PMC

(A). Representative images of different genotypes larvae by EdU staining. Arrows indicated the EdU (red) positive α-cell (green). The images are confocal projections; scale bar indicates 10 μm. (B). Quantification of EdU labeled α-cells. n= 7–10. All the values shown are means ± S.E., * P<0.05 by One-way ANOVA.

(A), Representative images of the principal islet of Tg(gcga:GFP), gcgra−/−;Tg(gcga:GFP), gcgrb−/−;Tg(gcga:GFP), gcgra−/−;gcgrb−/−;Tg(gcga:GFP) at 7 dpf. The images are confocal projections; scale bars indicate 10μm. (B), Quantification of the α-cell number in different genotypes of zebrafish at 7 dpf. N=8–12. ** P<0.01, *** P<0.001 by One-way ANOVA. (C), α-cell number in Tg(gcga:GFP), and gcgra−/−;gcgrb−/−;Tg(gcga:GFP) from 3 dpf to 8 dpf. n=8–16. The values shown are means ± S.E., ** P<0.01, *** P<0.001 by t-test.

(A–C). Real-time PCR analysis of gcga (A), gcgb (B), and insulin (C) mRNA in wildtype and mutant fish. (D). Total glucagon content in wildtype and mutant fish. (E). Total free glucose content in wildtype and mutant fish. (F–H) Quantitative RT-PCR analysis of the expression of key gluconeogenic genes pck1 (F), pck2(G) and g6pca.1 (H). Expression of β-actin was used as an internal control for all real-time PCR experiments. All the values shown are means ± S.E. from three independent experiments. * P<0.05, ** P<0.01, *** P<0.001 by One-way ANOVA.

(A–F) Schematic representations of TALEN targets and mutant alleles. Both gcgra (A) and gcgrb (D) consists of 13 exons (filled box). The TALEN pairs target exon 3 and exon 2, respectively. The sequences of the target regions are aligned to the selected alleles for gcgra (B) and gcgrb (E). The location of the truncation in the mutant gene product for gcgra (C) or gcgrb (F) are indicated with a dotted line. (G–H) Genotyping of wild, heterozygous and homozygous of gcgra (G) and gcgrb (H). (I). Real-time PCR analysis of gcgra and gcgrb mRNA levels. The results were from three independent experiments. All the values shown are means ± S.E., * P<0.05, *** P<0.001 by One-way ANOVA.

(A) Phylogenetic analysis of glucagon receptors. Full-length amino acid sequences of various receptors for glucagon-like peptides were analyzed using the neighbor-joining method. Posterior probability values (1,000 runs) for branches of the tree are given on nodes. GIPR = glucose-dependent insulinotropic polypeptide receptor; GLP1R, glucagon-like peptide 1 receptor; GLP2R, glucagon-like peptide 2 receptor. The prefixes used are: h, human; m, mouse; r, rat; c, chicken; x, Xenopus laevis; zf, zebrafish; gf, goldfish; lf, lungfish. *, predicted sequences. (B) Conserved synteny of gcgra and gcgrb to human GCGR. Zebrafish gcgra and gcgrb are located on Chromosome 3 and Chromosome 1, respectively. They share several syntenic genes of human GCGR on Chromosome 17.

(A) RT-PCR analysis gcgra and gcgrb mRNA at different stages of development. The upper panel is gcgra mRNA, the middle panel is gcgrb mRNA and lower panel is β-actin mRNA. The developmental stages are shown at the top as days (d) or weeks (wk) post fertilization. nc, negative control. (B) RT-PCR analysis gcgra and gcgrb mRNA in different tissues in adult fish. Total RNA was isolated from Liver (Li), intestine (In), muscle (Mu), heart (He) and brain (Br) from adult zebrafish. nc, negative control. (C–E), Dose-response curves of zebrafish and mouse glucagon receptors to zebrafish and mouse glucagon peptides as measured by cAMP-dependent CRE-luciferase expression. HEK293T cells were co-transfected with pCRE-luc and expression vector pcDNA3.1(+) containing zebrafish glucagon receptor-a (Gcgra) (C), zebrafish glucagon receptor-b (Gcgrb) (D) and mouse glucagon receptor (GCGR) (E). The cells were exposed to different concentrations of zebrafish glucagon-a (Gcga), glucagon-b (Gcgb) and mouse glucagon (mGCG) peptides 48 hours after transient transfection. Each data point is the mean of triplicates.