Knock-out of ADCY6 cause heart defects in zebrafish. a Bright-field images showing the morphology of 2 dpf uninjected, scramble, adcy6a and adcy6b F0 crispant zebrafish. Scale bars, 0.5 mm. b mRNA expression analysis of myl7 in 2dpf crispant hearts. Upper panels of (i) and (ii) imaged from control larvae. Lower panels imaged from (i) adcy6a and (ii) adcy6b crispants. Scale bars, 50 µm. c, d Quantification of cardiac defects observed in 2 dpf cadcy6a and dadcy6b crispants upon mRNA expression analysis of myl7. e–f Proportion of heart phenotypes observed in eadcy6a and fadcy6b crispants. Numbers central within bars indicate number of larvae in each classification. g–h Normalized heart rate measurements in beats per minute (bpm) analyzed at 2 dpf in gadcy6a and hadcy6b crispants. Two-way ANOVA (c, d), ordinary one-way ANOVA (g) and Kruskal–Wallis test (h) used for statistical analysis. Asterisk indicate P values: ***p < 0.001. n.s.: not significant

Vipa mRNA and peptide are detected in zebrafish testis. In situ hybridization using vipa antisense riboprobe (A, B) and sense riboprobe (C) on sections from WT testis. Arrows point to cells where the positive signal was observed with the antisense riboprobe. Immunostaining of WT (D, E) and vipa?/? (F) testis with anti-Vipa precursor, and WT with preimmune serum (G). LC, Leydig cells; Ps, prespermatogonia; Spc I, primary spermatocytes; Spg, spermatogonia; Sptd, spermatids.

Phylogenetic analysis of Glul across diverse species and the expression patterns of the Glul gene family in zebrafish. (A) The MEGA software (Version 11.0.13) was utilized to construct a phylogenetic tree based on amino acid sequences, encompassing a diverse range of species including H. sapiens, P. troglodytes, M. mulatta, C. lupus, B. taurus, M. musculus, R. norvegicus, G. gallus, D. rerio, D. melanogaster, A. gambiae, C. elegans, S. cerevisiae, K. lactis, E. gossypii, S. pombe, N. crassa, A. thaliana, O. sativa, and X. tropicalis. Branches of the same color are closer. Whole-mount in situ hybridization (WISH) was performed using glula, glulb, and glulc probes in wild-type zebrafish. (B) The results demonstrated that the expression of the glula gene was observed specifically in the otic vesicle. Green arrowheads indicate otic vesicle. Boxes indicate the magnified regions. (C) while the glulb gene exhibited expression in both the otic vesicle and lateral line. Green arrowheads indicate otic vesicle and neuromasts. Boxes indicate the magnified regions. (D) Conversely, no significant signal was detected for the glulc gene.

egr3 triggers EdC migration and Alcama expression(A) 3D reconstruction of representative hearts from control [Tg(fli1a:Gal4);Tg(UAS:Kaede)] and endothelial-specific egr3-overexpressing [Tg(fli1a:Gal4);Tg(UAS:Kaede);Tg(UAS:egr3-p2a-dTomato)] 72 hpf larvae; magnified regions show single z planes of the atrium from control and endothelial-specific egr3-overexpressing 72 hpf larvae; yellow arrowheads point to Tg(UAS:egr3-p2a-dTomato)+ atrial EdCs in the adjacent ECM. (B) Confocal images of representative AV VIC quantification through the spot render function in Imaris; VICs (magenta spots). (C) Quantification of EdCs present in the ECM per cardiac region in 72 hpf endothelial-specific egr3-overexpressing larvae; n = 9 control and 22 egr3 OE larvae, n = 0 and 0.14 EdCs (ventricle), n = 5 and 13.36 EdCs (AV canal), n = 0 and 0.59 EdCs (atrium). (D) Confocal images of representative hearts from control [Tg(fli1a:Gal4);Tg(UAS:Kaede)] and endothelial-specific egr3-overexpressing [Tg(fli1a:Gal4);Tg(UAS:Kaede);Tg(UAS:egr3-p2a-dTomato)] 54 hpf embryos immunostained for Alcama; magnified regions show Alcama+ cells in the AV canal of the control heart and in the atrium of the Tg(UAS:egr3-p2a-dTomato) heart. (E) Quantification of Alcama+ EdCs per cardiac region of control and endothelial-specific egr3-overexpressing embryos at 54 hpf; n = 13 control and 14 egr3 OE embryos, n = 0.07 and 3.28 EdCs (ventricle), n = 31.77 and 42.86 EdCs (AV canal), n = 0.38 and 4.71 EdCs (Atrium); three independent experiments. (F) Confocal images of representative hearts from control Pt(egr3:Gal4-VP16) and egr3-driven nr4a2b-overexpressing [Pt(egr3:Gal4-VP16); Tg(UAS:nr4a2b-p2a-dTomato)] egr3−/− larvae at 72 hpf. (G) Quantification of EdCs present in the AV canal ECM of control and nr4a2b-overexpressing egr3−/− larvae at 72 hpf; n = 16 and 15 larvae; two independent experiments. (C), (E), and (G) Student’s t test, mean ± SEM. Scale bars, 20 μm.