Similar variant impaired β-cell function in zebrafish. (A) Schematic representation of a similar HNF1a-Q125ter variant in zebrafish generated by CRISPR/Cas9. Mutation was caused via a two-base-pair deletion at the CRISPR target site for HNF1a. (B) The transcription level of hnf1a in WT and hnf1a+/−; n = 50 larvae for each genotype. (C) Survival curves of WT and hnf1a+/− zebrafish from 0 h to 24 h. (D,E) Representative confocal images (D) and quantification (E) of the β-cell number from Tg(−1.2ins:H2BmCherry) and hnf1a+/−; Tg(−1.2ins:H2BmCherry) zebrafish larvae. Scale bar indicates 25 μm; n = 20–35 larvae for each genotype. (F) Total free glucose level of WT and hnf1a+/− larvae at 6 dpf. (G) qRT-PCR analysis of the expression of insa and insb mRNA levels in WT and hnf1a+/− larvae at 6 dpf. (H) Representative confocal images of Tg(ins:H2BmCherry); Tg(pdx1:GFP) at 6 dpf for WT and hnf1a+/−. β-cells expressed insulin (mCherry+) without pdx1 (GFP−) are shown by white arrows. Scale bar: 10 μm. (I) Quantification of fluorescence intensity for insulin in WT and hnf1a+/−. (J) Quantification of double-positive cells’ (mCherry+/GFP+) rate in WT and hnf1a+/− larvae at 6 dpf; n = 3 larvae for each genotype. (K) Representative images of GCaMP6s response in β cells of Tg(Ins:H2BmCherry); Tg(Ins:GCaMP6s) and hnf1a−/−; Tg(Ins:H2BmCherry); Tg(Ins:GCaMP6s) by 5 or 20 mM glucose ECS solution; the green signal is GCaMP6s. Scale bar: 10 μm. (L) RT-qPCR quantification of mRNA levels for insulin-secretion markers in WT and hnf1a+/− larvae at 6 dpf: abcc8, scl2a2, gck, kcnj11, and kcnh6. (M) RT-qPCR quantification of mRNA levels for β-cell maturation and differentiation markers in WT and hnf1a+/− larvae at 6 dpf: mafa, pdx1, nkx6.1, and pax6b. (N,O) Representative electron micrographs of β cells (N) and quantification of insulin granules (O) in β cells from WT and hnf1a+/− larvae at 6 dpf. (N,O) Transmission electron microscopy for insulin granules. Islet isolation from WT and hnf1a+/− larvae at 6 dpf. The dotted blue lines represent intact individual β cells. Bar scale: 2 μm. n = 3 intact individual β cells for each genotype. Results are represented as means with standard errors; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Student’s t-test. All experiments were performed at least three times, unless otherwise indicated. WT, wild type.
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