Body Wall Formation Research Articles

Double-layered two-directional somatopleural cell migration during chicken body wall development revealed with local fluorescent tissue labeling.

The thoracic ventral body wall consists of the rib, the sternum, the intercostal muscles, and the connective tissues surrounding them. The ribs and the intercostal muscles are derived from the somite. The connective tissues are derived from the somatic layer of the lateral plate mesoderm, somatopleure. The lateral growth of the somatopleure forms the primary ventral body wall. The migration of somitic cells into the somatopleure generates the secondary body wall. As the migrating behavior of the somatopleural cells during secondary body wall formation is still unclear, we investigate here the migratory behavior of the somatopleural cells in the thorax during chicken ventral body wall development by labeling the thoracic somatopleural cells one-somite-wide by DiI labeling or gene transfection of the enhanced green fluorescent protein and observe their distribution assisted by the tissue-clearing technique FRUIT. Our labeling experiments revealed the rostral migration of the somatopleural cells into a deep part of the thoracic body wall in embryonic day 6.5 chickens. For embryonic day 8.5 chickens, these deep-migrating somatopleural cells were found around the sternal ribs. Thus, we identified the double-layered two-directional migrating pathways of the somatopleural cells: the rostral migration of the deep somatopleural cells and the lateral migration of the superficial somatopleural cells. Our findings imply that the rostral migration of deep somatopleural cells and the lateral migration of superficial ones might be associated with the developing sternal ribs and the innervation of the thoracic cutaneous nerves, respectively.

Teratogenesis in the chick embryo following post-gastrulation exposure to Y-27632 -effect of Y-27632 on embryonic development

The pyridine derivative Y-27632 inhibits Rho-associated coiled-coil-containing protein kinase (ROCK) signaling, which is involved in numerous developmental processes during embryogenesis, primarily by controlling actin-cytoskeleton assembly and cell contractility. Somite formation requires rearrangement of the cytoskeleton and assists in major morphological mechanisms, including ventral body wall formation. Administration of Y-27632 impairs cytoskeletal arrangements in post-gastrulation chick embryos leading to ventral body wall defects (VBWD) at later stages of development. The aim of this study was to investigate the effect of Y-27632 on somite development in post-gastrulation chick embryos during early embryogenesis. After 60 h incubation, embryos in shell-less culture were treated with Y-27632 or vehicle for controls. Following administration, abnormality rates were assessed. In treatment groups, embryos showed a kinked longitudinal body axis. Western blot confirmed impaired ROCK downstream signaling by decreased expression of phosphorylated cofilin-2. Histology, Lysotracker studies and RT-PCR demonstrated increased cell death in somites, the neural tube and the ectoderm. RT-PCR and Western blot of factors known to be involved during somitogenesis revealed reduced expression in the treatment group compared to controls. We hypothesize that administration of Y-27632 disrupts somite development causing axial kinking and embryo malformation, which may lead to VBWD.

A chicken embryo model for the study of umbilical and supraumbilical body wall malformations.

BACKGROUND/PURPOSE; The embryology of ventral body wall malformations is only partially understood, although their incidence is relatively common. As only few experimental data exist on the development of those defects, the aim of our study was to compare the teratogenic effect of trypan blue (TB) and suramin (SA) in their capability to induce umbilical and supraumbilical abdominal wall malformations in a chicken egg model. A total of 255 fertilized chicken eggs were incubated at 38 °C and 75% relative humidity. Embryos were treated in ovo on incubation day 2.5 (Hamburger/Hamilton (HH) stage 13). The eggshell was windowed, and solutions of TB or SA were injected into the coelomic cavity at the region of the umbilicus. The window was closed and the embryos reincubated until examination on day 8 (HH 34). A total of 60 embryos survived in each group. The largest number of embryos presented with defects in the umbilical and supraumbilical region (25% in the SA group and 40% in the TB group). A combination of both defects (thoracoabdominoschisis) was seen in 20% of the TB and 8.3% of the SA groups, respectively. Associated anomalies found in both groups were head and eye defects, abnormal pelvic configurations, leg deformities, and mild forms of cloacal exstrophies. TB and SA have both a high potential to induce umbilical and supraumbilical ventral body wall malformations in chicken embryos. This novel animal model might help to establish a more profound understanding of the developmental steps in ventral body wall formation and the embryology for its malformations.

Disturbance of SHH signalling pathway during early embryogenesis in the cadmium-induced omphalocele chick model

Administration of cadmium (Cd) after 60h (H) incubation induces ventral body wall defect (VBWD) similar to the omphalocele phenotype in the chick embryo. In this model, the earliest histological changes have been observed in somites commencing at 4-h post-treatment (4H). The molecular mechanism by which Cd acts in this critical period of embryogenesis still remains unclear. Sonic hedgehog (SHH) signalling plays an important role in vertebrate development, including somitogenesis and thus ventral body wall formation. Patched (PTCH), a cell membrane receptor for SHH, is expressed in somites and Patched knockout mice display somite dysfunction. Another transmembrane receptor, Smoothened (SMO), is also expressed in somites and transduces the SHH signal regulated by PTCH. We designed this study to test the hypothesis that SHH signalling is downregulated during the critical period of early embryogenesis in the Cd-induced omphalocele chick model. After 60h of incubation, chicks were exposed to either chick saline or 50μL of 50μM cadmium acetate and divided into two groups: control and Cd (n=24 for each group). Chicks were harvested 1, 4, and 8h post-treatment. Real-time RT-PCR was performed to evaluate the relative mRNA expression level of SHH, PTCH and SMO. Immunofluorescence confocal microscopy was then performed to evaluate protein expression/distribution of SHH, PTCH and SMO. The relative mRNA expression levels of SHH, PTCH and SMO were significantly downregulated in the Cd group compared to controls at 4H post treatment, whereas, there were no significant differences at the other time points. Immunohistochemistry revealed that the intensity of SHH, PTCH and SMO was markedly diminished at 4h in Cd-treated embryos compared to controls. Disturbance of the SHH signalling pathway as evidenced by SHH, PTCH and SMO downregulation during the narrow window of early embryogenesis may result in somite maldevelopment, contributing to the omphalocele phenotype in the Cd chick model.

Epigenetic Effect of Cadmium on Global De Novo DNA Hypomethylation in the Cadmium-Induced Ventral Body Wall Defect (VBWD) in the Chick Model

Administration of the heavy metal cadmium (Cd) induces ventral body wall defects (VBWD) in the chick embryo. In this model, the expression of most genes involved in body wall formation is altered 4h-posttreatment. However, the mechanism by which Cd results in the initiation of altered gene expression remains unclear. Epigenetic mechanisms can change genome function under exogenous influences. Moreover, Cd is one of the environmental factors that can affect epigenomic programming. De novo DNA methylation is essential for normal embryogenesis and is regulated by the DNA methyltransferases (DNMT)3A and DNMT3B. The objective of this study was to investigate the hypothesis that gene expression levels of DNMT3A/3B were altered, resulting in global DNA methylation changes during the critical period of embryogenesis in the Cd chick model. After 60-h incubation, chick embryos (n = 48) were harvested at 1, 4, and 8 h after treatment with saline or Cd, and divided into controls and Cd groups. Quantitative reverse transcription PCR was performed to evaluate the gene expression levels of DNMT3A/3B in the chick embryos and was statistically analyzed using Student's t-test. Immunohistochemistry was performed using a monoclonal antibody against 5-methylcytidine (5'MeC), which labels methyl-rich regions within the nucleus. DNMT3A/3B gene expression levels at 4 h were significantly downregulated in the Cd group compared with controls (p < 0.005/p < 0.00001, respectively). Immunoreactivity of 5'MeC was markedly diminished in the Cd group at 4 h. Our findings demonstrates for the first time that Cd impacts on the expression levels of DNMT3A/3B, which may underlie the pathogenesis of VBWD in the Cd chick model.

Altered PITX2 and LEF1 gene expression in the cadmium-induced omphalocele in the chick model

Although, recent studies have suggested that disruption of somitogenesis may be involved in ventral body wall (VBW) defects; the molecular mechanisms of VBW defects remain unclear. In the chick embryo, the administration of cadmium (Cd) induces VBW defects similar to the human omphalocele. In this model, the earliest histological change in the somite occurs commencing at 4 h post-Cd treatment (4 h). PITX2 is expressed in somites, and PITX2 mutants have been shown to display VBW defects. PITX2 interacts with lymphoid enhancer factor-1 (LEF1) to regulate somite myogenesis. We designed this study to investigate the hypothesis that PITX2 and LEF1 genes are downregulated during the critical period of early embryogenesis in the Cd-induced omphalocele chick model. Chick embryos were exposed to Cd or saline after 60 h incubation and harvested at 1, 4, and 8 h posttreatment. Chicks were then divided into two groups: control (n = 24), and Cd (n = 24). RT-PCR was performed and analyzed statistically (significant difference was accepted at p < 0.05). Immunohistochemistry was also performed to evaluate expression/distribution of those proteins. The mRNA expression levels of PITX2 and LEF1 at 4 h were significantly decreased in the Cd group compared with controls, whereas there were no differences at the other time points. Immunoreactivity of those proteins at 4 h was also markedly decreased in somites in the Cd-treated embryos compared with controls. Downregulation of PITX2 and LEF1 genes may interfere with ventral body wall formation in Cd chick model causing omphalocele by disrupting somite myogenesis.

BMPR1A-mediated BMP1 signalling is disrupted in the cadmium-induced omphalocele in the chick model

In the chick embryo, administration of cadmium (Cd) induces omphalocele phenotype. The earliest histological changes in this model are observed commencing at 4-h post treatment (4H). The molecular mechanisms by which Cd acts during the critical period of embryogenesis remain unclear. Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) regulate many fundamental biological processes and are involved in various embryonic morphogenesis, including ventral body wall (VBW) formation. Homozygous BMP1 mutant mice cause VBW defects. It has been reported that BMPR1A conditional knockouts also exhibit omphalocele phenotype. We designed this study to test the hypothesis that gene expression levels of BMP1 and BMPR1A are downregulated during the critical period of embryogenesis in the Cd chick model. After 60-h incubation, chick embryos were exposed to either Cd or saline and then harvested 1H, 4H and 8H. Chicks were divided into control (n = 24) and Cd (n = 24). RT-PCR was performed and differences between the two groups were tested statistically (significance was accepted at p < 0.05). Immunohistochemical study was also performed to evaluate those proteins expression/distribution. The gene expression levels of BMP1 and BMPR1A at 4H were significantly downregulated in Cd group compared to controls. Immunoreactivity of BMP1 and BMPR1A was also markedly decreased in Cd-treated embryos compared to controls. Disruption of BMPR1A-mediated BMP1 signalling during the narrow window of early embryogenesis may interfere with normal VBW formation, causing omphalocele phenotype in the Cd chick model.

Genetic Analysis of Hedgehog Signaling in Ventral Body Wall Development and the Onset of Omphalocele Formation

BackgroundAn omphalocele is one of the major ventral body wall malformations and is characterized by abnormally herniated viscera from the body trunk. It has been frequently found to be associated with other structural malformations, such as genitourinary malformations and digit abnormalities. In spite of its clinical importance, the etiology of omphalocele formation is still controversial. Hedgehog (Hh) signaling is one of the essential growth factor signaling pathways involved in the formation of the limbs and urogenital system. However, the relationship between Hh signaling and ventral body wall formation remains unclear.Methodology/Principal FindingsTo gain insight into the roles of Hh signaling in ventral body wall formation and its malformation, we analyzed phenotypes of mouse mutants of Sonic hedgehog (Shh), GLI-Kruppel family member 3 (Gli3) and Aristaless-like homeobox 4 (Alx4). Introduction of additional Alx4Lst mutations into the Gli3Xt/Xt background resulted in various degrees of severe omphalocele and pubic diastasis. In addition, loss of a single Shh allele restored the omphalocele and pubic symphysis of Gli3Xt/+; Alx4Lst/Lst embryos. We also observed ectopic Hh activity in the ventral body wall region of Gli3Xt/Xt embryos. Moreover, tamoxifen-inducible gain-of-function experiments to induce ectopic Hh signaling revealed Hh signal dose-dependent formation of omphaloceles.Conclusions/SignificanceWe suggest that one of the possible causes of omphalocele and pubic diastasis is ectopically-induced Hh signaling. To our knowledge, this would be the first demonstration of the involvement of Hh signaling in ventral body wall malformation and the genetic rescue of omphalocele phenotypes.

Disruption of GLI3–ZIC3 interaction in the cadmium-induced omphalocele chick model

Administration of heavy metal cadmium (Cd) after 60-h incubation induces omphalocele spectrum in the chick embryo. Although previous studies have shown that the earliest detectable histological changes in the chick Cd model occurs commencing at 4-h post-treatment (4H). However, the molecular mechanism by which Cd acts in the critical period of early embryogenesis still remains unclear. Zic3, a Gli superfamily transcription factor, is expressed in somites and plays an important role in vertebrate development, including somitogenesis and thus ventral body wall formation. Gli3 is also expressed in somites and interacts with Zic3 physically and functionally. It has been reported that Gli3 homozygous double mutants display omphalocele. Zic3 mutant mice have also been known to result in omphalocele phenotype. We designed this study to test the hypothesis that Gli3 and Zic3 gene expression is downregulated during the critical period of very early embryogenesis in the Cd-induced omphalocele in the chick model. After 60-h incubation, chick embryos were exposed to either saline or 50μM cadmium and divided into two groups: control and Cd (n=24 for each group). Real-time reverse transcription polymerase chain reaction was performed to evaluate the relative mRNA expression levels of Gli3 and Zic3 in the Cd-induced omphalocele chick model. Differences between the two groups at each time point were analyzed statistically and the significance was accepted at p<0.05. Immunohistochemistry was also performed to evaluate the expression/distribution of those proteins in chick embryo. The relative mRNA expression level of Gli3 and Zic3 was significantly decreased in the Cd group at 4H when compared with controls (p<0.05). However, there were no significant differences at the other time points. At 4H, immunoreactivity of GLI3 and ZIC3 was also markedly decreased in Cd-treated embryos, whereas strong expression of them was seen in the somite in controls. We provide evidence, for the first time, that Gli3 and Zic3 gene expression is downregulated during the narrow window of very early embryogenesis in Cd chick model. Disruption of Gli3-Zic3 interaction in the critical period for ventral body wall formation may contribute to omphalocele phenotype in Cd chick model.

HoxB2, HoxB4 and Alx4 genes are downregulated in the cadmium-induced omphalocele in the chick model

In the chick embryo, administration of cadmium (Cd) induces omphalocele phenotype. HoxB2 and HoxB4, expressed in cell types that contribute to ventral body wall (VBW) formation, act together to mediate proper closure of the VBW, involving a key downstream transcription factor, Alx4. HoxB2 and HoxB4 knockout mice display VBW defects with specific downregulation of Alx4 gene expression, while homozygous Alx4 knockouts show omphalocele phenotype. Although the earliest histological changes in the Cd chick model occur commencing at 4H post treatment, the exact timing and molecular mechanism by which Cd acts is still unclear. We hypothesized that HoxB2, HoxB4 and Alx4 genes are downregulated during the critical timing of very early embryogenesis in the Cd-induced omphalocele chick model. After 60H incubation, chick embryos were harvested at 1H, 4H and 8H after treatment with saline or Cd and divided into controls and Cd group (n = 24 for each group). RT-PCR was performed to investigate the gene expression of HoxB2, HoxB4 and Alx4 and statistically analyzed (significance was accepted at p < 0.05). Immunohistochemical staining was also performed to evaluate the protein expression/distribution of HoxB2, HoxB4 and Alx4 in the chick embryo. The expression levels of HoxB2, HoxB4 and Alx4 gene at 4H were significantly downregulated in the Cd group as compared to controls, whereas there were no significant differences at the other time points. Immunoreactivity of HoxB2, HoxB4 and Alx4 at 4H is also markedly decreased in the ectoderm and the dermomyotome in the Cd chick model as compared to controls. Downregulation of HoxB2, HoxB4 and Alx4 expression during the narrow window of early embryogenesis may cause omphalocele in the Cd chick model by interfering with molecular signaling required for proper VBW formation. Furthermore, these results support the concept that HoxB2, HoxB4 and Alx4 genes work together to mediate proper VBW formation.

The role of transforming growth factor- β 2 and 3 in formation of ventral body wall in the cadmium-induced omphalocele chick model

Purpose In the chick embryo, the administration of cadmium (Cd) induces ventral body wall defects (VBWDs) similar to the human omphalocele. Transforming growth factors β (TGFs- β) are involved in many developmental processes, including ventral body wall formation. The Tgf β2 −/− Tgf β3 −/− double knockout mice and Tgf β2 −/− Tgf β3 +/− mutants show VBWD, whereas Tgf β2 +/− Tgf β3 −/− mutants display normal ventral body wall fusion. We designed this study to investigate the messenger RNA (mRNA) levels of TGF- β2 and TGF- β3 in the Cd-induced omphalocele chick model during early embryogenesis. Methods Chick embryos were exposed to either Cd or saline, harvested 1 hour (1H), 4H, and 8H after treatment and then divided into 2 groups: control and Cd (n = 8 at each time-point, respectively). Reverse transcription polymerase chain reaction was performed to evaluate the mRNA levels of TGF- β2 and TGF- β3 and statistically analyzed. Results The mRNA expression levels of TGF- β2 at 1H were significantly decreased in the Cd group compared to controls ( P < .05). However, the levels of TGF- β3 were not altered at all the time-points studied. Conclusion We provide evidence, for the first time, that TGF- β2 gene expression is downregulated during a narrow window of early embryogenesis in the Cd chick model. Our data show that TGF- β2 is the key gene involved in the formation of ventral body wall.

Deficient Alk3-mediated BMP signaling causes prenatal omphalocele-like defect

BMP signaling plays important roles in many embryonic developmental processes. Alk3 is one of two BMP type I receptors that transduces BMP signal from the cell surface into cell. Conventional knockout of Alk3 resulted in early embryonic lethality around E7.5–E9.5. In this study, we have generated embryonic mesoderm-specific Alk3 conditional knockout by crossing Dermo1-Cre and floxed Alk3 mice. Abrogation of Alk3-mediated BMP signaling in this mouse resulted in severe defect of secondary ventral body wall formation, replicating the omphalocele phenotype in human. Our finding suggests that Alk3 plays an essential role in the formation of embryonic ventral abdominal wall, and abrogation of BMP signaling activity due to gene mutations in its signaling components could be one of the underlying causes of omphalocele at birth.

The role of inhibitory neurotransmission in ventral body wall formation

The role of inhibitory neurotransmission in ventral body wall formation

A new serine/threonine protein kinase,Omphk1, essential to ventral body wall formation

Here, we report a new serine/threonine protein kinase of the SNF1 subfamily Omphk1. Two Omphk homologues exist in each vertebrate species, and one homologue exists in Drosophila and Caenorhabditis elegans; the kinase domain is highly conserved among these homologues, and several domains are conserved among vertebrate Omphk. Omphk1 expression dynamically changes in the developing central nervous system, is found ubiquitously in epidermis, and is present uniquely in several other tissues. Its expression is also found in each tissue associated with the ventral body wall closure: the primary body wall composed of primitive ectoderm and each component of the secondary body wall. Concomitantly, its null mutant exhibits omphalocele with a failure in closure of the secondary body wall. There are no apparent gross morphological defects in brain, however, despite the unique Omphk1 expression in this tissue.

Ventral abdominal wall dysmorphogenesis of Msx1/Msx2 double‐mutant mice

Msx1 and Msx2 genes encode the homeodomain transcription factors. Several gene knockout mice and expression studies suggest that they possess functionally redundant roles in embryogenesis. In this study, we revealed that Msx1 and Msx2 were expressed during ventral body wall formation in an overlapping manner. Msx1/Msx2 double-mutant mice displayed embryonic abdominal wall defects with disorganized muscle layers and connective tissues. These findings indicate that Msx1 and Msx2 play roles in concert during embryonic ventral abdominal wall formation.

Requirement for Mab21l2 during development of murine retina and ventral body wall

The mab-21 gene was first identified because of its requirement for ray identity specification in Caenorhabditis elegans. It is now known to constitute a family of genes that are highly conserved from vertebrates to invertebrates, and two homologues Mab21l1 and Mab21l2 have been identified in many species. Here we describe the generation of Mab21l2-deficient mice, which have defects in eye and body wall formation. The mutant mouse eye has a rudimentary retina, as a result of insufficient invagination of the optic vesicle due to deficient proliferation, causing the absence of lens. The defects in optic vesicle development correlate with reduced expression of Chx10, which is also required for retina development; Rx, Lhx2, and Pax6 expression is not significantly affected. We conclude that Mab21l2 expression is essential for optic vesicle growth and formation of the optic cup, its absence causing reduced expression of Chx10. Mutant mice also display abnormal extrusion of abdominal organs, defects in ventral body wall formation, resulting in death in utero at mid-gestational stage. Our results reveal that Mab21l2 plays crucial roles in retina and in ventral body wall formation.

Hypaxial Muscle Migration during Primary Myogenesis in Xenopus laevis

In contrast to many vertebrates, the ventral body wall muscles and limb muscles of Xenopus develop at different times. The ventral body wall forms in the tadpole, while limb (appendicular) muscles form during metamorphosis to the adult frog. In organisms that have been examined thus far, a conserved mechanism has been shown to control migratory muscle precursor specification, migration, and differentiation. Here, we show that the process of ventral body wall formation in Xenopus laevis is similar to hypaxial muscle development in chickens and mice. Cells specified for the migratory lineage display an upregulation of pax3 in the ventro-lateral region of the somite. These pax3-positive cells migrate ventrally, away from the somite, and undergo terminal differentiation with the expression of myf-5, followed by myoD. Several other genes are selectively expressed in the migrating muscle precursor population, including neural cell adhesion molecule (NCAM), Xenopus kit related kinase (Xkrk1), and Xenopus SRY box 5 (sox5). We have also found that muscle precursor migration is highly coordinated with the migration of neural crest-derived melanophores. However, by extirpating neural crest at an early stage and allowing embryos to develop, we determined that muscle precursor migration is not dependent on physical or genetic interaction with melanophores.

Hoxb2 and Hoxb4 Act Together to Specify Ventral Body Wall Formation

Three different alleles of the Hoxb4 locus were generated by gene targeting in mice. Two alleles contain insertions of a selectable marker in the first exon in either orientation, and, in the third, the selectable marker was removed, resulting in premature termination of the protein. Presence and orientation of the selectable marker correlated with the severity of the phenotype, indicating that the selectable marker induces cis effects on neighboring genes that influence the phenotype. Homozygous mutants of all alleles had cervical skeletal defects similar to those previously reported for Hoxb4 mutant mice. In the most severe allele, Hoxb4PolII, homozygous mutants died eitherin utero at approximately E15.5 or immediately after birth, with a severe defect in ventral body wall formation. Analysis of embryos showed thinning of the primary ventral body wall in mutants relative to control animals at E11.5, before secondary body wall formation. Prior to this defect, both Alx3 and Alx4 were specifically down regulated in the most ventral part of the primary body wall in Hoxb4PolII mutants. Hoxb4loxp mutants in which theneo gene has been removed did not have body wall or sternum defects. In contrast, both the Hoxb4PolII and the previously described Hoxb2PolII alleles that have body wall defects have been shown to disrupt the expression of bothHoxb2 and Hoxb4 in cell types that contribute to body wall formation. Our results are consistent with a model in which defects in ventral body wall formation require the simultaneous loss of at least Hoxb2 and Hoxb4, and may involve Alx3 and Alx4.

The Development and Evolution of the Turtle Body Plan: Inferring Intrinsic Aspects of the Evolutionary Process from Experimental Embryology

Synopsis. The body plan of turtles is unique among tetrapods in the presence ofthe shell. The structure of the carapace involves a unique relationship between the axial and the appendicular skeletons. A common developmental mechanism, an epithelial-mesenchymal interaction, has been identified in the early stages of carapace development by means of basic histological and immunofluorescence techniques. By analogy to other structures ini? tiated by epithelial-mesenchymal interactions, it is hypothesized that carapace development is dependent on this interaction in the body wall. Surgical perturbations were designed to test the causal connection between the epithelial-mesenchymal interaction in the body wall and the unusual placement of the ribs in turtles. By comparison to data available on body wall formation in avian embryos, these experiments also shed light on the segregation of somitic and lateral plate cell populations and the embryonic origin ofthe scapula in turtles. This study specifically addresses the ontogeny of a unique tetrapod body plan. The onto? genetic information can be used to make inferences about the phylogeny of this body plan and how it could have evolved from the more typical primitive tetrapod. On a more general level this study explores the potential role of common developmental mechanisms in the generation of evolutionary novelties, and the developmental incongruities between homol? ogous skeletal elements in different groups of tetrapods.