Antisense miR-7 impairs insulin expression in developing pancreas and in cultured pancreatic buds.

MicroRNAs regulate gene expression by inhibiting translation or inducing target mRNA degradation. MicroRNAs regulate organ differentiation and embryonic development, including pancreatic specification and islet function. We showed previously that miR-7 is highly expressed in human pancreatic fetal and adult endocrine cells. Here we determined the expression profile of miR-7 in the mouse-developing pancreas by RT-PCR and in situ hybridization. MiR-7 expression was low between embryonic days e10.5 and e11.5, then began to increase at e13.5 through e14.5, and eventually decreased by e18. In situ hybridization and immunostaining analysis showed that miR-7 colocalizes with endocrine marker Isl1, suggesting that miR-7 is expressed preferentially in endocrine cells. Whole-mount in situ hybridization shows miR-7 highly expressed in the embryonic neural tube. To investigate the role of miR-7 in development of the mouse endocrine pancreas, antisense miR-7 morpholinos (MO) were delivered to the embryo at an early developmental stage (e10.5 days) via intrauterine fetal heart injection. Inhibition of miR-7 during early embryonic life results in an overall downregulation of insulin production, decreased ß-cell numbers, and glucose intolerance in the postnatal period. This phenomenon is specific for miR-7 and possibly due to a systemic effect on pancreatic development. On the other hand, the in vitro inhibition of miR-7 in explanted pancreatic buds leads to ß-cell death and generation of ß-cells expressing less insulin than those in MO control. Therefore, in addition to the potential indirect effects on pancreatic differentiation derived from its systemic downregulation, the knockdown of miR-7 appears to have a ß-cell-specific effect as well. These findings suggest that modulation of miR-7 expression could be utilized in the development of stem cell therapies to cure diabetes.

Insulin2 gene (Ins2) transcription by NOD bone marrow-derived cells does not influence autoimmune diabetes development in NOD-Ins2 knockout mice.

Insulin is a critical autoantigen for the development of autoimmune diabetes in non-obese diabetic (NOD) mice. About 80% of NOD females and 30-40% of NOD males develop diabetes. However, Insulin2 (Ins2) knockout NOD mice develop autoimmune diabetes with complete penetrance in both sexes, at an earlier age, and have stronger autoimmune responses to insulin. The severe diabetes phenotype observed in NOD-Ins2-/- mice suggests that lack of Ins2 expression in the thymus may compromise immunological tolerance to insulin. Insulin is a prototypical tissue specific antigen (TSA) for which tolerance is dependent on expression in thymus and peripheral lymphoid tissues. TSA are naturally expressed by medullary thymic epithelial cells (mTEC), stromal cells in peripheral lymphoid tissues and bone marrow (BM)-derived cells, mainly CD11c(+) dendritic cells. The natural expression of TSA by mTEC and stromal cells has been shown to contribute to self-tolerance. However, it is unclear whether this also applies to BM-derived cells naturally expressing TSA. To address this question, we created BM chimeras and investigated whether reintroducing Ins2 expression solely by NOD BM-derived cells delays diabetes development in NOD-Ins2-/- mice. On follow-up, NOD-Ins2-/- mice receiving Ins2-expressing NOD BM cells developed diabetes at similar rates of those receiving NOD-Ins2-/- BM cells. Diabetes developed in 64% of NOD recipients transplanted with NOD BM and in 47% of NOD mice transplanted with NOD-Ins2-/- BM (P = ns). Thus, NOD-Ins2-/- BM did not worsen diabetes in NOD recipients and Ins2 expression by NOD BM-derived cells did not delay diabetes development in NOD-Ins2-/- mice.