Foxc1 mutants display loss of lefty2 expression independent of changes to other left–right patterning genes. (A) In situ hybridization with southpaw (spaw) revealed no difference in the prevalence of normal left-sided expression (normal = blue, absent = black, bilateral = green, and right = red) in foxc1 mutants compared to controls (p > 0.1, Fisher’s exact test). (B) Conversely, lefty2 expression was significantly altered in foxc1 mutants. Normal left-sided lefty2 expression was absent more frequently in foxc1a−/− and double foxc1a−/−; foxc1b−/− homozygotes (p = 0.012 and 0.028, respectively) and trended to be absent in foxc1b−/− homozygotes without reaching statistical significance (p = 0.061, Fisher’s exact test) (normal = blue and absent = black). (C) lefty2 expression was significantly abnormal, with the overexpression of foxc1a or foxc1b (p = 0.0007 and 0.0030, respectively, ANOVA and Dunnett’s Test). (D) Analysis of the additional left–right patterning genes lefty1, pitx2c, and elvol6 revealed no differences between WT, and foxc1a−/−; foxc1b−/− double homozygotes. (* p < 0.05, ** p < 0.01, *** p < 0.001).
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