recombinant wnt9b  (R&D Systems)

Journal: bioRxiv

Article Title: Polycystin-1 acts as an atypical adhesion GPCR that responds to novel Wnt signaling and mechanical stimuli

doi: 10.1101/2025.05.21.655326

Figure Lengend Snippet: (a) Schematic diagram of the full length PC1 construct employed in these studies. The positions of the Flag tag, HA tag, GPS site and tethered agonist (TA) are indicated, as is the position of the lysine residues whose availability for biotinylation is assessed in 1e. (b) Immunofluorescence detection and quantification of surface PC1 NTF following 3 hrs treatment with Wnt9b. Flag staining (NTF) was performed under non-permeabilizing conditions. HA staining, following permeabilization, revealed total cell quantity of CTF and was used to normalize surface staining. Scale bar=5μM. Graph depicts ratio of Flag to HA staining, normalized to the mean of this value obtained after vehicle treatment. (c) PC1 and 2 expressing-HEK293 cells were treated with Wnt9b or subjected to an alkaline stripping protocol and surface biotinylation was performed. Proteins recovered by streptavidin pull-down were probed with anti-Flag. Cell lysates were probed with anti-HA antibodies. Surface NTF is detected by anti-Flag antibody, and total cell-associated PC1 is represented by the HA signal. The graph depicts ratio of Flag to HA signal, normalized to the mean of this value obtained after vehicle treatment. (d) Western blot detection of total PC1 NTF using anti-Flag antibody. Cells were treated with vehicle or Wnt9b overnight. Bar graphs represent Flag/HA signal ratio. (e) Western blot detection of biotinylated PC1 CTF in experiment where cell surface biotinylation was followed by HA pulldown and streptavidin blotting. Results show increased accessibility of the Lys residue in the PC1-CTF TM6-7 extracellular loop (extracellular loop 3) upon Wnt9b treatment or following alkaline stripping. Total cell-associated PC1 is represented by the HA signal. The graph depicts ratio of Streptavidin to HA signal, normalized to the mean of this value obtained after vehicle treatment. Data are shown as mean ±SEM, n≥3 for all experiments. To assess statistical significance unpaired Student t-Test analysis was performed for all of the panels except for the time course depicted in 1b, for which a paired Student t-Test was employed. P values <0.05 were considered significant. *=P<0.05; **=P<0.01; ***=P<0.001 Differences between mean values obtained for experimental conditions versus control conditions that produce minimal and/or maximal signals were evaluated.

Article Snippet: Recombinant Wnt9b (0.5ng/μL) (R&D Systems, 3669) was applied for 3 hrs or overnight.

Techniques: Construct, FLAG-tag, Immunofluorescence, Staining, Expressing, Stripping Membranes, Western Blot, Residue, Control

Journal: bioRxiv

Article Title: Polycystin-1 acts as an atypical adhesion GPCR that responds to novel Wnt signaling and mechanical stimuli

doi: 10.1101/2025.05.21.655326

Figure Lengend Snippet: (a) GSK3β co-immunoprecipitates with PC1 following NTF removal by exposure to high pH (strip) or Wnt9b. GSK3β associated with PC1 after 5 minutes of Wnt9b treatment. (b) GSK3β co-immunoprecipitates with PC1 from lysates prepared from Tg248 Pkd1 BAC-transgenic mouse kidneys. (c) Fluorescent biosensor assay of endogenous GSK3β kinase activity in HEK293 cells. PC1 and 2 inhibit GSK3β and this effect is enhanced by 3 hrs treatment with 13nM Wnt9b. Scale bar=100 μM. Graph depicts fluorescence intensity measured in each condition normalized to mean of that measured in cells that express GFP alone. (d) GSK3β kinase assay employing a peptide that electrophoretically migrates in agarose faster when phosphorylated. Lysates from PC1-expressing mouse kidney were compared to similar aged WT kidney lysates. Graph shows mean of the ratio of GSK3β-phosphorylated peptide (PP) to unphosphorylated peptide (P) under each condition. (e) Detection of S9 phosphorylated GSK3β in lysates from un-induced, pre-cystic and cystic kidneys. Blots were probed with anti pan-GSK3β, anti pS9-GSK3β and anti-actin antibodies. Graph depicts the mean of the ratio of the pS9-GSK3β signal to the pan GSK3β signal for each condition. (f) Fluorescent biosensor assay of endogenous GSK3β kinase activity in immortalized murine M113 cells. Untransfected cells were compared to cells expressing PC1 and PC2 as well as to PC1 and 2 expressing cells treated with Wnt9b for 3hrs Scale bar=100 μM. Graph depicts fluorescence intensity measured in each condition normalized to mean of that measured in cells that express GFP alone. Data are shown as mean ±SEM, n≥3 for all experiments. To assess statistical significance unpaired Student t-Test analysis was performed. P values <0.05 were considered significant. *=P<0.05; **=P<0.01; ***=P<0.001 Differences between mean values obtained for experimental conditions versus control conditions that produce minimal and/or maximal signals were evaluated.

Article Snippet: Recombinant Wnt9b (0.5ng/μL) (R&D Systems, 3669) was applied for 3 hrs or overnight.

Techniques: Stripping Membranes, Transgenic Assay, Biosensor Assay, Activity Assay, Fluorescence, Kinase Assay, Expressing, Control

Journal: bioRxiv

Article Title: Polycystin-1 acts as an atypical adhesion GPCR that responds to novel Wnt signaling and mechanical stimuli

doi: 10.1101/2025.05.21.655326

Figure Lengend Snippet: (a) PC1 ΔNTF co-immunoprecipitates with GSK3β to the same extent as Wnt9b-stimulated full length PC1. Less GSK3β co-immunoprecipitates with PC1 ΔNTF+ΔTA , which lacks a tethered agonist. The graph depicts the ratio of GSK3β signal to the HA signal, normalized to the value of this ratio for the HEK 1+2 condition. (b) GSK3β biosensor shows that PC1 ΔNTF inhibits GSK3β to the same extent as a maximal concentration (10 μM) of GSK3β inhibitor SB-216763. PC1 ΔNTF+ΔTA does not inhibit GSK3β. The graph depicts the ratio of GFP fluorescence in each condition, normalized to the mean of this value obtained with cells that express GFP alone. (c) Confocal Z-stack imaging of PC1 ΔNTF and PC1 ΔNTF+ΔTA detected with anti-HA antibody added to the media bathing unfixed and non-permeabilized cells shows that both proteins expressed in the absence of PC2 reach the surfaces of HEK293 cells. (d) PC1 ΔNTF+ΔTA , lacking the Stachel sequence, does not inhibit the GSK3β in the fluorescence biosensor assay. The ability of PC1 ΔNTF+ΔTA to inhibit GSK3β can be partially restored by addition of soluble Stachel peptide (300 μM). Scrambled Stachel sequence (scrm) did not produce this effect. Versions of the PC1 ΔNTF protein that carry successive groups of alanine substitutions in the sequences of their tethered agonists (PC1 ΔNTF -Ala1-4) do not inhibit GSK3β. Data are shown as mean ±SEM, n≥3 for all experiments. To assess statistical significance unpaired Student t-Test analysis was performed. P values <0.05 were considered significant *=P<0.05; **=P<0.01; ***=P<0.001.; ****=P<0.0001 Differences between mean values obtained for experimental conditions versus control conditions that produce minimal and/or maximal signals were evaluated. In panel 3d the values obtained for each condition were compared to those obtained in the GFP alone condition (indicated by the symbols above each bar) and pairwise comparisons of the values obtained in a subset of conditions related by a single experimental manipulation are indicated by the brackets above the bars.

Article Snippet: Recombinant Wnt9b (0.5ng/μL) (R&D Systems, 3669) was applied for 3 hrs or overnight.

Techniques: Concentration Assay, Fluorescence, Imaging, Sequencing, Biosensor Assay, Control

Journal: bioRxiv

Article Title: Polycystin-1 acts as an atypical adhesion GPCR that responds to novel Wnt signaling and mechanical stimuli

doi: 10.1101/2025.05.21.655326

Figure Lengend Snippet: (a) GSK3β activity measured +/− Gα13 or RhoA or control (Ctrl) siRNAs, or ROCK inhibitor Y27632. Suppressing Gα13, RhoA or ROCK dampened GSK3β inhibition by Wnt9b-stimulated PC1 or PC1ΔNTF. 10 μM SB-216763 (SB) treatment demonstrates fluorescence detected with maximal GSK3β inhibition. Graph depicts means of ratios of GFP signals normalized to mean of value obtained with cells transfected with GFP and Ctrl RNAi. (b) GTP-bound RhoA abundance was assessed by rotekin RhoA-GTP binding domain bead pull-down, followed by western blotting for RhoA. Wnt9b stimulation of PC1, or PC1ΔNTF expression, increased GTP-bound RhoA. Graph depicts means of ratios of RhoA-GTP signal, normalized to mean of value obtained with empty vector (EV) transfection. The quantity of RhoA-GTP is also substantially higher in lysates prepared from the kidneys of Tg248 Pkd1 BAC-transgenic mice as compared to that present in lysates of wild type mouse kidneys. (c) and (d) Detection of in vitro (c) and in vivo (d) quantity of Gα13 present in anti-HA precipitates of PC1 was assessed by western blotting. Wnt9b treatment modestly but significantly increased quantity of PC1-associated Gα13 PC1 (c). Graphs in c and d depict means of ratios of signal detected in Wnt9b-treated condition, normalized to mean of value obtained with vehicle-treated cells. Graph depicts the intensity of the Gα13 signal in each condition normalized to the corresponding HA signal. (e) Biosensor assay to detect importance of G-protein binding site of PC1 for regulation of GSK3β. PC1 or PC1 ΔNTF lacking its G-protein binding site, or PC1 carrying a mutation within the G-protein binding site sequence (ΔL4132), does not inhibit GSK3β. Similarly, soluble Stachel peptide does not induce GSK3β inhibition in cells expressing PC1 ΔNTF+ΔTA+ΔL4132 . Graph depicts ratio of GFP fluorescence, normalized to mean of value obtained with cells expressing GFP alone. Data are shown as mean ±SEM, n≥3 for all experiments. To assess statistical significance unpaired Student t-Test analysis was performed. P values ≤ 0.05 were considered significant *=P<0.05; **=P<0.01; ***=P<0.001.; ****=P<0.0001 Differences between mean values obtained for experimental conditions versus control conditions that produce minimal and/or maximal signals were evaluated. In panels 4a and 4f the values obtained for each condition were compared to those obtained in the GFP alone condition (indicated by the symbols above each bar) and pairwise comparisons of the values obtained in a subset of conditions related by a single experimental manipulation are indicated by the brackets above the bars.

Article Snippet: Recombinant Wnt9b (0.5ng/μL) (R&D Systems, 3669) was applied for 3 hrs or overnight.

Techniques: Activity Assay, Control, Inhibition, Fluorescence, Transfection, Binding Assay, Western Blot, Expressing, Plasmid Preparation, Transgenic Assay, In Vitro, In Vivo, Biosensor Assay, Protein Binding, Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: Polycystin-1 acts as an atypical adhesion GPCR that responds to novel Wnt signaling and mechanical stimuli

doi: 10.1101/2025.05.21.655326

Figure Lengend Snippet: (a) Immunofluorescent localization of the full length PC1 (green) in the primary cilia of LLCPK1+2 cells treated with wnt9b, or with a combination of Wnt9b and barbadin. Cilia are detected by immunostaining for Arl13b (orange) (b) Immunofluorescent localization of PC1 ΔNTF and PC1 ΔNTFΔTA in the primary cilia of LLCPK cells. Barbadin treatment results in localization of PC1 ΔNTF to cilia, while treatment with soluble TA peptide causes loss of PC1 ΔNTF+ΔTA from the cilia. (c) Immunofluorescence detection of the full length PC1 in the primary cilia of cells treated with mechanical stimulation, with medium from cells preconditioned by mechanical stimulation or with ATP added to the medium. Barbadin and Apyrase treatment blocked the effects of mechanical stimulation and ATP on PC1 ciliary localization. Data are shown as mean ±SEM, n≥3 for all experiments. Scale bar = 5μ. To assess statistical significance unpaired Student t-Test analysis was performed. P values <0.05 were considered significant *=P<0.05; **=P<0.01; ***=P<0.001.; ****=P<0.0001 Differences between mean values obtained for experimental conditions versus control conditions that produce minimal and/or maximal signals were evaluated.

Article Snippet: Recombinant Wnt9b (0.5ng/μL) (R&D Systems, 3669) was applied for 3 hrs or overnight.

Techniques: Immunostaining, Immunofluorescence, Control

Journal: bioRxiv

Article Title: Polycystin-1 acts as an atypical adhesion GPCR that responds to novel Wnt signaling and mechanical stimuli

doi: 10.1101/2025.05.21.655326

Figure Lengend Snippet: (a) Immunofluorescent detection of the localizations of PC1S3164A and PC1S3164D to the primary cilia in LLCPK cells. ATP treatment did not alter the ciliary distribution of PC1S3164A, whereas the ciliary exclusion of PC1 S3163D was reversed by Barbadin treatment. PKA inhibitor H89A treatment prevented the departure of PC1 from primary cilia that is induced by either ATP or Wnt9b treatment. (b) Biosensor assay was used to assess the importance of PC1 PKA phosphorylation site S3164 for PC1 activation-dependent inhibition of GSK3β kinase in HEK293 cells. Data are shown as mean ±SEM, n≥3 for all experiments. To assess statistical significance unpaired Student t-Test analysis was performed. P values <0.05 were considered significant *=P<0.05; **=P<0.01; ***=P<0.001.; ****=P<0.0001 Differences between mean values obtained for experimental conditions versus control conditions that produce minimal and/or maximal signals were evaluated. In panel 3d the values obtained for each condition were compared to those obtained in the GFP alone condition (indicated by the symbols above each bar) and pairwise comparisons of the values obtained in a subset of conditions related by a single experimental manipulation are indicated by the brackets above the bars.

Article Snippet: Recombinant Wnt9b (0.5ng/μL) (R&D Systems, 3669) was applied for 3 hrs or overnight.

Techniques: Biosensor Assay, Phospho-proteomics, Activation Assay, Inhibition, Control

Journal: Cell reports

Article Title: Geometric effects position renal vesicles during kidney development.

doi: 10.1016/j.celrep.2023.113526

Figure Lengend Snippet: Figure 1. Geometric effects position PTAs/RVs in developing kidneys (A) Regulation of PTA/RV formation. For details, see text. (B) Volumetric light-sheet microscopy data of an E12.5 embryonic kidney with surface segmentations for the PTAs/RVs (LHX1, yellow), the UB (HoxB7, green), and the CM (SIX2, orange). (C) A 3D simulation of the steady-state WNT9b distribution. Uniform secretion of WNT9b from the UB surface (Neumann boundary condition) leads to the highest ligand concentration (red) in the UB corner regions, coinciding with the position of PTAs/RVs (yellow). The geometries of the PTAs/RVs and the UB were extracted from light-sheet microscopy images. The WNT9b gradient length was set to l = 30 mm. The colormap shows the normalized, integrated WNT9b concentration profile along the normal direction of the UB toward the mesenchyme projected back on the surface of the UB. (D) The highest ligand upconcentration in PTAs/RVs is achieved for lz30 mm. The normalized WNT9b concentration is calculated as the difference between the mean concentration inside the PTAs/RVs, cPTA=RV, and the mean concentration inside the SIX2 population, cSIX2, relative to the highest concentration inside the kidney, cmax: cnorm = ðcPTA=RV cSIX2Þ=cmax: (E) Relative simulated WNT9b concentration inside the PTAs/RVs (yellow) and in the SIX2 population (gray). The predicted mean concentration in PTAs/RVs (yellow dashed line; c=cSIX2 = 1:99 ± 0:84 [SD]) is above the predicted mean concentration in the SIX2 population (gray dashed line; c=cSIX2 = 1:00 ± 0:62 [SD]) (p< 0:001, Welch’s two-sample t test). (F) The predicted WNT9b concentration inside the PTAs/RVs relative to the SIX2 population is higher in smaller PTAs/RVs. The red line and gray shade represent a linear fit with its standard error (R2 = 0:2). (G) A cross-section highlighting the relative location of the CM (orange), PTAs/RVs (yellow), and the simulated concentration gradient (blue-red colormap).

Article Snippet: After 24h the cells were treated with WNT9B (R&D, 3669-WN) as indicated.

Techniques: Microscopy, Concentration Assay

Journal: Cell reports

Article Title: Geometric effects position renal vesicles during kidney development.

doi: 10.1016/j.celrep.2023.113526

Figure Lengend Snippet: Figure 3. Concentration-dependent response of NPCs to WNT9b (A) Diffusion simulation on a simplified UB branch, illustrating ligand upconcentration and PTA/RV positioning by geometric effects. (B) The 3D Imaris surfaces of HoxB7/myr-Venus kidney explants (segmented UB in green, outer surface transparent) immunostained for LHX1 and SIX2 (control and 5 mM). Control, PTAs/RVs form in the corners of UB branches; treated with 5 mM CHIR99021 for 40 h, LHX1+ clusters form away from the UB, interspersed with SIX2+ progenitors; treated with 10 mM CHIR99021 for 24 h, high uniform WNT activation results in Lhx1 expression in most of the expanded metanephric mesenchyme. Scale bars, 100 mm. (C–E) Concentration-dependent response of Six2-positive primary NPCs, isolated from E12.5 kidneys, to recombinant WNT9b, added either in the medium (C) or on beads (E), as judged by qPCR of differentiation markers (Lef1, Wnt4) and renewal markers (Cited1, Pax8) (D). At high WNT9b concentrations, expression of Cited1, Pax8 was no longer detectable (n.d.). (F–L) HoxB7/myr-Venus kidney explants were cultured with control (G) or WNT9b-soaked (H and I) beads (dashed white circles). White arrowheads mark the position of RVs that are forming in close proximity to the WNT9b source. (I) UB branches (black lines) bend toward the WNT9b source over time, potentially following CM cells that are attracted to the WNT9b-soaked bead. Ectopic branching from the future ureter (white asterisk). (J–L) 3D rendered light-sheet fluo- rescence microscopy (LSFM) data of the explant culture endpoints, immunostained for LHX1 and SIX2 for control (G) and WNT9b-soaked (H and I) beads. The white asterisk marks the same branch as in (I).

Article Snippet: After 24h the cells were treated with WNT9B (R&D, 3669-WN) as indicated.

Techniques: Concentration Assay, Diffusion-based Assay, Control, Activation Assay, Expressing, Isolation, Recombinant, Cell Culture, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Canonical WNT/β-Catenin Signaling Activated by WNT9b and RSPO2 Cooperation Regulates Facial Morphogenesis in Mice

doi: 10.3389/fcell.2020.00264

Figure Lengend Snippet: (A) Expression of the Wnt9b and Rspo2 genes and a WNT reporter, TopGAL , in mouse embryos at E10.5. Frontal images are shown. Transverse section planes within the BA1 are marked by white-dotted lines, and the matching images of transverse sections are shown as a’, b’, and c’. Only the left side of the BA1 is shown. Orientation of the sections is indicated by dorsal/ventral (d/v) and lateral/medial (l/m) labels. (B) STF reporter assay. HEK293T cells were transfected with 20 ng of WNT signaling STF reporter and 10 ng TK- Renilla luciferase DNA in triplicate in 48-well plates. At 2 days after transfection, cells were treated with recombinant WNT9b and RSPO2 proteins with the indicated concentrations for 24 h. STF luciferase activities were normalized by Renilla luciferase activities. (C) Phosphorylated and total LRP6 protein levels were determined by Western blot analysis. HEK293T cells were treated with WNT9b (20 ng/ml) and RSPO2 (200 ng/ml) proteins for the indicated duration.

Article Snippet: Recombinant RSPO2 and WNT9B proteins were obtained from R&D Systems (Minneapolis, MN, United States) and treated at the concentrations of 200 and 20 ng/ml, respectively.

Techniques: Expressing, Reporter Assay, Transfection, Luciferase, Recombinant, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Canonical WNT/β-Catenin Signaling Activated by WNT9b and RSPO2 Cooperation Regulates Facial Morphogenesis in Mice

doi: 10.3389/fcell.2020.00264

Figure Lengend Snippet: (A) Expression of WNT/β-catenin signaling reporter, TopGAL transgene, in the facial processes at E10.5-11 embryos ( n = 3 for each embryonic age). To correctly visualize TopGAL expression, we used Rspo2 Δ ZN null mice in which the LacZ and neomycin-resistance genes inserted into the Rspo2 locus were removed. Embryos were incubated with X-gal substrate for 8 h. Red arrows indicate reduced or absent TopGAL expression. Red asterisk indicates a gap between lnp and mnp produced by a failure of nasal process fusion. lnp, lateral nasal process; mdBA1, mandibular process of the branchial arch 1; mnp, medial nasal process; mxBA1, maxillary process of the branchial arch 1. (B) qRT-PCR analysis for Axin2 expression in the facial process explants ( n = 4, NP/MxBA1, nasal process/maxillary branchial arch 1 and MdBA1, mandibular branchial arch 1) dissected from Wnt9b KO, Rspo2 KO, and DKO embryos at E10.5. (C) qRT-PCR analysis for Axin2 expression in the facial process explants ( n = 4, whole BA1, whole mandibular branchial arch 1 and BA1 Mes, mesenchymal part of the mandibular branchial arch 1) cultured in the presence of WNT9b (20 ng/ml) and/or RSPO2 (200 ng/ml) proteins. Error bars represent the standard error of the mean (SEM). * p < 0.05; ** p < 0.01.

Article Snippet: Recombinant RSPO2 and WNT9B proteins were obtained from R&D Systems (Minneapolis, MN, United States) and treated at the concentrations of 200 and 20 ng/ml, respectively.

Techniques: Expressing, Incubation, Produced, Quantitative RT-PCR, Cell Culture

Journal: Frontiers in Cell and Developmental Biology

Article Title: Canonical WNT/β-Catenin Signaling Activated by WNT9b and RSPO2 Cooperation Regulates Facial Morphogenesis in Mice

doi: 10.3389/fcell.2020.00264

Figure Lengend Snippet: (A) Whole mount in situ hybridization analysis for Fgf8 and Msx1 gene expressions in wild-type, Wnt9b KO, Rspo2 KO, and DKO embryos ( n = 3) at E10.5. Red arrows indicate the reduced gene expression. (B,C) qRT-PCR analysis of marker gene expression in the facial process explants ( n = 4) dissected from Wnt9b KO, Rspo2 KO, and DKO embryos at E10.5. (D,E) qRT-PCR analysis of marker gene expression in the facial process explants ( n = 4) cultured in the presence of WNT9b (20 ng/ml) and/or RSPO2 (200 ng/ml) proteins. * p < 0.05, ** p < 0.01, and *** p < 0.005.

Article Snippet: Recombinant RSPO2 and WNT9B proteins were obtained from R&D Systems (Minneapolis, MN, United States) and treated at the concentrations of 200 and 20 ng/ml, respectively.

Techniques: In Situ Hybridization, Gene Expression, Quantitative RT-PCR, Marker, Cell Culture

Journal: Frontiers in Cell and Developmental Biology

Article Title: Canonical WNT/β-Catenin Signaling Activated by WNT9b and RSPO2 Cooperation Regulates Facial Morphogenesis in Mice

doi: 10.3389/fcell.2020.00264

Figure Lengend Snippet: (A) Venn diagram of differential gene expression analysis in the facial processes of wild type, Wnt9b KO, Rspo2 KO, and DKO by RNA sequencing. The numbers of the differentially expressed genes (DEGs) with more than 1.5-fold change in Wnt9b KO, Rspo2 KO, and DKO compared to wild type ( n = 3 for each genotype). (B) The number of DEGs upregulated or downregulated in Wnt9b KO, Rspo2 KO, and DKO embryos. (C) Gene ontology analysis of the DEGs in Wnt9b KO, Rspo2 KO, and DKO embryos, respectively. The biological processes that are common in all three KO backgrounds and are only associated with Wnt9b KO and DKO are shown in blue and green, respectively. Gene numbers in each GOTERM are represented by circles with different diameters. (D) Gene ontology analysis for the biological processes for the subgroup DEGs in DKO mice regulated by Wnt9b:Rspo2 cooperative function. The number of genes belonging to the GOTERM are shown at the right end of each bar.

Article Snippet: Recombinant RSPO2 and WNT9B proteins were obtained from R&D Systems (Minneapolis, MN, United States) and treated at the concentrations of 200 and 20 ng/ml, respectively.

Techniques: Gene Expression, RNA Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Canonical WNT/β-Catenin Signaling Activated by WNT9b and RSPO2 Cooperation Regulates Facial Morphogenesis in Mice

doi: 10.3389/fcell.2020.00264

Figure Lengend Snippet: (A) Cell proliferation and apoptosis in the facial processes of wild-type, Wnt9b KO, Rspo2 KO, and DKO embryos at E10.5. Immunofluorescence staining of phospho-histone H3 for cell proliferation and TUNEL staining for cell apoptosis were performed on cryosections. DAPI (blue) was used for nuclei counterstaining. Red arrows point at the area showing changes in cell proliferation and apoptosis. Three embryos of each genotype were analyzed. (B) Quantitation of phospho-histone H3-positive cells presented as a percentage of total cells. Three embryos for each mutant background were used for collecting the data. (C) Quantitation of TUNEL-positive cells presented as a percentage of total cells. Three embryos for each mutant background were used for collecting the data. Error bars represent the standard error of the mean (SEM). * p < 0.05; ** p < 0.01; *** p < 0.005.

Article Snippet: Recombinant RSPO2 and WNT9B proteins were obtained from R&D Systems (Minneapolis, MN, United States) and treated at the concentrations of 200 and 20 ng/ml, respectively.

Techniques: Immunofluorescence, Staining, TUNEL Assay, Quantitation Assay, Mutagenesis

Journal: Frontiers in Cell and Developmental Biology

Article Title: Canonical WNT/β-Catenin Signaling Activated by WNT9b and RSPO2 Cooperation Regulates Facial Morphogenesis in Mice

doi: 10.3389/fcell.2020.00264

Figure Lengend Snippet: (A) Gross head morphology and skeleton (bottom panel) of wild-type, Wnt9b KO, Rspo2 KO, and DKO animals at E18.5 obtained from mating between compound Wnt9b ± ;Rspo2 ± male and female mice. The compound Wnt9b ± ;Rspo2 ± mice were normal and fertile. Lateral view (top and bottom panels) and frontal view (middle panel) are shown. The white eye color of Wnt9b KO is not a specific phenotype but is due to the mixed genetic background of Wnt9b KO mice. White arrows indicate severe defect in the lower jaw (top panel) and cleft lip phenotype in the upper jaw (middle panel). Yellow asterisk indicates the open-eye phenotype observed in DKO mice. Black and red arrows in the bottom panel specify maxillary and mandibular defects, respectively. (B) Measurement of various parameters of skull dimensions. Three to four fetuses were used for the measurement. Error bars represent the standard error of the mean (SEM). ** p < 0.01; *** p < 0.005.

Article Snippet: Recombinant RSPO2 and WNT9B proteins were obtained from R&D Systems (Minneapolis, MN, United States) and treated at the concentrations of 200 and 20 ng/ml, respectively.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Canonical WNT/β-Catenin Signaling Activated by WNT9b and RSPO2 Cooperation Regulates Facial Morphogenesis in Mice

doi: 10.3389/fcell.2020.00264

Figure Lengend Snippet: (A) Schematic structure of the RSPO2 protein and its derivatives. Mutated amino acid residues are labeled. (B) STF reporter assay to determine the cooperative activity between the WNT9b and RSPO2 mutant proteins. HEK293T cells were transfected, and reporter assay was performed as described in the caption. (C) LRP6 phosphorylation by Western blot analysis. Total LRP6 and α-tubulin expressions were also determined for normalization and loading controls. (D,E) qRT-PCR analysis of marker gene expression in the facial process explants ( n = 4, whole BA1, whole mandibular branchial arch 1; BA1 Mes, mesenchymal part of the mandibular branchial arch 1) cultured in the presence of WNT9b (20 ng/ml) and/or RSPO2 (200 ng/ml) proteins. Error bars represent the standard error of the mean. * p < 0.05; ** p < 0.01; *** p < 0.005.

Article Snippet: Recombinant RSPO2 and WNT9B proteins were obtained from R&D Systems (Minneapolis, MN, United States) and treated at the concentrations of 200 and 20 ng/ml, respectively.

Techniques: Labeling, Reporter Assay, Activity Assay, Mutagenesis, Transfection, Phospho-proteomics, Western Blot, Quantitative RT-PCR, Marker, Gene Expression, Cell Culture

Journal: bioRxiv

Article Title: Enhancer regulation for induced WNT3A expression during neuronal regeneration

doi: 10.1101/861153

Figure Lengend Snippet: Prediction of putative enhancers of WNT3A gene during neuronal regeneration. (a) Aggregation of normalized H3K4me3 signal density profiles of 88 WNT-related genes across the ± 4 kb promoter regions. H3K4me3 signals across control and injured samples are indicated by colored lines. (b) Upper: Schematic diagrams of gene tracks for rat WNT3A and WNT9B . Targeted loci for specific primers in the promoters used for ChIP assays are shown. Bottom: The fold-change of H3K4me3 at the promoters of WNT3A , WNT9B , or WNT10A in cortical neurons comparing DIV10 and iDIV10 were analyzed by ChIP-qPCR. The GAPDH promoter was used as a negative control. Data were normalized to IgG and then to DIV10 controls, indicated as fold change. Data are presented as mean ± SEM from three independent experiments. * P ≤ 0.05 (paired Student’s t -test). (c) Functional enrichment of chromatin states in rat genome performed by ChromHMM. Upper: Heatmap of the model parameter with chromatin states numbered in the emission order. The columns refer to relative enrichment for the indicated annotation in corresponding chromatin states. ChIP-seq data from four GEO datasets as well as data from this study were used to train the model. Bottom: Heatmap of the positional enrichment of annotated chromatin state. The genomic feature of the State2 elements is indicated in red boxes. (d) Snapshot of JBrowser Genome Browser demonstrating the region across 1.8 Mb flanking the WNT3A TSS of the rat genome (RCSC 6.0/rn6). Ten predicted genomic regions (e1-e10, indicated in boxed area) may be enhancers for the WNT3A gene based on the enrichment of clustered State2 elements assigned by ChromHMM. A diagram of WNT3A gene region is shown at the bottom. (e) Aggregation of normalized H3K27ac ChIP-seq profiles across the ± 2 kb central base pair of within each putative enhancers region (e2-e10) for control (DIV9, DIV10) and injured (iDIV9, iDIV10) samples.

Article Snippet: WNT8A (Cat#8419-WN-010/CF) and WNT9B (Cat#3669-WN-0.25/CF) were from R&D Systems (Minneapolis, MN).

Techniques: Control, ChIP-qPCR, Negative Control, Functional Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: Enhancer regulation for induced WNT3A expression during neuronal regeneration

doi: 10.1101/861153

Figure Lengend Snippet: Identification of regeneration-associated genes via RNA-seq and superarray analysis. (a, b) Scatter plots showing normalized gene expression levels on DIV9 versus iDIV9 and iDIV10 versus DIV10 respectively, as determined from RNA-seq. Data points representing TMM-normalized expression of WNT genes are marked as red dots. The blue lines indicate equal expression between uninjured control and injured samples. Right panels are the close-up view of the boxed regions on the left panels. (c) Differential expression profile of WNT-related genes by superarray assay is shown. Genes with an expression fold-change above 1.5 in response to injury are denoted. (d) Analysis of differential expression of WNT s by qPCR during neuronal regeneration. Data are presented as mean ± SEM from at least three independent experiments ( WNT3A , 8A : n = 4; WNT9B , 10A : n = 5). * P ≤ 0.05 (paired Student’s t -test).

Article Snippet: WNT8A (Cat#8419-WN-010/CF) and WNT9B (Cat#3669-WN-0.25/CF) were from R&D Systems (Minneapolis, MN).

Techniques: RNA Sequencing, Gene Expression, Expressing, Control, Quantitative Proteomics

Journal: bioRxiv

Article Title: Enhancer regulation for induced WNT3A expression during neuronal regeneration

doi: 10.1101/861153

Figure Lengend Snippet: Recombinant WNT3A promotes regeneration of injured cortical neurons and brain tissues. (a) Cortical neurons were pre-treated with PBS, WNT3A, WNT8A, WNT9B or WNT10A recombinant proteins prior to injury on DIV8. The percentages of gap closure were calculated from iDIV8 to iDIV11. Data are presented as mean ± SEM (n = 4 for WNT3A and WNT9B experiments; n = 5 for WNT10A experiment). * P ≤ 0.05 (paired Student’s t -test). (b) Cortical neurons with or without WNT3A (50 ng/ml) pre-treatment were fixed and subjected to immunofluorescence staining with anti-TUJ1 antibody (neurites) on iDIV11. (c) Cortical neurons were treated with 1% DMSO or IWR-1 (10 µM) prior to injury on DIV8. Neurons were subjected to immunofluorescence staining with anti-TUJ1 antibody and the representative images are shown. Dashed lines in (b) and (c) indicate the borders of the injured gap. Scale bar, 100 µm. (d) The effect of WNT3A on neuronal regeneration was assessed using organotypic brain slice culture. Brain slices were injured at the olfactory tubercle on DIV0, as indicated by the red dashed line in the upper left atlas map, and cultured with or without WNT3A (50 ng/ml) in AraC-containing medium. Brain tissues were fixed on iDIV4 and subjected to immunofluorescence staining with anti-TUJ1 (green) and anti-GFAP (cyan, glia) antibodies, and DAPI (blue). The border of injured sites is depicted by white dashed lines, and remaining brain tissue is underneath the lines in each panel. F: frontal lobe; O: occipital lobe. Scale bar, 100 µm. (e, f) The length of regenerating neurites from the brain slices treated with (e) PBS control or WNT3A and (f) AraC+PBS control or AraC+WNT3A were calculated. Data on the right panels in (e) and (f) is normalized to the PBS control, indicated as fold change. Data are presented as mean ± SEM (n = 4). * P ≤ 0.05 (paired Student’s t -test).

Article Snippet: WNT8A (Cat#8419-WN-010/CF) and WNT9B (Cat#3669-WN-0.25/CF) were from R&D Systems (Minneapolis, MN).

Techniques: Recombinant, Immunofluorescence, Staining, Slice Preparation, Cell Culture, Control