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. 2023 Dec 20;625(7993):126–133. doi: 10.1038/s41586-023-06850-7

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 6 — a. Schematic drawing illustrates the transplantation strategy from a WT or ror2^t13 donor to a ror2^t13 mutant host (for Extended Data Fig. 6v,w). b. Schematic drawing of principle of the JNK signaling KTR-reporter. c. Principle of measurements of cell rows (1–5), around the Wnt5b/Ror2-expressing clones. Scale Bar 20 µm. d,e,f,g,i,j,l,m,o,p,r,t. Wild-type zebrafish embryos were ubiquitously injected with KTR-mCherry and indicated constructs (ubiquitous, in the right box). At the 8-cell stage, one blastomere was injected with the indicated constructs (clonal, in the left box) to generate clones at the embryonic margin and imaged live at 6 hpf. White stars indicate the signaling-producing clonal cells expressing memGFP. Scale Bar 20 µm. g. N-terminal fusion of mCherry with Ror2 activates JNK signaling around the clone, similar to the C-terminal fusion. h,k,n,q,s,u. Relative JNK signaling within 5 cell rows distance from clone when injected with different amounts of Ror2 (n = 7, 7, 11, 10, n = numbers per embryo, 3 biological repeats), Cdc42^T17N (n = 6, 12, 11, 14, n = numbers per embryo, 3 biological repeats), with ubiquitous Ror2³ⁱ (n = 9, 10, 11, n = numbers of embryos, 3 biological repeats), ubiquitous Vangl2^10a (n = 11, 11, 6, n = numbers of embryos, 3 biological repeats), ubiquitous Dynamin2^K44A (n = 8,5, n = number of embryos, 3 biological repeats), with clonal Wnt8a (n = 5,7, n = number of embryos, three biological repeats). Significance is calculated by two-way ANOVA with Dunnett (h,n,k,q) and Sidak’s (s,u) multiple comparisons test. p-values as indicated. v,w. Transplantation assay. ror2^t13 cells or WT cells or WT cells expressing IRSp53^4K were labeled with memGFP (white asterisks) and grafted into a ror2^t13 mutant embryo microinjected with the KTR JNK reporter. Yellow arrows highlight cells targeted by cytonemes in which KTR mCherry is shifted from the nucleus to the cytoplasm, indicating JNK activation, asterisks represent the location of grafted cells. w. Quantification of the cytoplasmic/nuclear ratio of KTR mCherry in the neighboring cells of 2–5 grafted cells from 7 different embryos per treatment. Significance is calculated by a one-way ANOVA together with a Dunnett multiple comparisons test. p-values as indicated, Scale bars 20 µm.

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