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Influence of Hyperglycaemoa on 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. Some malformations are presumably due to teratogenic influence of high glucose concentrations which may act by altering expression of developmental genes. Our intention was to test whether and to what extent hyperglycemia influences the expression of Pax3 and NeuroD1 genes encoding transcription factors involved in neural embryonic development in mice. For that purpose we recovered embryos on embryonic day 9.5 (E9.5) from two healthy and two diabetic NOD (non-obese diabetic) female mice. Some animals of NOD strain spontaneously develop type I diabetes. Preliminary results were obtained by measuring gene expression by semi quantitative RT-PCR performed on RNA isolated from individual embryos. NOD mice were mated and embryos from two diabetic and two non-diabetic mothers 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 assay (ThermoTrace). RNA was extracted from individual embryos from each mother by RNeasy Mini Kit (Qiagen). Genomic DNA was removed by treatment with RNase-Free DNase Set (Qiagen). Total RNA (0.5 μ g) was reverse transcribed by Super Sript II Reverse Transcriptase (Invitrogen) after treatment with DNase I, Amplification Grade (Invitrogen). PCR was performed by using HotStarTaq DNA Polymerase (Qiagen) and sets of specific primers representing Pax3, NeuroD1 and Tbp (housekeeping). Pax3 gene is extremely unevenly expressed in individual embryos, but there is no correlation between Pax3 gene expression and diabetic condition of mothers or embryo morphology (Fig. 1, 2 ABCD). In contrast, NeuroD1 gene display dramatic down regulation in embryos of the diabetic mother with the highest glucose value (20.1 mM) (Fig. 1, mother C). On the other hand, in embryos of the mother with milder hyperglycemia (15 mM) (Fig. 1 mother D), NeuroD1 expression is only slightly decreased in comparison to embryos of non-diabetic mothers with normal glucose values (6.6 mM) (Fig. 1, mother A and B). As well as Pax3, expression level of NeuroD1 does not stand in correlation with the external morphology of embryos (Fig. 1, 2 ABCD). In contrast to results of previous experiments performed on ICR mice, Pax3 expression in NOD mice we used is not extremely reduced / lost by the E9.5 in embryos exposed to hyperglycemia during embryonic development. It could be that impact of hyperglycemia on Pax3 expression is strain-specific, since it is known that different strains differ substantially in response to maternal diabetes. Furthermore, diabetes in ICR mice was chemically induced with the drug streptozotocin, while development of diabetes in NOD mice depends on genetic background. In the first case there is a possibility that streptozotocin has an influence on Pax3 expression. In the second case, genetic factors that predispose NOD model to type I diabetes could contribute to altered Pax3 expression independently of maternal hyperglycemia. It is also possible that variability of Pax3 expression that we observed might be due to asynchronous development of embryos in utero, as the transcription of Pax3 begins around E9.5 (exactly the age of analyzed embryos). Strong down regulation of NeuroD1 in diabetic pregnancy is potentially very interesting result never observed before. Studies of NeuroD1 null-mutants revealed essential role of that transcription factor in differentiation of pancreatic beta cells, proper insulin expression and function of Langerhans islets. These animals develop severe diabetes and die perinatally. We intent to investigate consequences of diminished NeuroD1expression observed in E9.5 embryos on further pancreatic development (particularly between E14.5 and 17.5, a period of major expansion of the β cell population). Another interesting finding in neuroD1 deficient mice is that they do not display any obvious brain abnormalities although NeuroD1 is expressed abundantly in the developing nervous system. We intent to analyze the influence of NeuroD1 loss on neural development and potential malformations in animals with diabetes-prone genetic background.

diabetes; embryonic stem cells

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