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<italic>alk5</italic> expression and function in zebrafish embryos and larvae.(A–B’) Confocal images showing TgBAC(alk5b:EGFP) expression (green/white) in 30 (A) and 52 (B) hpf zebrafish embryos. Expression is high in the neural tube (NT, B). (C–D’) in situ hybridization showing the expression of alk5b in 30 and 72 hpf animals. Arrowhead, gut; boxed areas shown in C’ and D’. (E) Schematics of wild-type and mutated Alk5 proteins. Black and white stars indicate the site of the corresponding mutations in alk5a and alk5b, respectively, and the black rectangle represents new sequence downstream of the mutation. (F) alk5a and alk5b mRNA levels in 30 hpf alk5+/+, alk5a-/- and alk5b-/- embryos; means ± SD; p values from Mann Whitney tests; average Ct values are listed in Supplementary file 2. (G–J) Brightfield images of 96 hpf alk5+/+, alk5a-/-, alk5b-/-, and alk5-/- larvae. Arrowheads, lack of swim bladder; arrow, pericardial edema. (K–L) Confocal images of OFT and connecting vessels in 78 hpf alk5+/+ and alk5-/- larvae (n > 30). Asterisk, absence of the VA in alk5 mutants. (M) Quantification of DA diameter in 56 and 96 hpf alk5+/+ and alk5-/- animals. The same animals were analyzed at the two different time-points; means ± SD. (N, O) Confocal images of 78 hpf alk5+/+ and alk5-/- hearts (n > 30). (P) Quantification of heart rate in 78 hpf alk5+/+ (n = 6) and alk5-/- (n = 11) larvae; means ± SD; p values from Mann Whitney test. SP- signal peptide, RD- receptor domain, TM- transmembrane domain, GS- glycine-serine rich domain, STK- serine-threonine kinase domain, A- atrium, V- ventricle, NT- neural tube, DA- dorsal aorta, PCV- posterior cardinal vein, VA- ventral artery, AA1- aortic arch 1. Scale bars: (A, A’, C-D’ G-J) 200 μm; (B, B’) 50 μm; (K–O) 20 μm.
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