Whole-mount in situ hybridization (WISH) analysis against fam222 homologues and cardiac markers adprhl1 and myl7 in wt and fam222ba/bb dd-KO zfl at 48 hpf. (A) Expression of fam222 homologues at 48 hpf using three different probes against each zf fam222 gene: fam222ba (a-c), fam222bb (d-f), fam222aa (g-i). Staining in wt zfl (a,d,g) compared to fam222ba/bb dd-KO zfl (c,f,i). Blue WISH staining is apparent in the zf head region as well as in the pericardium region including the heart. Additional red staining of the heart was performed using a probe against cardiac adprhl1 (j). Double staining was realized in wt zfl (b,e,h) in order to test for overlay of fam222 in blue and adprhl1 in red. Scale bar at the bottom applies for whole row above: 500 µm. (B) WISH-analysis to examine heart laterality defects. Myl7 indicates cardiac looping in wt and fam222ba/bb dd-KO zfl at 48 hpf. Analysis of various ways of cardiac looping: d-loop (right, normal), no loop (mid), l-loop (left, inversed). Scale bar 100 µm. (C) Direction and amount of false directed heart looping in wt and fam222ba/bb dd-KO zfl (N = 6, n = 148–173 in each group, in total), no loop in purple, l-loop in lavender. Two-sided Chi2 test: p-value: 0.0017, ** = very significant. (D) Comparative analysis of whole-mount Tg(kdrl:EGFP) wt and Tg(kdrl:EGFP) fam222ba/bb dd-KO zfl at 48 hpf showed no difference in fluorescent in vivo imaging concerning morphological and cardiovascular development between both groups. Lateral view of whole-mount zfl in bright field (a,d). (b,e) Lateral view of whole-mount zfl in fluorescence mode (b,e). Lateral view of zoomed in cranial head & heart region of whole-mount zfl in fluorescence mode (c,f). Abbreviations: asterisks* = heart, AT = atrium, V = ventricle. Scale bar for (a,b,d,e): 500 µm. Scale bar for (c,f): 200 µm. (E) Comparison of heartbeats per minute reveals no difference between wt and dd-KO zfl (N = 3, n = 30 in each group). Median (bpm): wt: 115, dd-KO: 116.5. Unpaired two-tailed t-test: p-value: 0.938, ns = not significant.
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