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The Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic movements of the axial mesoderm and neuroectoderm via Wnt signaling

Hiroki Hikasa*, Mikihito Shibata, Ichiro Hiratani and Masanori Taira{dagger}

Department of Biological Sciences, Graduate School of Science, University of Tokyo, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan
* Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, and Molecular Medicine Unit, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA



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  		Fig. 1. Structure of Xror2 and its mutant constructs. (A) Comparison of predicted amino acid sequences of Xror2 and mouse Ror2 and Ror1. X_ror2, Xror2 accession no. AB087137; M_Ror2, mouse Ror2 (NM013846); M_Ror1, mouse Ror1 (NM013845). The domain structure of the Ror family is indicated by overlines with the domain names. Conserved residues shared by all or by two of them are in black or shaded boxes, respectively. Asterisks, conserved cysteines in the Frizzled-like domain; dashes, spaces for alignment; dots, predicted ATP-binding motifs. (B) Schematic diagrams of Xror2 constructs. SP, signal peptide; IG, immunoglobulin-like domain; FZ, Frizzled-like domain; KR, kringle domain; TM, transmembrane domain; TK, tyrosine kinase domain. See text for details.

 


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  		Fig. 2. Northern blot analysis. (A) Xlim-1/3m (3m) and Xlim-1 plus Ldb1 initiate expression of Xror2 and chordin in animal caps. (B) BMP antagonists, chordin (chd) and noggin (nog), initiate expression of Xror2 in animal caps. (C) Activation of Xror2 expression by Xlim-1 requires protein synthesis. Animal caps (stages 8-8.5) were treated with CHX and 30 minutes later with DEX until the equivalent of stage 11. In A-C, Amounts of injected mRNA (pg/embryo): globin, 250-1000; 3m (H) or 3m, 250; 3m (L), 125; Xlim1+Ldb1(H), 250 each; Xlim1+Ldb1(L), 125 each; chordin, 250; noggin, 125; GR-{Delta}NA, 250. (D) Developmental expression pattern of Xror2. st., developmental stage; whole, whole embryos. Probes are indicated on the right-hand side. Ethidium bromide-stained 18S rRNA is for loading control.

 


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  		Fig. 3. Localization of Xror2 transcripts visualized by whole-mount in situ hybridization. (A) Vegetal view of the gastrula (dorsal is upwards). (B) Hemisectioned early gastrula (animal is upwards; dorsal towards the right). Embryos were bisected sagittally before whole-mount in situ hybridization. (C) Comparison of expression domains between Xror2 (left panel) and Xlim-1 (right panel) at the gastrula stage. Embryos were bisected into left and right halves and subjected to in situ hybridization for Xlim-1 and Xror2, respectively. Dorsal is towards the right (Xror2) or the left (Xlim-1). (D) Late gastrula bisected sagittally (animal is upwards; dorsal towards the right). (E) Dorsal view of late gastrula (anterior is upwards). (F) Transverse section of stained embryos. Xror2 transcripts are detected in the notochord and neuroectoderm. (G,H) Dorsal view of mid-neurula embryos cleared with benzyl benzoate/benzyl alcohol. Xror2 transcripts were detected in the notochord and neuroectoderm with a clear anterior limit in the neuroectoderm. (I) Double whole-mount in situ analysis of Xror2 (magenta) and en2 (turquoise). Arrowheads, en2 expression. (J) Transverse section of neurula embryos. (K) Lateral view of tailbud-stage embryos. Numbers 1, 2, 3 and 4 indicate Xror2 expression in mandibular crest, hyoid crest, anterior branchial crest and posterior branchial crest segments, respectively. st., developmental stages.

 


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  		Fig. 4. Overexpression of Xror2 and its mutant constructs causes a shortened anteroposterior axis. Two blastomeres of four-cell stage embryos were injected in the ventral (/V) or dorsal (/D) equatorial region with mRNAs (1 ng/embryo) as indicated (A-G). Injected embryos were cultured until stage 38. (A,C) Globin mRNA-injected control embryos. (B) Ventral overexpression of Xror2 causes malformation in posterior structures. (D-F) Dorsal overexpression of Xror2 and kinase domain mutants (Xror2-3I, Xror2-TM) causes a shortened body axis with dorsal bending and abnormalities in head structures, including one-eyed phenotypes. (G) Xror2-KR shows much weaker phenotypes than the other constructs of Xror2.

 


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  		Fig. 5. Analysis of convergent extension and neural plate closure after overexpression of Xror2 constructs. mRNA (1 ng/embryo) of globin (A,G), Xror2 (B,B',H), Xror2-3I (C,I), Xror2-TM (D,J) or Xror2-KR (E,K) as indicated was injected together with nß-gal mRNA (20 pg/embryo) for a tracer into one blastomere on the dorsoanimal and right side at the four-cell stage, and subjected to ß-gal staining (red). (A-F) Stage 13 embryos; dor, dorsal view; pos, posterior view; a, archenteron; b, blastocoel. (G-K) Stage 18 embryos (dorsal view; anterior is upwards). (A) Globin-expressing control embryos and a section. As a result of normal convergent extension, nß-galpositive cells were restrictedly distributed along the midline in the trunk region (see also G). (B,B') Xror2-expressing embryos (and section in B) with and without open yolk plug, respectively. nß-gal-stained cells were laterally expanded on the right side of both mesoderm and ectoderm layers as a result of the inhibition of convergent extension. (C) Xror2-3I-expressing embryos showing weaker phenotypes than did wild type. (D) Xror2-TM-expressing embryos. nß-gal-stained cells were laterally expanded, and condensation of pigmented cells was observed in nß-gal-positive regions of ectoderm, as indicated by the arrow and arrowhead, each of which show the corresponding position in a whole embryo (second panel from the left) and in sections (third or fourth panel). When nß-gal-positive cells were laterally expanded, the archenteron was not formed (B,D). (E) Xror2-KR-expressing embryo and section showing normal-looking cell movements. (F) Globin-expressing embryos with open yolk plug. In a few embryos that had open yolk plugs as a result of overexpressing globin, nß-gal-positive cells still converged to the midline. (G) Globin-expressing embryos at stage 18. nß-gal-positive cells were restrictedly distributed along the midline in the trunk region. (H) Xror2-expressing embryos which failed to close the neural plate in nß-gal-positive regions (white arrows). Right panel, higher magnification. (I) Xror2-3I-expressing embryos showing weaker phenotypes than those of wild-type Xror2-expressing embryos. (J) Xror2-TM-expressing embryos. Expression of Xror2-TM resulted in neural groove-like formation with pigmented cells (white arrowheads). Right panel shows a higher magnification. (K) Xror2-KR-expressing embryos. Xror2-KR does not have apparent phenotypes, similar to globin control.

 


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  		Fig. 6. Wild-type and its intracellular mutants of Xror2 affect convergent extension of neural tissue and notochord but not cell differentiation markers. Embryos were injected with mRNA as indicated, and were subjected at stage 18 to ß-gal staining (red) and whole-mount in situ hybridization with pan-neural marker nrp1 (A-C) or notochord marker XPA26 (D-F) as probes. Right panel, sections of stained embryos; inset, higher magnification of neural tissue or notochord region. Expression domains of nrp1 were widened laterally and shortened anteriorly in Xror2- (B) and Xror2-TM- (C) expressing embryos, compared with globin-expressing embryos (A). nrp1 expression was not inhibited in nß-gal-positive regions of Xror2- (B) and Xror2-TM- (C) expressing embryos (right panel, inset). Expression domains of XPA26 failed to elongate anteriorly and were located near the blastopore in Xror2- (E) and Xror2-TM- (F) expressing embryos, compared with those expressing globin (D) (left panel). Areas of XPA26 expression domains on section are much larger in Xror2- (E) and Xror2-TM- (F) expressing embryos than in globin-expressing embryos (D) (right panel, inset), whereas there is no inhibition of XPA26 expression in nß-gal-positive regions. a, archenteron; arrowhead, collapsed archenteron; arrow, thickened pigmented epithelial layers of the ectoderm.

 


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  		Fig. 7. Effects of Xror2 and its mutant constructs on morphogenetic movements of animal caps stimulated with activin. Two-cell stage embryos were injected with mRNAs of globin (A,F), Xror2 (B,G), Xror2-3I (C), Xror2-TM (D,I), Xror2-KR (E) or a mixture of Xror2 and Xror2-TM (H) in the animal pole region of both blastomeres. Doses of injected mRNA (ng/embryo) are indicated in parentheses. Note that doses of mRNA used in F-I are lower than in A-E. Animal caps (stages 8-8.5) were treated with (right panels in A-E; F-I) or without (left panels in A-E) activin A as indicated and cultured until sibling stage 18. A-E and F-I are separate experiments. Activin treatment initiated elongation of control animal caps (A,F). Xror2 (B), Xror2-3I (C) and Xror2-TM (D) suppressed elongation of animal caps by activin. In Xror2-TM-expressing animal caps, a neural groove-like structure with pigmented cells (black arrowheads) was observed in activin-treated ones. Elongation of Xror2-KR-expressing animal caps treated with activin was slightly reduced (E), compared with globin-expressing animal caps. Xror2 or Xror2-TM with lower doses show moderate inhibition of activin-induced elongation of animal caps (G,I). Co-expression of Xror2 and Xror2-TM shows cumulative effects on the inhibition of activin-induced elongation, indicating that wild-type and Xror2-TM do not compete with each other (H).

 


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  		Fig. 8. Effects of Xror2 on activin-induced elongation of animal caps can be cancelled by Cdc42T17N, a dominant-negative Cdc42 mutant. mRNA injection and animal cap assay were performed as described in Fig. 7. All animal caps were treated with activin A. (A) Xror2 inhibits elongation of animal caps by activin. (B) Co-expression of Cdc42T17N with Xror2 partially rescues the extent of explant elongation. (C) Xwnt11 inhibits elongation of animal caps. (D) Co-expression of Cdc42T17N with Xwnt11 partially rescues explant elongation. (E) Globin-expressing animal caps (negative control) treated with activin show elongation. Amounts of mRNA (ng/embryo):Xwnt11, 0.5; Cdc42T17N, 0.6; Xror2, 0.2; globin, 1. (F) Summary of activin-induced elongation assay. The extent of animal cap elongation induced by activin (A-E) was classified by blind scoring as follows. -, no elongation; +, weak elongation; ++ moderate elongation; +++, strong elongation.

 


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  		Fig. 9. Xror2 can associate with Xwnt11, Xwnt5a and Xwnt8. Proteins were extracted from embryos injected with mRNA as indicated, immunoprecipitated (IP) with anti-FLAG antibody, and subjected to western blotting (WB) using anti-Myc antibody (top). The equivalent amounts of proteins generated from injected mRNAs were confirmed by western blotting of lysates using anti-Myc (middle) or anti-FLAG (bottom) antibody. White arrowheads, Wnt proteins. Amounts of mRNA (ng/embryo): globin, 1 (with Xror2KR-FLAG) or 2; Xror2KR-FLAG, 2; Exfz7-FLAG, 0.5; Xwnt11-Myc, 0.5 (with Exfz7-FLAG) or 1; Xwnt5a-Myc, 1; Xwnt8-Myc, 1.

 

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