Project description:Diabetic embryopathy can affect any developing organ system, although cardiovascular malformations, neural tube defects and caudal dysgenesis syndrome are the most prominent congenital malformations. We hypothesize that the metabolic imbalance occurring in diabetic pregnancy de-regulates tissue specific gene expression programs in the developing embryo. In order to identify genes whose expression is affected by maternal diabetes, we analyzed gene expression profiles of diabetes-exposed mouse embryos by using Affymetrix microarrays. We identified 129 genes with altered expression levels; 21 genes had increased and 108 genes had decreased expression levels in diabetes-exposed embryos relative to controls. A substantial fraction of these genes (35) are essential for normal embryonic development as shown by functional studies in mouse models. The largest fraction of diabetes-affected genes was in transcription factor and DNA-binding/chromatin remodeling functional categories (19%), which directly affect transcription. These findings suggest that transcriptional regulation in the developing embryos is perturbed by maternal diabetes and that transcriptional regulation plays a major role in the responses of embryos to intrauterine exposure to diabetic conditions. Interestingly, we found the expression of hypoxia-inducible factor 1 α (Hif1α) deregulated in the embryos exposed to the conditions of maternal diabetes. Since hypoxic stress is associated with the complications of diabetic pregnancy, we performed a post-hoc analysis of our microarray data with a specific focus on known HIF1 target genes. Of 39 genes detected in our microarrays, the expression changes of 22 genes (20 were increased and two genes were decreased in diabetes-exposed embryos) were statistically significant. These results indicate that HIF1-regulated pathways are affected in diabetes-exposed embryos. These results strongly suggest that de-regulation of hypoxia/HIF1 activated pathways could be the one of the key molecular events associated with the exposure to the teratogenic intrauterine environment of a diabetic mother. Keywords: Comparative studies of embryonic response to maternal diabetes

Project description:Maternal diabetes is a recognized risk factor for both short-term and long-term complications in offspring. Beyond the direct teratogenicity of maternal diabetes, the intrauterine environment can influence offspring cardiovascular health. Abnormalities in the cardiac sympathetic system are implicated in conditions such as sudden infant death syndrome, cardiac arrhythmic death, heart failure, and certain congenital heart defects in children from diabetic pregnancies. However, the mechanisms by which maternal diabetes affect the development of the cardiac sympathetic system and consequently, heightening health risks and predisposing to cardiovascular disease remain poorly understood. In this study, we present a comprehensive analysis of the combined impact of a Hif1a-deficient sympathetic system and the maternal diabetes environment on both heart development and the formation of the cardiac sympathetic system. The synergic negative effect of exposure to maternal diabetes and Hif1a deficiency resulted in the most pronounced deficit in cardiac sympathetic innervation and the development of adrenal medulla. Abnormalities in the cardiac sympathetic system were accompanied by a smaller heart, reduced ventricular wall thickness, dilated subepicardial veins, and coronary arteries in the myocardium, along with anomalies in the branching and connections of the main coronary arteries. Transcriptional profiling by RNA-seq revealed significant transcriptome changes in Hif1a-deficient sympathetic neurons, primarily associated with cell cycle regulation, proliferation, and mitosis, explaining the shrinkage of the sympathetic neuron population. Our data demonstrate that a failure to adequately activate HIF-1α regulatory pathway, particularly in the context of maternal diabetes, may contribute to abnormalities in the cardiac sympathetic system. In conclusion, our findings indicate that the interplay between deficiencies in the cardiac sympathetic system and subtle structural alternations in the vasculature, microvasculature, and myocardium during heart development not only increases the risk of cardiovascular disease but also diminishes the adaptability to the stress associated with the transition to extrauterine life, thus, increasing the risk of neonatal death.

Project description:Diabetic embryopathy can affect any developing organ system, although cardiovascular malformations, neural tube defects and caudal dysgenesis syndrome are the most prominent congenital malformations. We hypothesize that the metabolic imbalance occurring in diabetic pregnancy de-regulates tissue specific gene expression programs in the developing embryo. In order to identify genes whose expression is affected by maternal diabetes, we analyzed gene expression profiles of diabetes-exposed mouse embryos by using Affymetrix microarrays. We identified 129 genes with altered expression levels; 21 genes had increased and 108 genes had decreased expression levels in diabetes-exposed embryos relative to controls. A substantial fraction of these genes (35) are essential for normal embryonic development as shown by functional studies in mouse models. The largest fraction of diabetes-affected genes was in transcription factor and DNA-binding/chromatin remodeling functional categories (19%), which directly affect transcription. These findings suggest that transcriptional regulation in the developing embryos is perturbed by maternal diabetes and that transcriptional regulation plays a major role in the responses of embryos to intrauterine exposure to diabetic conditions. Interestingly, we found the expression of hypoxia-inducible factor 1 Î± (Hif1Î±) deregulated in the embryos exposed to the conditions of maternal diabetes. Since hypoxic stress is associated with the complications of diabetic pregnancy, we performed a post-hoc analysis of our microarray data with a specific focus on known HIF1 target genes. Of 39 genes detected in our microarrays, the expression changes of 22 genes (20 were increased and two genes were decreased in diabetes-exposed embryos) were statistically significant. These results indicate that HIF1-regulated pathways are affected in diabetes-exposed embryos. These results strongly suggest that de-regulation of hypoxia/HIF1 activated pathways could be the one of the key molecular events associated with the exposure to the teratogenic intrauterine environment of a diabetic mother. Experiment Overall Design: Total RNA was isolated from whole embryos at embryonic day 10.5 (E10.5). Five diabetes-exposed embryos (2 of 5 embryos had neural tube defects; NTD) were compared to two control embryos. Diabetes was induced in female FVB mice (aged 7-9 weeks) by streptozotocin Embryos were isolated only from dams that were classified as diabetic with the blood glucose levels > 250 mg/dl from the mating day until the day of embryo harvest. Five ug of total RNA was reverse-transcribed, and biotin-labeled cRNA probe generated using the Affymetrix labeling kit and protocol per manufacturers instructions. Probes were then hybridized to the Affymetrix 430A 2.0 genome chip with 14,338 genes. Following washing and staining, the chips were scanned using the Affymetrix GeneChip 3000 scanner in Genetic Microarray Core Facility at the University of Nebraska Medical Center. Images were analyzed using Affymetrix GCOS imaging software. Quality metric parameters including noise level, background, and the efficiency of reverse transcription were ascertained for all hybridizations. The expression levels and the âcallâ of âPresentâ, âMarginalâ, âAbsentâ for each probe set were determined by the comparison of the signal intensity of matched and mismatched oligonucleotide probes for the gene sequence.

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Single-cell transcriptome profiling of mouse cardio-pharyngeal development under maternal diabetes mellitus

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