(A-F) Representative images of embryos injected with control tdTomato or sox4 mRNA. (A-B) Representative images of the head were taken at 72 hpf from the ventral side. (C-F) Lateral views of body showing the somites. Insets in (C) and (D) were enlarged in (E) and (F). (G) Quantification of the percentage of cyclopia in embryos injected with different combinations of control and sox4 mRNA. 1.0 ng/embryo Td-tomato: 0.76± 1.31%, n= 1/108; 0.2 ng sox4a/b + 0.6 ng Td-tomato/embryo: 9.35± 1.17%, n= 13/139; 0.3 ng sox4a/b + 0.4 ng Td-tomato/embryo: 17.03± 3.69%, n= 11/67; 0.4 ng sox4a/b + 0.2 ng Td-tomato/embryo: 25.56± 2.20%, n= 25/97. (H) qPCR analysis of ihhb and shha expression in control and sox4 overexpressing heads at 18 and 24 hpf. In sox4 mRNA injected embryos, ihhb expression was significantly decreased by 1.61-fold at 18 hpf (Student's t-test, P<0.05) and 1.51-fold at 24 hpf (Student's t-test, P=0.057). Shha expression was also significantly reduced following sox4 mRNA injection at both 18 and 24 hpf (by 1.82-fold and 3.57-fold, respectively; Student's t-test, P<0.05).