Journal: Nature Communications

Article Title: Decreased scleral Wnt5a hi fibroblasts exacerbate myopia progression by disrupting extracellular matrix homeostasis in mice

doi: 10.1038/s41467-025-67246-x

Figure Lengend Snippet: a Distribution of fibroblast subtypes expressed as a difference between FD-treated and FD-fellow sclera (two-sided chi-square test with Bonferroni correction for multiple testing). The X-axis indicates the subtypes corresponding to the uniform manifold approximation and projection plot. The Y-axis shows the log-transformed P -value obtained by the chi-square test between FD-treated and FD-fellow eyes’ scleral fibroblast subtypes. Positive values indicate a decrease, whereas negative values indicate an increase in the FD-treated eyes when compared with the FD-fellow eyes. b Heat map of the ECM outgoing (left) and incoming (right) of cell-cell interaction in all fibroblast subtypes. c Heat map of the outgoing (left) and incoming (right) cell-cell interaction signals of all fibroblast subtypes. The row indicates the relative strength of each interaction pathway. The column indicates the total interaction strength of each fibroblast subtype. d Ligand-receptor pairs of the ncWNT signalling pathway of all fibroblast subtypes. The relative contribution of each ligand-receptor pair of the ncWNT signalling pathway among all scleral fibroblast subtypes. e Expression profile of Wnt5a of ncWNT signalling in each subtype of fibroblast. f Expression profile of Wnt11 of ncWNT signalling in each fibroblast subtype. g Wnt5a mRNA level in FD-48h treated eyes and FD-48h fellow eyes (two-sided Wilcoxon signed-rank test). h Information flow of each cell–cell interaction pathway in FD treatment for 24 h or 48 h. FD form deprivation. Data are expressed as mean ± SEM.

Article Snippet: The sections were blocked with 6% normal donkey serum, 1% BSA, and 0.2% Triton X-100 in 0.1 M PBS for 2 h at room temperature, then incubated with rabbit polyclonal antibodies against WNT5A (1:100, #bs-1948R, Bioss, Woburn, MA, USA) and SPARC (1:300, #15274-1-AP, Proteintech, Rosemont, IL, USA) for 24 h at 4 °C.

Techniques: Transformation Assay, Expressing

Journal: Nature Communications

Article Title: Decreased scleral Wnt5a hi fibroblasts exacerbate myopia progression by disrupting extracellular matrix homeostasis in mice

doi: 10.1038/s41467-025-67246-x

Figure Lengend Snippet: a Locations of regionally differentiated sclera: iPPS, oPPS, and peripheral sclera. b Alteration of the number and percentage of Wnt5a + A2m + cells and Wnt5a + puncta in the temporal and nasal iPPS, oPPS, and peripheral sclera in normal murine eyes at age 3 weeks. n = 18 micrographs; Friedman test with Dunn’s post hoc test (two-sided) or RM-ANOVA with Bonferroni’s post hoc test (two-sided). c , d The numbers and percentages of Wnt5a + A2m + cells and Wnt5a + puncta in the temporal/nasal iPPS, oPPS, and peripheral sclera in NC eyes, FD-F eyes, and FD-T eyes. n = 9 micrographs for NC eyes; for FD eyes, n = 12 micrographs in FD-24h and n = 9 micrographs in FD-48h, one-way ANOVA with Bonferroni’s post hoc test or Kruskal-Wallis non-parametric test with Dunn’s post hoc test (two-sided). e The Wnt5a mRNA level in NC eyes, FD-F eyes, and FD-T eyes treated by FD-48 h or FD-2W. For NC eyes, n = 10 in FD-48 h and n = 18 in FD-2W; for FD-F eyes and FD-T eyes, n = 9; one-way ANOVA with Bonferroni’s post hoc test. WNT5A protein immunofluorescence intensity ( f ) and distribution ( g ) in the temporal/nasal iPPS, oPPS, and peripheral sclera in normal murine eyes. n = 11, Friedman tests with Dunn’s post hoc tests (two-sided). WNT5A protein immunofluorescent intensity ( h ) and change ( i ) in the temporal/nasal iPPS, oPPS, and peripheral sclera in NC eyes, FD-F eyes, and FD-T eyes. For NC eyes, n = 5 in FD-48 h and n = 6 in FD-2W; for FD-F eyes and FD-T eyes, n = 5, one-way ANOVA with Bonferroni’s post hoc test or Kruskal–Wallis non-parametric test with Dunn’s post hoc test (two-sided). Western blots ( j ) and densitometric quantification ( k ) of scleral WNT5A protein in FDM mice. n = 6, one-way ANOVA with Bonferroni’s post hoc test (two-sided). red: Wnt5a ; green: A2m ; blue: 4’, 6-diamidino-2-phenylindole (DAPI), iPPS the inner peripapillary sclera, oPPS the outer peripapillary sclera, peripheral the peripheral sclera. NC normal control eyes, FD-F untreated contralateral eyes of form deprivation mice, FD-T form-deprived eyes. Data are expressed as mean ± SEM.

Article Snippet: The sections were blocked with 6% normal donkey serum, 1% BSA, and 0.2% Triton X-100 in 0.1 M PBS for 2 h at room temperature, then incubated with rabbit polyclonal antibodies against WNT5A (1:100, #bs-1948R, Bioss, Woburn, MA, USA) and SPARC (1:300, #15274-1-AP, Proteintech, Rosemont, IL, USA) for 24 h at 4 °C.

Techniques: Immunofluorescence, Western Blot, Control

Journal: Nature Communications

Article Title: Decreased scleral Wnt5a hi fibroblasts exacerbate myopia progression by disrupting extracellular matrix homeostasis in mice

doi: 10.1038/s41467-025-67246-x

Figure Lengend Snippet: a sh Wnt5a -AAV injection and refraction and ocular biometrics measurements were carried out at different time points. b AAV infection in the whole murine sclera after sh Wnt5a sub-Tenon’s injection. c The scleral Wnt5a mRNA level declined by sh Wnt5a -AAV injection. n = 6 for sh Scramble -AAV injected group and n = 7 for sh Wnt5a -AAV injected group, Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 post hoc test (two-sided). d , e WNT5A protein levels were significantly downregulated by sh Wnt5a -AAV injection. n = 7 mice per group, Friedman test with Dunn’s post hoc test (two-sided). f The refraction of sh Wnt5a -AAV injected eyes underwent a greater myopic shift than did the shScramble-AAV injected mice. n = 15 for the shScramble-AAV injected group and n = 13 for the sh Wnt5a -AAV injected group, two-way repeated measures ANOVA with Bonferroni’s post hoc test. g Compared with shScramble-AAV infection, sh Wnt5a -AAV infection caused a significant increase in axial length. n = 15 for the shScramble-AAV injected group and n = 13 for the sh Wnt5a -AAV injected group, two-way repeated measures ANOVA with Bonferroni’s post hoc test. Col1a1 and Acta2 mRNA ( h , for Col1a1 , n = 6 for shScramble-AAV injected group and n = 10 for sh Wnt5a -AAV injected group, Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 post hoc test, two-sided; for Acta2 , n = 7 for shScramble-AAV injected group and n = 9 for sh Wnt5a -AAV injected group, one-way ANOVA with Bonferroni’s post hoc test, two-sided) and protein ( i , j, n = 7 for COL1A1 and n = 3 for α-SMA, RM-ANOVA with Bonferroni’s post hoc test (two-sided) for COL1A1 and Friedman test with Dunn’s post hoc test (two-sided) for α-SMA) level in shScramble-AAV, sh Wnt5a -AAV injected mice. k scleral fibril ultrastructure and scleral fibril diameter of the fellow eyes and the treated eyes in shScramble-AAV injected mice and sh Wnt5a -AAV injected mice ( n = 3 mice per group). Data are expressed as mean ± SEM.

Article Snippet: The sections were blocked with 6% normal donkey serum, 1% BSA, and 0.2% Triton X-100 in 0.1 M PBS for 2 h at room temperature, then incubated with rabbit polyclonal antibodies against WNT5A (1:100, #bs-1948R, Bioss, Woburn, MA, USA) and SPARC (1:300, #15274-1-AP, Proteintech, Rosemont, IL, USA) for 24 h at 4 °C.

Techniques: Injection, Infection

Journal: Nature Communications

Article Title: Decreased scleral Wnt5a hi fibroblasts exacerbate myopia progression by disrupting extracellular matrix homeostasis in mice

doi: 10.1038/s41467-025-67246-x

Figure Lengend Snippet: Six weeks after sub-Tenon injection of OE Wnt5a -AAV, Wnt5a was overexpressed in the sclera at mRNA ( a , n = 5 for vector -AAV injected group and n = 9 for OE Wnt5a -AAV injected group, Kruskal–Wallis non-parametric test with Dunn’s post hoc test) and protein levels ( b , c, n = 3, RM-ANOVA with Bonferroni’s post hoc test, two-sided). d Schematic schedule of FD treatment and Wnt5a overexpression with AAV injection. In the FDM mouse model, OE Wnt5a -AAV injected + FDM mice developed less myopic shift than did the vector-AAV injected mice ( e ). In comparison with the vector-AAV injected eyes, OE Wnt5a -AAV injected + FD eyes exhibited a significant difference for axial length ( f, n = 13 for vector-AAV injected group and n = 16 for OE Wnt5a -AAV injected group, two-way repeated measures ANOVA with Bonferroni’s post hoc tests, two-sided, for refraction and for axial length). g , h Wnt5a overexpression can partially rescue the COL1A1 protein level, which is downregulated by FD. n = 7, ordinary two-way ANOVA with Bonferroni’s post hoc test (two-sided). In mice undergoing normal refractive development ( i ), sub-Tenon’s injection of OE Wnt5a -AAV did not affect refraction ( j ) or axial length ( k ). n = 11 for the vector-AAV injected group and n = 15 for the OE Wnt5a -AAV injected group, two-way repeated measures ANOVA with Bonferroni’s post hoc test (two-sided). FD form deprivation; FDM form deprivation myopia. Data are expressed as mean ± SEM.

Article Snippet: The sections were blocked with 6% normal donkey serum, 1% BSA, and 0.2% Triton X-100 in 0.1 M PBS for 2 h at room temperature, then incubated with rabbit polyclonal antibodies against WNT5A (1:100, #bs-1948R, Bioss, Woburn, MA, USA) and SPARC (1:300, #15274-1-AP, Proteintech, Rosemont, IL, USA) for 24 h at 4 °C.

Techniques: Injection, Plasmid Preparation, Over Expression, Comparison

Journal: Nature Communications

Article Title: Decreased scleral Wnt5a hi fibroblasts exacerbate myopia progression by disrupting extracellular matrix homeostasis in mice

doi: 10.1038/s41467-025-67246-x

Figure Lengend Snippet: a , b Gene ontology (GO) and Reactome pathway analysis of differentially expressed genes (DEGs) from scleral bulk RNA-seq after sub-Tenon’s injection of sh Wnt5a -AAV ( n = 2 each group, one-sided hyper-geometric test with Benjamini-Hochberg correction for multiple testing). c DEGs heatmap of the ECM pathway from scleral bulk RNA-seq analysis. d Reverse Transcription quantitative Polymerase Chain Reaction validation of the DEGs ( Sparc , Adamts2 , Pcolce2 , Pcolce , Bmp1 , Itga2 ) from bulk RNA-seq analysis. For Sparc, n = 4 for shScramble-AAV injected group and n = 6 for sh Wnt5a -AAV injected group; for Adamts2 , Pcolce2 and Bmp1, n = 5 for both shScramble-AAV injected group and sh Wnt5a -AAV injected group; for Pcolce, n = 3 for shScramble-AAV injected group and n = 5 for sh Wnt5a -AAV injected group; for Itga2 , n = 4 for shScramble-AAV injected group and n = 5 for sh Wnt5a -AAV injected group. One-way ANOVA with Bonferroni’s post hoc test (two-sided) for Sparc, Adamts2 , and Pcolce , Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 post hoc tests (two-sided) for Pcolce2, Bmp1 , and Itga2 . e , f Network of the 20 hub genes and functional enrichment (one-sided hyper-geometric test with Benjamini–Hochberg correction for multiple testing) of the Wnt5a assigned gene module. g Western blots and densitometric quantification of scleral WNT5A and SAPRC proteins; representative blots are shown for sh Wnt5a -AAV injected mice. n = 5, Friedman tests with Dunn’s post hoc test (two-sided) for WNT5A; RM-ANOVA with Bonferroni’s post hoc test (two-sided) for SAPRC. h , i Immunofluorescence intensity and quantification of SPARC protein expression in the temporal/nasal iPPS, oPPS, and peripheral sclera of NC eyes. In FD-48h, n = 6 mic per group; in FD-2W, n = 3 for NC eyes and n = 4 for both FD-F and FD-T eyes. One-way ANOVA with Bonferroni’s post hoc test (two-sided), Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 post hoc test (two-sided) or Kruskal–Wallis non-parametric test with Dunn’s post hoc test (two-sided) were used. Red: SPARC; blue: 4’, 6-diamidino-2-phenylindole (DAPI); iPPS the inner peripapillary sclera, oPPS the outer peripapillary sclera, peripheral, the peripheral sclera. NC normal control eyes, FD-F untreated contralateral eyes of form deprivation mice, FD-T form deprivation treated eyes. Data are expressed as mean ± SEM.

Article Snippet: The sections were blocked with 6% normal donkey serum, 1% BSA, and 0.2% Triton X-100 in 0.1 M PBS for 2 h at room temperature, then incubated with rabbit polyclonal antibodies against WNT5A (1:100, #bs-1948R, Bioss, Woburn, MA, USA) and SPARC (1:300, #15274-1-AP, Proteintech, Rosemont, IL, USA) for 24 h at 4 °C.

Techniques: RNA Sequencing, Injection, Reverse Transcription, Real-time Polymerase Chain Reaction, Biomarker Discovery, Functional Assay, Western Blot, Immunofluorescence, Expressing, Control

Journal: Nature Communications

Article Title: Decreased scleral Wnt5a hi fibroblasts exacerbate myopia progression by disrupting extracellular matrix homeostasis in mice

doi: 10.1038/s41467-025-67246-x

Figure Lengend Snippet: Abnormal visual stimulation propagating via the retina-choroid-sclera signalling cascade reduces Wnt5a hi fibroblast numbers and/or activity, and thus Wnt5a secretion. Subsequently, the expression of genes for ECM molecules such as Col1a1 , Sparc , Pcolce , Pcelce2 , Adamts2 , Bmp1 , and Igta2 is downregulated via the noncanonical Wnt signalling pathway (Wnt5a-Ca 2+ -CaMKII). This response may disrupt ECM structural organisation, decrease collagen I level, and reduce the collagen fibril diameter, finally resulting in myopia progression due to increased distension of the posterior sclera under constant intraocular pressure.

Article Snippet: The sections were blocked with 6% normal donkey serum, 1% BSA, and 0.2% Triton X-100 in 0.1 M PBS for 2 h at room temperature, then incubated with rabbit polyclonal antibodies against WNT5A (1:100, #bs-1948R, Bioss, Woburn, MA, USA) and SPARC (1:300, #15274-1-AP, Proteintech, Rosemont, IL, USA) for 24 h at 4 °C.

Techniques: Activity Assay, Expressing

Journal: Bone Research

Article Title: Skeletal progenitor LRP1 deficiency causes severe and persistent skeletal defects with Wnt pathway dysregulation

doi: 10.1038/s41413-024-00393-x

Figure Lengend Snippet: LRP1 mediates endocytosis of Wnt5a, a core non-canonical WNT/planar cell polarity (PCP) pathway component. a Schematic diagram illustrating the long bone phenotype of Lrp1 flox/flox /Prrx1 Cre mice. Full-length sLRP1 was coated onto microtiter plates and the binding of 0–200 nmol/L Wnt5a ( b ), Wnt11 ( c ), Wnt3a ( d ) was measured using specific antibody for each Wnt as described under “Materials and Methods”. Mean values of technical duplicates for none-coating, LRP1-coating and after normalisation were shown as circles, squares and triangles, respectively. Extrapolated K D,app values were estimated based on one-phase decay nonlinear fit analysis (black lines). WT and LRP1 KO MEFs ( n = 3) were incubated with 40 nmol/L Wnt5a for 0.5–60 min and Wnt5a in the cell lysate was detected by Western blotting ( e ). The relative amount of Wnt5a was expressed by taking the amount of Wnt5a after 60-min incubation as 1 ( f ). Circles represent individual mice and bars show the mean ± S values for the amount of Wnt5a after incubation for 5–30 min in WT versus LRP1 KO MEFs were evaluated by two-way ANOVA. * P < 0.05. Representative images of confocal microscopy analysis for Wnt5a and LRP1 in WT and LRP1 KO MEFs ( n = 3) ( e ) or human normal chondrocytes ( n = 3) f Cells were incubated with 20 nmol/L Wnt5a for 3 h in the absence ( g , h ) or presence of 500 nmol/L RAP ( h ). Wnt5a, LRP1, cytoskeleton and nucleus were visualised as described under “Materials and Methods”. Scale bar, 10 µm. Regions delineated by the white squares in the panels have been magnified in the top right ( g ). WT and LRP1 KO MEFs ( n = 3) were incubated with 20 nmol/L Wnt5a for 3–24 h and Wnt5a in the medium and cell lysate were detected by Western blotting ( i ). Densitometric analysis of immunoreactive Wnt5a bands was carried out. The relative amount of Wnt5a in the media, cell lysate and both media and cell lysate (total) were expressed by taking the amount of Wn5a after 3-h incubation in WT MEFs as 1 ( j ). k human normal chondrocytes ( n = 3) were incubated with 20 nmol/L Wnt5a for 1–24 h and analysed as in a and b . The relative amount of Wnt5a after 24-h incubation was expressed by taking the amount of Wn5a after 1-h incubation as 1. Circles represent individual experiment and bars show the mean ± SD

Article Snippet: The primary antibodies used were as follows; rabbit monoclonal anti-LRP1 (1:200)(ab92544, Abcam), rabbit polyclonal anti-Sox9 (1:500)(AB5535, SIGMA), rabbit polyclonal anti-Wnt5a (1:200)(bs1948R, Bioss).

Techniques: Binding Assay, Incubation, Western Blot, Confocal Microscopy

Journal: Bone Research

Article Title: Skeletal progenitor LRP1 deficiency causes severe and persistent skeletal defects with Wnt pathway dysregulation

doi: 10.1038/s41413-024-00393-x

Figure Lengend Snippet: LRP1 partially colocalises with Wnt5a and its deficiency alters abundance and distribution of Wnt5a in the developing limbs. Representative images of confocal microscopy analysis for Wnt5a and LRP1 ( a and b ), and total ( c and e ) and phosphorylated Vangl2 ( d and f ) in E13.5 ( a , c and d ) or E16.5 ( b , e and f ) hind limb sections of WT and Lrp1 flox/flox /Prrx1 Cre (cKO) mice ( n = 3). Wnt5a, LRP1, Vangl2, phospho-Vangl2 and nucleus were visualised as described under “Materials and Methods”. Regions delineated by the white squares have been magnified in the top right of each panel. PF proliferative flattened chondrocytes; PC perichondrium. Scale bar, 50 µm

Article Snippet: The primary antibodies used were as follows; rabbit monoclonal anti-LRP1 (1:200)(ab92544, Abcam), rabbit polyclonal anti-Sox9 (1:500)(AB5535, SIGMA), rabbit polyclonal anti-Wnt5a (1:200)(bs1948R, Bioss).

Techniques: Confocal Microscopy

Journal: Stem Cell Reviews and Reports

Article Title: HucMSCs-derived Exosomes Promote Lung Development in Premature Birth via Wnt5a/ROCK1 Axis

doi: 10.1007/s12015-024-10824-1

Figure Lengend Snippet: Term-Exos promoted the expression of lung development related proteins. A : WB examined the expressions of SPC and RhoA in E13.5 lung tissues. In term-Exos group, the expression of SPC and RhoA were obviously increasing. B : WB examined the expressions of SPC and RhoA in E18.5 lung tissues. Like which in E13.5 group, the expression of SPC and RhoA were improved in term-Exos groups. C : The expression of ABCA3 and SPB were examined by WB, which showed the expressions of ABCA3 in E13.5 and E18.5 stage were increasing after term-Exos injection, while the expressions of SPB precursor and mature SPB in E18.5 were obviously promoted after term-Exos injection. D , E , F : Real-time PCR examined the mRNA expressions of SFTPC, RhoA and ABCA3 in E13.5 and E18.5 lung tissue. Among E13.5 groups, the expression of ABCA3 mRNA was obviously increasing after term-Exos injection compared with the control while there were no significant differences in E18.5 ( n =3, *P<0.05, **P<0.01 ). Among E18.5 groups, the expression of SFTPC mRNA was increasing after term-Exos injection, while in E13.5, the result was opposite ( n =3, *P<0.05,**P<0.01 ). The expression of RhoA mRNA, however, was seemed to be inhibited after preterm-Exos use which was lower than that of term-Exos group, and there was no significant differences between the control and term-Exos group (n=3, *P<0.05,**P<0.01 )

Article Snippet: Overnight at 4 °C, the membranes were incubated with primary antibodies, which included monoclonal antibodies against SPC (Sigma, USA), ROCK1 (Cell Signaling Technology, USA) and p-ROCK1 (Invitrogen, USA), polyclonal antibodies against RhoA (Cell Signaling Technology, USA), Wnt5a (abcam, UK), LC3B (Cell Signaling Technology, USA), Atg5 (Abcam, UK), SPB (Santa cruz biotechnology, USA) and ABCA3 (Bioss, CHN), as well as a monoclonal antibody against β-actin (Millipore, US).

Techniques: Expressing, Injection, Real-time Polymerase Chain Reaction, Control

Journal: Stem Cell Reviews and Reports

Article Title: HucMSCs-derived Exosomes Promote Lung Development in Premature Birth via Wnt5a/ROCK1 Axis

doi: 10.1007/s12015-024-10824-1

Figure Lengend Snippet: A schematic model showing the proposed mechanism for the role of exosomal Wnt5a in lung development. Term-Exos: Term infant umbilical cord mesenchymal stem cells-derived exosomes, Ror: Receptor tyrosine kinase-like orphan receptor; FZD 6 : Frizzled receptor 6; ROCK1: Rho-associated protein kinase 1; RhoA: Ras homolog gene family, member A; LC3B: Microtubule-associated proteins 1A/1B light chain 3B; P: Phosphorylation; α-SMA: alpha smooth muscle actin

Article Snippet: Overnight at 4 °C, the membranes were incubated with primary antibodies, which included monoclonal antibodies against SPC (Sigma, USA), ROCK1 (Cell Signaling Technology, USA) and p-ROCK1 (Invitrogen, USA), polyclonal antibodies against RhoA (Cell Signaling Technology, USA), Wnt5a (abcam, UK), LC3B (Cell Signaling Technology, USA), Atg5 (Abcam, UK), SPB (Santa cruz biotechnology, USA) and ABCA3 (Bioss, CHN), as well as a monoclonal antibody against β-actin (Millipore, US).

Techniques: Derivative Assay, Phospho-proteomics