Embryonic Heart and Skin Defects in Mice Lacking Plakoglobin

Plakoglobin is the only component common to both the desmosomal plaque and the cadherin–catenin cell adhesion complex in the adherens junction. It is highly homologous to vertebrate β-catenin and toDrosophilaarmadillo protein and may—like these proteins—be also involved in signaling pathways. To analyze the role of plakoglobin during mouse development we inactivated theplakoglobingene by homologous recombination in embryonic stem cells and generated transgenic mice.Plakoglobinnull-mutant embryos died from Embryonic Day 10.5 onward, due to severe heart defects. Some mutant embryos developed further, especially on a C57BL/6 genetic background, and died around birth, presumably due to cardiac dysfunction, and with skin blistering and subcorneal acantholysis. Ultrastructural analysis revealed that here desmosomes were greatly reduced in number and structurally altered. Thus, using reversed genetics we demonstrate that plakoglobin is an essential structural component for desmosome function. The skin phenotype in plakoglobin-deficient mice is reminiscent of the human blistering disease, epidermolytic hyperkeratosis.     

Plakoglobin has both structural and signalling roles in zebrafish development

Plakoglobin, or gamma-catenin, is found in both desmosomes and adherens junctions and participates in Wnt signalling. Mutations in the human gene are implicated in the congenital heart disorder, arrhythmogenic right ventricular cardiomyopathy (ARVC), but the signalling effects of plakoglobin loss in ARVC have not been established. Here we report that knockdown of plakoglobin in zebrafish results in decreased heart size, reduced heartbeat, cardiac oedema, reflux of blood between heart chambers and a twisted tail. Wholemount in situ hybridisation shows reduced expression of the heart markers nkx2.5 at 24 hours post fertilisation (hpf), and cmlc2 and vmhc at 48 hpf, while there is lack of restriction of the valve markers notch1b and bmp4 at 48 hpf. Wnt target gene expression was examined by semi-quantitative RT-PCR and found to be increased in morphant embryos indicating that plakoglobin is antagonistic to Wnt signalling. Co-expression of the Wnt inhibitor, Dkk1, rescues the cardiac phenotype of the plakoglobin morphant. β-catenin protein expression is increased in morphant embryos as is its colocalisation with E-cadherin in adherens junctions. Endothelial cells at the atrioventricular boundary of morphant hearts have an aberrant morphology, indicating problems with valvulogenesis. Morphants also have decreased numbers of desmosomes and adherens junctions in the intercalated discs. These results establish the zebrafish as a model for ARVC caused by loss of plakoglobin function and indicate that there are signalling as well as structural consequences of this loss.     

Filamin C plays an essential role in the maintenance of the structural integrity of cardiac and skeletal muscles, revealed by the medaka mutant zacro

Filamin C is an actin-crosslinking protein that is specifically expressed in cardiac and skeletal muscles. Although mutations in the filamin C gene cause human myopathy with cardiac involvement, the function of filamin C in vivo is not yet fully understood. Here we report a medaka mutant, zacro (zac), that displayed an enlarged heart, caused by rupture of the myocardiac wall, and progressive skeletal muscle degeneration in late embryonic stages. We identified zac to be a homozygous nonsense mutation in the filamin C (flnc) gene. The medaka filamin C protein was found to be localized at myotendinous junctions, sarcolemma, and Z-disks in skeletal muscle, and at intercalated disks in the heart. zac embryos showed prominent myofibrillar degeneration at myotendinous junctions, detachment of myofibrils from sarcolemma and intercalated disks, and focal Z-disk destruction. Importantly, the expression of γ-actin, which we observed to have a strong subcellular localization at myotendinous junctions, was specifically reduced in zac mutant myotomes. Inhibition of muscle contraction by anesthesia alleviated muscle degeneration in the zac mutant. These results suggest that filamin C plays an indispensable role in the maintenance of the structural integrity of cardiac and skeletal muscles for support against mechanical stress.     

Embryonic atrial function is essential for mouse embryogenesis, cardiac morphogenesis and angiogenesis

The requirement for atrial function in developing heart is unknown. To address this question, we have generated mice deficient in atrial myosin light chain 2 (MLC2a), a major structural component of the atrial myofibrillar apparatus. Inactivation of the Mlc2a gene resulted in severely diminished atrial contraction and consequent embryonic lethality at ED10.5-11.5, demonstrating that atrial function is essential for embryogenesis. Our data also address two longstanding questions in cardiovascular development: the connection between function and form during cardiac morphogenesis, and the requirement for cardiac function during vascular development. Diminished atrial function in MLC2a-null embryos resulted in a number of consistent secondary abnormalities in both cardiac morphogenesis and angiogenesis. Our results unequivocally demonstrate that normal cardiac function is directly linked to normal morphogenic development of heart and vasculature. These data have important implications for the etiology of congenital heart disease.     

A novel dominant mutation in plakoglobin causes arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disorder associated with arrhythmias and sudden death. A recessive mutation in the gene encoding plakoglobin has been shown to cause Naxos disease, a cardiocutaneous syndrome characterized by ARVC and abnormalities of hair and skin. Here, we report, for the first time, a dominant mutation in the gene encoding plakoglobin in a German family with ARVC but no cutaneous abnormalities. The mutation (S39_K40insS) is predicted to insert an extra serine residue at position 39 in the N-terminus of plakoglobin. Analysis of a biopsy sample of the right ventricle from the proband showed markedly decreased localization of plakoglobin, desmoplakin, and connexin43 at intercalated discs in cardiac myocytes. A yeast-two-hybrid screen revealed that the mutant protein established novel interactions with histidine-rich calcium-binding protein and TGF beta induced apoptosis protein 2. Immunoblotting and confocal microscopy in human embryonic kidney 293 (HEK293) cell lines transfected to stably express either wild-type or mutant plakoglobin protein showed that the mutant protein was apparently ubiquitylated and was preferentially located in the cytoplasm, suggesting that the S39_K40insS mutation may increase plakoglobin turnover via proteasomal degradation. HEK293 cells expressing mutant plakoglobin also showed higher rates of proliferation and lower rates of apoptosis than did cells expressing the wild-type protein. Electron microscopy showed smaller and fewer desmosomes in cells expressing mutant plakoglobin. Taken together, these observations suggest that the S39_K40insS mutation affects the structure and distribution of mechanical and electrical cell junctions and could interfere with regulatory mechanisms mediated by Wnt-signaling pathways. These results implicate novel molecular mechanisms in the pathogenesis of ARVC.     

Rescuing desmoplakin function in extra-embryonic ectoderm reveals the importance of this protein in embryonic heart, neuroepithelium, skin and vasculature

Desmosomes mediate intercellular adhesion through desmosomal cadherins, which interface with plakoglobin (PG) and desmoplakin (DP) to associate with the intermediate filament (IF) cytoskeleton. Desmosomes first assemble in the E3.5 mouse trophectoderm, concomitant with establishment of epithelial polarity and appearance of a blastocoel cavity. Increasing in size and number, desmosomes continue their prominence in extra-embryonic tissues, but as development proceeds, they also become abundant in a number of embryonic tissues, including heart muscle, epidermis and neuroepithelium. Previously, we explored the functional importance of desmosomes by ablating the Dsp gene. Homozygous Dsp mutant embryos progressed through implantation, but did not survive beyond E6.5, owing to a loss or instability of desmosomes and tissue integrity. We have now rescued the extra-embryonic tissues by aggregation of tetraploid (wild-type) and diploid (Dsp mutant) morulae. These animals survive several days longer, but die shortly after gastrulation, with major defects in the heart muscle, neuroepithelium and skin epithelium, all of which possess desmosomes, as well as the microvasculature, which does not. Interestingly, although wild-type endothelial cells of capillaries do not form desmosomes, they possess unusual intercellular junctions composed of DP, PG and VE-cadherin. The severity in phenotype and the breadth of defects in the Dsp mutant embryo is greater than PG mutant embryos, substantiating redundancy between PG and other armadillo proteins (e.g. beta-catenin). The timing of lethality is similar to that of the VE-cadherin null embryo, suggesting that a participating cause of death may be a defect in vasculature, not reported for PG null embryos.     

Differences in the distribution of synemin, paranemin, and plectin in skeletal muscles of wild-type and desmin knock-out mice

Mice lacking the gene encoding for the intermediate filament protein desmin have a surprisingly normal myofibrillar organization in skeletal muscle fibers, although myopathy develops in highly used muscles. In the present study we examined how synemin, paranemin, and plectin, three key cytoskeletal proteins related to desmin, are organized in normal and desmin knock-out (K/O) mice. We show that in wild-type mice, synemin, paranemin, and plectin were colocalized with desmin in Z-disc-associated striations and at the sarcolemma. All three proteins were also present at the myotendinous junctions and in the postsynaptic area of motor endplates. In the desmin K/O mice the distribution of plectin was unaffected, whereas synemin and paranemin were partly affected. The Z-disc-associated striations were in general no longer present in between the myofibrils. In contrast, at the myotendinous and neuromuscular junctions synemin and paranemin were still present. Our study shows that plectin differs from synemin and paranemin in its binding properties to the myofibrillar Z-discs and that the cytoskeleton in junctional areas is particularly complex in its organization.     

Requirement of plakophilin 2 for heart morphogenesis and cardiac junction formation

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.     

Absence of the tight junctional protein AF-6 disrupts epithelial cell-cell junctions and cell polarity during mouse development.

BACKGROUND: The establishment, maintenance and rearrangement of junctions between epithelial cells are extremely important in many developmental, physiological and pathological processes. AF-6 is a putative Ras effector; it is also a component of tight and adherens junctions, and has been shown to bind both Ras and the tight-junction protein ZO-1. In the mouse, AF-6 is encoded by the Af6 gene. As cell-cell junctions are important in morphogenesis, we generated a null mutation in the murine Af6 locus to test the hypothesis that lack of AF-6 function would cause epithelial abnormalities. RESULTS: Although cell-cell junctions are thought to be important in early embryogenesis, homozygous mutant embryos were morphologically indistinguishable from wild-type embryos through 6.5 days post coitum (dpc) and were able to establish all three germ layers. The earliest morphological abnormalities were observed in the embryonic ectoderm of mutant embryos at 7.5 dpc. The length of the most apical cell-cell junctions was reduced, and basolateral surfaces of those cells were separated by multiple gaps. Cells of the embryonic ectoderm were less polarized as assessed by histological criteria and lateral localization of an apical marker. Mutant embryos died by 10 dpc, probably as a result of placental failure. CONCLUSIONS: AF-6 is a critical regulator of cell-cell junctions during mouse development. The loss of neuroepithelial polarity in mutants is consistent with a loss of efficacy of the cell-cell junctions that have a critical role in establishing apical/basolateral asymmetry.     

Plakoglobin is required for maintenance of the cortical actin skeleton in early Xenopus embryos and for cdc42-mediated wound healing

Early Xenopus embryos are large, and during the egg to gastrula stages, when there is little extracellular matrix, the cytoskeletons of the individual blastomeres are thought to maintain their spherical architecture and provide scaffolding for the cellular movements of gastrulation. We showed previously that depletion of plakoglobin protein during the egg to gastrula stages caused collapse of embryonic architecture. Here, we show that this is due to loss of the cortical actin skeleton after depletion of plakoglobin, whereas the microtubule and cytokeratin skeletons are still present. As a functional assay for the actin skeleton, we show that wound healing, an actin-based behavior in embryos, is also abrogated by plakoglobin depletion. Both wound healing and the amount of cortical actin are enhanced by overexpression of plakoglobin. To begin to identify links between plakoglobin and the cortical actin polymerization machinery, we show here that the Rho family GTPase cdc42, is required for wound healing in the Xenopus blastula. Myc-tagged cdc42 colocalizes with actin in purse-strings surrounding wounds. Overexpression of cdc42 dramatically enhances wound healing, whereas depletion of maternal cdc42 mRNA blocks it. In combinatorial experiments we show that cdc42 cannot rescue the effects of plakoglobin depletion, showing that plakoglobin is required for cdc42-mediated cortical actin assembly during wound healing. However, plakoglobin does rescue the effect of cdc42 depletion, suggesting that cdc42 somehow mediates the distribution or function of plakoglobin. Depletion of alpha-catenin does not remove the cortical actin skeleton, showing that plakoglobin does not mediate its effect by its known linkage through alpha-catenin to the actin skeleton. We conclude that in Xenopus, the actin skeleton is a major determinant of cell shape and overall architecture in the early embryo, and that plakoglobin plays an essential role in the assembly, maintenance, or organization of this cortical actin.     

Afadin: A key molecule essential for structural organization of cell-cell junctions of polarized epithelia during embryogenesis.

Afadin is an actin filament-binding protein that binds to nectin, an immunoglobulin-like cell adhesion molecule, and is colocalized with nectin at cadherin-based cell-cell adherens junctions (AJs). To explore the function of afadin in cell-cell adhesion during embryogenesis, we generated afadin(-/-) mice and embryonic stem cells. In wild-type mice at embryonic days 6.5-8.5, afadin was highly expressed in the embryonic ectoderm and the mesoderm, but hardly detected in the extraembryonic regions such as the visceral endoderm. Afadin(-/-) mice showed developmental defects at stages during and after gastrulation, including disorganization of the ectoderm, impaired migration of the mesoderm, and loss of somites and other structures derived from both the ectoderm and the mesoderm. Cystic embryoid bodies derived from afadin(-/-) embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm. Cell-cell AJs and tight junctions were improperly organized in the ectoderm of afadin(-/-) mice and embryoid bodies. These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis.     

Conditional gene targeting of connexin43: exploring the consequences of gap junction remodeling in the heart

Abnormalities in cardiac gap junction expression have been postulated to contribute to arrhythmias and ventricular dysfunction. We investigated the role of cardiac gap junctions by generating a heart-specific conditional knock-out (CKO) of connexin43 (Cx43), the major cardiac gap junction protein. While the Cx43 CKO mice have normal heart structure and contractile function, they die suddenly from spontaneous ventricular arrhythmias. Because abnormalities in gap junction expression in the diseased heart can be focal, we also generated chimeric mice formed from Cx43-null embryonic stem (ES) cells and wildtype recipient blastocysts. Heterogeneous Cx43 expression in the chimeric mice resulted in conduction defects and depressed contractile function. These novel genetic murine models of Cx43 loss of function in the adult mouse heart define gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate and an important factor in heart dysfunction.     

Conditional Gene Targeting of Connexin43: Exploring the Consequences of Gap Junction Remodeling in the Heart

Abnormalities in cardiac gap junction expression have been postulated to contribute to arrhythmias and ventricular dysfunction. We investigated the role of cardiac gap junctions by generating a heart-specific conditional knock-out (CKO) of connexin43 (Cx43), the major cardiac gap junction protein. While the Cx43 CKO mice have normal heart structure and contractile function, they die suddenly from spontaneous ventricular arrhythmias. Because abnormalities in gap junction expression in the diseased heart can be focal, we also generated chimeric mice formed from Cx43-null embryonic stem (ES) cells and wildtype recipient blastocysts. Heterogeneous Cx43 expression in the chimeric mice resulted in conduction defects and depressed contractile function. These novel genetic murine models of Cx43 loss of function in the adult mouse heart define gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate and an important factor in heart dysfunction.     

Cardiac abnormalities cause early lethality of jumonji mutant mice

jumonji (jmj) mutant mice, obtained by a gene trap strategy, showed several morphological abnormalities including neural tube and cardiac defects, and died in utero around embryonic day 11.5 (E11.5). It is unknown what causes the embryonic lethality. Here, we demonstrate that exogenous expression of jmj gene in the heart of jmj mutant mice rescued the morphological phenotypes in the heart, and these embryos survived until E13.5. These results suggest that there are at least two lethal periods in jmj mutant mice, and that cardiac abnormalities may cause the earlier lethality. In addition, the rescue of the cardiac abnormalities by the jmj transgene provided solid evidence that the cardiac abnormalities resulted from mutation of the jmj gene.     

Transgenic Expression of the Formin Protein Fhod3 Selectively in the Embryonic Heart: Role of Actin-Binding Activity of Fhod3 and Its Sarcomeric Localization during Myofibrillogenesis

Fhod3 is a cardiac member of the formin family proteins that play pivotal roles in actin filament assembly in various cellular contexts. The targeted deletion of mouse Fhod3 gene leads to defects in cardiogenesis, particularly during myofibrillogenesis, followed by lethality at embryonic day (E) 11.5. However, it remains largely unknown how Fhod3 functions during myofibrillogenesis. In this study, to assess the mechanism whereby Fhod3 regulates myofibrillogenesis during embryonic cardiogenesis, we generated transgenic mice expressing Fhod3 selectively in embryonic cardiomyocytes under the control of the β-myosin heavy chain (MHC) promoter. Mice expressing wild-type Fhod3 in embryonic cardiomyocytes survive to adulthood and are fertile, whereas those expressing Fhod3 (I1127A) defective in binding to actin die by E11.5 with cardiac defects. This cardiac phenotype of the Fhod3 mutant embryos is almost identical to that observed in Fhod3 null embryos, suggesting that the actin-binding activity of Fhod3 is crucial for embryonic cardiogenesis. On the other hand, the β-MHC promoter-driven expression of wild-type Fhod3 sufficiently rescues cardiac defects of Fhod3-null embryos, indicating that the Fhod3 protein expressed in a transgenic manner can function properly to achieve myofibril maturation in embryonic cardiomyocytes. Using the transgenic mice, we further examined detailed localization of Fhod3 during myofibrillogenesis in situ and found that Fhod3 localizes to the specific central region of nascent sarcomeres prior to massive rearrangement of actin filaments and remains there throughout myofibrillogenesis. Taken together, the present findings suggest that, during embryonic cardiogenesis, Fhod3 functions as the essential reorganizer of actin filaments at the central region of maturating sarcomeres via the actin-binding activity of the FH2 domain.     

The Na+-Ca2+ Exchanger Is Essential for Embryonic Heart Development in Mice

The cardiac Na+ -Ca2+ exchanger 1 (NCX1) is thought to be the major calcium extrusion mechanism and to play an important role in the regulation of intracellular calcium in the heart. The Na+ -Ca2+ exchanger is particularly abundant in the heart, although it is found in a variety of other tissues. To investigate the role of NCX1, we have generated NCX1-deficient mice. Mice heterozygous for the NCX1 mutation showed no discernable phenotype, grew normally, and were fertile; however, no viable homozygote was observed among 175 offspring obtained from intercrosses of heterozygotes. All the homozygous mutant mice died in utero before E10.5. Morphological analysis indicated that homozygotes of NCX1 mutation at E9.5 died with an underdeveloped heart with a dilated pericardium. Microscopic analysis of these embryos showed myocardial cell loss due to apoptosis. The apoptosis was first observed in E8.5 mutant heart. Areas outside the heart appeared normal in the mutant embryos at E8.5. In contrast, at E9.0, various regions of mutant embryos showed extensive cell loss. These results suggest that mutant embryos die owing to cardiac abnormalities caused by apoptotic cell loss, indicating that NCX1 is essential for normal development of the heart.