Maternal Diabetes alters Transcriptional Programs in the Developing Embryo

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 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 “call” of “Present”, “Marginal”, “Absent” for each probe set were determined by the comparison of the signal intensity of matched and mismatched oligonucleotide probes for the gene sequence.