figures 4C,D), which is opposite to the published Gli1 mRNA expression in Ptch1 ^−/− and Ptch2 ^−/− MEFs (Binder et al., 2019). Unexpectedly, Gli2 expression exhibited stronger nuclear staining in wild-type MEFs compared to either mutant cell populations (Figures 4E,F). However, previous studies have shown that Hh pathway activity depends on the ratio of full-length Gli2 protein (the activator form) and repressor form that is generated through proteolytic processing of full-length protein (Pan et al., 2006; Li et al., 2011). Our immunoblotting analysis shows evidence of Gli2 proteolysis in wild-type and Ptch2 ^−/− but not Ptch1 ^−/− MEFs (Figure 4G), which suggests higher Hh pathway activity in Ptch1 ^−/− MEFs compared to the two other MEF cultures. The observed differences in the regulation of Gli1 and Gli2 expression suggest that Ptch2 role in the Hh signaling is independent and mainly exerted through regulation of Gli1.