Journal: Biology Open

Article Title: Planar polarization of endogenous ADIP during Xenopus neurulation

doi: 10.1242/bio.062452

Figure Lengend Snippet: Response of endogenous ADIP to wound healing. (A-C) Immunostaining of stage 12 embryos reveals ADIP puncta in ectoderm cells (A). Cell borders are marked by Wtip (A′); merged image (A″) shows partial colocalization of ADIP and Wtip puncta in the apical domain (white arrows). (B) Segmented cell outlines show random orientation of ADIP polarity vectors (arrows). (C) Rose plot confirms random distribution. (D-F) Stage 12 embryos were wounded on the ventral animal side and allowed to heal for 30 min in 0.7× MMR. ADIP becomes polarized in the cells proximal to the wound margin (D). Wtip labels cell borders (D′); merged image (D″). Arrows in D indicate ADIP puncta and polarized Wtip at cell junction facing the wound. Scale bar in D″: 20 μm; the same scale applies to panels A-A′′, B, E, G-G′, H-H′. Cell segmentation (E) and rose plot (F) show ADIP polarity vectors (arrows in E) oriented toward the wound site (0°). Rose plot represents pooled polarity from two embryos. Chi-square test indicates a non-random distribution, P <0.05. (G-I) Anti-Wtip staining of stage 10.5 embryos co-injected with 20 ng of control MO (G-G′) or Wtip MO (H-H′) and 50 pg H2B-GFP RNA as lineage tracer. MO-injected cells are marked by rabbit polyclonal antibody for GFP; merged images in G′,H′. (I) Immunoblotting of endogenous Wtip in stage 13 embryo lysates injected with control MO or Wtip MO. Blots were probed with mouse anti-Wtip (DA2B11) antibody (upper panel) and anti-β-catenin antibody as a loading control (lower panel). Data shown are representative of three independent experiments, each performed on 10-15 embryos.

Article Snippet: Data and resource availability The monoclonal antibody DA2B11 has been deposited in Developmental studies hybridoma bank (DSHB).

Techniques: Immunostaining, Staining, Injection, Control, Western Blot

Journal: Biology Open

Article Title: Planar polarization of endogenous ADIP during Xenopus neurulation

doi: 10.1242/bio.062452

Figure Lengend Snippet: Response of endogenous ADIP to wound healing. (A-C) Immunostaining of stage 12 embryos reveals ADIP puncta in ectoderm cells (A). Cell borders are marked by Wtip (A′); merged image (A″) shows partial colocalization of ADIP and Wtip puncta in the apical domain (white arrows). (B) Segmented cell outlines show random orientation of ADIP polarity vectors (arrows). (C) Rose plot confirms random distribution. (D-F) Stage 12 embryos were wounded on the ventral animal side and allowed to heal for 30 min in 0.7× MMR. ADIP becomes polarized in the cells proximal to the wound margin (D). Wtip labels cell borders (D′); merged image (D″). Arrows in D indicate ADIP puncta and polarized Wtip at cell junction facing the wound. Scale bar in D″: 20 μm; the same scale applies to panels A-A′′, B, E, G-G′, H-H′. Cell segmentation (E) and rose plot (F) show ADIP polarity vectors (arrows in E) oriented toward the wound site (0°). Rose plot represents pooled polarity from two embryos. Chi-square test indicates a non-random distribution, P <0.05. (G-I) Anti-Wtip staining of stage 10.5 embryos co-injected with 20 ng of control MO (G-G′) or Wtip MO (H-H′) and 50 pg H2B-GFP RNA as lineage tracer. MO-injected cells are marked by rabbit polyclonal antibody for GFP; merged images in G′,H′. (I) Immunoblotting of endogenous Wtip in stage 13 embryo lysates injected with control MO or Wtip MO. Blots were probed with mouse anti-Wtip (DA2B11) antibody (upper panel) and anti-β-catenin antibody as a loading control (lower panel). Data shown are representative of three independent experiments, each performed on 10-15 embryos.

Article Snippet: Primary antibodies used were rabbit anti-ADIP, mouse anti-Wtip [DA2B11, Developmental studies hybridoma bank (DSHB)] and mouse anti-ZO1-FITC (Invitrogen) to label cell membranes, rabbit anti-β-catenin antibody C2206 (Sigma-Aldrich), mouse anti-HA antibody (12CA5) and rabbit anti-GFP antibody A6455 (Invitrogen) to detect the HA-myrBFP or H2B-eGFP tracer in MO-injected cells.

Techniques: Immunostaining, Staining, Injection, Control, Western Blot

Journal: Biology Open

Article Title: Planar polarization of endogenous ADIP during Xenopus neurulation

doi: 10.1242/bio.062452

Figure Lengend Snippet: ADIP polarization in the neural and epidermal ectoderm of Xenopus embryos. En face views of the dorsal surface of representative embryos at stages 14-16 that were double stained with rabbit anti-ADIP and mouse anti-Wtip antibodies. (A,E,I) Low magnification view. Scale bars: 20 μm. (B,B′,F,F′,J,J′) High magnification views of the areas that are boxed in A,E,I, respectively. Scale bars: 20 µm. LNE, left anterior neuroectoderm; RNE, right anterior neuroectoderm; NNE, nonneural ectoderm. Wtip marks cell borders. A-P axis is indicated. Dashed line indicates the dorsal midline in A,E,I and the anterior border of the neural plate in J,K. Following cell segmentation in C,G,K, ADIP puncta enrichment was quantified by rose plots (D,H,L). ADIP puncta are oriented to the right to the midline in A-D, to the left towards the midline in E-H, and to the anterior border of the neural plate in I-L. Polarity vectors were quantified in (C,G,K), and angular distribution is shown in the corresponding rose plot (L). Rose plots represent data from three embryos. High significance of ADIP orientation is confirmed by Chi-square test, P <0.001 for all. Each experiment was repeated three times with 10-15 embryos per group.

Article Snippet: Primary antibodies used were rabbit anti-ADIP, mouse anti-Wtip [DA2B11, Developmental studies hybridoma bank (DSHB)] and mouse anti-ZO1-FITC (Invitrogen) to label cell membranes, rabbit anti-β-catenin antibody C2206 (Sigma-Aldrich), mouse anti-HA antibody (12CA5) and rabbit anti-GFP antibody A6455 (Invitrogen) to detect the HA-myrBFP or H2B-eGFP tracer in MO-injected cells.

Techniques: Staining