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