(A,B) Axis elongation was quantified at bud stage by measuring the axis extension angle α using the marker genes shhb (A, expressed in the notochord) and foxa3 (B, expressed in notochord and prechordal plate). Axis extension is reduced following injection of mib1 morpholino (MO mib1) or RNAs encoding Mib1ΔRF123 or Mib1ΔRF3. Lateral views of bud stage embryos, anterior up, dorsal to the right. (C) Dorsal views of bud stage embryos stained for foxa3 also reveal a significant widening of the Notochord. Boxes represent mean values ± SD. See Figure 1—source data 1 for complete statistical information.

(A,B) Analysis of bud stage axis extension (A) and mib1 in situ hybridization (B) show that the mib1^ta52b mutation does not impair Convergent Extension (CE) or mib1 transcript abundance (B, n = 4 mutant embryos analyzed). (C,D) In contrast mib1^nce2a mutants present a weak reduction of CE (C) and a loss of mib1 transcripts (D, n = 6 mutant embryos). (E) qPCR analysis of mib1 transcript levels (30 hfp stage). mib1^tfi91 and mib1^nce2a homozygous mutants present a reduction in mib1 transcripts that is not observed for the mib1^ta52b allele. Error bars represent SE from three biological replicates. (F) mib1^nce2a mutants present a CrisprCas-induced InDel that causes a frame shift and leads to premature protein truncation. (A,C) depict lateral views of bud stage embryos, anterior up, dorsal to the right. A quantitative analysis of the corresponding data sets is provided in Figure 1D and G. (B,D) Represent dorsal views of bud stage embryos, anterior up. To warrant identical acquisition conditions, two embryos were photographed on a single picture. Scalebars: 200 µm.

(A) Mib1 morphants display reduced convergent extension (quantified through the measure of the axis extension level α) that is not rescued by constitutively activated Notch (NICD). (B) cDNA sequence from wild-type embryos injected with mib1 exon/intron1 splice morpholino. Morpholino injection causes a retention of intron 1. As a consequence, the Mib1 morphant proteins comprises only the first 76 amino acids of WT Mib1 followed by 14 intronically encoded residues and a premature Stop codon. (C) Mib1 morphant axis extension can be restored through the overexpression of Notch-signaling-deficient mib1^ta52b. (A,C) depict lateral views of bud stage embryos, anterior up, dorsal to the right. A quantitative analysis of the corresponding data sets is provided in Figure 1E and F. Scalebars: 200 µm.

(A) RhoA overexpression rescues mib1 morphant axis extension. (B,C) Mib1 proteins lacking all (Mib1ΔRF123, B) or only the last (Mib1ΔRF3, C) RING finger impair axis extension in mib1 morphant or WT embryos. Lateral views of bud stage embryos, anterior up, dorsal to the right. Scalebars: 200 µm. Boxes represent mean values ± SD. Figure 2—source data 1 for complete statistical information.

Figure 2—source data 1.

(A) Coinjection of RNAs encoding Rab5-GFP and Flag-Ryk-Myc reveals that 70.7% of Ryk-expressing intracellular compartments are positive for the early endosomal marker Rab5 (n = 75 cells from eight embryos analyzed). Arrowheads in A’’ indicate compartments that are positive for Rab5 only (green), Ryk only (magenta), or present both markers (white). (B,C) Mib1 overexpression promotes the internalization of Ryk-GFP but not the one of the plasma membrane marker GAP43-RFP (n = 4). (D,E) RNAs encoding Ryk-GFP and Histone2B-mRFP were injected with increasing amounts of mib1 RNA. While a low dose of Mib1 relocalizes Ryk from the plasma membrane to intracellular compartments (n = 6), high amounts of Mib1 cause an overall loss of Ryk signal (n = 6). D’-F’, The Histone2B-mRFP signal was used to ascertain that embryos had received a comparable amount of injected material. (D-F and D’-F’) are sum projections of three consecutive slices from confocal stacks. All pictures depict dorsal views of 90% epiboly stage embryos, anterior up. Scalebars: 10 µm in A-C, 20 µm in D-F.

(A,B) mib1 morpholino (MO mib1) injection reduces Ryk endocytosis. (C,D) A more pronounced inhibition of Ryk endocytosis is observed upon coinjection of MO mib1 and RNA encoding dominant-negative Mib1 (mib1DRF123). (E,F) Ryk endocytosis is reduced in mib1^tfi91 mutant embryos. All pictures depict dorsal views of 90% epiboly stage embryos, anterior up. A’-F’, The Histone2B-mRFP signal was used to ascertain that control and mib1-depleted embryos had received a comparable amount of injected material. Embryos depicted in A-F were injected with 12 pg Ryk-GFP RNA. (C-F) correspond to the display items also shown in Figure 3F–I. Scalebars: 10 µm.

(A,B) vangl2 RNA injection does not rescue axis extension in mib1 morphants. (A) Lateral views of bud stage embryos, anterior up, dorsal to the right. Scalebar 200 µm. In (B) boxes represent mean values ± SD. See Figure 3—source data 1 for complete statistical information.

(A,B) ryk morphants present Convergent Extension (CE) defects that can be rescued by WT ryk (A) but not ryk^nce4g mutant (B) RNA. (C) CE is similar in Zygotic (Z) ryk^nce4g mutants and their WT siblings. (D) In situ hybridization reveals that ryk transcript levels are reduced in Z ryk^nce4g mutants (n = 24) compared to WT siblings (n = 24). 12 somite stage embryos, anterior to the left, dorsal up. To warrant identical acquisition conditions, two embryos were photographed on a single picture. (E–F) foxa3 in situ hybridization shows that Maternal Zygotic (MZ) ryk mutants present a reduced axial elongation (E) and an increased width of the notochord (F, for quantification see Figure 4I). (G) qPCR analysis of bud stage embryos reveals that MZ ryk^nce4g mutants present reduced ryk transcript levels. (H) In contrast to Z ryk^nce4g mutants, Maternal Zygotic (MZ) ryk^nce4g mutants present CE defects. To exclude any defects due to genetic background variation, the parental fish used to obtain the embryos for this experiment were ryk[+/+] and ryk[nce4g/nce4g] siblings obtained from the same incross. ryk WT RNA injection allows to rescue MZ ryk^nce4g mutant CE defects. (I) ryk morpholino injection has no effect in MZ ryk^nce4g mutants. Lateral (A,B,C,E,H,I) or dorsal (F) views of bud stage embryos, anterior up. Scalebars 200 µm. Boxes in (E,I) boxes represent mean values ± SD. Error bars in G represent SE from three biological replicates. Quantitative analysis of the data sets displayed in (A,B,C,H) is provided in Figure 4D, E, G and H respectively. See Figure 4—source data 1 for complete statistical information.

(A,B) 17 out of 18 Wnt5b-injected embryos present severely reduced Ryk-GFP signals compared to WT controls (n = 12). (C,D) A similar Wnt5b-dependent decrease of Ryk-GFP levels is observed in mib1^tfi91 homozygous mutants that were additionally injected with dominant-negative Mib1ΔRF123 (n = 14 mutant and n = 16 WT control embryos). All pictures depict dorsal views of 90% epiboly stage embryos, anterior up. The Histone2B-mRFP signal (A’-D’) was used to ascertain that control and Wnt5b-expressing embryos had received comparable amounts of injected material. Scalebars: 10 µm.