Chromatin immunoprecipitation assays using primary MEM cells from wild type embryos demonstrated that ALX3 binds to this region of the endogenous Foxo1 gene in the context of native chromatin in vivo when cells are incubated in high glucose (30 mM), but not in the presence of low glucose (2.8 mM) (Fig. 4I). Using electrophoretic mobility shift assays, binding of ALX3 to the target site of the Foxo1 promoter was only detected when primary MEM cells were cultured in high rather than low glucose concentrations (Fig. 4J). Consistent with these findings, glucose induced an increase in the levels of FOXO1 protein in cells from wild type embryos, but not in cells from Alx3-null embryos (Fig. 4E).