Eya1 and Eya2 gene expression is down-regulated during somitic myogenesis in the cadmium-induced omphalocele chick model.

PURPOSE: The molecular mechanisms underlying omphalocele are still largely unknown. Recently, established cadmium (Cd)-induced omphalocele chick model has been used to investigate the pathogenesis of omphalocele. The earliest histologic changes in this model has been observed in somites 4 hours posttreatment, leading us to hypothesize that disruption of migration of somite-derived cells ventrally may cause omphalocele phenotype. Eyes absent (Eya) genes are expressed in the somite (dermomyotome) and play a key role in somitic myogenesis. We designed this study to investigate the hypothesis that Eya1 and Eya2 gene expression is down-regulated during the critical period of early embryogenesis in the Cd-induced omphalocele chick model. METHODS: After 60 hours of incubation, chicks were exposed to either chick saline or Cd and divided into control and Cd (n = 24 for each group). Chicks were then harvested 1 hour, 4 hours, and 8 hours posttreatment. Real-time quantitative polymerase chain reaction was performed to evaluate gene expression levels of Eya1 and Eya2 in the chick embryo, and they were statistically analyzed. Immunofluorescence confocal microscopy was also performed to evaluate protein expression and distribution pattern of Eya1 and Eya2. RESULTS: At 4 hours posttreatment, the relative messenger RNA expression levels of Eya1 and Eya2 were significantly down-regulated in the Cd group compared with controls (P < .05). The intensity of Eya1 and Eya2 immunofluorescence was also markedly diminished at 4 hours in the Cd-treated embryos, whereas in control embryos, strong intensity of immunofluorescence of them was expressed in the dermomyotomal cells. CONCLUSION: Down-regulation of Eya genes during the critical period of early embryogenesis may contribute to omphalocele phenotype in the Cd chick model, interfering with migration of embryonic body wall ventrally.

EphB2/B3 gene expression is down-regulated during early embryogenesis in the cadmium-induced omphalocele chick model.

PURPOSE: In the chick embryo, the administration of cadmium (Cd) induces omphalocele phenotype. The earliest histologic change in this model is observed in the somite 4 hours (H) post treatment, postulating that disruption of somite development in embryogenesis may cause omphalocele phenotype. EphB2 and EphB3 are involved in many embryonic developmental processes, including somitogenesis. EphB2(-/-)EphB3(-/-) double knockouts display omphalocele phenotype. We hypothesized that EphB2/B3 genes are down-regulated in the Cd chick model during the critical period of embryogenesis. METHODS: After 60H incubation, chicks were harvested 1H, 4H, and 8H post treatment with saline or Cd and divided into control and Cd groups. Reverse transcriptase-polymerase chain reaction was performed to evaluate gene expression levels of EphB2/B3. Immunofluorescence confocal microscopy was performed to evaluate protein expression/distribution of EphB2/B3. RESULTS: At 4H post treatment, the messenger RNA expression levels of EphB2/B3 were significantly down-regulated in the Cd group compared with controls (P < .05). The intensity of EphB2/B3 immunofluorescence was markedly diminished at 4H in the Cd-treated embryos, whereas strong immunoreactivity was observed in the somite in controls. CONCLUSION: Downregulation of EphB2/B3 during the narrow window of early embryogenesis may interfere with normal somitogenesis, preventing migration of embryonic body wall ventrally and thus causing omphalocele.

Presenilin genes are downregulated during somitogenesis in the cadmium-induced omphalocele chick model.

PURPOSE: Although the precise pathogenesis of ventral body wall (VBW) defects is not clearly understood, it has recently postulated that disruption of somite development during early embryogenesis may cause failure of proper VBW formation. The administration of cadmium (Cd) after 60 h of incubation induces omphalocele spectrum in the chick embryo. Previous studies have shown that one of the earliest histological changes seen in this model is abnormal cell death in the somite, occurring at 4 h post treatment (4H). However, the molecular mechanism by which Cd acts in this critical period of embryogenesis still remains unclear. Presenilins are expressed in somites and play an important role in vertebrate development, including somitogenesis and thus VBW formation. We designed this study to test the hypothesis that gene expression levels of presenilin 1 (PSEN1) and presenilin 2 (PSEN2) are downregulated during the critical period of early embryogenesis in the Cd-induced omphalocele in the chick model. METHODS: After 60 h of incubation, chick embryos were exposed to either saline or 50 µM cadmium and divided into two groups: control and Cd (n = 8 at each time point for each group). Real-time RT-PCR was performed to evaluate the relative mRNA expression levels of PSEN1 and PSEN2 in the Cd-induced omphalocele chick model. Differences between two groups at each time point were analysed statistically and the significance was accepted at p < 0.05. Immunofluorescence confocal microscopy was performed to evaluate the protein expression/distribution of presenilins in the somite of chick embryo. RESULTS: The relative mRNA expression levels of PSEN1 and PSEN2 were significantly downregulated in the Cd group at 4H compared with controls (p < 0.005) (Table). However, there were no significant differences at the other time points. At 4H, immunofluorescence of presenilins (green) was markedly diminished in the Cd-treated embryos, whereas strong immunofluorescence of them was seen in the somite (dermomyotome) in controls (Fig. 1). 1 Immunofluorescence Confocal Microscopy for PSEN1 and PSEN2 in the dermomyotome of the somite in the trunk level of chick embryo 4H post treatment. Intensity of PSEN1 immunofluorescence (green) was markedly diminished in Cd-treated embryos, whereas strong PSEN1 expression was seen in the dermomyotome in controls. PSEN2 immunofluorescence was also decreased in the Cd-treated embryos, whereas strong PSEN2 immunofluorescence (green) was observed predominantly in the dermomyotome in controls. Immunofluorescence in orange is DNA counter staining by DAPI CONCLUSION: We provide evidence, for the first time, that gene expression of presenilins is downregulated during the narrow window of very early embryogenesis in the Cd chick model. Decreased expression of presenilins may contribute to omphalocele phenotype in Cd chick model, by disrupting somite development.

Alteration of gene expression of IQGAP1 and Rho-family GTPases in the cadmium-induced ventral body wall defects in the chick model.

Cadmium (Cd) induces ventral body wall defects (VBWD) in the chick embryo, with adherens junctions (AJs) breakdown at 4h post treatment (4H). Signalling by which Cd disrupts AJs in this model remains unclear. IQGAP1 regulates AJs via binding to Cdc42 and Rac1, Rho-family GTPases. Activation of IQGAP1-Cdc42 interaction regulates AJs positively, whereas Rac1 activation inhibits AJs. We hypothesised that IQGAP1 and Cdc42 are downregulated and Rac1 is upregulated during embryogenesis in the Cd chick model. Chick embryos were explanted and treated with saline or Cd after 60 h incubation. Chicks were harvested at 1H, 4H and 8H post treatment and RT-PCR and immunohistochemistry were performed. Gene expression levels of IQGAP1 and Cdc42 were significantly downregulated and Rac1 was upregulated in Cd group compared to controls only at 4H. Immunoreactivity of IQGAP1 and Cdc42 was also markedly decreased, whereas Rac1 was increased in Cd group compared to controls at 4H. Alteration of IQGAP and Rho-family GTPases may cause VBWD in Cd chick model by inducing the dissociation of cadherin-mediated AJs.

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.

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

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: Downregulation of PITX2 and LEF1 genes may interfere with ventral body wall formation in Cd chick model causing omphalocele by disrupting somite myogenesis.

Pre-treatment with N-acetylcysteine upregulates superoxide dismutase 2 and catalase genes in cadmium-induced oxidative stress in the chick omphalocele model.

PURPOSE: In the chick embryo, administration of the heavy metal Cadmium (Cd) induces omphalocele phenotype. Cd is a potent inhibitor of antioxidant enzymes and causes accumulation of reactive oxygen species (ROSs) such as hydrogen peroxide. Previous work with the Cd chick model has demonstrated that increased levels of MDA, as a marker for oxidative stress, 24 h post Cd treatment (24H) are identical in chick embryos exposed to Cd. Furthermore, of the several antioxidants assessed, only N-acetylcysteine (NAC) has been shown to reduce MDA levels to control values in the Cd-treated chick embryo. However, the molecular mechanisms by which NAC acts to maintain oxidative stress in the Cd-induced ventral body wall defect chick model remains to be unclear. We designed this study to investigate the hypothesis that gene expression levels of antioxidant enzymes are downregulated in malformed embryos exposed to Cd compared to controls and to determine the effect of pre-treatment with NAC on the expression levels of genes encoding antioxidant enzymes. METHODS: After 60 h incubation, chick embryos were pre-treated with NAC and exposed to either chick saline or Cd. Chicks were then harvested at 24H and divided into five groups: control, Cd group without malformation [Cd(-)], Cd group with malformation [Cd(+)], NAC + Cd(-) and NAC + Cd(+). Real-time PCR was performed to evaluate the relative mRNA expression levels of antioxidant enzymes, including superoxide dismutase (SOD)-1, SOD2, catalase (CAT) and glutathione peroxidase (GPX)-4. Differences between five groups were tested by Tukey-Kramer post-hoc test following one-way ANOVA. Statistical significance was accepted at p < 0.05. Immunohistochemistry was also performed to evaluate protein expression. RESULTS: The mRNA expression levels of SOD2 and CAT were significantly decreased in Cd(+) as compared to controls, whereas there was no significant difference between controls and Cd(-) (p < 0.05 vs. controls). In addition, gene expression levels of SOD2 and CAT were significantly increased in NAC + Cd(-) as compared to Cd(+) and NAC + Cd(+) (p < 0.05 vs. controls). However, there were no significant differences in the expression levels of SOD1 and GPX4 among any groups. Increased immunoreactivity of SOD2 and CAT was also observed in NAC + Cd(-) as compared to Cd(+) and NAC + Cd(+). CONCLUSION: Our results suggest that SOD2 and CAT may play an important role in preventing Cd-induced teratogenesis. Prenatal treatment with drugs which can upregulate SOD2 and CAT transcripts may have a therapeutic potential in preventing omphalocele phenotype.

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

PURPOSE: 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. METHODS: After 60-h incubation, chick embryos were exposed to either saline or 50 µM 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. RESULTS: 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. CONCLUSION: 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.

Disruption of noncanonical Wnt/CA2+ pathway in the cadmium-induced omphalocele in the chick model.

PURPOSE: Cadmium (Cd) has been found to cause ventral body wall defects (VBWDs) in the chick embryo similar to human omphalocele. The earliest detectable histologic changes in Cd-induced VBWD chick model have been observed 4 hours posttreatment. The exact mechanism by which Cd acts in the early embryogenesis remains unclear. Wnt proteins play a key role during embryogenesis, and altered Wnt signaling has been linked to developmental defects. Noncanonical Wnt/Ca(2+) pathway has been implicated in regulating embryogenesis by controlling cell movement and adhesion. Wnt11 can activate protein kinase C (PKC) and calcium/calmodulin-dependent kinase II (CaMKII) in the Wnt/Ca(2+) pathway. We hypothesized that the Wnt11, PKCalpha, and CaMKII gene expression is downregulated in the Cd-induced VBWD during early embryogenesis. METHODS: After 60 hours of incubation, chick embryos were harvested 1 hour (1H), 4H, and 8H after treatment of saline or cadmium and divided into 2 groups: control and Cd (n = 8 at each time-point, respectively). Real-time polymerase chain reaction was performed to evaluate the messenger RNA (mRNA) expression of Wnt11, PKCalpha, and CaMKII in the Cd-induced VBWD chick model. RESULTS: The mRNA expression levels of Wnt11, PKCalpha, and CaMKII were significantly decreased at 1H in Cd group compared to controls (P < .05). However, there were no significant differences in the other time-points. CONCLUSION: Downregulation of Wnt11, PKCalpha, and CaMKII gene expression during the narrow window of early embryogenesis may cause VBWD, interfering with cell movement and adhesion, disrupting Wnt/Ca(2+) pathway.