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Requirement of plakophilin 2 for heart morphogenesis and cardiac junction formation.

Grossmann KS, Grund C, Huelsken J, Behrend M, Erdmann B, Franke WW, Birchmeier W - J. Cell Biol. (2004)

Bottom Line: Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease.By contrast, embryonic epithelia show normal junctions.Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.

View Article: PubMed Central - PubMed

Affiliation: Max Delbrueck Center for Molecular Medicine (MDC), D-13092 Berlin, Germany.

ABSTRACT
Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease. We have ablated the plakophilin 2 gene in mice. The resulting mutant mice exhibit lethal alterations in heart morphogenesis and stability at mid-gestation (E10.5-E11), characterized by reduced trabeculation, disarrayed cytoskeleton, ruptures of cardiac walls, and blood leakage into the pericardiac cavity. In the absence of plakophilin 2, the cytoskeletal linker protein desmoplakin dissociates from the plaques of the adhering junctions that connect the cardiomyocytes and forms granular aggregates in the cytoplasm. By contrast, embryonic epithelia show normal junctions. Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.

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Strategy used to disrupt the plakophilin 2 gene. (a) Schematic representation of the plakophilin 2 targeting vector and the wt and mutant (Δpkp2) plakophilin 2 locus. Exon sequences are represented by gray boxes; the neo cassette has been fused in opposite transcriptional orientation to codon 43 of the first exon. Probes used for Southern hybridization are indicated by black horizontal boxes (ext, neo). The sizes of restriction fragments by XbaI and HindIII digests are indicated. (b) Southern blot analysis of the plakophilin 2 locus from wt (+/+) and plakophilin 2 heterozygous (+/−) and homozygous (−/−) E10.75 embryos, as examined by external (ext) and neo probes. (c) PCR analysis of the plakophilin 2 locus of wt (+/+), heterozygous (+/−), and homozygous (−/−) mutant embryos. (d) Western blot analyses for plakophilin 2 synthesis in wt, heterozygous, and homozygous mutant embryos of E10.75. The loading control was performed with an Erk antibody. (e and f) Immunofluorescence microscopy for plakophilin 2 of wt hearts at (e) E10.75 and (f) E13.75, using antibodies against the COOH terminus of plakophilin 2. Counterstaining of nuclei was with DAPI. For clarity, a dotted line marks the tissue border between cardiomyocytes and epicard. at, atrium; v, ventricle. Bars, 200 μm.
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fig1: Strategy used to disrupt the plakophilin 2 gene. (a) Schematic representation of the plakophilin 2 targeting vector and the wt and mutant (Δpkp2) plakophilin 2 locus. Exon sequences are represented by gray boxes; the neo cassette has been fused in opposite transcriptional orientation to codon 43 of the first exon. Probes used for Southern hybridization are indicated by black horizontal boxes (ext, neo). The sizes of restriction fragments by XbaI and HindIII digests are indicated. (b) Southern blot analysis of the plakophilin 2 locus from wt (+/+) and plakophilin 2 heterozygous (+/−) and homozygous (−/−) E10.75 embryos, as examined by external (ext) and neo probes. (c) PCR analysis of the plakophilin 2 locus of wt (+/+), heterozygous (+/−), and homozygous (−/−) mutant embryos. (d) Western blot analyses for plakophilin 2 synthesis in wt, heterozygous, and homozygous mutant embryos of E10.75. The loading control was performed with an Erk antibody. (e and f) Immunofluorescence microscopy for plakophilin 2 of wt hearts at (e) E10.75 and (f) E13.75, using antibodies against the COOH terminus of plakophilin 2. Counterstaining of nuclei was with DAPI. For clarity, a dotted line marks the tissue border between cardiomyocytes and epicard. at, atrium; v, ventricle. Bars, 200 μm.

Mentions: We generated a mutation of the plakophilin 2 gene by homologous recombination in embryonic stem (ES) cells. In the targeting vector, a neo cassette inserted in opposite transcriptional orientation replaced a 7.8-kb genomic fragment from the NotI site in exon 1 to the BamHI site in intron 1 (Fig. 1 a). Homologous recombination events were identified by Southern blot analyses (Fig. 1 b): an external probe (ext) yielded a novel Xba fragment of 10 kb in the mutant, Δpkp2 (the wild-type [wt] fragment is 16 kb), and the neo probe produced a 4.5-kb HindIII fragment. We generated a mutation in the plakophilin 2 gene after homologous recombination because insertion of neo prevents splicing between exon 1 and 2 and leads to an early stop of plakophilin 2 translation after 43 aa. Using two lines of mutant ES cells, we produced plakophilin 2 mutant chimeric and heterozygous mice that were healthy and fertile. However, matings between heterozygous mice produced no live plakophilin 2–deficient offspring, implying that the mutant embryos died during embryogenesis. To determine the time of death, embryos from different developmental stages were genotyped by PCR (Fig. 1 c) and inspected visually. Up to day 10.75 of embryogenesis (E10.75), the expected Mendelian ratio of homozygous mutant embryos was observed (Table I); however the mutant embryos showed blood accumulation in the pericardial and peritoneal cavities. At E11.5, the number of viable plakophilin 2 −/− embryos declined, as judged by PCR genotyping and lack of heart beating. Western blot analyses using an antibody against the COOH terminus of plakophilin 2 indicated absence of full-length or truncated protein in E10.75 plakophilin 2 −/− embryos (Fig. 1 d). In wt embryos at E10.75, plakophilin 2 was prominent in cardiomyocytes of the atrium and the ventricle of the heart (Fig. 1 e), and expression was lost in the mutation embryos (see also below, Fig. 4 Bd′, and Fig. 8). At E13.75, plakophilin 2 was intensely synthesized in the wt cardiomyocytes, but less in the surrounding epicard (Fig. 1 f).


Requirement of plakophilin 2 for heart morphogenesis and cardiac junction formation.

Grossmann KS, Grund C, Huelsken J, Behrend M, Erdmann B, Franke WW, Birchmeier W - J. Cell Biol. (2004)

Strategy used to disrupt the plakophilin 2 gene. (a) Schematic representation of the plakophilin 2 targeting vector and the wt and mutant (Δpkp2) plakophilin 2 locus. Exon sequences are represented by gray boxes; the neo cassette has been fused in opposite transcriptional orientation to codon 43 of the first exon. Probes used for Southern hybridization are indicated by black horizontal boxes (ext, neo). The sizes of restriction fragments by XbaI and HindIII digests are indicated. (b) Southern blot analysis of the plakophilin 2 locus from wt (+/+) and plakophilin 2 heterozygous (+/−) and homozygous (−/−) E10.75 embryos, as examined by external (ext) and neo probes. (c) PCR analysis of the plakophilin 2 locus of wt (+/+), heterozygous (+/−), and homozygous (−/−) mutant embryos. (d) Western blot analyses for plakophilin 2 synthesis in wt, heterozygous, and homozygous mutant embryos of E10.75. The loading control was performed with an Erk antibody. (e and f) Immunofluorescence microscopy for plakophilin 2 of wt hearts at (e) E10.75 and (f) E13.75, using antibodies against the COOH terminus of plakophilin 2. Counterstaining of nuclei was with DAPI. For clarity, a dotted line marks the tissue border between cardiomyocytes and epicard. at, atrium; v, ventricle. Bars, 200 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172504&req=5

fig1: Strategy used to disrupt the plakophilin 2 gene. (a) Schematic representation of the plakophilin 2 targeting vector and the wt and mutant (Δpkp2) plakophilin 2 locus. Exon sequences are represented by gray boxes; the neo cassette has been fused in opposite transcriptional orientation to codon 43 of the first exon. Probes used for Southern hybridization are indicated by black horizontal boxes (ext, neo). The sizes of restriction fragments by XbaI and HindIII digests are indicated. (b) Southern blot analysis of the plakophilin 2 locus from wt (+/+) and plakophilin 2 heterozygous (+/−) and homozygous (−/−) E10.75 embryos, as examined by external (ext) and neo probes. (c) PCR analysis of the plakophilin 2 locus of wt (+/+), heterozygous (+/−), and homozygous (−/−) mutant embryos. (d) Western blot analyses for plakophilin 2 synthesis in wt, heterozygous, and homozygous mutant embryos of E10.75. The loading control was performed with an Erk antibody. (e and f) Immunofluorescence microscopy for plakophilin 2 of wt hearts at (e) E10.75 and (f) E13.75, using antibodies against the COOH terminus of plakophilin 2. Counterstaining of nuclei was with DAPI. For clarity, a dotted line marks the tissue border between cardiomyocytes and epicard. at, atrium; v, ventricle. Bars, 200 μm.
Mentions: We generated a mutation of the plakophilin 2 gene by homologous recombination in embryonic stem (ES) cells. In the targeting vector, a neo cassette inserted in opposite transcriptional orientation replaced a 7.8-kb genomic fragment from the NotI site in exon 1 to the BamHI site in intron 1 (Fig. 1 a). Homologous recombination events were identified by Southern blot analyses (Fig. 1 b): an external probe (ext) yielded a novel Xba fragment of 10 kb in the mutant, Δpkp2 (the wild-type [wt] fragment is 16 kb), and the neo probe produced a 4.5-kb HindIII fragment. We generated a mutation in the plakophilin 2 gene after homologous recombination because insertion of neo prevents splicing between exon 1 and 2 and leads to an early stop of plakophilin 2 translation after 43 aa. Using two lines of mutant ES cells, we produced plakophilin 2 mutant chimeric and heterozygous mice that were healthy and fertile. However, matings between heterozygous mice produced no live plakophilin 2–deficient offspring, implying that the mutant embryos died during embryogenesis. To determine the time of death, embryos from different developmental stages were genotyped by PCR (Fig. 1 c) and inspected visually. Up to day 10.75 of embryogenesis (E10.75), the expected Mendelian ratio of homozygous mutant embryos was observed (Table I); however the mutant embryos showed blood accumulation in the pericardial and peritoneal cavities. At E11.5, the number of viable plakophilin 2 −/− embryos declined, as judged by PCR genotyping and lack of heart beating. Western blot analyses using an antibody against the COOH terminus of plakophilin 2 indicated absence of full-length or truncated protein in E10.75 plakophilin 2 −/− embryos (Fig. 1 d). In wt embryos at E10.75, plakophilin 2 was prominent in cardiomyocytes of the atrium and the ventricle of the heart (Fig. 1 e), and expression was lost in the mutation embryos (see also below, Fig. 4 Bd′, and Fig. 8). At E13.75, plakophilin 2 was intensely synthesized in the wt cardiomyocytes, but less in the surrounding epicard (Fig. 1 f).

Bottom Line: Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease.By contrast, embryonic epithelia show normal junctions.Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.

View Article: PubMed Central - PubMed

Affiliation: Max Delbrueck Center for Molecular Medicine (MDC), D-13092 Berlin, Germany.

ABSTRACT
Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease. We have ablated the plakophilin 2 gene in mice. The resulting mutant mice exhibit lethal alterations in heart morphogenesis and stability at mid-gestation (E10.5-E11), characterized by reduced trabeculation, disarrayed cytoskeleton, ruptures of cardiac walls, and blood leakage into the pericardiac cavity. In the absence of plakophilin 2, the cytoskeletal linker protein desmoplakin dissociates from the plaques of the adhering junctions that connect the cardiomyocytes and forms granular aggregates in the cytoplasm. By contrast, embryonic epithelia show normal junctions. Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.

Show MeSH
Related in: MedlinePlus