A Schematic depiction of dand5 reporter assay. dand5 3′-UTR sequences fused to luciferase coding were injected either with or without bicc1 mRNA into the animal region of four-cell embryos. At st. 10, the animal cap region was excised and assayed for luciferase activity. Adapted from refs. ⁶⁵ and ⁶⁶. B Animal cap reporter assay following injections of dand5 S- or L 3′-UTRs alone or together with Xenopus (bicc1) or mouse Bicc1 (mbicc1) effector mRNAs. Note that both alloalleles were equally repressed. Note also that mbicc1 was efficient as a repressor as well. C Luciferase reporter constructs harboring different regions of the dand5 (S-allele) 3′-UTR. D Repression of translation is mediated through a proximal 139 nucleotides (nt) sequence element in the dand5 3′-UTR. E Schematic depiction of medial and distal target protector MOs (m-tpMOs or d-tpMOs) binding to the minimal Bicc1 responsive element (Bicc1RE) in the dand5 3′-UTR (L or S). F m- and d-tpMOs (0.4 or 0.5 pmol/embryo, respectively) interact differently with the luciferase reporter expression. m-tpMO blocked and d-tpMO boosted luciferase activity. Co-injection of d-tpMOs prevented Bicc1-dependent repression of the full-length dand5 reporters (L and S) and further enhanced their expressivity. N in B, D, and F represents the number of independent experiments. A pool of 10 animal caps was analyzed per experiment and treatment. Results from reporter mRNAs alone served as reference and were set to 100% RLU. Relative values of single experiments are depicted as blue dots. Data of at least 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 (Bonferroni corrected) using the values of at least three individual experiments. p values, values for individual experiments, the mean values, and standard deviations are found in the source data file. n.s. not significant p > 0.05; * significant, p < 0.05; ** highly significant p < 0.01; ***, very highly significant p < 0.001; RLU relative luciferase units; Lucluciferase.

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.

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.

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. Bgdf3 mRNA was not affected by bicc1 LoF. C Quantification of gdf3 expression in bicc1 morphants at the LRO margin. Dgdf3 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.

A Quantification of right-sided pitx2 induction by co-injecting a low, ineffective m-tpMO dosage with single allele-specific bicc1 SBMO. Controls (co), m-tpMO (S or L, low), or allele (S or L) specific bicc1 SBMO alone showed wt pitx2 asymmetry. B Co-injecting m-tpMO (low) with bicc1 TPMO (L or S) impacted dand5 mRNA stability. Treatment with low concentrations of m-tpMO, single allele-specific bicc1 TPMO, and uninjected co showed wt dand5 expression at post-flow stages. C Quantification of dand5 expression. D Quantification of pitx2 asymmetry. Only in combination both suboptimal dosages of d-tpMO (low) or single allele-specific bicc1 SBMO (S or L) prevented left pitx2 expression. Wt expression was found in controls (co) and in embryos that were left-sided injected with one MO alone. MO pmol/embryo: bicc1 SBMO (L or S, 1); m-tpMO low (L or S, 0.4); d-tpMO low (L or S, 0.5). Asterisks in B mark injected side. Numbers (n) in A, C, and D 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 (A, D) or Bonferroni–Holm (C). n.s., not significant p > 0.05; ***, very highly significant p < 0.001. p values and listing of individual experiments can be found in the source data file. Scale bar in B represents 100 µm. st. stage, a anterior, l left, r right, p posterior.

A Expression of dicer1 in sensory (s) LRO cells ( N = 3; n = 30) of the frog (GRP; gastrocoel roof plate). Whole-mount in situ hybridizations of stage 18 dorsal explant with a dicer1-specific antisense RNA probe. (A’) The transverse histological section (indicated in A) reveals mRNA expression in sLRO cells, somites (som), and deep cells of the notochord (no), but absence of signals from central (c) flow-generating LRO and lateral endodermal cells (end). B Quantification of MO-mediated inhibition of dicer. Note knockdown in left, but not right sLRO cells prevented pitx2 asymmetry in the left LPM, which was rescued by co-injecting dand5 MO. C mRNA expression of nodal in control (Dicer^flox/+) and dicer conditional knockout (Dicer^flox/floxNoto^CreERT2/+) mouse embryos at E8.0. Note that Nodal asymmetry in the left LPM (arrowhead) was lost in mutants. D Absence of flow-induced dand5 mRNA decay at the left LRO margin in post-flow dicer1 morphants (st. 20). Representative dorsal explants of wt (left) and dicer1 morphant (right) specimens hybridized with a dand5 antisense RNA probe. E Quantification of dand5 results. F Flow-induced Dand5 mRNA downregulation in left crown cells of the murine node was lost in Dicer conditional knockout (Dicer^flox/floxNoto^CreERT2/+) mouse embryos at E7.5. G Lack of dand5 repression in 10 somite stage (ss) MZdicer mutant zebrafish embryos. H Absence of dand5 mRNA by RNAseq reads in 24hpf wt zebrafish embryos, but maintenance in MZdicer mutants. Ibicc1 and dicer1 interact in LR asymmetry. Wt pitx2 expression upon isolated left-sided injections of allele-specific bicc1 SBMOs and moderate effects upon dicer1 TBMO1 injection. Asymmetric pitx2 was significantly inhibited by co-injecting dicer1 and bicc1 MOs. MO pmol/embryo: dicer1 TBMO1 (1.5); dicer1 TBMO2 (1); bicc1 TBMO (L or S, each 1); bicc1 SBMO (L or S, each 1). Asterisks in D mark injected side. Numbers (n) in B, E, and I represent analyzed specimens from three independent experiments. Statistical analyses were done with one-sided Pearson’s chi-square test, which was adjusted for multiple comparisons by Bonferroni (B, I) or Bonferroni–Holm (E). n.s. not significant p > 0.05; * significant, p < 0.05; ***, very highly significant p < 0.001. p values and listing of individual experiments can be found in the source data file. Scale bars in A, A’, C, F, and D represent 100 µm. st. stage, a anterior, d dorsal, l left, r right, v ventral, p posterior.

A Absence of dand5 repression in maternal zygotic (MZ) pkd2 mutant zebrafish at 10 somite stage (ss). B Quantification of dand5 asymmetry in controls (co) and pkd2 morphant (1–4 ng) zebrafish. Asymmetry was determined by picture analysis using ImageJ. Number (n) represents the number of analyzed specimens. Statistical analyses were done with one-sided Pearson’s chi-square test. C Animal cap luciferase reporter assay of full-length dand5.S 3′-UTR (cf. Figure 1A). The reporter construct was injected as mRNA either alone or in combination with high or low dose bicc1 mRNA, pkd2 mRNA or pkd2 TBMO. Gradual repression upon co-injection of high or low concentrations of bicc1 mRNA was observed. Administering only pkd2 mRNA or pkd2 TBMO (1 pmol) efficiently blocked or boosted luciferase expression, respectively. The data further indicate that in AC cells endogenous dand5 mRNA is post-transcriptionally regulated in a Pkd2-dependent manner. In the presence of a lower amount of bicc1 mRNA high-level, strong repression was achieved when pkd2 mRNA was co-injected, or further diminished upon knockdown of pkd2 using TBMO. N represents the number of independent experiments. A pool of 10 animal caps was analyzed per experiment and treatment. The results from reporter mRNA alone served as reference and were 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 (Bonferroni corrected) using the values of three individual experiments. p values, values for individual experiments, mean values, and standard deviations are found in the source data file. n.s. not significant, p < 0.05; ** highly significant p < 0.01; ***, very highly significant p < 0.001, RLU relative luciferase units, Lucluciferase.

In the early neurula pre-flow stages, Bicc1 has two functions. Bicc1 assures gdf3 mRNA translation and thereby indirectly ensures nodal1 transcription by Gdf3 signaling. Simultaneously Bicc1 mediates dand5 mRNA stability via the medial (m) sub-region of the Bicc1RE. Thus, Dand5 protein levels are sustained on both sides, keeping Nodal in tight repression. Leftward flow activates the Pkd2 channel in left flow sensor cells, resulting in an asymmetric calcium signal. In post-flow stages, a calcium-dependent mechanism activates/modifies Bicc1 to become a repressor of dand5 translation, which is relayed by the distal (d) sub-region of the Bicc1RE. Subsequently, dand5 mRNA gets degraded in a Dicer1 (miR) dependent manner. Attenuated Dand5 expression lifts repression of Nodal and defines leftness by induction of the LPM Nodal signaling cascade. For details, see text.