Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development

Abstract

Several vertebrate microRNAs (miRNAs) have been implicated in cellular processes such as muscle differentiation, synapse function, and insulin secretion. In addition, analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis. Nonetheless, only a few loss-of-function phenotypes for individual miRNAs have been described to date. Here, we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic development. Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and temporally knock down miRNAs. Morpholinos can block processing of the primary miRNA (pri-miRNA) or the pre-miRNA, and they can inhibit the activity of the mature miRNA. We used this strategy to knock down 13 miRNAs conserved between zebrafish and mammals. For most miRNAs, this does not result in visible defects, but knockdown of miR-375 causes defects in the morphology of the pancreatic islet. Although the islet is still intact at 24 hours postfertilization, in later stages the islet cells become scattered. This phenotype can be recapitulated by independent control morpholinos targeting other sequences in the miR-375 precursor, excluding off-target effects as cause of the phenotype. The aberrant formation of the endocrine pancreas, caused by miR-375 knockdown, is one of the first loss-of-function phenotypes for an individual miRNA in vertebrate development. The miRNA knockdown strategy presented here will be widely used to unravel miRNA function in zebrafish.

Role of islet microRNAs in diabetes: which model for which question?

“…These mice display hyperglycaemia and hyperglucagonaemia and, if crossed with ob/ob mice, a model of obesity and insulin resistance, they develop a severe diabetic state because of the inability of beta cells to expand and compensate for the increased insulin needs. These, together with other studies [16][17][18][19], highlight the central role played by miR-375 in endocrine pancreas development and in the regulation of insulin gene expression and release.…”

MiR-375 frequently downregulated in gastric cancer inhibits cell proliferation by targeting JAK2

“…MiR-375 gene knockout mice revealed that miR-375 is not only required for normal glucose homeostasis but also influences pancreatic α-and β-cell mass [24]. Inhibition of miR-375 maturation with morpholinos in zebrafish has revealed an essential role for miR-375 in the formation of insulin-secreting pancreatic islet [25]. By far, functional studies of miR-375 appear to be restricted to the regulation of pancreas.…”

Regulation of zebrafish fin regeneration by microRNAs

“…S1). The increased rate of regeneration at this temperature allows disruption of gene function by morpholinos, which can persist for up to 72 h (31). Because the array data were obtained from fish maintained at 27°C, we first verified, by northern blot analysis, that the patterns of expression for differentially expressed miRNAs, as determined using arrays, were similarly altered when regeneration proceeded at 33°C Microarrays were performed to identify differentially expressed miRNAs during caudal fin regeneration.…”

“…2B). We also co-injected antisense morpholinos complementary to miR-203 (i.e., miR-203 MO ) that function to block the activity of endogenous miR-203 and thus should lead to increased fluorescence (31,33). As a control, GFP lacking the lef1 3Ј UTR also was injected into single-cell zebrafish embryos with and without exogenous miR-203 and miR-203 MO (Fig.…”