Fig. 4: Bicc1 indirectly regulates nodal1 expression via Gdf3 signaling.

A Animal cap assay using a luciferase reporter mRNA which contained gdf3 3′-UTR sequences. Translation of gdf3 reporter was efficiently blocked by co-injecting bicc1 mRNA. N represents the number of independent experiments. A pool of 10 animal caps was analyzed per experiment and treatment. The result from reporter mRNA alone served as a reference and was set to 100% RLU. Relative values of single experiments are depicted as blue dots. Data of three experiments are presented as mean value (bar) ±standard deviation (error bar, SD). Statistical analyses were done with a one-sided Student’s t test for two independent means using the values of three individual experiments. B gdf3 mRNA was not affected by bicc1 LoF. C Quantification of gdf3 expression in bicc1 morphants at the LRO margin. D gdf3 GoF rescues nodal1 expression in bicc1 morphants. Representative nodal1 staining in left sLRO cells is shown for control (co), bicc1 morphant, and rescued specimens. E Quantification of the bicc1 MO rescue of nodal1 expression by gdf3. F, G Left-asymmetric pitx2 expression (arrowhead) is restored in bicc1 morphants by co-injecting gdf3 mRNA. MO pmol/embryo: bicc1 SBMO (L and S, each 1). Asterisks in B and D mark injected side. Numbers (n) in C, E, and G represent analyzed specimens from more than independent experiments. Statistical analyses were done with one-sided Pearson’s chi-square test, which was adjusted for multiple comparisons by Bonferroni–Holm (C, E) or Bonferroni (G). n.s. not significant, p > 0.05; ** highly significant p < 0.01; ***, very highly significant p < 0.001. p-values, mean values, SD and listing of individual experiments can be found in the source data file. Scale bars in B and D represent 100 µm and in F 1 mm. RLU relative luciferase units, st. stage, a anterior, l left, r right, p posterior, d dorsal, v ventral.