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Deletion of Porcn in mice leads to multiple developmental defects and models human focal dermal hypoplasia (Goltz syndrome).

Liu W, Shaver TM, Balasa A, Ljungberg MC, Wang X, Wen S, Nguyen H, Van den Veyver IB - PLoS ONE (2012)

Bottom Line: Conditional Porcn inactivation by EIIa-driven or Hprt-driven Cre recombinase results in increased early embryonic lethality.Mesenchyme-specific Prx-Cre-driven inactivation of Porcn produces FDH-like limb defects, while ectodermal Krt14-Cre-driven inactivation produces thin skin, alopecia, and abnormal dentition.Furthermore, cell-based assays confirm that human PORCN mutations reduce WNT3A secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT

Background: Focal Dermal Hypoplasia (FDH) is a genetic disorder characterized by developmental defects in skin, skeleton and ectodermal appendages. FDH is caused by dominant loss-of-function mutations in X-linked PORCN. PORCN orthologues in Drosophila and mice encode endoplasmic reticulum proteins required for secretion and function of Wnt proteins. Wnt proteins play important roles in embryo development, tissue homeostasis and stem cell maintenance. Since features of FDH overlap with those seen in mouse Wnt pathway mutants, FDH likely results from defective Wnt signaling but molecular mechanisms by which inactivation of PORCN affects Wnt signaling and manifestations of FDH remain to be elucidated.

Results: We introduced intronic loxP sites and a neomycin gene in the mouse Porcn locus for conditional inactivation. Porcn-ex3-7flox mice have no apparent developmental defects, but chimeric mice retaining the neomycin gene (Porcn-ex3-7Neo-flox) have limb, skin, and urogenital abnormalities. Conditional Porcn inactivation by EIIa-driven or Hprt-driven Cre recombinase results in increased early embryonic lethality. Mesenchyme-specific Prx-Cre-driven inactivation of Porcn produces FDH-like limb defects, while ectodermal Krt14-Cre-driven inactivation produces thin skin, alopecia, and abnormal dentition. Furthermore, cell-based assays confirm that human PORCN mutations reduce WNT3A secretion.

Conclusions: These data indicate that Porcn inactivation in the mouse produces a model for human FDH and that phenotypic features result from defective WNT signaling in ectodermal- and mesenchymal-derived structures.

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Phenotype of Porcn-ex3-7-Neo-flox chimeric mice.(A) Hypoplastic, fused, and missing digits on right (R) and left (L) fore- (F) and hindlimbs (H) in different chimeras (C1–C4) compared to wild type (WT). (B) Skeletal preparations of extremities shown in panel A. (C) Vertebral abnormalities in the tail of chimera 1 (C1). (D) Hydronephrosis of the right kidney and normal left kidney in C1. (E) Hypoplastic testicle in C1 and normal testicle in C5. (F) Uterine abnormalities: asymmetrical hypoplastic uterine horn in C6. (G) Gel picture showing amplification of the targeted allele in various tissues of C1. H, heart; K(L), left kidney; LU, lung; SK, skin; SP, spleen; T, testis; K(R), right kidney; LI, liver; TG, targeted allele; WT, wild type allele.
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pone-0032331-g002: Phenotype of Porcn-ex3-7-Neo-flox chimeric mice.(A) Hypoplastic, fused, and missing digits on right (R) and left (L) fore- (F) and hindlimbs (H) in different chimeras (C1–C4) compared to wild type (WT). (B) Skeletal preparations of extremities shown in panel A. (C) Vertebral abnormalities in the tail of chimera 1 (C1). (D) Hydronephrosis of the right kidney and normal left kidney in C1. (E) Hypoplastic testicle in C1 and normal testicle in C5. (F) Uterine abnormalities: asymmetrical hypoplastic uterine horn in C6. (G) Gel picture showing amplification of the targeted allele in various tissues of C1. H, heart; K(L), left kidney; LU, lung; SK, skin; SP, spleen; T, testis; K(R), right kidney; LI, liver; TG, targeted allele; WT, wild type allele.

Mentions: We next inserted an FRT-flanked neomycin (Neo) gene, a loxP site in intron 2, and a second loxP site in intron 7 of Porcn (Figure S1) and electroporated them into mouse ES cells to generate XPorcn-ex3-7Neo-flox/Y ES cell clones. We obtained 5/288 (1.7%) correctly targeted clones from which 3 independent lines of chimeric mice were generated after successful blastocyst injections with different clones. Of 17 chimeric mice, there were 9 high-level male and female chimeras from 2 different clones that displayed a mild phenotype reminiscent of human FDH (Figure 2A–G). These mice had absent, fused, and shortened digits on 1 to all 4 of their extremities (Figure 2A and B), vertebral anomalies of the tail (Figure 2C), hydronephrosis (Figure 2D), small testicles in males (Figure 2E), and uteri with rudimentary uterine horns in females (Figure 2F). We confirmed the presence of the targeted allele in various tissues by PCR amplification with primers P1 and P2 (Figure 2G). Therefore, intronic integration of the Neo gene and loxP sites created at least a hypomorphic Porcn allele. The phenotype observed in chimeric mice further confirmed that as in human FDH, mosaic expression of mutant Porcn is sufficient to generate developmental abnormalities. We excised the Neo gene by mating XPorcn-ex3-7Neo-flox/Y chimeras with mice expressing Flp recombinase to generate XPorcn-ex3-7flox/X and XPorcn-ex3-7flox/Y mice (Figure S1D), who had no observable phenotype and were fertile.


Deletion of Porcn in mice leads to multiple developmental defects and models human focal dermal hypoplasia (Goltz syndrome).

Liu W, Shaver TM, Balasa A, Ljungberg MC, Wang X, Wen S, Nguyen H, Van den Veyver IB - PLoS ONE (2012)

Phenotype of Porcn-ex3-7-Neo-flox chimeric mice.(A) Hypoplastic, fused, and missing digits on right (R) and left (L) fore- (F) and hindlimbs (H) in different chimeras (C1–C4) compared to wild type (WT). (B) Skeletal preparations of extremities shown in panel A. (C) Vertebral abnormalities in the tail of chimera 1 (C1). (D) Hydronephrosis of the right kidney and normal left kidney in C1. (E) Hypoplastic testicle in C1 and normal testicle in C5. (F) Uterine abnormalities: asymmetrical hypoplastic uterine horn in C6. (G) Gel picture showing amplification of the targeted allele in various tissues of C1. H, heart; K(L), left kidney; LU, lung; SK, skin; SP, spleen; T, testis; K(R), right kidney; LI, liver; TG, targeted allele; WT, wild type allele.
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Related In: Results  -  Collection

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pone-0032331-g002: Phenotype of Porcn-ex3-7-Neo-flox chimeric mice.(A) Hypoplastic, fused, and missing digits on right (R) and left (L) fore- (F) and hindlimbs (H) in different chimeras (C1–C4) compared to wild type (WT). (B) Skeletal preparations of extremities shown in panel A. (C) Vertebral abnormalities in the tail of chimera 1 (C1). (D) Hydronephrosis of the right kidney and normal left kidney in C1. (E) Hypoplastic testicle in C1 and normal testicle in C5. (F) Uterine abnormalities: asymmetrical hypoplastic uterine horn in C6. (G) Gel picture showing amplification of the targeted allele in various tissues of C1. H, heart; K(L), left kidney; LU, lung; SK, skin; SP, spleen; T, testis; K(R), right kidney; LI, liver; TG, targeted allele; WT, wild type allele.
Mentions: We next inserted an FRT-flanked neomycin (Neo) gene, a loxP site in intron 2, and a second loxP site in intron 7 of Porcn (Figure S1) and electroporated them into mouse ES cells to generate XPorcn-ex3-7Neo-flox/Y ES cell clones. We obtained 5/288 (1.7%) correctly targeted clones from which 3 independent lines of chimeric mice were generated after successful blastocyst injections with different clones. Of 17 chimeric mice, there were 9 high-level male and female chimeras from 2 different clones that displayed a mild phenotype reminiscent of human FDH (Figure 2A–G). These mice had absent, fused, and shortened digits on 1 to all 4 of their extremities (Figure 2A and B), vertebral anomalies of the tail (Figure 2C), hydronephrosis (Figure 2D), small testicles in males (Figure 2E), and uteri with rudimentary uterine horns in females (Figure 2F). We confirmed the presence of the targeted allele in various tissues by PCR amplification with primers P1 and P2 (Figure 2G). Therefore, intronic integration of the Neo gene and loxP sites created at least a hypomorphic Porcn allele. The phenotype observed in chimeric mice further confirmed that as in human FDH, mosaic expression of mutant Porcn is sufficient to generate developmental abnormalities. We excised the Neo gene by mating XPorcn-ex3-7Neo-flox/Y chimeras with mice expressing Flp recombinase to generate XPorcn-ex3-7flox/X and XPorcn-ex3-7flox/Y mice (Figure S1D), who had no observable phenotype and were fertile.

Bottom Line: Conditional Porcn inactivation by EIIa-driven or Hprt-driven Cre recombinase results in increased early embryonic lethality.Mesenchyme-specific Prx-Cre-driven inactivation of Porcn produces FDH-like limb defects, while ectodermal Krt14-Cre-driven inactivation produces thin skin, alopecia, and abnormal dentition.Furthermore, cell-based assays confirm that human PORCN mutations reduce WNT3A secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT

Background: Focal Dermal Hypoplasia (FDH) is a genetic disorder characterized by developmental defects in skin, skeleton and ectodermal appendages. FDH is caused by dominant loss-of-function mutations in X-linked PORCN. PORCN orthologues in Drosophila and mice encode endoplasmic reticulum proteins required for secretion and function of Wnt proteins. Wnt proteins play important roles in embryo development, tissue homeostasis and stem cell maintenance. Since features of FDH overlap with those seen in mouse Wnt pathway mutants, FDH likely results from defective Wnt signaling but molecular mechanisms by which inactivation of PORCN affects Wnt signaling and manifestations of FDH remain to be elucidated.

Results: We introduced intronic loxP sites and a neomycin gene in the mouse Porcn locus for conditional inactivation. Porcn-ex3-7flox mice have no apparent developmental defects, but chimeric mice retaining the neomycin gene (Porcn-ex3-7Neo-flox) have limb, skin, and urogenital abnormalities. Conditional Porcn inactivation by EIIa-driven or Hprt-driven Cre recombinase results in increased early embryonic lethality. Mesenchyme-specific Prx-Cre-driven inactivation of Porcn produces FDH-like limb defects, while ectodermal Krt14-Cre-driven inactivation produces thin skin, alopecia, and abnormal dentition. Furthermore, cell-based assays confirm that human PORCN mutations reduce WNT3A secretion.

Conclusions: These data indicate that Porcn inactivation in the mouse produces a model for human FDH and that phenotypic features result from defective WNT signaling in ectodermal- and mesenchymal-derived structures.

Show MeSH
Related in: MedlinePlus