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Fig. 3: Bicc1-dependent dand5 and nodal1 expression in sLRO cells. A Absence of left LPM pitx2 expression in bicc1 morphants, unilaterally injected on the left, was rescued by parallel knockdown of dand5. Specificity of TBMO or SBMO was shown by co-injecting rescue mRNAs, i.e., mouse bicc1 or Xenopus bicc1, respectively. Note dand5 knockdown on the right efficiently induced pitx2 expression, as published. B Loss of dand5 mRNA at post-flow stages (st. 20) following left- and right-sided bicc1 SBMO injections. Controls (co) showed wt expression. dand5 expression was restored by co-injecting bicc1 rescue mRNA. Note enhanced dand5 staining in rescued specimens. C Quantification of dand5 expression after knockdown of bicc1. The effect was observed in the left and right sLRO cells. D, E Downregulation of nodal1 in bicc1 morphants. D Quantification of results. E Wt specimens show bilateral nodal1 mRNA. Left or right bicc1 SBMO injections reduced nodal1, which was restored by adding rescue bicc1 mRNA. MO pmol/embryo: bicc1 SBMO (L and S, each 1); bicc1 TBMO, (L and S, each 1); dand5 TPMO (0.5). Asterisks in B and E mark the injected side. Numbers (n) in A, C, and D represent analyzed specimens from more than three independent experiments. Statistical analyses were done with one-sided Pearson’s chi-square test, which was adjusted for multiple comparisons by Bonferroni (B) or Bonferroni–Holm (C, D). n.s. not significant p > 0.05; * significant, p < 0.05; ** highly significant p < 0.01; ***, very highly significant p < 0.001. p values and listing of individual experiments can be found in the source data file. st., stage. Scale bars in B and E represent 100 µm. st. stage, a anterior, l left, r right, p posterior. |
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Supplementary Figure 4. nodal1 transcription in sLRO cells requires Gdf3 signaling.
(A) nodal1 sLRO expression at stage 20 in controls (co) and gdf3 TBMO (0,3 pmol/embryo) unilaterally left injected specimens. Note reduced nodal1 staining on the targeted side.
(B) Quantification of nodal1 signals.
(C) pitx2 asymmetry in the LPM was lost when gdf3 MO was injected on the left.
(D) Quantification of pitx2 expression in co and gdf3 morphants.
Numbers (n) in (B and D) represent analyzed specimens from >3 independent experiments. Statistical analyses were done with a one-sided Pearson’s chi-square test. n.s., not significant; ***, very highly significant, p < 0.001; **, highly significant, p < 0.01. p-values and listing of individual experiments can be found in the source data file.Asterisk in (A) marks injected side. Scale bars represent in (A) represents 100 μm and in (C) 1 mm. st., stage; a, anterior; l, left; r, right; p, posterior; d, dorsal; v, ventral; |
