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Fig. 6: Dicer interacts with Bicc1 in dand5 repression. 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 (Dicerflox/+) and dicer conditional knockout (Dicerflox/flox NotoCreERT2/+) 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 (Dicerflox/flox NotoCreERT2/+) 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. I bicc1 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.
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