Deletion of Mouse Porcn Blocks Wnt Ligand Secretion and Reveals an Ectodermal Etiology of Human Focal Dermal Hypoplasia/Goltz Syndrome

Deletion of mouse Porcn blocks Wnt ligand secretion and reveals an ectodermal etiology of human focal dermal hypoplasia/Goltz syndrome

Jared J. Barrotta, Gabriela M. Casha, Aaron P. Smithb, Jeffery R. Barrowb, and L. Charles Murtaugha,1

aDepartment of Human Genetics, University of Utah, Salt Lake City, UT 84112; and bDepartment of Physiology and Developmental Biology, Brigham Young University, Provo, UT 84602

Edited* by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved June 16, 2011 (received for review May 12, 2010) The Drosophila porcupine gene is required for secretion of wingless drome is never transmitted to male offspring, suggesting male- and other Wnt proteins, and sporadic mutations in its unique specific embryonic lethality. The congenital abnormalities asso- human ortholog, PORCN, cause a pleiotropic X-linked dominant ciated with FDH are highly pleiotropic and variable, including disorder, focal dermal hypoplasia (FDH, also known as Goltz syn- multiple aspects of skin and skeletal development (20), and con- drome). We generated a conditional allele of the X-linked mouse siderably overlap defects observed in mouse Wnt pathway Porcn gene and analyzed its requirement in Wnt signaling and em- mutants (Table S1). Given the connections between porcupine/ bryonic development. We find that Porcn-deficient cells exhibit Porcn and Wnt signaling, we developed a conditional allele of a cell-autonomous defect in Wnt ligand secretion but remain re- mouse Porcn that provides a unique genetic tool to block Wnt sponsive to exogenous Wnts. Consistent with the female-specific ligand biogenesis. inheritance pattern of FDH, Porcn hemizygous male embryos arrest during early embryogenesis and fail to generate mesoderm, a phe- Results notype previously associated with loss of Wnt activity. Heterozy- Deletion of Mouse Porcn Abolishes Wnt Production but Not Respon- gous Porcn mutant females exhibit a spectrum of limb, skin, and siveness. The Porcn targeting vector places loxP sites around body patterning abnormalities resembling those observed in hu- exons 2 and 3, such that Cre-mediated deletion will eliminate the man patients with FDH. Many of these defects are recapitulated by Porcn protein start codon and the first three predicted trans- ectoderm-specific deletion of Porcn, substantiating a long-standing membrane domains (Fig. 1A and Fig. S1 A and B). Homologous hypothesis regarding the etiology of human FDH and extending recombination inserts an Flp recognition target (FRT)-flanked, previous studies that have focused on downstream elements of promoterless neoR selection cassette downstream of the first R Wnt signaling, such as β-catenin. Conditional deletion of Porcn thus (noncoding) exon; neo should not be expressed if the targeting provides an experimental model of FDH, as well as a valuable tool vector integrates randomly into the genome (21). Although rel- to probe Wnt ligand function in vivo. atively few colonies were obtained after electroporation and selection, 12 of 18 clones analyzed had undergone targeting, epidermis | dermis | hair follicle | skeletal development thereby disrupting the only Porcn allele in these X/Y ES cells (Fig. S1C). We obtained clones either incorporating or omitting ingless/Wnt signaling has been implicated in the devel- the distal loxP site, based on the position of the 3′ crossover neo2lox Wopment of nearly all animal tissues as well as human dis- event (Fig. S1 A and C), and one clone of each (Porcn /Y or neo1lox eases, such as diabetes and cancer (1, 2). Although the Wnt Porcn /Y) was used for further study (Fig. S1D). R signaling pathway was first delineated in Drosophila, translating As depicted in Fig. S1D, the neo cassette was excised by insights from this species to vertebrates has been complicated by transient expression of Flp, and matched sublines were derived genetic redundancy among its components (2). β-catenin is one in which the Porcn coding sequence was left intact (referred to as lox of the few nonredundant components of the “canonical” Wnt Porcn ) or subjected to Cre-mediated deletion of exons 2 and 3 Δ lox Δ pathway, and its genetic manipulation is widely used to study (Porcn ). Because all Porcn and Porcn sublines behaved Wnt signaling in the mouse (3). A requirement for β-catenin is identically, they are henceforth described collectively rather than not necessarily the same as a requirement for Wnt, however, referring to individual subclones. given that each can function independently of the other (4, 5). A custom anti-Porcn antiserum, raised against an epitope Drosophila wingless/Wnt secretion and activity require the dedi- encoded by exons 9 and 10, detected a specific band of ∼40 kDa lox cated function of an endoplasmic reticulum (ER)-localized in lysates from WT and Porcn ES cells that was absent from Δ acyltransferase enzyme, porcupine (6–8). Porcupine has a sin- Porcn lysate (Fig. 1B). This protein comigrated with a specific gle mammalian ortholog, Porcn (9), and inhibiting this molecule band present in lysates of HeLa cells overexpressing mouse by RNAi or small molecule antagonists impairs the palmitoyla- Porcn, suggesting that it represents endogenous Porcn. The ab- Δ tion, secretion, and activity of multiple vertebrate Wnts (10–12). sence of lower molecular-weight bands in Porcn lysate suggests Although Porcn is one of several related membrane-bound that alternative translation initiation sites are not used and that Δ O-acyltransferase (MBOAT) enzymes (13), functional studies deletion of exons 2 and 3 produces a null allele. Porcn ES cells reveal no substrate overlap between Porcn and other MBOATs grew normally under standard conditions and maintained ex- (12, 14–16). These observations suggest that Porcn represents a genetic “bottleneck” in the vertebrate Wnt pathway, compa- β rable to -catenin but regulating ligand production rather than Author contributions: J.R.B. and L.C.M. designed research; J.J.B., G.M.C., A.P.S., J.R.B., and response. Nonetheless, the relationship of Porcn to Wnt function L.C.M. performed research; J.J.B., G.M.C., A.P.S., J.R.B., and L.C.M. analyzed data; and J.J.B. has yet to be analyzed genetically in a vertebrate model organism. and L.C.M. wrote the paper. In fact, the first loss-of-function phenotype of this gene was The authors declare no conflict of interest. described in humans, as the X-linked dominant syndrome focal *This Direct Submission article had a prearranged editor. dermal hypoplasia (FDH, also known as Goltz syndrome; Online 1To whom correspondence should be addressed. E-mail: [email protected]. – Mendelian Inheritance in Man (OMIM) no. 305600) (17 19). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Most patients with FDH are female heterozygotes, and the syn- 1073/pnas.1006437108/-/DCSupplemental.

12752–12757 | PNAS | August 2, 2011 | vol. 108 | no. 31 www.pnas.org/cgi/doi/10.1073/pnas.1006437108 Downloaded by guest on September 30, 2021 Fig. 1. Generating and characterizing a conditional mouse Porcn allele. (A) Schematic diagram of WT, loxP-targeted, and deletion alleles of Porcn. Exons are boxed and numbered, with the coding region indicated in black. (B) Fig. 2. Wnt activity and processing in Porcn-deficient cells. (A) TOPFlash Western blot of whole-cell lysates from parental and targeted ES cells (Left) luciferase reporter assays comparing the response of Porcnlox/Y (blue) and Δ and HeLa cells transfected with empty vector or mouse Porcn (Right) using Porcn /Y (red) ES cells with exogenous Wnt3a (10% (vol/vol) L-Wnt3a–con- a polyclonal antiserum against a C-terminal Porcn epitope. The diamond ditioned medium) or endogenously overexpressed Wnt3a (cells transfected indicates the band corresponding to overexpressed mouse Porcn and is with Wnt3a expression plasmid). Where indicated, cells were cotransfected lox present only in parental and Porcn ES cells. (C and D) Whole-mount in situ with expression plasmids for WT or H341L mutant human PORCN. Relative Δ hybridization for Brachyury (purple) on E6.5 control or Porcn /Y embryos. light units indicate TOPFlash activity, normalized to an internal transfection (Scale bar: 100 μm.) (E and F) Sections through Brachyury whole-mount control and plotted on a log scale as fold change relative to untreated stained embryos, with approximate positions indicated by the dotted lines in Porcnlox/Y cells (n =3–8 independent experiments per condition). RLU, rel- C and D. Note that sections were taken from an independent representative ative light unit. *P < 0.05 by Welch’s two-tailed t test. (B) Firefly luciferase pair of embryos, overstained to preserve signal in sections (asterisks indicate assays of 10T1/2 cells stably infected with a TOPFlash-based lentiviral re- Δ Δ nonspecific background). Porcn /Y embryos consistently contain a hollow porter, cocultured with immortalized Porcnlox/Y (blue) and Porcn /Y (red) lumen at this stage, indicating a lack of gastrulation. MEFs that were previously infected with an empty retroviral vector (LNCX) or LNC-Wnt3a-HA. Relative light units are plotted as fold change relative to reporter-transduced cells cultured alone (black) (n = 3 independent experi- pression of the pluripotency marker Oct4 (Fig. S2 A–D), in- ments). *P < 0.05 by Welch’s two-tailed t test. (C) Western blots (with dicating that targeted disruption of Porcn does not prevent ES antibodies indicated to left of panels) on whole-cell lysates from control and cell self-renewal. Wnt3a-HA–expressing MEFs (Left) and on anti-HA immunoprecipitates of We analyzed the effects of Porcn disruption on Wnt signaling conditioned media from the same cells (Right, asterisk indicates rabbit IgG ∼ by transient transfection of a β-catenin/T-cell factor (TCF)- heavy chain). Note that the immunoprecipitates represent 10-fold more Porcnlox PorcnΔ input material than the corresponding cellular lysates. Tubulin serves as dependent TOPFlash reporter gene (22, 23). and a loading control. (D) Western blots (antibodies indicated on left) on lysates ES cells were assayed for their response to Wnts either added from control and Wnt3a-HA–expressing MEFs, either unprocessed (whole) or exogenously, via conditioned medium of L-Wnt3a cells (24), or separated into aqueous and detergent phases by Triton X-114 extraction. overexpressed endogenously, by transfection with Wnt expres- GAPDH and β-catenin serve as controls for recovery of nonacylated aqueous- lox Δ sion plasmids. Although both Porcn and Porcn ES cells phase proteins, and Ras serves as a control for detergent-phase recovery of lox responded robustly to exogenous Wnt3a, only Porcn cells acylated proteins. exhibited TOPFlash activation when transfected with Wnt3a or Wnt1 (Fig. 2A and Fig. S2E). The defective endogenous Wnt PorcnΔ cells (n = 12 EBs per genotype; Fig. S2 F–H). Neuronal differ- response of cells was fully rescued by cotransfection with + PORCN entiation, indicated by βIII-tubulin cells, occurred normally in WT human , whereas a missense mutant observed in I J a patient with FDH, affecting the putative active site histidine all genotypes (Fig. S2 and ). A Having established that Porcn was required for Wnt signaling (H341L) (19), had no rescuing activity (Fig. 2 ). This functional Porcnlox Porcn2lox test of a human PORCN mutant supports the hypothesis that in vitro, we generated mice from (i.e., ) ES cells D Porcnlox/Y Porcnlox/lox FDH is a disease of impaired Wnt signaling. (Fig. S1 ). and mice are viable and fertile, and they are indistinguishable from WT littermates. To delete

lox BIOLOGY Porcn Is Required for Mesoderm Induction in ES Cells and Embryos. Porcn in vivo, we crossed Porcn females to males carrying Sox2- Wnt signaling is required for mesoderm formation in the mouse Cre, an epiblast-speciﬁc deleter transgene (30). Embryos were DEVELOPMENTAL (25–27), and this requirement is recapitulated in ES cell-derived harvested at an early gastrulation stage [embryonic day (E) 6.5] embryoid bodies (EBs) (28, 29). As an assay of endogenous Wnt and analyzed for expression of the early mesoderm marker lox activity, we examined the effect of Porcn deletion on EB de- Brachyury. Brachyury was expressed normally in Porcn /Y em- lox velopment. Consistent with a defect in mesoderm development, bryos but was undetectable in Porcn /Y; Sox2-Cre+ littermates Δ Δ individually picked Porcn EBs failed to form beating myosin (henceforth referred to as Porcn /Y) (Fig. 1 C–F). Porcn is thus heavy chain (MHC)-expressing cardiomyocytes after 13 d of at- essential for mesoderm formation in vivo, like other Wnt com- Δ tachment culture; by that time, beating foci of MHC+ cells had ponents (25–27). We have not recovered Porcn /Y males in late lox developed in all EBs derived from parental WT and Porcn ES embryogenesis (E15.5–E18.5), and a recently described gene

Barrott et al. PNAS | August 2, 2011 | vol. 108 | no. 31 | 12753 Downloaded by guest on September 30, 2021 trap allele of Porcn was found to cause developmental arrest at gastrulation (31). We therefore conclude that the female-speciﬁc inheritance of human FDH results from early loss of PORCN- deﬁcient male embryos.

Loss of Porcn Causes a Cell-Autonomous Defect in Wnt Ligand Secretion. Drosophila porcupine is essential for wingless protein secretion (7), and inhibiting Porcn function by RNAi or a small molecule antagonist prevents Wnt3a secretion (10, 12). Because transient transfection of ES cells did not produce sufficient Wnt proteins for biochemical analysis, we isolated and immortalized lox Δ Porcn /Y mouse embryo fibroblasts (MEFs), generated Porcn /Y lox derivatives by Cre transduction, and stably infected Porcn and Δ Porcn cells with either an empty retroviral vector (LNCX) or a vector encoding HA-tagged Wnt3a (LNC-Wnt3a-HA), previously shown to be biologically active (32). TOPFlash assays indicated lox robust TCF activity in Porcn /Wnt3a cells, which was absent Δ in Porcn /Wnt3a cells but fully restored by transfection with human PORCN (Fig. S3A). To confirm that Porcn acts cell- autonomously, we stably transduced C3H 10T1/2 fibroblasts with a TOPFlash-based lentivirus (33), generating a WT “responder” cell line, and cocultured these with the above-described MEFs. lox Coculture with Porcn /LNCX cells produced a modest induction of LEF/TCF activity (∼8-fold over responder cells cultured alone), consistent with endogenous Wnt production by MEFs (34), lox whereas Porcn /Wnt3a cells induced >100-fold activation (Fig. Δ 2B). Porcn /Wnt3a MEFs failed to induce significant LEF/TCF activity in responder cells (Fig. 2B), confirming that Porcn functions specifically in Wnt ligand-producing cells. lox Western blotting of whole-cell lysates indicated that Porcn Δ and Porcn MEFs expressed similar levels of Wnt3a-HA, whereas anti-HA immunoprecipitates of conditioned media revealed that lox only Porcn cells produced soluble Wnt3a-HA (Fig. 2C). Stain- ing MEFs for colocalization of Wnt3a-HA with secretory pathway markers, we found in both genotypes that anti-HA immunofluo- rescence completely overlapped the ER marker protein disulfide Δ isomerase but did not detectably overlap the Golgi marker giantin Fig. 3. FDH-like phenotypes in Porcn /+ heterozygous embryos. (A–C) Δ (Fig. S3 B–S). These results suggest that Wnt3a-HA is trafficked Comparing E17.5 Porcn /+ embryos with WT reveals a range of phenotypes relatively inefficiently through the secretory pathway but that its that include cleft palate, tail hypoplasia, omphalocele, atrophic dermis secretion nonetheless depends on Porcn function (Fig. 2C). This through which the liver is visible, and tail/posterior axis truncation. cp, cleft conclusion is in agreement with previous in vitro studies of Porcn palate; de, atrophic dermis; om, omphalocele; tr, tail/posterior axis trunca- function, in which impaired Wnt secretion correlated with de- tion; tl, tail hypoplasia. (D–F) Alcian blue/alizarin red skeletal stains of WT Δ/+ fective fatty acid modification of the ligand (10–12). Using a Tri- and Porcn forelimbs. The arrow in F indicates lack of autopod, accompanied by absence of ulna. (G–H) Alcian blue/nuclear fast red staining to ton X-114 partitioning assay to assess Wnt acylation (11, 12), we Δ reveal skeletal elements, including sternum, of E15.5 WT and Porcn /+ ven- were surprised to find that detergent partitioning of Wnt3a-HA PorcnΔ D tral body walls sectioned at the level of the heart. he, heart; st, sternum. (I was reduced but not eliminated in cells (Fig. 2 ). Because and J) Immunostaining of sections semiadjacent to G and H (approximate this minor change could not account for the >100-fold reduction Δ positions indicated by red boxes) for E-cadherin (red) and the dermis/mes- in Wnt3a signaling activity of Porcn cells, we hypothesize that enchyme marker PDGF receptor-α (green). de, dermis; ep, epidermis. (Scale Wnt3a is subject to Porcn-independent acylation events that are bars: G and H, 500 μm; I and J,50μm.) insufficient for ligand secretion.

Porcn Heterozygosity Provides a Mouse Model of FDH Defects. To visible through an epidermal monolayer (Fig. 3C). In severe cases, determine the in vivo requirements for Porcn after gastrulation, these defects were accompanied by hypoplasia of the sternum as Δ we used Sox2-Cre to generate Porcn /+ females, all of which well as ventral body wall closure defects (Fig. 3 G–J). These were recognizably abnormal in late embryogenesis (E15.5–E18.5, phenotypes recapitulate those of dermal-speciﬁc β-catenin KOs n = 42 total). The phenotypes, summarized in Table S1, closely (35), as well as those attributed to severe cases of human FDH resembled human FDH and partially recapitulated one or more (20, 36). previously described Wnt pathway mutants (Fig. 3). As in the Other grossly obvious defects have not been reported in FDH Δ human syndrome, Porcn /+ phenotypes varied widely in severity, but resembled those of other mouse Wnt pathway mutants Δ most likely attributable to stochastic X-inactivation (20). For (Table S1). For example, almost all Porcn /+ embryos exhibited example, almost every heterozygous embryo examined exhibited a range of tail defects (Fig. 3 B and C) similar to those of Wnt5a one or more abnormal limbs, with defects ranging from digit loss nulls (37), Wnt3a and Lrp6 hypomorphs (38, 39), and Dvl2;Dvl3 Δ or fusion to complete absence of autopod and ulna (Fig. 3 D–F). double mutants (40). Taken together, the concordance of Porcn /+, The deﬁning phenotype of FDH in humans is thin or absent FDH, and mouse Wnt mutant phenotypes provides independent dermis, which typically manifests at birth in discrete lesions support for a central role of Porcn in Wnt signaling. ranging in size from millimeters to centimeters (20). Similarly, we Δ observed large areas of dermal atrophy in a subset of Porcn /+ Porcn Is Required for Ectodermal Expression of Lef1 and Hair Follicle embryos, such that internal organs, including liver and heart, were Development. FDH is commonly associated with localized defects

12754 | www.pnas.org/cgi/doi/10.1073/pnas.1006437108 Barrott et al. Downloaded by guest on September 30, 2021 in ectodermal appendages, such as hair, teeth, and nails (20), the Dkk1, a canonical Wnt inhibitor (41), suggesting that Porcn development of which requires canonical Wnt signaling (41, 42). mediates the Wnt-dependent up-regulation of epidermal Lef1. Our efforts to study postnatal development of these tissues were fi Porcn frustrated by perinatal lethality, because several litters produced Tissue-Speci c Deletion Implicates Ectodermal Wnt Defects in Δ only a single viable but runted Porcn /+ pup. This mouse exhibited FDH Etiology. FDH has been speculatively attributed to defective gene function in the ectoderm (44), a hypothesis that we have focal hairlessness on its ventral skin but was otherwise WT in lox/lox A B begun to test by crossing Porcn females to additional Cre appearance (Fig. 4 and ), suggesting that postnatal survival fi selects for individuals at the mild end of a phenotypic spectrum. transgenic males to produce tissue-speci c KO male offspring. The first hair follicle primordia are detected at E14.5 and are Our initial experiments focused on the limb, because of the marked by Wnt-dependent expression of the TCF factor Lef1, availability of Cre lines and well-characterized Wnt KO phenotypes (Table S1). Using a mesenchyme-specific Prx1-Cre driver which, in turn, is required for hair development (41, 43). Whole- (45), we obtained a dramatically shortened limb phenotype at mount in situ hybridizations on embryos of this stage revealed + E17.5, which almost perfectly reproduced that of Wnt5a null mice a decreased density of Lef1 placodes in the dorsolateral skin of lox Δ (37) (n =10of10Porcn /Y; Prx1-Cre embryos; Fig. 5 A and Porcn /+ embryos (Fig. 4 C and D). This phenotype was more Δ/+ B). In addition to confirming that Porcn is required for a well- pronounced at E17.5; at that stage, Porcn embryos exhibited “ ” fi characterized noncanonical Wnt signaling process, this result large patches of abnormally smooth, hair follicle-de cient epi- suggests that the direct effects of Wnt5a on limb outgrowth are E–G dermis (Fig. 4 ). In WT E17.5 embryos, Lef1 was strongly mediated by its mesenchymal expression domain rather than its expressed throughout the basal epidermis as well as in nascent expression in the overlying apical ectodermal ridge (AER) (37). H J lox hair follicles (Fig. 4 and ), and both of these expression Like Wnt5a nulls, Porcn /Y; Prx1-Cre mice exhibit loss of distal Porcn domains were reduced or absent in hairless patches of digits but otherwise preserve all individual skeletal elements, and heterozygotes (Fig. 4 I and K). Other basal markers, such as therefore do not reproduce the syndactyly or truncation phe- Δ keratin-14 and p63, were expressed normally in these regions, as notypes commonly seen in patients with FDH and in Porcn /+ was the suprabasal marker keratin-10, indicating normal kerati- mice (Table S1). nocyte differentiation (Figs. 4 L and M and Fig. S4). These Fusion and loss of skeletal elements are observed when Wnt3 is phenotypes closely resemble those induced by overexpression of deleted in the hind-limb ectoderm with Msx2-Cre (46, 47). Based on that finding, we used Msx2-Cre to ablate Porcn and observed a variably penetrant Wnt3-like phenotype at E18.5, including

Fig. 5. Tissue-speciﬁc Porcn deletion phenotypes. (A and B) Alcian blue/ alizarin red-stained forelimbs of E17.5 control or Porcnlox/Y; Prx1-Cre (Prx1KO) embryos. Mutant embryos exhibit shortening of all skeletal ele-

ments and loss of distal digits. hu, humerus; ra, radius; sc, scapula; ul, ulna. BIOLOGY (C–F) Hind-limb skeleton preparations of E18.5 control or Porcnlox/Y; Msx2-

Δ DEVELOPMENTAL Fig. 4. Focal absence of hair follicles in Porcn /+ heterozygotes. (A and B) Cre (Msx2KO) embryos and whole-mount in situ hybridization for the AER Δ Ventral view of WT and Porcn /+ littermates at weanling stage (postnatal marker Fgf8 on E11.5 hind limbs. Note the almost complete absence of day 22). (C–E) Whole-mount in situ hybridization for Lef1 (brown) on autopod in E18.5 mutant and focal loss of Fgf8 expression at E11.5. (G and H) Δ shoulder region of E14.5 control or Porcn /+ embryos. The asterisk indicates H&E-stained sections of the ventral body wall from E18.5 control or Msx2KO a fragment of skin accidentally removed during dissection. (E) Dorsal view of embryos. The brackets in H indicate an area of severe dermal thinning in an E17.5 PorcnΔ/+ embryo, indicating patches of unusually smooth skin mutant body wall, such that liver almost directly abuts surface ectoderm. li, (arrowheads). (F–M) Semiadjacent sections of dorsal skin from E17.5 WT or liver. (I and J) Lef1 immunostaining of dorsal skin from E18.5 control or Δ Porcn /+ embryos, stained with H&E or immunostained for Lef1 or p63. The Msx2KO embryos, revealing extensive domain of hairless Lef1-devoid epi- brackets in I indicate patches devoid of hair follicles and expressing low or no dermis in mutant (arrowhead in J indicates isolated patch of Lef1+ basal Lef1. hf, hair follicle. (Scale bars: 100 μm.) cells). (Scale bars: G and H, 500 μm; I and J, 100 μm.)

Barrott et al. PNAS | August 2, 2011 | vol. 108 | no. 31 | 12755 Downloaded by guest on September 30, 2021 lox Δ syndactyly and autopod truncation (n = 6 of 14 Porcn /Y; Msx2- Porcn /+ mice appear to die perinatally, and the only postnatal Cre hind limbs; Fig. 5 C and E). Similar variability is observed with survivor yet obtained had an extremely mild phenotype com- Δ Wnt3 ablation because of inefficient deletion across the dorsal- pared with embryos. As in FDH, the defects in Porcn /+ embryos ventral boundary of the distal limb ectoderm (46). As with Wnt3 are typically discrete and asymmetrical, presumably reflecting deletion, the variable skeletal phenotype of Porcn deletion was domains of Porcn-deficient cells established by X-inactivation. prefigured by mosaic AER defects in the hind limb at E11.5 (Fig. 5 The characteristic skin defects of patients with FDH frequently D F Msx2-Cre and ). In addition to the limb, drives mosaic ecto- follow the so-called “lines of Blaschko,” stripes and whorls hy- dermal recombination throughout the trunk (48), where we ob- pothesized to represent the clonal descendants of embryonic served focal skin and dermal defects at E18.5 that reproduced Δ/+ lox ectoderm progenitors (20). In turn, this observation suggests that those of Porcn mice (n = 3 of 3 Porcn /Y; Msx2-Cre embryos; PORCN G–J fi fi FDH results from loss of function in the ectoderm, with Fig. 5 ). Skin-speci c deletion therefore appears to be suf - non-cell-autonomous effects on underlying mesodermal cells cient to account for many of the structural and differentiation (44). Our tissue-specific deletion studies provide experimental defects of human and mouse Porcn heterozygotes, supporting support for this hypothesis and, in addition to illuminating the a major role for this gene in the ectoderm. lox etiology of FDH, exemplify the utility of Porcn as a tool to Discussion dissect Wnt signaling in vivo. Given that Porcn is X-linked, tissue- specific KO males can be obtained in a single generation by In the past 2 decades, homologs of nearly every developmental Porcnlox gene identified in Drosophila have been mutated in the mouse, crosses between Cre-transgenic males and females. It with porcupine representing an unusual exception. We have now should therefore be facile and economical to test the role of Porcn generated a conditional mutant allele of its unique mouse -dependent Wnt signals in any tissue for which an appro- ortholog, Porcn, for which our studies indicate a conserved role priate Cre driver is available. in Wnt ligand secretion (7, 8). Our genetic analyses confirm and extend previous studies, using RNAi or small molecules to knock Materials and Methods lox down Porcn function in vitro (10–12), and indicate that Porcn Detailed methods are provided in SI Materials and Methods. In brief, the neo2lox represents a potent tool to probe Wnt ligand function in vivo. Porcn targeting vector (Fig. S1A) was constructed via recombineering This study does not address the scope and specificity of Porcn (52, 53), and ES cells were electroporated, selected, and analyzed using function: Is it required for all 19 mammalian Wnts, and does it standard techniques (54). ES cells used to generate mice were cultured regulate any other ligands? With respect to the first question, all continuously on MEF feeders, whereas ES cells used for in vitro experiments were grown under feeder-free conditions, in the presence of serum and mammalian Wnts contain the conserved serine residue that is the Drosophila leukemia inhibitory factor (LIF) (54). A polyclonal rabbit anti-Porcn antise- substrate for Porcn palmitoylation in Wnt3a (10). rum was generated by Covance against a C-terminal peptide epitope WntD, an orphan family member, is the only Wnt that lacks this (TEEKDHLEWDLTVSR, encoded by exons 9 and 10). Wnt reporter gene assays residue and the only Wnt that has been shown to be secreted in × × Δ used the pSuper8 TOPFlash and pSuper8 FOPFlash plasmids (23), as well the absence of porcupine (49). Porcn cells can be used directly as the 7TFP lentiviral reporter construct (33). Triton X-114 partitioning assays to test the role of Porcn in the biogenesis of diverse Wnt ligands, were performed essentially as described (12, 55). Chimeric mice were pro- as well as to address open questions regarding additional Wnt duced by the University of Utah Transgenic Core Facility, via ES cell injection modifications and their involvement in ligand trafficking and into C57BL/6 blastocysts. In all experiments, mutant embryos were com- activity (6). We were surprised to find that although Wnt3a-HA pared with littermate controls retaining full Porcn coding regions (i.e., Δ secretion is abolished in Porcn cells, its acylation is only slightly WT or floxed) on all alleles. Sox2-Cre (30) and Prx1-Cre mice (45) were reduced. Prior studies have identified multiple sites of Wnt3a obtained from The Jackson Laboratory, and Msx2-Cre mice (47) were pro- acylation (10, 24), and we speculate that loss of Porcn arrests vided by Mark Lewandoski (National Cancer Institute, Frederick, MD). All ligand trafficking in MEFs only after completion of a Porcn- animal experiments were performed according to protocols approved by Δ independent acylation step. Porcn cells may provide a useful institutional committees of the University of Utah and Brigham Young system to identify the site, nature, and biological relevance of this University. posttranslational modification. Note Added in Proof. While this paper was in press, Biechele et al. (56) With respect to Porcn substrates beyond Wnts, it has been published a study characterizing ES cells harboring a Porcn gene trap loss-of- shown that porcupine is dispensable for hedgehog and bone Drosophila function allele, and demonstrated defects in Wnt production and mesoderm morphogenetic protein (BMP) signaling in (8), and we differentiation very similar to those described here. find that limb mesenchyme-specific deletion of Porcn causes de- Wnt5a fective outgrowth, mimicking loss of (37), without ablation ACKNOWLEDGMENTS. We thank the following individuals for reagents, of specific skeletal elements as seen in mesenchyme-specific Shh equipment, and advice: Mario Capecchi, Mike Howard, Kristen Kwan, Kirk Δ or Bmp2/Bmp4 mutants (50, 51). Future studies, in Porcn cells as Thomas, Yukio Saijoh, Sabine Fuhrmann, Diane Ward, Aubrey Chan, Neal fi Copeland, Mark Lewandoski, and Randall Moon. We are particularly grate- well as tissue-speci c KOs, will address the possible requirement ful to Susan Tamowski for deriving germ-line-chimeric mice. We thank Suzi for this gene in other pathways. Mansour, Daniel Kopinke, and Kristen Kwan for comments on the manu- The Porcn heterozygous phenotype closely resembles human script. L.C.M. thanks Norbert Perrimon for originally drawing his interest to FDH, particularly its more severe manifestations (20). Studies of porcupine. This work was supported by Grant 06-B-116 from the Searle Scholars Foundation (to L.C.M.), Grant R01-DK075072 from the National inheritance and X-inactivation in human patients have suggested Institutes of Health (to L.C.M.), the University of Utah Seed Grant Program that only the least-affected patients with FDH survive beyond (to L.C.M.), and Grant R15-HD060087 from the National Institutes of Health birth (19), and our data strongly support this model: Most (to J.R.B.).

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