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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 chimeric Porcn genetrap mouse.(A) Porcn genetrap construct CSD256. Top: A genetrap cassette with a strong splice acceptor (SA) upstream of an engrailed2 leader sequence (En2) fused to a β-galactosidase-neomycin (β-geo) gene is inserted in intron 2 of Porcn. Bottom: resulting genetrapped fusion transcript that truncates Porcn after exon 2 (green arrow, translation initiation site, A(n) poly-A tail). (B) Fused and hypoplastic shortened digits. (C) Areas of hypoplastic skin with alopecia (black arrows) and swelling at lower back. (D) Intra-abdominal cystic structure (likely dilated vas deferens), right hydronephrosis (arrow), testicle and seminiferous tubules (*). (E) Skeletal microCT images showing fusion and hypoplasia of digits in mutant mouse (WT, wild type mouse; CSD, mouse generated from CSD256; inset, enlargement of hindpaw). (F) Hypoplastic skin with dermoid cyst (*). Scale bar represents 1 mm. (G) Top panel: ovary and oviducts (scale bar represents 200 µm). Bottom panel: hypoplastic testis (scale bar represents 100 µm). (H) Top panel: hydronephrotic kidney with thin medulla. Bottom panel: contralateral normal kidney (scale bars represent 1 mm). (I) Gel picture showing amplification of lacZ (364 bp fragment) in various tissues. M, 1 kb ladder; 1, skin with lesion; 2, normal skin; 3, liver; 4, tail; P, positive control (CSD256 ES cell DNA); N, negative control (C57BL6 gDNA).
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pone-0032331-g001: Phenotype of chimeric Porcn genetrap mouse.(A) Porcn genetrap construct CSD256. Top: A genetrap cassette with a strong splice acceptor (SA) upstream of an engrailed2 leader sequence (En2) fused to a β-galactosidase-neomycin (β-geo) gene is inserted in intron 2 of Porcn. Bottom: resulting genetrapped fusion transcript that truncates Porcn after exon 2 (green arrow, translation initiation site, A(n) poly-A tail). (B) Fused and hypoplastic shortened digits. (C) Areas of hypoplastic skin with alopecia (black arrows) and swelling at lower back. (D) Intra-abdominal cystic structure (likely dilated vas deferens), right hydronephrosis (arrow), testicle and seminiferous tubules (*). (E) Skeletal microCT images showing fusion and hypoplasia of digits in mutant mouse (WT, wild type mouse; CSD, mouse generated from CSD256; inset, enlargement of hindpaw). (F) Hypoplastic skin with dermoid cyst (*). Scale bar represents 1 mm. (G) Top panel: ovary and oviducts (scale bar represents 200 µm). Bottom panel: hypoplastic testis (scale bar represents 100 µm). (H) Top panel: hydronephrotic kidney with thin medulla. Bottom panel: contralateral normal kidney (scale bars represent 1 mm). (I) Gel picture showing amplification of lacZ (364 bp fragment) in various tissues. M, 1 kb ladder; 1, skin with lesion; 2, normal skin; 3, liver; 4, tail; P, positive control (CSD256 ES cell DNA); N, negative control (C57BL6 gDNA).

Mentions: We performed two sets of injections into blastocysts of mouse ES cells (line CSD256) that were targeted with a genetrap cassette containing a splice-acceptor sequence upstream of a β-galactosidase-neomycin fusion (β-geo) reporter gene inserted 3′ to exon 2, which contains the translation initiation site of Porcn (Figure 1A). Consistent with the prediction that this allele would result in inactivation of Porcn, only a single liveborn male chimera (Figure 1B–I) was found in 28 offspring. This animal had fused and hypoplastic digits on all 4 extremities (Figure 1B and 1E), 2 midline ventral wall defects, and a lower dorsal skin defect with alopecia (Figure 1C). The skin lesions showed a thin epidermis with presence of a dermoid cyst in one of the ventral lesions (Figure 1F). Interestingly, this mouse also had external male genitalia, but was a hermaphrodite with a single ovary with oviduct and a hypoplastic testis (Figure 1G). It developed a cystic intra-abdominal structure, suspected to be a dilated obstructed vas deferens (Figure 1D), and had a hydronephrotic kidney (Figure 1H). We confirmed the presence of the targeted allele in skin, liver, and tail of this mouse by PCR amplification of lacZ (Figure 1I). The phenotype in this mosaic male chimeric animal is consistent with the clinical presentation of FDH in males, who have somatic mosaic mutations [8], [9], [28]. The low survival of male chimeras is also in agreement with recent data showing that injection of CSD256 ES cells into tetraploid blastocysts results in early embryonic lethality and extensive gastrulation defects [27], [29]. This data provided the rationale for generation of a conditionally targeted Porcn allele to study in vivo effects of mammalian Porcn inactivation.


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 chimeric Porcn genetrap mouse.(A) Porcn genetrap construct CSD256. Top: A genetrap cassette with a strong splice acceptor (SA) upstream of an engrailed2 leader sequence (En2) fused to a β-galactosidase-neomycin (β-geo) gene is inserted in intron 2 of Porcn. Bottom: resulting genetrapped fusion transcript that truncates Porcn after exon 2 (green arrow, translation initiation site, A(n) poly-A tail). (B) Fused and hypoplastic shortened digits. (C) Areas of hypoplastic skin with alopecia (black arrows) and swelling at lower back. (D) Intra-abdominal cystic structure (likely dilated vas deferens), right hydronephrosis (arrow), testicle and seminiferous tubules (*). (E) Skeletal microCT images showing fusion and hypoplasia of digits in mutant mouse (WT, wild type mouse; CSD, mouse generated from CSD256; inset, enlargement of hindpaw). (F) Hypoplastic skin with dermoid cyst (*). Scale bar represents 1 mm. (G) Top panel: ovary and oviducts (scale bar represents 200 µm). Bottom panel: hypoplastic testis (scale bar represents 100 µm). (H) Top panel: hydronephrotic kidney with thin medulla. Bottom panel: contralateral normal kidney (scale bars represent 1 mm). (I) Gel picture showing amplification of lacZ (364 bp fragment) in various tissues. M, 1 kb ladder; 1, skin with lesion; 2, normal skin; 3, liver; 4, tail; P, positive control (CSD256 ES cell DNA); N, negative control (C57BL6 gDNA).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3295752&req=5

pone-0032331-g001: Phenotype of chimeric Porcn genetrap mouse.(A) Porcn genetrap construct CSD256. Top: A genetrap cassette with a strong splice acceptor (SA) upstream of an engrailed2 leader sequence (En2) fused to a β-galactosidase-neomycin (β-geo) gene is inserted in intron 2 of Porcn. Bottom: resulting genetrapped fusion transcript that truncates Porcn after exon 2 (green arrow, translation initiation site, A(n) poly-A tail). (B) Fused and hypoplastic shortened digits. (C) Areas of hypoplastic skin with alopecia (black arrows) and swelling at lower back. (D) Intra-abdominal cystic structure (likely dilated vas deferens), right hydronephrosis (arrow), testicle and seminiferous tubules (*). (E) Skeletal microCT images showing fusion and hypoplasia of digits in mutant mouse (WT, wild type mouse; CSD, mouse generated from CSD256; inset, enlargement of hindpaw). (F) Hypoplastic skin with dermoid cyst (*). Scale bar represents 1 mm. (G) Top panel: ovary and oviducts (scale bar represents 200 µm). Bottom panel: hypoplastic testis (scale bar represents 100 µm). (H) Top panel: hydronephrotic kidney with thin medulla. Bottom panel: contralateral normal kidney (scale bars represent 1 mm). (I) Gel picture showing amplification of lacZ (364 bp fragment) in various tissues. M, 1 kb ladder; 1, skin with lesion; 2, normal skin; 3, liver; 4, tail; P, positive control (CSD256 ES cell DNA); N, negative control (C57BL6 gDNA).
Mentions: We performed two sets of injections into blastocysts of mouse ES cells (line CSD256) that were targeted with a genetrap cassette containing a splice-acceptor sequence upstream of a β-galactosidase-neomycin fusion (β-geo) reporter gene inserted 3′ to exon 2, which contains the translation initiation site of Porcn (Figure 1A). Consistent with the prediction that this allele would result in inactivation of Porcn, only a single liveborn male chimera (Figure 1B–I) was found in 28 offspring. This animal had fused and hypoplastic digits on all 4 extremities (Figure 1B and 1E), 2 midline ventral wall defects, and a lower dorsal skin defect with alopecia (Figure 1C). The skin lesions showed a thin epidermis with presence of a dermoid cyst in one of the ventral lesions (Figure 1F). Interestingly, this mouse also had external male genitalia, but was a hermaphrodite with a single ovary with oviduct and a hypoplastic testis (Figure 1G). It developed a cystic intra-abdominal structure, suspected to be a dilated obstructed vas deferens (Figure 1D), and had a hydronephrotic kidney (Figure 1H). We confirmed the presence of the targeted allele in skin, liver, and tail of this mouse by PCR amplification of lacZ (Figure 1I). The phenotype in this mosaic male chimeric animal is consistent with the clinical presentation of FDH in males, who have somatic mosaic mutations [8], [9], [28]. The low survival of male chimeras is also in agreement with recent data showing that injection of CSD256 ES cells into tetraploid blastocysts results in early embryonic lethality and extensive gastrulation defects [27], [29]. This data provided the rationale for generation of a conditionally targeted Porcn allele to study in vivo effects of mammalian Porcn inactivation.

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