ORIGINAL ARTICLE

Patched 1 and Patched 2 Redundancy Has a Key Role in Regulating Epidermal Differentiation Christelle Adolphe1,5, Erica Nieuwenhuis2,5, Rehan Villani1,5,ZhuJuanLi2,3, Pritinder Kaur4,Chi-chungHui2,3 and Brandon J. Wainwright1

The Patched 1 (Ptch1) receptor has a pivotal role in inhibiting the activity of the Hedgehog (Hh) pathway and is therefore critical in preventing the onset of many human developmental disorders and tumor formation. However, the functional role of the mammalian Ptch2 paralogue remains elusive, particularly the extent to which it contributes to regulating the spatial and temporal activity of Hh signaling. Here we demonstrate in three independent mouse models of epidermal development that in vivo ablation of both Ptch receptors results in a more severe phenotype than loss of Ptch1 alone. Our studies indicate that concomitant loss of Ptch1 and Ptch2 activity inhibits epidermal lineage specification and differentiation. These results reveal that repression of Hh signaling through a dynamic Ptch regulatory network is a crucial event in lineage fate determination in the skin. In general, our findings implicate Ptch receptor redundancy as a key issue in elucidating the cellular origin of Hh-induced tumors. Journal of Investigative Dermatology (2014) 134, 1981–1990; doi:10.1038/jid.2014.63; published online 27 February 2014

INTRODUCTION cells by binding to the Ptch1 receptor and stimulating The Hedgehog (Hh) signaling pathway has a central role in internalization and lysosomal degradation of the Hh–Ptch1 regulating appropriate embryonic development and maintain- complex (Mastronardi et al., 2000), thus relieving the repres- ing adult tissue homeostasis. Disruption to the spatiotemporal sive effect on Smo. Activated Smo subsequently translocates to activation of the Hh pathway can lead to a number of deve- the primary cilium (Rohatgi et al., 2007; Ingham et al., 2011), lopmental defects, including craniofacial, limb, and skeletal where it activates Gli transcription factors to enter the nucleus anomalies, whereas persistent pathway activity is linked to the and initiate transcription of target genes, among them Ptch1 formation of various cancers, the most common being basal itself. cell carcinoma (BCC) of the skin and medulloblastoma of Various components of the Hh pathway have been manipu- the cerebellum (Adolphe et al., 2006; Yang et al., 2008b). lated in the epidermis of murine skin, resulting in pathway In the absence of Hh ligand, the Patched 1 (Ptch1) protein activation and the subsequent formation of epidermal lesions functions to maintain the Hh pathway in an inactive state by reminiscent of human BCCs (Oro et al., 1997; Xie et al., 1998; inhibiting translocation of the signaling effector Smoothened Grachtchouk et al., 2000; Adolphe et al., 2004; Youssef et al., (Smo) to the primary cilium (Rohatgi et al., 2007; Ingham 2010; Grachtchouk et al., 2011; Kasper et al., 2011; Wang et al., 2011). Hh ligands activate pathway signaling in target et al., 2011; Wong and Reiter, 2011). In such murine models, BCC formation often occurs at the expense of hair follicles 1Division of Molecular Genetics and Development, Institute for Molecular (HFs), suggesting that pathway activity diverts follicular cells Bioscience, The University of Queensland, Brisbane, Queensland, Australia; toward a tumorigenic fate. The Hh pathway activation in the 2 Program in Developmental and Stem Cell Biology, The Hospital for Sick skin also results in hyperproliferation of the epidermal basal Children, Toronto, Ontario, Canada; 3Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada and 4Epithelial Stem Cell cell compartment, where the transient amplifying progenitor Biology Laboratory, Research Division, Peter MacCallum Cancer Center, cell population resides, and in a subsequent delay in the onset Melbourne, Victoria, Australia of interfollicular epidermis (IFE) differentiation (Adolphe et al., 5These authors contributed equally to this work. 2004; Adolphe et al., 2006; Villani et al., 2010). The endo- Correspondence: Brandon J. Wainwright, Division of Molecular Genetics and genous Hh pathway activity in the skin is essential for the Development, Institute for Molecular Bioscience, The University of downgrowth of HFs during both embryonic development (St- Queensland, Brisbane, Queensland 4072, Australia. E-mail: [email protected] or Chi-chung Hui, Program in Jacques et al., 1998; Chiang et al., 1999) and the adult HF Developmental and Stem Cell Biology, The Hospital for Sick Children, and cycle (Sato et al., 1999; Wang et al., 2000). The appropriate Department of Molecular Genetics, University of Toronto, Toronto, Ontario, spatiotemporal activation of the Hh pathway is achieved by a Canada M5G1 8. E-mail: [email protected] complex network of positive and negative regulators acting at Abbreviations: HF, hair follicle; Hh, Hedgehog; IFE, interfollicular epidermis; various tiers of the pathway (Ingham et al., 2011), in which Ptch, Patched Ptch has a paramount role. Received 5 July 2013; revised 5 December 2013; accepted 30 December 2013; accepted article preview online 3 February 2014; published online Ptch1 encodes a 12-pass transmembrane glycoprotein that 27 February 2014 exhibits dual regulatory activities. Not only does Ptch1

& 2014 The Society for Investigative Dermatology www.jidonline.org 1981 CAdolpheet al. Ptch1 and Ptch2 Redundancy in the Skin

function to maintain Smo in an inactive state, but transcrip- and/or Ptch2 activity. Immunohistochemical detection of tional activation of Ptch1 as a Hh target gene also functions as b-galactosidase expression of E18.5 Ptch1lacZ/ þ (Goodrich a critical negative feedback regulatory loop, which serves to et al., 1997) skin revealed that Ptch1 is strongly expressed in attenuate pathway activity through sequestration of Hh ligand the dermal papillae of developing HFs and in a small number (Chen and Struhl, 1996). Although Ptch1 is the most exten- of follicular and IFE keratinocytes (Supplementary Figure S1a sively studied Hh receptor, there exists another important yet online, arrows). We also observe the evidence of weak Ptch1 rarely acknowledged receptor, Patched 2 (Ptch2). This second expression in cells spanning the length of the developing HF mammalian Ptch paralogue shares a significant degree of and in a subset of basal cells within the IFE (Supplementary sequence and structural homology with Ptch1 (Motoyama Figure S1a online). Although previous studies have published et al., 1998). Both Ptch receptors have been shown to exhibit a more restricted expression profile via X-gal detection, Ptch1 Hh ligand–binding activity, and there is evidence to suggest expression in a broad population of IFE and HF basal cells is that both receptors can interact with Smo (Carpenter et al., consistent with our previous publication data (Adolphe et al., 1998). Similar to Ptch1, transcriptional activation of Ptch2 2006). In order to detect the endogenous expression of Ptch2 occurs in response to Shh (Takabatake et al., 1997), is by analogous means, we generated a Ptch2lacZ/ þ reporter downregulated in the absence of Gli (Motoyama et al., mouse (Supplementary Figure S2 online). Immunohistochemical 1998), and its expression is high in both human BCC and detection of b-galactosidase expression of E18.5 Ptch2lacZ/ þ skin medulloblastoma (Zaphiropoulos et al., 1999; Lee et al., revealed that Ptch2 is likewise expressed throughout the HF 2006). In addition, analyses of zebrafish ptc1;ptc2 double but not in the IFE (Supplementary Figure S1b online, asterisks), mutants have indicated an increase in the level of Hh target indicating that there is an overlap in the expression of both gene activation upon loss of both receptors (Koudijs et al., Ptch1 and Ptch2. On the basis of their expression patterns in 2008; Hammond et al., 2010). However, unlike the broad the developing epidermis, both Ptch1 and Ptch2 may function developmental defects and early embryonic lethality in regulating the activity of the Hh pathway in cells of the associated with loss of Ptch1 activity in murine embryonic developing HF. development (Goodrich et al., 1997), we have previously To examine functional overlap between the two Ptch demonstrated that the sole defect observed in Ptch2-deficient receptors, we determined whether the loss of one Ptch mice is a loss of HF structures and epidermal hyperplasia receptor activity resulted in transcriptional activation of in aged male mice (Nieuwenhuis et al., 2006). These data the other Ptch paralogue. As previously published, E18.5 suggest that mammalian Ptch2 has a functional role in the Ptch2tm1/tm1 embryos are wild type in appearance with no skin. Although the skin phenotype of Ptch2-deficient mice is evidence of aberrant Hh pathway activity (as represented by subtle, it is possible that Ptch2 activity is obscured due to the Gli1 transcriptional activation) in the developing epidermis persistence of Ptch1-receptor activity. Despite evidence to (Supplementary Figure S1g online). We propose that this is suggest that Ptch2 is a downstream target of the Hh pathway likely due to sustained negative regulation of the pathway as a and contributes to its negative regulation, the degree of result of ectopic Ptch1 transcription in the IFE of Ptch2-defi- functional compensation and/or redundancy between the cient skin (Supplementary Figure S1j online) (Nieuwenhuis two Ptch receptors remains uncharacterized. et al., 2006). By contrast, we show that following loss of Ptch1 In order to investigate the degree of functional redundancy in the developing epidermis of K5Cre:Ptch1lox/loxmice, clear between the two Ptch receptors, we concurrently ablated both embryonic epidermal defects arise, and E18.5 skin exhibits Ptch1 and Ptch2 activity in the basal cells of adult and pathway activation through upregulation of the canonical Hh embryonic mouse epidermis using three distinct Cre-recombi- target genes Gli1 and Ptch1 (Supplementary Figure S1h online nase transgenes. Here, we show that, for each mouse model and data not shown). Consistent with the transcriptional described, the phenotype of Ptch1;Ptch2-deficient epidermis is activation of Ptch1 in Ptch2-deficient skin, we observed distinct from the loss of Ptch1 alone. Surprisingly, we show ectopic transcriptional activation of Ptch2 in Ptch1-deleted that combined loss of Ptch receptor activity in developing skin (Supplementary Figure S1k online). These data further epidermal keratinocytes results in a previously undescribed support the contention that Ptch2 is a target of the Hh pathway phenotype, namely, complete inhibition of HF, sebaceous activity and suggest that functional compensation between gland (SG), and IFE lineage specification as well as differentia- Ptch1 and Ptch2 receptor activity in the skin may be in tion. Collectively, this study shows that Ptch2 functions in operation. It is important to note that ectopic Hh pathway collaboration with Ptch1 in regulating Hh signal transduction activation is observed in Ptch1-deficient mice despite tran- in the epidermis and that Ptch receptor activity is required for scriptional activation of Ptch2, indicating that Ptch2 is unable keratinocytes to respond to lineage specification and differ- to functionally compensate for the loss of the Ptch1 pathway entiation signals. repression.

RESULTS Concomitant loss of Ptch1 and Ptch2 accelerates the onset of The Hh pathway activity in the epidermis uses both Ptch1 and Ptch1-derived skin lesions Ptch2 as negative feedback regulators As one approach to address whether Ptch1 and Ptch2 exhibit In order to gain an understanding of the functional relationship overlapping activity in the skin, we followed a similar between Ptch1 and Ptch2 in the skin, we set out to identify approach to that taken by Lee et al. (2006), which describes which epidermal cell populations exhibit endogenous Ptch1 an increase in medulloblastoma incidence upon loss of both

1982 Journal of Investigative Dermatology (2014), Volume 134 C Adolphe et al. Ptch1 and Ptch2 Redundancy in the Skin

Ptch1 and Ptch2 activity. In order to specifically induce Ptch1 analyses (K14, K10, or Loricrin) (Figure 1d, h, and l). In order ablation in adult skin, we took advantage of the tamoxifen to characterize the follicular nature of the mutant invaginating (TM)-inducible K14CreERT2 model. No overt phenotype was structures, we screened a cohort of HF lineage markers and observed in vehicle-treated skin (Supplementary Figure S3a observed these cells to express p-cadherin (Figure 2b), a and b online) or in TM treatment of control epidermis (data not marker of HF induction. We also observed the expression of shown). Consistent with the preliminary stages of skin tumor- CDP (Figure 2d) and Sox9 (Figure 2f), markers of an early hair igenesis generated by loss of Ptch1, 4 weeks after TM germ stage of follicular development. In wild-type epidermis, treatment, K14CreERT2:Ptch1lox/lox mice presented with mild cells undergoing HF morphogenesis (specifically those pene- epidermal hyperplasia and intermittent regions in which basal trating down into the dermis) express the transcription factor cells began to invade the dermis (Supplementary Figure S3c Lef1, marking acquisition of a HF multi-potent progenitor online: arrows). By contrast, the neoplastic transformation of (matrix) cell fate committed to terminally differentiating K14CreERT2:Ptch1lox/lox;Ptch2tm1/tm1 TM-treated skin was along the hair shaft lineage (Figure 2g: arrows). In contrast, more advanced, with features reminiscent of early BCC K14Cre:Ptch1lox/lox;Ptch2tm1/tm1-deficient keratinocyteswithin formation. Four weeks after TM treatment of K14CreERT2:Pt- the mutant follicular-like structures failed to express Lef1 ch1lox/lox;Ptch2tm1/tm1mice, we observed epidermal nests ori- (Figure 2h: asterisks). Nor did these structures express other ginating from both the IFE and HF structures that had markedly HF lineage markers, including AE15 and AE13 (data not invaded the dermis, with the characteristic BCC feature of shown). Furthermore, we show that K14Cre:Ptch1;Ptch2 adult cells palisading the periphery of the invading mass skin grafts (9 weeks) develop BCC with complete absence of (Supplementary Figure S3d online: asterisk). Given that pro- mature HF structures (Figure 2j). These data further support the gression toward a BCC-like phenotype was more advanced in fact that, despite HF lineage initiation, K14Cre:Ptch1;Ptch2 K14CreERT2:Ptch1lox/lox;Ptch2tm1/tm1 adult skin, these data null cells do not proceed along the HF and hair shaft provide in vivo evidence to indicate an overlap in the activity differentiation program. Consistent with being maintained in of both Ptch1 and Ptch2 in restraining the tumor-inducing an early primitive HF cell fate, K14Cre:Ptch1lox/lox;Ptch2tm1/tm1 signals of the Hh pathway in epidermal tissue. epidermal invaginations continue to express markers charac- teristic of the G1/S phase of the cell cycle (cyclin D1) Embryonic HF lineage differentiation requires Ptch receptor (Figure 1p) and proliferation (proliferating cell nuclear antigen activity (PCNA)) (Figure 1n). In order to address the HF-restricted After having established a likely functional role for Ptch2 in phenotype of K14Cre:Ptch1lox/lox;Ptch2tm1/tm1 epidermis, we adult epidermis, we addressed whether Ptch2 biological performed detailed analyses of the spatial and temporal acti- activity is restricted to adult epidermis or whether it also vity of the K14Cre transgene using both K14Cre:ROSAlacZ/ þ functions in regulating embryonic development of the skin. and K14Cre:ROSAEYFP/ þ models. These data revealed that We have previously observed that conditional ablation of Cre-mediated deletion (lacZ- positive cells) was predomi- Ptch1 using a K14Cre transgene, moderately active from E15.0 nately localized within the HF structures, with only intermittent in embryonic epidermis (Supplementary Figure S4a online), recombined interfollicular keratinocytes (Supplementary results in viable fertile mice (Villani et al., 2010). More Figure S4a and c online). To further confirm the follicular- specifically, the embryonic epidermis of E18.5 K14Cre: restricted activity of K14Cre, we screened for the distribution Ptch1lox/lox mice is indistinguishable from that of Ptch2tm1/tm1 of the Hh pathway activity in K14Cre:Ptch1lox/lox;Ptch2tm1/tm1 and control mice. Histologically, control, Ptch2tm1/tm1,and epidermis and observed high levels of Gli1 target gene K14Cre:Ptch1lox/lox epidermis all present with the appropriate transcription in the mutant follicular-like structures density of developing HFs and a fully stratified IFE (Figure 1a–c). (Supplementary Figure S4e online), with no evidence of IFE The typical distribution of a single basal cell layer (keratin 14, Hh pathway activity. K14, Figure 1e–g), and successive squamous (K10, Figure These data suggest that, in the absence of any Ptch receptor 1i–k) and cornified (loricrin, data not shown) layers, confirms activity in developing HFs, follicular cells become unrespon- the normal differentiation and stratification of the IFE in the sive to the HF differentiation program and are instead main- absence of either Ptch1 or Ptch2 alone. Together, these results tained in an actively proliferating state. Furthermore, these indicate that in the presence of either Ptch1 or Ptch2 receptor data indicate that Ptch receptor activity is necessary for HF activity, embryonic HF morphogenesis and IFE stratification lineage morphogenesis, such that basal keratinocytes require are unaffected. the functional activity of either Ptch1 or Ptch2 for HF lineage We next assessed whether the loss of both Ptch1 and Ptch2 specification and differentiation. receptor activity would perturb the development of embryonic epidermis. Concomitant ablation of both Ptch1 and Ptch2 Ptch receptor activity is required by epidermal keratinocytes for activity (K14Cre:Ptch1lox/lox;Ptch2tm1/tm1) resulted in perinatal epidermal lineage specification and differentiation to occur lethality, which was clearly distinct from unaffected, viable After having established Ptch-deficiency results in complete K14Cre:Ptch1lox/lox or Ptch2tm1/tm1 mice. The most obvious loss of embryonic HF cell fate, we next addressed whether histological defect in E18.5 K14Cre:Ptch1lox/lox;Ptch2tm1/tm1 Ptch receptor activity is required for IFE lineage differentiation. epidermis was the presence of hyperplastic epidermal invagi- For this approach, we used another well-characterized nations (Figure 1d, arrows). IFE stratification appeared grossly embryonic skin model, K5Cre (Tarutani et al., 1997). normal via histology and standard IFE differentiation marker Analyses of K5Cre:ROSAlacZ/ þ and K5Cre:ROSAEYFP/ þ

www.jidonline.org 1983 CAdolpheet al. Ptch1 and Ptch2 Redundancy in the Skin

K14Cre:Ptch1lox/lox; Control Ptch2 tm1/tm1 K14Cre:Ptch1lox/lox Ptch2 tm1/tm1 abc d

E18.5 hf

efgh

bcl K14

ijkl

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K14Cre:Ptch1lox/lox; K14Cre:Ptch1lox/lox; K14Cre:Ptch1lox/lox K14Cre:Ptch1lox/lox Ptch2 tm1/tm1 Ptch2 tm1/tm1 mno p K14/ PCNA

CyclinD1/K14 CyclinD1/K14

Figure 1. K14Cre:Patched-deficient skin is maintained in a hyperproliferative state. (a–d) Histology of E18.5 transgenic epidermis. Note loss of typical elongated follicular structures in K14Cre:Ptch1lox/lox; Ptch2tm1/tm1 invaginations (d:arrows).(e–h) Basal (K14), (i–l) suprabasal (K10), and (m, n) cornified (loricrin) cell layers present in E18.5 transgenic skin. Expanded PCNA (proliferating cell nuclear antigen; n) and maintained expression of (p: arrows) cycling D1 in K14Cre:Ptch1lox/lox;Ptch2tm1/tm1 invaginations compared with (m:arrowsando) K14Cre:Ptch1lox/lox control skin. bcl: basal cell layer, cl: cornified layer; sbl: suprabasal cell layer. Bar ¼ 50 mm.

reporter mice revealed intense b-galactosidase and EYFP hyperproliferation (Figure 4w) and delayed onset of IFE staining in the vast majority of epidermal basal keratinocytes differentiation (Figure 4g and j). K5Cre:Ptch1lox/lox embryos and developing HF cells from E15.0 (Supplementary Figure thus faithfully model the mitogenic effects commonly attrib- S4b and d online) compared with the predominately follicular- uted to the Hh pathway activation during embryonic devel- like activity of K14Cre described above. Wide-ranging K5Cre opment. This embryonic phenotype is clearly distinct from the activity is further supported by evidence of the pathway in all normal development observed in K14Cre:Ptch1lox/lox epider- epidermal cells of K5Cre:Ptch1;Ptch2 skin (Supplementary mis, as described above. Figure S4f online), with respect to follicular-restricted activity We next asked whether loss of both Ptch1 and Ptch2 would in K14Cre: Ptch1;Ptch2 epidermis (Supplementary Figure S4e amplify the severity of this mitogenic phenotype, potentially online). Consistent with widespread gene deletion by the shedding light on the functional role of Ptch2 during embryo- K5Cre transgene during embryonic epidermal development, nic epidermal development. K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 we observed perinatal lethality upon inactivation of Ptch1 embryos are readily distinguishable upon embryonic dissec- alone (K5Cre:Ptch1lox/lox), and the E18.5 epidermis revealed tion as they exhibit a taut and highly transparent epidermis perturbed HFs (Figure 3d) alongside modest K14 basal cell (Figure 3a) with no evidence of HF protrusions (Figure 4a).

1984 Journal of Investigative Dermatology (2014), Volume 134 C Adolphe et al. Ptch1 and Ptch2 Redundancy in the Skin

K14Cre:Ptch1lox/lox; (Figure 4e). Unlike the primordial HF fate attributed to K14Cre:Ptch1lox/lox lox/lox tm1/tm1 Ptch2 tm1/tm1 K14Cre:Ptch1 ;Ptch2 -deficient keratinocytes, we ab observed no evidence of Sox9, CDP, or nuclear b-Catenin expression in K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 epidermis (Supplementary Figure S5 online). Moreover, there was no evidence of p-cadherin expression (Supplementary Figure S5

p-cad online). These data support a complete loss of follicular induction and differentiation in K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 epidermis. Most unexpectedly, however, K5Cre:Ptch1lox/lox; Ptch2tm1/tm1 epidermis also presented with an apparent loss of cd IFE differentiation (Figure 3e). Subsequent immunostaining analyses confirmed a complete absence of terminally differ- entiated squamous (K10) and cornified (loricrin) layers * (Figure 3h and k, arrows). Consistent with a loss of IFE differ- CDP entiation, K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 epidermis exhibits a * complete lack of epidermal barrier function (Figure 4b). This epidermal barrier defect is restricted to K5Cre:Ptch1lox/lox; Ptch2tm1/tm1 epidermis and is not evident in all other mouse ef models described herein (K14Cre:Ptch1, K14Cre:Ptch1;Ptch2, and K5Cre:Ptch1). Although epidermal barrier defects have been identified in a number of epidermal mouse models, such defects have been predominantly attributed to delays in IFE Sox9 differentiation and/or cell adhesion defects. That K5Cre: Ptch1lox/lox;Ptch2tm1/tm1 embryos fail to exhibit any degree of IFE lineage specification of differentiation is an unprecedented mouse model phenotype. ghcl Consistent with a lack in epidermal lineage differentiation, K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 keratinocytes fail to exit the proliferating basal cell compartment, maintaining the expres- * sion of proliferative (PCNA, Ki67, CyclinD1) (Figure 4c, f, Lef1/ Lor * and i) and progenitor-like markers (p63/K17/K15) (Figure 4o, r, and u). However, it is interesting to note that, although undifferentiated and actively proliferating, the K5Cre:Pt- ch1lox/lox;Ptch2tm1/tm1 keratinocytes express relatively low ij levels of the keratins typically present in resident basal cells (K5 and K14) (Figure 4i and x). In summary, these data demonstrate that loss of both Ptch1 and Ptch2 receptor activity in developing epidermal keratinocytes results in a complete

Graft lack of HF and SG lineage fate specification, and, strikingly, a complete absence of IFE stratification and differentiation. This is a previously unreported mouse model phenotype presenting with total inhibition of IFE stratification and clearly illustrates that either Ptch1 or Ptch2 receptor activity is essential for Figure 2. K14Cre:Patched-deficient follicular structures exhibit a primordial hair follicle (HF)–like state. Hair germ stages of follicular development keratinocytes to initiate and commit to differentiate along all in control epidermis express (a) p-cadherin, (c) CDP, and (e) Sox9. epidermal lineages. K14Cre:Ptch1lox/lox;Ptch2tm1/tm1 invaginations also express (b) p-cadherin, (d)CDP,and(f) Sox9. A proportion of K14Cre:Ptch1lox/lox;Ptch2tm1/tm1 DISCUSSION invaginations did not express CDP (d: asterisks). (g: yellow arrows) Lef1 Here we show that Ptch1 and Ptch2 exhibit a partial overlap expression in the HF downgrowths of E18.5 control epidermis. (h: asterisks) in function. Importantly, a pivotal role for Ptch receptor Mutant follicular-like structures invading the dermis fail to express Lef1. (i)HF activity in epidermal morphogenesis has been revealed. This structures present in control skin grafts (9 weeks). (j) Presence of basal cell carcinoma (BCC) lesions and lack of HF structures in K14Cre:Ptch1lox/ study furthers our understanding of the functional relationship lox;Ptch2tm1/tm1 skin grafts. Dermal papillae structures associated with control between the two mammalian Ptch receptors in several ways. It HFs (white arrows: a, c,ande) are absent in mutant epidermis. Bar ¼ 50 mm. demonstrates that Ptch1 and Ptch2 are paralogous genes that do not appear to have undergone complete functional diver- gence, but rather both can act to negatively regulate the Hh Histologically, E18.5 K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 epidermis pathway activity in the skin. More precisely, Ptch1 and Ptch2 presented as a hyperplastic mass of invaginating interfollicular can partially compensate for each other but are not function- keratinocytes, which lacked any HF or SG structures ally redundant. Our results also reveal that the degree of

www.jidonline.org 1985 CAdolpheet al. Ptch1 and Ptch2 Redundancy in the Skin

a a′ a″ b E18.5

K5Cre:Ptch1lox/lox; Control K5Cre:Ptch1lox/lox Ptch2 tm1/tm1 cde

hf E18.5

E185 f g h

sbl E10

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cl Lor

Figure 3. Loss of glandular, follicular, and interfollicular epidermal lineage specification and differentiation in K5Cre:Patched-deficient skin. Gross morphology indicating epidermal transparency of (a: LHS and a’) control and (a: RHS and a’’) K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 E18.5 embryos. Epidermal barrier assay revealing penetration of dye in (b) K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 epidermis. (d) Histology of E18.5 transgenic epidermis revealing perturbed hair follicles (hf) in K5Cre:Ptch1lox/lox epidermis and (e: arrows) loss of all epidermal appendages and hyperplastic epithelial invaginations in K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 epidermis compared with (c) controls. Immunohistochemical staining of interfollicular differentiation markers (f–h)K10and(i–k) loricrin revealing absence of suprabasal (sbl) (h: arrows) and (k: arrows) cornified cell layers (cl) in K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 epidermis. Bar ¼ 50 mm.

pathway activity that occurs in the absence of Ptch receptor Ptch2 activity results in a more severe epidermal phenotype repression and attenuation blocks the process of keratinocyte than loss of Ptch1 alone, providing direct evidence that Ptch2 cell fate determination. This finding highlights the importance has an important role in the skin. Our previous study showed of intact Ptch repression in model systems aimed at elucidat- that Ptch2 is not essential for embryonic development and ing the molecular clues involved in epidermal lineage cell fate suggested that Ptch1 might functionally compensate for the determination. loss of Ptch2, thus potentially masking Ptch2 receptor activity (Nieuwenhuis et al., 2006). Indeed, we establish here that the Ptch2 has a significant role in regulating skin morphogenesis activity of Ptch2 can only be revealed in the absence of Ptch1. Here we describe the epidermal phenotype of three distinct Our data also support the hypothesis that the transcriptional Ptch1-deficient mouse model systems, which exhibit pheno- activation of Ptch2 upon Ptch1 ablation in the epidermis types consistent with previous reports of epidermal hyperpla- occurs potentially as a compensatory mechanism, whereby sia and skin neoplasia formation (Adolphe et al., 2006; Villani Ptch1-deficient cells sense disequilibrium in negative et al., 2010). Importantly, we show that loss of both Ptch1 and feedback inhibition and thereby recruit the functional

1986 Journal of Investigative Dermatology (2014), Volume 134 C Adolphe et al. Ptch1 and Ptch2 Redundancy in the Skin

K5Cre:Ptch1lox/lox; K5Cre:Ptch1lox/lox; Control K5Cre:Ptch1lox/lox Control K5Cre:Ptch1lox/lox Ptch2 tm1/tm1 Ptch2 tm1/tm1 ab c mno p63 PCNA

de f pqr K17 Ki67

gh i stu K15 Cyclin D1

jk l vw x bcl

K5 ors * K14 dp

Figure 4. Proliferation and progenitor cell state of K5Cre:Patched-deficient epidermis. Protein expression of the proliferation markers (a–c) PCNA (proliferating cell nuclear antigen), (d–f) Ki67, and transcriptional expression of (g–i) cyclin D1 mRNA. Protein expression of epidermal stem/progenitor cell markers (m–o) p63 and transcriptional expression of (p–r)K17and(s–u) K15 mRNA. Immunohistochemical staining of the basal cell markers (k–l)K5and(v–x) K14. Note decrease in the expression of basal cell markers in K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 epidermis (l: arrows, x: arrows). Bar ¼ 50 mm. activity of Ptch2. Our three epidermal mouse model systems, from that of Ptch1 during epidermal development. On the described here, clearly showed that Ptch2 has a functional other hand, high Ptch2 expression was observed in the testis role in Ptch1-deficient epidermis, which is contrary to (Carpenter et al., 1998) and peripheral nerve cells (Rahnama previous in vitro studies, suggesting that the upregulation of et al., 2004; Bajestan et al., 2006), leading to the hypothesis Ptch2 in Ptch1-deficient cells is not indicative of functional that Ptch proteins might have undergone functional diver- compensation (Bailey et al., 2002). It is therefore possible that gence (more specifically, specialization), whereby each a role for Ptch2 in regulating the morphogenesis and paralogue exerts Ptch activity in specific tissues. Contrary to differentiation of a broader range of mammalian tissues may this notion, the genetic studies presented here indicate that become apparent following the generation of other tissue- Ptch1 and Ptch2 likely function as degenerate components, specific Ptch1;Ptch2 null models. whereby they compensate for one another under certain conditions. A partial overlap in Ptch receptor activity has Ptch1 and Ptch2 are not functionally equivalent also been suggested in other vertebrate species, such as during Our data clearly show that Ptch2 does not exhibit complete zebrafish ear and somite development. (Koudijs et al., 2008; redundancy with Ptch1, but rather that Ptch1 and Ptch2 Hammond et al., 2010). overlap in their ability to repress the Hh pathway activity, The data we present herein show that Ptch2 has a crucial and Ptch1 appears to have the predominant role. In our initial role in attenuating pathway function following reduced Ptch1 characterization of Ptch2, we found that Ptch2 is co-expressed receptor activity in the skin. The signaling activity of the Hh with Shh in the follicular keratinocytes of developing epider- pathway in Ptch1;Ptch2-deficient keratinocytes results in loss mal structures, whereas Ptch1 is highly expressed in comple- of a protective IFE barrier and is incompatible with survival. It mentary structures (namely the mesenchymal-derived dermal is therefore likely that the Ptch gene duplication event arose as papillae) (Motoyama et al., 1998). These observations an evolutionary measure to provide robustness to the recep- suggested that Ptch2 might have acquired a function distinct tion and attenuation of the Hh signal. This is analogous to an

www.jidonline.org 1987 CAdolpheet al. Ptch1 and Ptch2 Redundancy in the Skin

already established evolutionary mechanism referred to as rendering the skin dysfunctional. This study therefore defines mutational robustness, which represents the ability of an the importance of having at least one functional Ptch receptor organism to withstand mutations (Wagner, 2008). That Ptch1 during epidermal development to ensure IFE stratification and is frequently mutated in epidermal tissue yielding high barrier viability. incidence of human skin cancer (BCC) supports the evolu- tion of Ptch2 in preventing lethal adverse effects of Hh The role of Ptch receptors in regulating BCC development signaling and promoting fitness of an organism. Previous studies have shown that BCC development does not One possible mechanism by which Ptch2 may exert its occur in mouse models exhibiting low levels of pathway crucial role as a Hh pathway regulator is via increased protein activation, such as overexpressing SmoM2 using a truncated stability. Previous studies have identified a conserved sequence K5 promoter (Grachtchouk et al., 2003), low-level expression present in the cytoplasmic C-terminal domain of Ptch1 pro- of GLI2DN (Grachtchouk et al., 2011), and a proportion of K5- teins that contributes to rapid protein turnover (Lu et al., 2006; Gli1 overexpression mice (Nilsson et al., 2000). BCC develop- Kawamura et al., 2008). One of the main topological differ- ment is only seen to arise in mouse models exhibiting high ences between mammalian Ptch paralogues is that Ptch2 levels of the Hh pathway activation, such as K5-Gli2 proteins exhibit a truncated C-terminal domain that lacks the (Grachtchouk et al., 2000), K14Cre:SmoM2, K14CreER: protein turnover regulatory sequence, thus rendering Ptch2 SmoM2 (Youssef et al., 2012), and high-level expression of more stable (Kawamura et al., 2008). In light of the data shown GLI2DN (Grachtchouk et al., 2011). Here we show that adult here, it is possible that Ptch2 protein stability could ensure a K14Cre:Ptch1;Ptch2 skin grafts develop BCC lesions. These more durable effect in limiting the spatial and temporal signal- data support the fact that loss of both Ptch1 and Ptch2 mimics ing activity of the pathway and that Ptch2 might function in the a similar level of pathway activation as achieved following reinforcement of the Hh pathway attenuation. high-level expression of Gli and mutant Smo. However, it is important to note that the levels of pathway activity induced in Inhibition of pathway activity is required for epidermal lineage both K14Cre:Ptch1 (Villani et al., 2010) and K14Cre: specification and differentiation Ptch1;Ptch2 models are sufficient to induce BCC formation. Dissecting the relationship between the Hh pathway activity Taken together, these data suggest that the level of pathway and epidermal development/BCC formation has been activity achieved upon loss of Ptch1 alone, loss of Ptch1 and addressed by targeting various tiers of the pathway, including Ptch2, and overexpression of SmoM2 or GLI2DN in K5/K14 but not limited to: loss of Ptch1 receptor activity (Adolphe models all drive BCC development. Our data lend support to et al., 2006; Villani et al., 2010; Wang et al., 2011); the proposed model that high levels of Hh signaling activity overexpression or inhibition of Shh ligand (Fan et al., 1997; are required to yield a BCC tumor–initiating cell, by conferring Adolphe et al., 2004) (St-Jacques et al., 1998; Chiang et al., epidermal basal cells with a primordial HF-like fate. 1999); constitutive activation or ablation of Smo signaling (Xie et al., 1998; Grachtchouk et al., 2003; Youssef et al., 2010; MATERIALS AND METHODS Wong and Reiter, 2011) (Gritli-Linde et al., 2007); and Mouse strains overexpression of the downstream transcription factors Gli1 K14CreERT2 mice (Li et al., 2000), K14Cre mice (Jonker et al.,2004), and Gli2 (Dahmane et al., 1997) (Grachtchouk et al., 2000; K5Cre mice (Tarutani et al., 1997), Ptch1lox mice (Ellis et al.,2003), Sheng et al., 2002). and Ptch2tm1 mice (Nieuwenhuis et al., 2006) have been previously Here we show that restricted loss of Ptch1 and Ptch2 in the described. K5Cre, Ptch1, Ptch1;Ptch2 strains, and K5Cre:Ptch1 and developing HFs (K14Cre:Ptch1lox/lox;Ptch2tm1/tm1) leads to a K5Cre:Ptch1;Ptch2 embryos were all maintained in the Hui block in the HF differentiation. More specifically, laboratory on a mixed outbred background of CD1, and 129/Sv. K14:Ptch1;Ptch2-deficient follicular cells arrest in the early K14Cre, Ptch1 strains and K14Cre:Ptch1 and K14Cre:Ptch1;Ptch2 stages of HF development and are devoid of committed hair embryos were all maintained in the Wainwright laboratory on mixed shaft progenitors and a recognizable dermal papillae structure, genetic backgrounds. Despite the mixed genetic backgrounds of all both required for later stages of HF differentiation. Our data mice, we observe no phenotype/genotype variation. The phenotypes therefore show that high levels of constitutive pathway have been completely consistent across this study. All animal studies activation in developing HFs acts to maintain developing HF were reviewed and approved by the University of Queensland animal cells in a highly proliferative primordial HF-like cell fate. ethics committee. These data are consistent with previous studies indicating that During our analyses of the phenotypes described within this overexpression of SmoM2 in K5 or K14 epithelia faithfully manuscript, we always observed Ptch1 heterozygote and Ptch2- recapitulates a primordial HF-like fate (Yang et al., 2008a; deficient embryos to be indistinguishable from wild-type embryos. Youssef et al., 2012). ‘‘Control’’ animals therefore refer to either wild-type, Ptch1 hetero- We also show that developing basal keratinocytes that are zygote, and Ptch2-deficient embryos. deficient in both Ptch1 and Ptch2 activity (K5Cre:Ptch1lox/lox; Ptch2tm1/tm1) fail to undergo any evidence of epidermal Immunohistochemistry, in situ hybridization, and alkaline lineage commitment. K5Cre;Ptch-deficient keratinocytes phosphatase analysis express all the features of proliferating progenitor cell activity Flank skin specimens from E14.5 to E18.5 embryos were fixed while exhibiting no structural or protein markers of HF, SG, or overnight in 4% paraformaldehyde in phosphate-buffered saline and IFE lineages, thus leaving the skin devoid of an IFE barrier and embedded in paraffin. Histological analysis and antibody markers

1988 Journal of Investigative Dermatology (2014), Volume 134 C Adolphe et al. Ptch1 and Ptch2 Redundancy in the Skin

were analyzed on 4-mm tissue sections via standard immunofluores- Ellis T, Smyth I, Riley E et al. (2003) Patched 1 conditional null allele in mice. cence and immunohistochemistry techniques using the following Genesis 36:158–61 antibodies: K14, K5, K10, loricrin (Covance, Princeton, NJ), Ki67 Fan H, Oro AE, Scott MP et al. (1997) Induction of basal cell carcinoma (DAKO, Agilent Technologies, Santa Clara, CA), PCNA (Invitrogen, features in transgenic human skin expressing Sonic Hedgehog. Nat Med 3:788–92 Carlsbad, CA and Santa Cruz Biotechnologies, Santa Cruz, CA), p63, Goodrich LV, Milenkovic L, Higgins KM et al. (1997) Altered neural cell fates CDP (Santa Cruz), Lef1 (Cell Signalling Technologies, Danvers MA), and medulloblastoma in mouse patched mutants. Science 277:1109–13 Sox9 (R&D Systems, Minneapolis, MN), p-cadherin (Life Technolo- Grachtchouk M, Mo R, Yu S et al. (2000) Basal cell carcinomas in mice gies, Carlsbad, CA), and cyclinD1 (Abcam, Cambridge, UK). In situ overexpressing Gli2 in skin. Nat Genet 24:216–7 hybridization and alkaline phosphatase analysis were performed on Grachtchouk M, Pero J, Yang SH et al. (2011) Basal cell carcinomas in mice 5-mm paraffin sections as previously described (Li et al., 2012). arise from hair follicle stem cells and multiple epithelial progenitor populations. J Clin Invest 121:1768–81 TM treatment Grachtchouk V, Grachtchouk M, Lowe L et al. (2003) The magnitude of Mice aged between 6–8 weeks were anesthetized with isoflourane, hedgehog signaling activity defines skin tumor phenotype. EMBO J 22:2741–51 and a patch of skin was shaved from both their left and right flanks. Gritli-Linde A, Hallberg K, Harfe BD et al. (2007) Abnormal hair development The right flank was treated with vehicle (acetone) alone. Left flank and apparent follicular transformation to mammary gland in the absence was treated with 500 mg of TM. Treatment was performed for 2 days. of hedgehog signaling. Dev Cell 12:99–112 Four-weeks post-treatment skin was collected and processed for Hammond KL, van Eeden FJ, Whitfield TT (2010) Repression of Hedgehog histology. signalling is required for the acquisition of dorsolateral cell fates in the zebrafish otic vesicle. Development 137:1361–71 Skin grafts Ingham PW, Nakano Y, Seger C (2011) Mechanisms and functions of Hedge- hog signalling across the metazoa. Nat Rev Genet 12:393–406 E18.5 epidermis was grafted onto severe combined immunodefi- Jonker L, Kist R, Aw A et al. (2004) Pax9 is required for filiform papilla ciency mice as previously described (Adolphe et al., 2004) development and suppresses skin-specific differentiation of the mamma- lian tongue epithelium. Mech Dev 121:1313–22 Kasper M, Jaks V, Are A et al. (2011) Wounding enhances epidermal CONFLICT OF INTEREST tumorigenesis by recruiting hair follicle keratinocytes. Proc Natl Acad The authors state no conflict of interest. Sci USA 108:4099–104 Kawamura S, Hervold K, Ramirez-Weber FA et al. (2008) Two patched protein ACKNOWLEDGMENTS subtypes and a conserved domain of group I proteins that regulates We thank Paul Joosa for technical assistance, and Vicki Metzis and Laura turnover. JBiolChem283:30964–9 Genovesi for intellectual input. This work was supported by research grants Koudijs MJ, den Broeder MJ, Groot E et al. (2008) Genetic analysis of the two from the Australian Research Council (ARC) and National Health and Medical zebrafish patched homologues identifies novel roles for the hedgehog Research Council of Australia Project Grant 517948 (to BW) and the Canadian signaling pathway. BMC Dev Biol 8:15 Cancer Society Research Institute (2011–700774 to CCH). Confocal micro- scopy was performed at the Australian Cancer Research Foundation Dynamic Lee Y, Miller HL, Russell HR et al. (2006) Patched2 modulates tumorigenesis in Imaging Centre for Cancer Biology. patched1 heterozygous mice. 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