Velayudhan SS et al. (2025), Mechanosensitive localization of Diversin highl...

XB-ART-61421

Biol Open 2025 Aug 15;148:. doi: 10.1242/bio.062128.

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Mechanosensitive localization of Diversin highlights its function in vertebrate morphogenesis and planar cell polarity.

Velayudhan SS , Chu CW , Itoh K , Sokol SY .

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Diversin is a vertebrate homolog of the core planar cell polarity (PCP) protein Diego. Here we studied the function of Diversin in Xenopus embryo morphogenesis and its subcellular localization at different locations in superficial ectoderm cells. Depletion of Diversin in the neuroectoderm inhibited apical domain size and neural tube closure and disrupted the polarized localization of endogenous Vangl2, another PCP protein. Whereas Diversin puncta were randomly distributed in early ectoderm, they acquired planar polarity in the neuroectoderm in a stage- and position-specific manner. We find that Diversin is accumulated at the cell junctions adjacent to apically constricting cells at the Xenopus neural plate border and the gastrula blastopore lip. Moreover, Diversin cytoplasmic puncta redistributed in the direction of the pulling forces from the cells with constricting apical domains, suggesting a mechanosensitive process. PCP complexes of Dishevelled (Dvl2) and Diversin or the mechanosensitive adaptor ADIP exhibited planar polarity in the neural plate and the wound edge and promoted wound healing. We propose that Diversin- and Dvl2-containing PCP complexes control morphogenesis in a tension-dependent manner.

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	Fig. 1. Diversin depletion impairs apical constriction and leads to planar polarity defects in the neuroectoderm. (A) Experimental scheme. Diversin (Div) morpholino (MO) or control MO (CoMO) was injected with HA-myr-BFP RNA as lineage tracer into a single dorsal blastomere of four-cell Xenopus embryos. (B,C) Diversin depletion inhibits neural plate folding, reduces F-actin and expands the apical domain. (B) Representative stage 16 embryo shows unilaterally expanded neural plate and reduced F-actin intensity in DivMO-injected cells marked by HA-myr-BFP (magenta). (B′) Magnified view of phalloidin-stained cells illustrating the expanded apical domain in DivMO-injected cells (asterisks) on the right side, as indicated by the dashed magenta line. (C) Quantification of the apical domain area; total numbers of cells per group are indicated. ****P<0.0001, Student's t-test. (D-E′) Representative phenotypes of stage 16 embryos unilaterally injected at the four-cell stage with 30 ng of either control morpholino (CoMO) or Diversin MO (DivMO). Arrowheads indicate the injected side. Compared to the normal neural tube closure in CoMO-injected embryos (D), DivMO-injected embryos exhibited a wide range of defects, from mild abnormalities with weak or discontinuous pigmentation (E) to severe neural tube folding defects (E′). (F-I′) Defective Vangl2 polarity in Diversin-depleted embryos. Cells of stage 16 embryo injected with DivMO are marked with HA-myr-BFP (magenta). Anteroposterior (A–P) axis is indicated. (F,H) Vangl2 immunostaining in the magnified regions of CoMO- (F) and DivMO-injected (H) embryos (dashed boxes). (G-G′) Vangl2 is polarized in CoMO-injected cells. (I-I′) Vangl2 polarity is reduced in DivMO-injected cells (asterisks). Anti-HA staining for HA-myr-BFP (F,G′,H,I′), and anti-Vangl2 staining (G,I) are shown. HA-myr-BFP staining is variable for F and H, due to distinct locations of the injection sites. Scale bars: 20 μm. Data represent six embryos from three independent experiments.
	Fig. 2. Planar polarization of Diversin in ectoderm cells during neurulation. (A) Schematic of RNA injection: HA-RFP-Diversin RNA (200 pg) was co-injected with myr-BFP RNA (50 pg) as cell border and lineage tracer into four-cell embryos to target neural or non-neural ectoderm. (B-D) Animal pole ectoderm at stage 12. (E-H) Anterior neural plate at stages 14. (I-L) Nonneural ectoderm adjacent to anterior neural plate border. Yellow arrows indicate Diversin accumulation at cell corners. Dashed boxes highlight regions with polarized Diversin that are magnified in F and J. Neural plate border is marked by dashed lines or intense F-actin staining (magenta in I). Anterior–posterior axis is shown in (E,I). NE and NNE, neural and non-neural ectoderm, respectively. Scale bar: 20 µm. (C,G,K) Cell segmentation. Red arrows indicate cell polarity marked by Diversin puncta enrichment. Rose plots (D,H,L) show Diversin puncta enrichment relative to the posterior axis (0° in H), neural plate midline (H), or anterior neural plate border (L). Data are from three embryos. Chi-square tests indicate non-random Diversin distribution.
	Fig. 3. Diversin polarizes in the cells adjacent to the blastopore lip during gastrulation. (A) Schematic of RNA injections. RFP-Diversin (200 pg) and myr-BFP (50 pg) mRNAs were co-injected into the animal pole or the marginal zone of Xenopus four-cell embryos. (B) Animal view of early gastrula, (C-D′) vegetal view of stage 10.5 gastrula with red dashed boxes corresponding to the dorsal and ventral marginal zones (DMZ and VMZ) at the blastopore lip. (B-D″) RFP-Diversin localization in animal ectodermal cells (B,B′), or cells adjacent to the DMZ (C,C′) and VMZ (D,D′) blastopore lip at stage 10.5. Yellow arrows indicate asymmetric Diversin accumulation at cell corners. Scale bar: 50 μm. Data represent five to ten embryos per group.
	Fig. 4. Diversin puncta orient towards apically constricting neighboring cells. (A) Experimental scheme. Clones expressing control myr-GFP (20 pg), mNeonGreen-Shroom3 (20 pg), or GFP-Plekhg5 (20 pg) mRNA. RFP-Diversin-expressing cells are marked with myr-BFP (cyan). (B-B″,D-D″,F,F′) Random distribution of RFP-Diversin (red) in cells adjacent to control mem-GFP clones at stage 12.5. (B,B′) Polarized accumulation of RFP-Diversin toward neighboring mNeonGreen-Shroom3 (D,D′), and GFP-Plekhg5 (F,F′) expressing cells. Yellow arrows show the enrichment of Diversin puncta. Scale bar: 20 µm. (C,E,G) Rose plots show Diversin puncta orientation relative to the border of adjacent control cells (C), mNeonGreen-Shroom3-expressing cells (E) and GFP-Plekhg5-expressing cells (G), respectively. (0° is perpendicular to the border). Data represent three embryos per condition. Chi–square analysis confirms random distribution in controls (B-B″,C) but not in the cells adjacent to Shroom3 (D-D″,E) or Plekhg5-expressing cells (F-F″,G), respectively.
	Fig. 5. Diversin colocalizes with Dvl2 in neural and epidermal ectoderm. (A) Injection scheme. GFP-Dvl2 (200 pg) together with myr-BFP (50 pg) or RFP-Diversin (200 pg), GFP-Dvl2 (200 pg) with myr-BFP (50 pg) mRNAs were co-injected as indicated into one dorsal blastomere of four-cell stage Xenopus embryos. Myr-BFP marks cell borders. Neural plate border is outlined by yellow dashed lines, Phalloidin staining is shown (magenta), and the anterior–posterior (A–P) axis is indicated. White boxes mark stage 14 anterior neural ectoderm (NE) in (B-B′) and (E-E″) or adjacent stage 15 non-neural ectoderm (NNE) in (G-G′′). (B,B′) Stage 14 anterior neural ectoderm, dorsal view. GFP-Dvl2 (B); merged with myr-BFP (B′). GFP-Dvl2 puncta weakly oriented in few cells relative to midline of the neural plate. (C-D′) Stage 12 ectoderm. RFP-Diversin (C); GFP-Dvl2 (C′); merged with myr-BFP (C′′). Rose plots show random orientation of Diversin (D) and Dvl2 (D′). (E-F′) Stage 14 anterior neural ectoderm embryo show the localization of RFP-Diversin (E), GFP-Dvl2 (E′); merged with myr-BFP (E′′). Rose plots show orientation of Diversin (F) and Dvl2 (F′) to the midline of neural plate. (G-H′) Stage 15 anterior non-neural ectoderm, dorsal view. RFP-Diversin (G), GFP-Dvl2 (G′); merged with myr-BFP in G′′. White arrows show the orientation of Diversin and Dvl2. Scale bar: 20 µm. (E-G′), Rose plots show Diversin (H) and Dvl2 puncta (H′) polarization in tissue plane, 0° corresponds to the posterior axis. Quantification is based on three independent embryos. Chi-squire test shows non-random distribution of Diversin and Dvl2 puncta.
	Fig. 6. Polarization of the Dvl and ADIP complex during epidermal wound healing. (A-A″) Colocalization of RFP-Dvl2 (A) and GFP-ADIP (A′) in the control unwounded stage 11 Xenopus ectoderm. (B-B″) Dvl2 (B) and ADIP (B′) puncta polarize relative to the wound edge. Scale bars: 20 µm. Cell borders are outlined by myrBFP. Data are representative of two independent experiments. (C) Physical association of Dvl2-GFP and HARFP-ADIP revealed by the pulldown with GFP-trap. Non-specific band (asterisk) demonstrates equal protein loading. Molecular weight markers (kD) are indicated on the left. (D,E) Inhibition of epidermal wound healing by the dominant-interfering Dvl2 construct. Ectoderm explants were isolated from stage 9.5 embryos injected earlier with Dvl-DEP+ RNA (1 ng, E) or control embryos (D). Wound healing was assessed after 1 h post wounding as the area of the epidermis covering the wound (marked by the dotted line). Data are representative of three experiments with 58 explants in each group.