Supplementary MaterialsS1 Fig: Fzd3a includes a cell-autonomous function in FBMN migration. rhombomere positon. Scale bar: 50 m.(TIF) pgen.1005934.s001.tif (1.0M) GUID:?B9916093-D705-4218-B0EF-81EC9EA7F92A S2 Fig: Post-mitotic FBMNs require PCP signaling for migration. (A) Live confocal image showing the dorsal view of a mutant embryo hindbrain at 48 hpf after transplantation of post-mitotic FBMNs from a wild type donor. Cascade blue-dextran marks all donor-derived cells (blue), marks host FBMNs (green) and marks donor-derived FBMNs (magenta). (B) Histogram indicates the percent of donor-derived FBMNs at 48 hpf that failed to migrate, (rhombomere (r)4), partially migrated (r5) or fully migrated (r6) and numbers indicate the number of FBMNs represented in each bar. White arrows indicate migrated donor derived FBMNs. While post-mitotic FBMNs in general migrate poorly after being transplanted, they do sometimes migrate in WT and mutant hosts but never in mutant hosts (see Fig 2). Brackets indicate rhombomere positon. Scale bar: 50m.(TIF) pgen.1005934.s002.tif (723K) GUID:?7E7A28D3-E441-41E7-BEED-EE2104028397 S3 Fig: PCP-DN expression in the floorplate disrupts planar polarity. (A-C) driven expression of in the notochord and floorplate of a 14 hpf (A) 24 hpf embryo (B) and a 48 DLL4 hpf embryo (C). Anterior is to the left. Images are live lateral views in A-C and live dorsal views at the hindbrain level, A,B. (D-F) Confocal images showing floorplate planar polarity of the anterior spinal cord in 48 KAG-308 hpf zebrafish embryos. Anterior is to the top. Anti-ZO-1 marks subapical tight junctions (white), anti-Cc2d2a marks the basal bodies of the primary cilia (magenta, arrows), and anti-GFP indicates dominant negative protein expression (green). Scale bar: 10m. Whereas basal bodies are localized toward the posterior membrane in wild type embryos (D), this polarity is disrupted in floorplate cells expressing Xdd1-GFP (E) or Fzd3aC-GFP (F) (arrows in E and F). (G) Schematic of the method used to quantify floorplate planar polarity. Total cell length (x) is measured as the distance between the anterior and posterior membranes (white) at the level of the basal body (magenta). Basal body position (y) is measured as the distance between the anterior membrane as well as the basal body. Cellular planar polarity is certainly quantified because the proportion of x/con. (H) Quantitation of ordinary basal body placement in the ground bowl of 48 hpf embryos. Each data stage represents the suggest basal body placement for everyone cells quantitated within a embryo. WT: N = 34 embryos, 411 cells; Xdd1-GFP: N = KAG-308 14 embryos, 207 expressing cells; FzdC-GFP: N = 29 embryos, 484 expressing cells; embryos is roofed for evaluation. Graph represents data as mean SD. **p 0.0001 in comparison to wild-type control.(TIF) pgen.1005934.s003.tif (2.6M) GUID:?9DF56324-F11B-4DCE-AEBC-0083A04B9FAF S4 Fig: Vangl2 is not needed within the mouse floorplate for FBMN migration. (A-B) Dorsal watch of KAG-308 E13.5 mouse hindbrains with FBMNs (magenta) tagged with anti-Isl1 staining. Dotted lines reveal length of cosmetic motor nucleus. To boost the possibilities a Cre-expressing cell shall possess a biallelic deletion of Vangl2, in these tests we utilized the null allele, which we uncovered belatedly to result in a minor FBMN migration defect in substance heterozygotes using the floxed allele. Even so, deleting the floxed allele with did not enhance the partial migration defect in controls. For the experiments using shown in Fig 1 we did not use the allele. (A) FBMNs in a control embryo. N = 6 embryos. (B) FBMNs in embryo. Addition of does not further disrupt FBMN migration. N = 4 embryos. (C) Quantitation of FBMN migration stream length in control embryos and embryos. Scale bar: 100m(TIF) pgen.1005934.s004.tif (1.0M) GUID:?D56790DF-58E7-4128-953E-FB568BE80E53 S5 Fig: Specificity of the anti-Vangl2 antibody. (A-B) Dorsal view of wild type (A) and mutant (B) 24 hpf neural tubes immunostained with anti-Vangl2-NT (green). The neuroepithelial membrane staining visible in wild type is usually absent in the mutant. (C) Western blot analysis of whole embryo lysates with anti-Vangl2 antibody. Anti-alpha-tubulin was used as a loading control. Zebrafish Vangl2 is usually expected to run at approximately 60kDa. For the anti-Vangl2 blot there is a band that is present in the wild type and absent in the mutant, see asterisk.(TIF) pgen.1005934.s005.tif (737K) GUID:?B028834A-9594-4CB9-8072-9E3079C79130 S6 Fig: Migrating FBMNs display polarized protrusions that fail to polarize in mutants. (A,C,E) Representative frames of mTFP expressing FBMNs from time-lapse images taken at 24 hpf to 32 hpf. (B,D,F) Each natural data point for protrusion angle is usually plotted around the circular graph below. Each division is usually.