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. 2022 Feb 10;11:e71928. doi: 10.7554/eLife.71928

Figure 4. mib1 loss of function has no effect on convergent extension in maternal zygotic ryk mutants.

(A) ryknce4g mutants present an 11 base pair insertion in exon 6. The RYK-nce4g mutant protein comprises only part of the extracellular (blue) and lacks the entire transmembrane (yellow) and intracellular (green) domains. (B,C) Accordingly, a C-terminal HA tag that allows to localize WT Ryk (B, n = 12) becomes undetectable upon introduction of the ryknce4g mutation (C, n = 14). Dorsal views of 90% epiboly stage embryos, anterior up. Scalebar 20 µm. (D,E) The Convergent Extension (CE) phenotypes of ryk morphant animals can be rescued using 1.5 pg WT ryk (D) but not ryknce4g mutant (E) RNA. (F) Overexpressing high levels (25 pg) WT ryk RNA causes severe embryonic malformations while no effect is observed using ryknce4g mutant RNA. 32 hpf embryos, anterior to the left, dorsal up (n = 24 embryos/condition). (G) Zygotic (Z) ryk loss of function does not impair CE. (H–J) In contrast, Maternal Zygotic (MZ) ryk mutants present characteristic CE phenotypes such as a reduced axial elongation (H, shhb in situ hybridization) and an increased width of the notochord (I, foxa3 in situ hybridization, see also Figure 4—figure supplement 1F). (J) ryk WT RNA injection allows a significant rescue of MZ ryk mutant CE defects. (K) Similar CE defects are observed in MZ ryk single mutants and MZ ryk; mib1 double mutants. (H,J,K) Lateral views of bud stage embryos, anterior up, dorsal to the right. Scalebars 200 µm. In (D,E,G–K) boxes represent mean values ± SD. See Figure 4—source data 1 for complete statistical information.

Figure 4—source data 1. Complete statistical information for the experiments reported in Figure 4 and Figure 4—figure supplement 1.

Figure 4.

Figure 4—figure supplement 1. Maternal zygotic ryknce4g mutants present Convergent Extension defects.

Figure 4—figure supplement 1.

(A,B) ryk morphants present Convergent Extension (CE) defects that can be rescued by WT ryk (A) but not ryknce4g mutant (B) RNA. (C) CE is similar in Zygotic (Z) ryknce4g mutants and their WT siblings. (D) In situ hybridization reveals that ryk transcript levels are reduced in Z ryknce4g 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 ryknce4g mutants present reduced ryk transcript levels. (H) In contrast to Z ryknce4g mutants, Maternal Zygotic (MZ) ryknce4g 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 ryknce4g mutant CE defects. (I) ryk morpholino injection has no effect in MZ ryknce4g 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.