Santhi Velayudhan S et al. (2026), Planar polarization of endogenous ADIP during X...

XB-ART-61696

Biol Open 2026 Feb 15;152:. doi: 10.1242/bio.062452.

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Planar polarization of endogenous ADIP during Xenopus neurulation.

Santhi Velayudhan S, Itoh K, Chu CW, Alfandari D, Sokol SY.

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Coordinated cell polarity and force-responsive protein localization are essential for tissue morphogenesis, yet how embryonic cells sense forces and respond to mechanical cues remains a challenging question. Afadin- and α-actinin-binding protein (ADIP) has been implicated in microtubule minus-end anchoring, centrosome maturation and ciliogenesis. ADIP is also proposed to associate with the actomyosin cortex and regulate collective cell migration. ADIP behaves as a mechanosensitive planar cell polarity (PCP) protein when overexpressed in Xenopus embryos, but the distribution and regulation of endogenous ADIP has been unknown. Here we show that ADIP is present in early ectoderm as randomly distributed puncta that rapidly reorganize and polarize during epithelial wound repair. Endogenous ADIP also becomes enriched and planar polarized in the anterior neural plate towards the midline, consistent with its regulation by mechanical forces that operate during neural tube closure. ADIP polarization is attenuated by depletion of the core PCP component Diversin/Ankrd6, in agreement with the proposed interaction between the two proteins during PCP establishment. Finally, pharmacological disruption of microtubules, F-actin, and nonmuscle myosin II eliminates ADIP polarization in the neuroectoderm, indicating roles for microtubules and actomyosin networks in PCP. Together, these findings suggest that endogenous ADIP senses mechanical cues via the cytoskeletal machinery and functions in a context-dependent manner to control collective cell behaviors during vertebrate morphogenesis.

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Species referenced: Xenopus laevis
Genes referenced: ssx2ip
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	Fig. 1. Distribution of endogenous ADIP in Xenopus ectoderm. (A-E′) Double staining of stage 12 Xenopus ectoderm with anti-ADIP and anti-ZO1 (A-C′) or anti-HA (D-E′) antibodies. (A-A′) Background staining with rabbit IgG (A). ZO1 marks cell borders in the merged image (A′). (B-C′) ADIP is detected predominantly as peri-junctional puncta in stage 10 ectoderm (B) and cytoplasmic puncta in stage 12 (C). (B′,C′) ZO1 marks cell borders. (D-D′) Control MO (20 ng) co-injected with 50 pg HA-myrBFP RNA does not reduce ADIP staining (D), HA-myrBFP labels cell borders in D′. (E-E′) ADIP MO (20 ng) co-injected with 50 pg of HA-myrBFP RNA reduces ADIP puncta (E), HA-myrBFP marks cell borders in E′. Scale bar: 20 µm. (F) Quantification of ADIP fluorescence intensity in uninjected embryos and in embryos injected with control MO and ADIP MO and cultured until at stage 13. Data represent mean fluorescence intensities from three embryos. Welch's t-test, P<0.001. (G) Anti-ADIP antibody detects 67 kDa protein in stage 12 animal cap lysates by immunoblotting. The 67 kDa band is reduced by ADIP MO but not control MO. Non-specific band (asterisk) is unaffected by ADIP MO. Actin is a loading control (lower panel). Experiments were repeated three times, with 10-15 embryos analyzed per experiment.
	Fig. 2. 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.
	Fig. 3. 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.
	Fig. 4. Diversin knockdown disrupts ADIP polarization. (A) Experimental scheme. CoMO, ADIP MO, or Diversin-MO (DivMO), 20 ng each, were injected into the left dorsal blastomere of four-cell X. laevis embryos to target neural ectoderm. MyrBFP-HA RNA was co-injected as a membrane lineage tracer. (B) En face low magnification view of stage 16 embryo immunostained for ADIP and HA. Scale bar: 20 μm. The anterior-posterior (AP) axis and neural plate boundary are marked; dashed box indicates the region magnified in panels C,E,G. (C-C″) Control MO. Arrows mark clearly polarized cells. (E-E″) ADIP MO reduces ADIP fluorescence (E) and expands apical domain. (G-G″) DivMO. Cells lacking ADIP polarity are marked by asterisks. Scale bar: 25 µm. (D,F,H) Rose plots quantify ADIP polarity. (I) Quantification of ADIP intensity in cells injected with DivMO (n=74 cells), and ADIP MO (n=58 cells), as compared to control MO (n=107 cells). Means and s.d. are shown on graphs; one-way ANOVA, ****P<0.0001. Results are representative of three experiments, with more than ten embryos in each group.
	Fig. 5. Planar polarity of ADIP in neuroectoderm requires microtubules. (A,D,G) Neural plates of stage 16 embryos were treated with DMSO (negative control, A), 2 µM Taxol (positive control, D), or 2.5 µM nocodazole (G) from stage 12 to stage 16 and immunostained for ADIP and Wtip. The anterior–posterior (AP) axis is labeled, and the neural plate is outlined with dashed lines. Scale bar: 20 μm. (B,E,H) Boxed regions correspond to the anterior neural ectoderm. Wtip marks cell borders in B′-B″,E′-E″,H′-H″. Colocalization of ADIP and Wtip puncta is visible. Scale bar: 20 µm. Fluorescent images represent three independent experiments, each consisting of 15 embryos per group. (C,F,I) Rose plots quantify ADIP polarity vectors after the DMSO (C), taxol (F) and nocodazole (I) treatments, respectively. Data were obtained for three embryos per group. Chi-square test indicates non-random ADIP puncta orientation in C and F, P<0.0001.
	Fig. 6. Actomyosin contractility and F-actin integrity are necessary for ADIP planar polarization in neuroectoderm. (A) Experimental scheme. Embryos were treated with different drugs to inhibit the RhoA–ROCK–Myosin pathway and were double stained at stage 16 for ADIP and Wtip. (B) Low magnification dorsal view of stage 16 neurulae. Scale bar: 20 μm. Dashed boxes denote the anterior neural ectoderm shown at higher magnification in C-C″,E-E″,G-G″ and I-I″. The anterior-posterior (AP) axis is labeled, and the neural plate is outlined with dashes. Images (C-I″) represent three independent experiments with 10-15 embryos per group. (C-C″) Control embryo (0.01% DMSO). (D) Rose plot indicates significant planar polarization of ADIP. (E-E″) ROCK inhibitor Y-27632 (50 µM) disrupts ADIP polarity. (F) Rose plot reveals loss of ADIP polarity. (G-G″) Blebbistatin (Blebb, 25 µM) similarly abolishes ADIP polarity. (H) Rose plot confirms loss of polarity. (I-J) Depolymerization of F-actin with 2.5 µM cytochalasin D (Cyto D) diminishes ADIP orientation. ADIP (I) and Wtip (I′) staining, along with merged image (I″), show reduced medial enrichment; scale bar: 20 µm. (J) Rose plot shows loss of polarity.