Abstract Body (Do not enter title and authors here): Introduction: Pregestational diabetes is an established risk factor for congenital heart disease (CHD), including heterotaxy syndrome, which results from abnormal left-right (L-R) axis. The mechanisms by which hyperglycemia disturbs the L-R axis and gives rise to a variety of heterotaxy defects have remained unknown.
Methods: Gene expression in mouse embryos of dams with streptozotocin-induced diabetes was examined by in situ hybridization, reporter, and RNA-sequencing analyses. Intracellular pH (pH_i) was investigated with a fluorescent indicator.

Results: Various Pitx2 expression patterns indicative of disruption of L-R axis formation were apparent in embryos of diabetic dams. Expression of Nodal at the node, which triggers a Nodal-Pitx2 expression cascade during L-R axis formation, showed marked regression. This regression was similar to that apparent in Wnt3a^–/– embryos, and canonical Wnt signaling was found to be downregulated in embryos of diabetic dams. pH_i was lowered in the primitive streak of such embryos, leading to the suppression of Wnt signaling. Maternal vitamin A intake increased the incidence and diversity of L-R axis defects in embryos of diabetic dams, with dysregulation of retinoic acid metabolism being apparent in these embryos and in Wnt3a^–/– embryos.

Conclusions: Hyperglycemia downregulates canonical Wnt signaling by lowering pH_i, which not only impairs L-R axis formation but also dysregulates retinoid metabolism. Consequently, embryos of diabetic dams become vulnerable to fluctuation of maternal vitamin A intake, resulting in diverse L-R axis defects. These findings provide insights into the etiology of CHD and suggest the importance of diet for prevention of heterotaxy during diabetic pregnancy.