Figure S2.
Wnt11f2- and Wnt5b-tagged constructs are functional.(A) Schematic diagram illustrating mosaic labeling approach to assess the localization of tagged Wnt’s in vivo. At the 1-cell stage, embryos were injected with mem-mCherry RNA to label the plasma membrane of all cells; at the 16-cell stage, a single blastula cell was injected with RNAs encoding wnt5b or wnt11f2 (with distinct tags), together with H2A-mCherry, to express Wnt’s in a subset of cells (whose nuclei are labeled with mCherry). At 50% epiboly (50%E), embryos expressing Wnt-Myc were fixed for immunostaining using an anti–C-MYC antibody; and embryos expressing Wnt-mNeonGreen were subjected for live imaging. (B–E′) Confocal images of zebrafish embryos at 50%E, following mosaic injection. The expression of tagged Wnt constructs (green) is shown; all cells of the embryo are labeled with mem-mCherry (magenta). White arrowheads indicate Wnt-labeled puncta outside the expressing cells (asterisks). (B–C′) Confocal images showing the expression of Wnt5b-Myc (B and B′) and Wnt11f2-Myc (C and C′), as detected by immunostaining. (D–E′) Live confocal images showing the expression of Wnt5b-mNeonGreen (D and D′) and Wnt11f2-mNeonGreen (E and E′). (F-I) Expression of hgg1 (white arrowheads), dlx3, and krox20 at 3 somite (s) stage, as detected by WISH in control and wnt11f2−/− embryos (uninjected or injected with the wnt11f2 RNA). The expressing domain of hgg1 islagging behind that of dlx3 in wnt11f2−/− embryos (G). Injecting wnt11f2 RNA did not impact hgg1 expression in control siblings (H) but largely rescued the defects of hgg1 expression pattern in wnt11f2−/− embryos (I). (J) Percentage of wnt11f2−/− control embryos and counterparts injected with RNAs encoding WT wnt11f2 or various tagged forms of wnt11f2 that display defects in hgg1 expression. (K–L′′) Bright-field images of live embryos, showing control WT embryos and counterparts injected with wnt5b RNA, showing C&E defects of different severity. 12 s, 12 somite stage. (M) Percentage of C&E defects of varying severity in embryos injected with RNAs encoding mem-mCherry or wnt5b or various tagged forms of wnt5b.

Wnt11f2- and Wnt5b-tagged constructs are functional. (A) Schematic diagram illustrating mosaic labeling approach to assess the localization of tagged Wnt’s in vivo. At the 1-cell stage, embryos were injected with mem-mCherry RNA to label the plasma membrane of all cells; at the 16-cell stage, a single blastula cell was injected with RNAs encoding wnt5b or wnt11f2 (with distinct tags), together with H2A-mCherry, to express Wnt’s in a subset of cells (whose nuclei are labeled with mCherry). At 50% epiboly (50%E), embryos expressing Wnt-Myc were fixed for immunostaining using an anti–C-MYC antibody; and embryos expressing Wnt-mNeonGreen were subjected for live imaging. (B–E′) Confocal images of zebrafish embryos at 50%E, following mosaic injection. The expression of tagged Wnt constructs (green) is shown; all cells of the embryo are labeled with mem-mCherry (magenta). White arrowheads indicate Wnt-labeled puncta outside the expressing cells (asterisks). (B–C′) Confocal images showing the expression of Wnt5b-Myc (B and B′) and Wnt11f2-Myc (C and C′), as detected by immunostaining. (D–E′) Live confocal images showing the expression of Wnt5b-mNeonGreen (D and D′) and Wnt11f2-mNeonGreen (E and E′). (F-I) Expression of hgg1 (white arrowheads), dlx3, and krox20 at 3 somite (s) stage, as detected by WISH in control and wnt11f2−/− embryos (uninjected or injected with the wnt11f2 RNA). The expressing domain of hgg1 islagging behind that of dlx3 in wnt11f2−/− embryos (G). Injecting wnt11f2 RNA did not impact hgg1 expression in control siblings (H) but largely rescued the defects of hgg1 expression pattern in wnt11f2−/− embryos (I). (J) Percentage of wnt11f2−/− control embryos and counterparts injected with RNAs encoding WT wnt11f2 or various tagged forms of wnt11f2 that display defects in hgg1 expression. (K–L′′) Bright-field images of live embryos, showing control WT embryos and counterparts injected with wnt5b RNA, showing C&E defects of different severity. 12 s, 12 somite stage. (M) Percentage of C&E defects of varying severity in embryos injected with RNAs encoding mem-mCherry or wnt5b or various tagged forms of wnt5b.

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