Laterality defects of the brain and heart in zebrafish embryos. (A) Ventra lviews of the parapineal (arrow) and the myocardium in 48 hpf Tg(foxd3:EGFP/myl7:EGFP) embryos (a–e) visualized using 3D SPIM, which revealed their laterality defect at the cellular level. 10× lens; 1.5× zoom; scale bar, 50 µm. (B) Dorsal views of left-right asymmetry of the pineal complex (a–d) and ventral views of the heart (a’–d’) were imaged at 4 days post-fertilization using a fluorescent stereomicroscope. Scale bar, 50 µm. (C) Embryos treated with and without cold shock at 22.5°C (n = 226) and 28.5°C (n = 795), respectively, were examined for their laterality defects of the parapineal (arrow) and cardiac looping at 72hpf. LD, left parapineal(L)and dextral heart looping (D); LL, left parapineal(L)and leftward heart looping (L); RD, right parapineal (R) and dextral heart looping (D); RL, right parapineal (R) and leftward heart looping (L); NU, no parapineal (N) and unloop heart (U); pp, parapineal; R-L, right-left sides; L-R, left-right sides; A, atrium; V, ventricle. *P< .05, **P< .01, and ***P< .001 by Student’s t-test.

Laterality defects of the brain and heart in zebrafish embryos. (A) Ventra lviews of the parapineal (arrow) and the myocardium in 48 hpf Tg(foxd3:EGFP/myl7:EGFP) embryos (a–e) visualized using 3D SPIM, which revealed their laterality defect at the cellular level. 10× lens; 1.5× zoom; scale bar, 50 µm. (B) Dorsal views of left-right asymmetry of the pineal complex (a–d) and ventral views of the heart (a’–d’) were imaged at 4 days post-fertilization using a fluorescent stereomicroscope. Scale bar, 50 µm. (C) Embryos treated with and without cold shock at 22.5°C (n = 226) and 28.5°C (n = 795), respectively, were examined for their laterality defects of the parapineal (arrow) and cardiac looping at 72hpf. LD, left parapineal(L)and dextral heart looping (D); LL, left parapineal(L)and leftward heart looping (L); RD, right parapineal (R) and dextral heart looping (D); RL, right parapineal (R) and leftward heart looping (L); NU, no parapineal (N) and unloop heart (U); pp, parapineal; R-L, right-left sides; L-R, left-right sides; A, atrium; V, ventricle. *P< .05, **P< .01, and ***P< .001 by Student’s t-test.

Cardiac looping defects and hypoplastic ventricle. (A) 3D SPIM imaging of the developing endocardium of Tg(fli1:EGFP) zebrafish embryos revealed cardiac defects in 50 hpf embryos from multiple in-crosses and cold shock: D-loop (a), incomplete loop (b), L-loop (c), and unloop (d) hearts. A, atrium; V, ventricle. Scale bar, 50 µm. (B) Total cell number in the endocardium of Tg(fli1:EGFP) zebrafish embryos at 50 hpf was counted using the 3D images (n = 6 of embryos with D-loop, n = 17 of embryos with laterality defects). Endocardial ventricles show more severe hypoplasia in the experimental embryos (E) compared with that in controls (C). Values are presented as mean ± SEM of total cell numbers. **P< .001 by two-sample Student’s t-test.

Cardiac looping defects and hypoplastic ventricle. (A) 3D SPIM imaging of the developing endocardium of Tg(fli1:EGFP) zebrafish embryos revealed cardiac defects in 50 hpf embryos from multiple in-crosses and cold shock: D-loop (a), incomplete loop (b), L-loop (c), and unloop (d) hearts. A, atrium; V, ventricle. Scale bar, 50 µm. (B) Total cell number in the endocardium of Tg(fli1:EGFP) zebrafish embryos at 50 hpf was counted using the 3D images (n = 6 of embryos with D-loop, n = 17 of embryos with laterality defects). Endocardial ventricles show more severe hypoplasia in the experimental embryos (E) compared with that in controls (C). Values are presented as mean ± SEM of total cell numbers. **P< .001 by two-sample Student’s t-test.

Situs solitus, situs inversus, and situs ambiguus in zebrafish visceral organ development (A) Whole mount double in situ hybridization reveals laterality of the developing liver (arrow) using a foxa3 RNA probe (purple) in the dorsal view of control (a) and experimental embryos (b–d) and the cardiac looping using the myocardial marker myl7 (brown) in the ventral view of control (a’) and experimental embryos (b’–d’). A, atrium; V, ventricle. Scale bars, 150 µm. (B) Ventral views of 6 dpf larvae stained with Streptavidin-Cy3 (Strep-Cy3) to visualize situs solitus (a), situs inversus (b), and situs ambiguus (c) of the heart (black arrow), liver (white arrow) and gut (yellow arrow). Scale bar, 150 µm.

Disruption of the nodal signalling and left-right organizer development in zebrafish embryos in the experimental conditions of over-crosses and temporary cold shock during early development. (A) In situ hybridization reveals defect in nodal signalling. Asymmetrical expressions of dand5, spaw and lft2 are often randomized in the experimental embryos (b-d, b’-d’, and b”-d”) compared to controls (a, a’, and a”) respectively. B, bilateral; L, left-sided; R, right-sided; S, symmetrical. Scale bars, 150 µm. (B) The experimental condition causes reduced size of the Kuffer’s vesicle and lower number of cilia. (a-c) Confocal imaging showing primary cilia (green) labelled with an anti-acetylated α-tubulin (Ac-a-tubulin) antibody, Kuffer’s vesicle (yellow dotted circle), and the nuclei of neighbouring cells (red, SYTO17 staining). Scale bar, 20 µm. (d-f) Organizer size, cilia number and the cilia length were compared between the control (C) and experimental embryos (E). Statistical difference between groups was evaluated by the Student’s t-test.­­ **P< .0005.

Disruption of the nodal signalling and left-right organizer development in zebrafish embryos in the experimental conditions of over-crosses and temporary cold shock during early development.(A)In situ hybridization reveals defect in nodal signalling. Asymmetrical expressions of dand5, spaw and lft2 are often randomized in the experimental embryos (b-d, b’-d’, and b”-d”) compared to controls (a, a’, and a”) respectively. B, bilateral; L, left-sided; R, right-sided; S, symmetrical. Scale bars, 150 µm. (B) The experimental condition causes reduced size of the Kuffer’s vesicle and lower number of cilia. (a-c) Confocal imaging showing primary cilia (green) labelled with an anti-acetylated α-tubulin (Ac-a-tubulin) antibody, Kuffer’s vesicle (yellow dotted circle), and the nuclei of neighbouring cells (red, SYTO17 staining). Scale bar, 20 µm. (d-f) Organizer size, cilia number and the cilia length were compared between the control (C) and experimental embryos (E). Statistical difference between groups was evaluated by the Student’s t-test.­­ **P< .0005.

Disruption of the nodal signalling and left-right organizer development in zebrafish embryos in the experimental conditions of over-crosses and temporary cold shock during early development.(A)In situ hybridization reveals defect in nodal signalling. Asymmetrical expressions of dand5, spaw and lft2 are often randomized in the experimental embryos (b-d, b’-d’, and b”-d”) compared to controls (a, a’, and a”) respectively. B, bilateral; L, left-sided; R, right-sided; S, symmetrical. Scale bars, 150 µm. (B) The experimental condition causes reduced size of the Kuffer’s vesicle and lower number of cilia. (a-c) Confocal imaging showing primary cilia (green) labelled with an anti-acetylated α-tubulin (Ac-a-tubulin) antibody, Kuffer’s vesicle (yellow dotted circle), and the nuclei of neighbouring cells (red, SYTO17 staining). Scale bar, 20 µm. (d-f) Organizer size, cilia number and the cilia length were compared between the control (C) and experimental embryos (E). Statistical difference between groups was evaluated by the Student’s t-test.­­ **P< .0005.

Histopathological assessment of heterotaxy in juvenile zebrafish. (A) Haematoxylin and eosin–stained transverse paraffin sections of 5-week-old zebrafish showing situs solitus, situs inversus, and situs ambiguus of the heart (a–c) and other visceral organs (a’–c’) in each fish. Atrium tip, red arrowhead; intestinal bulb, red arrow; two anterior and posterior intestines, black arrows. Malrotated guts (arrows) are shown in the fish. Scale bars, 100 µm. Laterality of visceral organs in the selected fish are summarized in Table 1. (B) L-looped heart in 48 hpf embryo developed to dextrocardia (a) or mesocardia (b) at 5 weeks of age, while unlooped heart developed to mesocardia (c). Additionally, some experimental fish had transposition of the bulbo-ventricular valve (b, red arrowhead), non-apex ventricle (c, black arrowhead), and abnormal atrioventricular canal (c, black arrow). Scale bar, 100 µm. (C) Abnormal structure and laterality defect of the outflow tract (bulbus arteriosus–ventricle) valve (black arrow), blood flow defect in the outflow tract (red arrowhead), and ectopic membrane (red arrow) are shown in Fish #12. Scale bar, 50 µm. (D) Coarctation of the outflow tract (arrow) with defective smooth muscle layer (arrowhead) was observed in Fish #12. Scale bar, 50 µm.

Histopathological assessment of heterotaxy in juvenile zebrafish. (A) Haematoxylin and eosin–stained transverse paraffin sections of 5-week-old zebrafish showing situs solitus, situs inversus, and situs ambiguus of the heart (a–c) and other visceral organs (a’–c’) in each fish. Atrium tip, red arrowhead; intestinal bulb, red arrow; two anterior and posterior intestines, black arrows. Malrotated guts (arrows) are shown in the fish. Scale bars, 100 µm. Laterality of visceral organs in the selected fish are summarized in Table 1. (B) L-looped heart in 48 hpf embryo developed to dextrocardia (a) or mesocardia (b) at 5 weeks of age, while unlooped heart developed to mesocardia (c). Additionally, some experimental fish had transposition of the bulbo-ventricular valve (b, red arrowhead), non-apex ventricle (c, black arrowhead), and abnormal atrioventricular canal (c, black arrow). Scale bar, 100 µm. (C) Abnormal structure and laterality defect of the outflow tract (bulbus arteriosus–ventricle) valve (black arrow), blood flow defect in the outflow tract (red arrowhead), and ectopic membrane (red arrow) are shown in Fish #12. Scale bar, 50 µm. (D) Coarctation of the outflow tract (arrow) with defective smooth muscle layer (arrowhead) was observed in Fish #12. Scale bar, 50 µm.