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Hyperglycemia and Gene Expression during Embryonic Development

Diabetic embryopathy is a complication of diabetes in which the embryo of a diabetic mother develops congenital malformations, mostly of the hart and the neural tube. These malformations arise at the beginning of organogenesis during the first 8 weeks of gestation in human embryos, and during the first 7-10 days in mouse and rat embryos. Our intention was to test whether and to what extent hyperglycemia influences the expression of several genes involved in neural embryonic development in NOD (non-obese diabetic) mice. In order to asses if altered gene expression detected in vivo is due to hyperglycemia, we tested in vitro system that mimics early embryonic development (differentiation of mouse embryonic stem cells as embryoid bodies in culture) at different glucose concentrations. In vitro R1 mouse embryonic stem cell line was cultured and propagated on mitotically inactive mouse primary fibroblasts in a culture medium supplemented with leukemia inhibitory factor (LIF). Differentiation was triggered by omitting LIF from culture medium and growing embryonic stem cells as aggregates (embryoid bodies) in non-adherent Petri dishes. Embryoid bodies were cultured under three glucose concentrations (5, 5 mmol/L ; 25 mmol/L ; 36 mmol/L). At different time points embryoid bodies were harvested, homogenized and RNA was extracted. Semi quantitative RT-PCR was performed by three sets of primers representing following genes: pax3, neuroD1and ngn3. In vivo Hyperglycemic and normoglycemic NOD mice were mated and embryos were recovered 9.5 days post coitum. Blood of the female mice was extracted just before mating and immediately after the sacrifice, and the glucose level was determined by glucose oxydase. Embryos from each mother were pooled and RNA was extracted. Semi quantitative RT-PCR was performed by nine sets of primers representing following genes: pax3, pax4, pax6, neuroD1, ngn3, sod1, sod2, gpx1, and cat. Preliminary results of our study confirmed previous findings that the expression of transcription factor pax3 gene is significantly inhibited in embryos of diabetic NOD mice (in contrast to embryos of normoglycemic NOD mice). Gene for transcription factor neuroD1 displayed even more dramatic down regulation. In contrast, embryoid bodies grown in culture at various glucose concentrations (5.5 mM, 24.8 mM, 35.8 mM) and harvested for RNA isolation after 3, 12 and 14 days displayed strong and equal neuroD1 expression in all tested samples, while pax3 expression was completely undetectable in the same samples. Gene for transcription factor ngn3 could not be detected in any sample both in vivo and in vitro. Since all other tested genes (transcription factors pax4 and pax6 as well as genes coding antioxydative enzymes sod1, sod2, gpx1 and cat) were expressed equally in all embryos regardless of mother’ s glycemic status, they have not been tested in vitro. Our preliminary study indicates strong down regulation of neuroD1 expression in embryos of diabetic mothers. Since NeuroD1 transcription factor is required during development of the central as well as the peripheral nervous system, consequences of its loss should be investigated further. Since we did not observe the similar effect in vitro, higher glucose concentrations for culturing embryoid bodies should be applied. Morover, analysis of the protein expression is needed.

diabetes; embrionic stem cells; NOD mice

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