Bicc1 responsive element (Bicc1RE) of the dand5 3′-UTR is required for LR asymmetry.A Uninjected control (co), m-tpMO, or d-tpMO-injected embryos showed left, bilateral, or absent pitx2 expression, respectively. Lateral views (left and right) of embryos are presented. Arrowheads mark pitx2-positive lateral plate mesoderm. B Quantification of pitx2 results of m-tpMO-treated specimens. C Quantification of pitx2 asymmetry by d-tpMO injections. Note administration of dand5 TPMO together with d-tpMOs restored wt pitx2 expression. D Diminished dand5 mRNA expression by left-sided and right-sided m-tpMO injections compared with control. E Quantification of dand5 expression at post-flow stages (st.20) following m-tpMO treatment. F Quantification of dand5 expression in pre-flow specimens injected with m-tpMO. G Wildtype dand5 repression in control (co) and left- or right-sided d-tpMO injected specimens. H Quantification of dand5 asymmetry. Note flow-induced dand5 mRNA decay was observed in controls and following d-tpMO application. I Quantification of dand5 staining of pre-flow specimens (st.16) following d-tpMO injections. MO pmol/embryo: m-tpMO (L or S, 0.8); d-tpMO (L or S, 1). Asterisks in D and G mark injected side. Scale bars in D and G represent 100 µm. Numbers (n) in B, C, E, F, H, and I 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, C) or Bonferroni–Holm (E, F, H, I). p values and listing of individual experiments can be found in the source data file. n.s. not significant p > 0.05; **highly significant p < 0.01; ***very highly significant p < 0.001; st. stage; a anterior; l left; r right; p posterior.
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