FIGURE 4

Expression of sox17, sox32, cdh1, and cdh2. (A) GFP is expressed in endodermal cells and DFCs at 80% epiboly in Tg (sox17:GFP) transgenic line (DFCs are circled with a white square). (B) Expression of sox17 was examined using WISH at 80% epiboly. b1, normal sox17 at 28.5°C (100%, n = 67); b2, normal sox17 at 22°C (42.8%, n = 74, p < 0.002); b3 dispersed expression of sox17 at 22°C (57.2%, n = 74, p < 0.001). DFCs (white arrow). (C) Number DFCs and migration situation of DFCs. C1, DFCs with normal migration at 28.5°C (91.6%, n = 12); c2, DFCs with normal migration at 22°C (41.7%, n = 12, p < 0.003); c3, dispersed DFCs at 22°C (38.3%, n = 12, p < 0.002); c4: dispersed DFCs at 22°C (20%, n = 12, p < 0.004). Scale bar, 40 μm. (D) Statistical analysis of DFC number. n = 12; ns, not significant (E) Expression of sox32 at 80% epiboly. e1, normal sox17 at 28.5°C (98.9%, n = 64); e2, normal sox17 at 22°C (63.2%, n = 70, p < 0.01); e3, dispersed expression of sox17 at 22°C (36.8%, n = 70, p < 0.03). DFCs (white arrow). (F) Expression of cdh1 at 80% epiboly. f1, normal cdh1 at 28.5°C (97.3%, n = 107); f2, decreased expression of cdh1 in the DFC field at 22°C (73.5%, n = 111, p < 0.001); f3, weak expression of cdh1 in the DFC field at 22°C (26.5%, n = 111, p < 0.01). DFC field (white circle). (G) Percentages of normal, decreased, and weak expression of cdh1 in embryos treated at 28.5°C or 22°C. A statistically significant difference (p < 0.01) could be seen between the embryos treated at 28.5 vs 22°C. (H) Expression of cdh2 in 80% epiboly. h1, normal cdh2 at 28.5°C (97%, n = 122); h2, decreased expression of cdh1 in the DFC field at 22°C (68.4%, n = 127, p < 0.001); h3, weak expression of cdh1 in the DFC field at 22°C (31.6%, n = 127, p < 0.003). DFC field (white circle). (I) Percentages of normal, decreased, and weak expression of cdh2 in embryos treated at 28.5°C or 22°C. A statistically significant difference (p < 0.003) could be seen between the embryos treated at 28.5 vs 22°C. (J) Percentage of normal looping, reversed looping, and no looping of the heart in embryos treated at 22°C (as control) and embryos treated at 22°C plus overexpression of cdh1/cdh2 mRNA in DFCs. A statistically significant difference (p < 0.05) could be seen between control embryos and embryos injected with cdh1/cdh2 mRNA. (K) Percentage of normal liver, reversed liver, and liver bifida in embryos treated at 22°C and embryos treated at 22°C plus overexpression of cdh1/cdh2 mRNA in DFCs. A statistically significant difference (p < 0.05) could be seen between them. (L) Mechanism diagram. Ns, not significant; “*“p < 0.05; “**“p < 0.03; and “***“p < 0.001.