Ror2 receptor mediates Wnt11 ligand signaling and affects convergence and extension movements in zebrafish

Abstract

The receptor-tyrosine kinase Ror2 acts as an alternative receptor or co-receptor for Wnt5a and mediates Wnt5a-induced convergent extension movements during embryogenesis in mice and Xenopus as well as the polarity and migration of several cell types during development. However, little is known about whether Ror2 function is conserved in other vertebrates or is involved in other non-canonical Wnt ligands in vivo. In this study we demonstrated that overexpression of dominant-negative ror2 (ror2-TM) mRNA in zebrafish embryos resulted in convergence and extension defects and incompletely separated eyes, which is consistent with observations from slb/wnt11 mutants or wnt11 knockdown morphants. Moreover, the co-injection of ror2-TM mRNA and a wnt11 morpholino or the coexpression of ror2 and wnt11 in zebrafish embryos synergetically induced more severe convergence and extension defects. Transplantation studies further demonstrated that the Ror2 receptor responded to the Wnt11 ligand and regulated cell migration and cell morphology during gastrulation. DnRor2 inhibited the action of Wnt11, which was revealed by a decreased percentage of Wnt11-induced convergence and extension defects. Ror2 physically interacts with Wnt11. Theintracellular Tyr-647andSer-863 sites ofRor2are essential for mediating the action of Wnt11. Dishevelled and RhoA act downstream of Wnt11-Ror2 to regulate convergence and extension movements. Overall, our data suggest an important role of Ror2 in mediating Wnt11 signaling and in regulating convergence and extension movements in zebrafish.

FIGURE 1.

Zebrafish Ror2 is similar to ROR2 proteins in other vertebrates and is ubiquitously expressed during gastrulation. A, domain structure of zebrafish Ror2 protein and comparison of zebrafish Ror2 domains with those of humans, mice, and Xenopus. Ig, immunoglobulin domain; Kr, Kringle domain; TM, transmembrane domain; Tyrosine kinase, tyrosine kinase domain; PRD, proline-rich domain; Ser/Thr, Ser/Thr-rich domains. B, phylogenetic analysis of the Ror2 family. Full-length sequences of Ror were analyzed using the MEGA4 software with a JTT matrix and the neighbor-joining method. The reliability of each node was assessed by the bootstrap method with 1000 replications. The numbers on the branches represent the percentages for the branching of two clades as sisters. The clade with >50% bootstrap support is shown on each node. Fruit fly and worm Ror were used as outgroups. C, semiquantitative RT-PCR revealed continuous ror2 expression at different developmental stages (hpf). β-actin, internal control. D, whole-mount in situ hybridization revealed the expression of ror2 at different developmental stages.

FIGURE 2.

Knockdown or overexpression of Ror2 in zebrafish embryos impairs convergence and extension movements during gastrulation. A, structure of the zebrafish dominant-negative Ror2, Ror2-TM. Ig, immunoglobulin domain; Kr, Kringle domain; TM, transmembrane domain; Tyrosine kinase, tyrosine kinase domain; PRD, proline-rich domain; Ser/Thr, Ser/Thr-rich domains. B, representative images of gfp- and ror2-TM mRNA-injected embryos at 12 hpf. Lateral view reveals the A-P angle between the anterior- and posterior-most embryonic structures; the dorsal view reveals the width of the somites. C, statistics for convergence and extension defects in embryos injected with 800 pg of gfp, 400 pg of ror2-TM, or 800 pg of ror2-TM. The embryo number of each group is shown at the top of each associated column. Results are from three independent microinjection experiments. Two-tailed t tests were used to determine significant differences. The values are presented as the mean ± S.E. (n = 3). ***, p < 0.001. **, p < 0.01. D, embryos are shown at 24 hpf and separated into four categories according to the distance of the separate eyes: normal (wild type), class I (eyes close together but not touching), class II (eyes partly fused), and class III (eyes fused). E, quantitation of eye phenotypes in zebrafish embryos as described in D. The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments. F, WISH of marker genes (hgg, dlx3b, and ntl) in ror2-TM mRNA-injected embryos. The black double-headed arrow indicates the width of the neural plate, and the embryos were separated into three categories according to the width: normal, moderate, and severe. The embryos were also separated into three categories according to the position of hgg anterior to dlx3b: normal, moderate, and severe. The asterisk indicates the position of hgg. G, width statistics of the neural plate of gfp- and ror2-TM-injected embryos as described in F. The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments. H, position statistics of hgg anterior to dlx3b of gfp- and ror2-TM-injected embryos as described in F. The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments. I, representative images of gfp- and ror2 mRNA-injected embryos at 12 hpf (lateral view and dorsal view are shown, respectively). J, quantitation of convergence and extension defect phenotypes in zebrafish embryos injected with 800 pg of gfp, 400 pg of ror2, and 600 pg of ror2. The results are from three independent microinjection experiments. The values are presented as the mean ± S.E. (n = 3). Two-tailed t tests were used to determine significant differences. ***, p < 0.001. *, p < 0.05. K, WISH of marker genes (hgg, dlx3b, and ntl) in gfp- and ror2 mRNA-injected embryos. The phenotype definition is described in F. L, width statistics of the neural plate of gfp- and ror2-injected embryos, as described in (K). The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments. M, position statistics of hgg anterior to dlx3b of gfp- and ror2-injected embryos, as described in K. The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments.

FIGURE 3.

Co-knockdown or coexpression of Wnt11 and Ror2 synergistically induced severe convergence and extension defects. A, quantitation of convergence and extension defects in zebrafish embryos injected with the indicated mRNA and/or MO at 12 hpf. The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments. B, WISH of marker genes (hgg, dlx3b, and ntl) in gfp-injected and wnt11-, ror2 knockdown or double knockdown embryos. The black double-headed arrow indicates the width of the neural plate, and the embryos were separated into three categories according to the width: normal, moderate, and severe. According to the position of hgg anterior to dlx3b, the embryos were also separated into three categories: normal, moderate, and severe. The asterisk indicates the hgg position. C, width statistics of the neural plate of each indicated group, as described in B. The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments. D, position statistics of hgg anterior to dlx3b of each indicated group, as described in B. The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments. E, quantitation of eye phenotypes in each indicated group at 24 hpf. The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments. F, quantitation of convergence and extension defect phenotypes in zebrafish embryos injected with the indicated mRNA at 12 hpf. The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments. G, WISH of marker genes (hgg, dlx3b, and ntl) in gfp, ror2, wnt11, and ror2 + wnt11 mRNA-injected embryos. The phenotype definition is described in B. H, width statistics of the neural plate of each indicated group, as described in G. The embryo number of each group is shown at the top of each associated column. The results are from three independent microinjection experiments. I, position statistics of hgg, anterior to dlx3b of each indicated group, as described in G. The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments.

FIGURE 4.

Knockdown of Ror2 and Wnt11 changes cell movement during gastrulation. A, donor embryos were injected with the control MO labeled with Alexa Fluor 488 (green) or Alexa Fluor 546 (red). Donor cells from ∼3.3 hpf embryos were transplanted into the center of the lateral mesoderm of uninjected host embryos at the shield stage. A representative embryo is shown at ∼11 hpf. B, control MO-injected (green) and ror2-TM-injected (red) donor cells were transplanted into uninjected host embryos. C, control MO-injected (green) and ror2-TM-injected (red) donor cells were transplanted into Wnt11 MO-injected host embryos. D, quantitation of the distance between Alexa Fluor 546 and Alexa Fluor 488 tracers. In each group 10 embryos were scored. One-way analysis of variance followed by Tukey's multiple comparison test was used to determine significant differences. The values are presented as the mean ± S.E. (n = 10). ***, p < 0.001. ns, non-significant.

FIGURE 5.

Wnt11 stimulates elongation of Ror2-expressing cells. A–D, representative views of cells in 90% epiboly stage embryos; lateral view. Scale bar = 5 μm. A, donor embryo injected with ror2 mRNA and wnt11 MO combined with membrane gfp mRNA. B, donor embryo injected with ror2-TM mRNA and wnt11 MO combined with membrane gfp mRNA. C, Ror2-overexpressing donor cells in Wnt11-overexpressing host embryos. D, Ror2-TM-overexpressing donor cells in Wnt11-overexpressing host embryos. E and F, analysis of cell roundness of transplanted cells, as described in C and D. E, a column chart shows the roundness for each group. Values are the mean ± S.E. ***, p < 0.001; unpaired t test. F, a cumulative frequency chart shows the distribution of roundness values for each group. Approximately 100 cells were measured for each group.

FIGURE 6.

Inhibition of Ror2 attenuates Wnt11 activity in convergence and extension movements, and Wnt11 and Ror2 interact physically. A, quantitation of convergence and extension defect phenotypes in zebrafish embryos injected with wnt11, ror2-TM, and wnt11 + ror2-TM mRNAs at 12 hpf. The embryo number of each group is shown at the top of each associated column. The results are from three independent microinjection experiments. The values are presented as the mean ± S.E. (n = 3). Two-tailed t tests were used to determine significant differences. *, p < 0.05. ns, non-significant. B, quantitation of the width of the neural plate in each indicated group. The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments. C, quantitation of hgg anterior to dlx3b in each indicated group. The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments. D, quantitation of eye phenotypes in each indicated group at 24 hpf. The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments. E, proteins were extracted from the lysates of cells that coexpressed Ror2 and Wnt11, immunoprecipitated (IP), and subjected to Western blot (WB) analysis using the indicated antibodies. Input, positive control; IP, IgG, negative control.

FIGURE 7.

Tyr-647 and Ser-863 are important functional sites of Ror2 in convergence and extension movements. A, structure of zebrafish Ror2; single vertical lines indicate the mutant sites. B, quantitation of convergence and extension defect phenotypes in zebrafish embryos at 12 hpf injected with the indicated mRNA: gfp, 100 pg; wnt11, 25 pg; ror2, 100 pg; ror2-Y647F, 100 pg; ror2-S863A, 100 pg; ror2-Y647F-S863A, 100 pg. The embryo number of each group is shown at the top of each associated column. The results are from three to six independent microinjection experiments. The values are presented as the mean ± S.E. Two-tailed t tests were used to determine significant differences. ***, p < 0.001. ns, non-significant.

FIGURE 8.

Dsh and RhoA act downstream of the Wnt11-Ror2 signaling pathway. A, quantitation of eye phenotypes in zebrafish embryos injected with ror2-TM or ror2-TM + XDsh-ΔDIX mRNA (100 pg) at 24 hpf. The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments. B, quantitation of eye phenotypes in zebrafish embryos injected with ror2-TM or ror2-TM + rhoA mRNA (10 pg) at 24 hpf. The embryo number of each group is shown at the top of each related column. The results are from three independent microinjection experiments. C, schematic diagram of the interaction between the Ror2 and Wnt11 signaling pathways.