Journal: Development (Cambridge, England)

Article Title: Deletion of an enhancer that controls Wnt gene expression following tissue injury produces increased adipogenesis in regenerated muscle

doi: 10.1242/dev.204933

Figure Lengend Snippet: Wnt1 and Wnt10b are induced by tissue injury following BaCl 2 injection. (A,B) Wnt expression post-BaCl 2 -induced injury in skeletal muscle, assessed by RNAscope mRNA in situ hybridization. Wnt1 (A) and Wnt10b (B) expression in TA muscle at 1, 3, 5 and 7 days following BaCl 2 injection are shown. Uninjured contralateral muscle at 1 day post-injury is shown for comparison. Wnt1 - and Wnt10b -positive cells are infrequently observed in uninjured muscle. Transcripts appear in single cells post-injury and then in nascent myofibers, which display centrally located nuclei. Boxes mark regions enlarged under each panel. Arrows mark red mRNA signals. Yellow dashed lines outline myofibers. (C,D) Quantification of Wnt1 and Wnt10b expression at days 1 and 3 post-injury. (C) The number of Wnt1 -positive nuclei increases significantly at 1 and 3 days post-injury compared to uninjured contralateral muscles. * P =0.0155, ** P =0.0026 (one-way ANOVA, Kruskal–Wallis test). n =8 uninjured represents n =4 d1 uninjured, n =4 d3 uninjured; n =6 d1 BaCl 2 , n =5 d3 BaCl 2 . (D) Number of Wnt10b -positive nuclei increases significantly at 1 and 3 days post-injury compared to uninjured contralateral muscles. * P =0.0378, ** P =0.0076 (Brown–Forsythe and Welch one-way ANOVA tests). n =6 uninjured consists of n =3 d1 uninjured, n =3 d3 uninjured; n =4 d1 BaCl 2 , n =5 d3 BaCl 2 . (E,F) Duplex mRNA in situ hybridization showing Wnt1 (E) and Wnt10b (F) signals in Pdgfra- , Pax7- and Adgre- positive cells, which mark the FAPs, satellite cells and macrophages, respectively, at 3 days post-injury. Arrows point to examples of double-positive nuclei. n =2 animals. (G) Duplex mRNA in situ hybridization showing that Wnt1 and Wnt10b can be colocalized. Representative examples from 1, 3 and 5 days post-injury are shown. White numbered boxes mark regions magnified below each image. Yellow dashed lines mark individual nuclei. Arrows mark Wnt double-positive cells. Box limits mark the 25th and 75th percentiles, whiskers mark minimum to maximum values, and horizontal line marks the median. d, days post-injury; ns, not significant. Scale bars: 20 µm (A,B); 5 µm (E-G).

Article Snippet: Probes utilized for assays were: Srp14 (Mm00726104_s1), Pax7 (Mm01354484_m1), Pdgfra (Mm00440701_m1), Cebpb (Mm00843434_s1), Wnt1 (Mm01300555_g1), Wnt10b (Mm00442104_m1), Wnt5a (Mm00437347_m1), Cebpa (Mm00514283_s1), Pparg (Mm00440940_m1), Axin2 (Mm_00443610).

Techniques: Injection, Expressing, RNAscope, In Situ Hybridization, Comparison, Muscles

Journal: Development (Cambridge, England)

Article Title: Deletion of an enhancer that controls Wnt gene expression following tissue injury produces increased adipogenesis in regenerated muscle

doi: 10.1242/dev.204933

Figure Lengend Snippet: Regulatory sequences residing between Wnt1 and Wnt10b are injury responsive. (A) UCSC Genome Browser data showing that Wnt1 and Wnt10b reside on the same chromosome and are transcribed in opposite directions (arrows). Three highly conserved sequences between Wnt1 and Wnt10b are labeled: 1 (light blue), 2 (yellow), 3 (red). Multiple genome alignment tracks (Multiz Align) and conserved peaks from placental mammal alignments are shown. Below, schematics of constructs containing putative Enhancers 2, 3, and 2+3 fused to the hsp68 - LacZ gene are provided [minimal hsp68 promoter (green), lacZ reporter (blue)]. (B) BaCl 2 injury of adult Enh2-LacZ, Enh3-LacZ and Enh2+3-LacZ mice reveal that all three sequences drive reporter expression following injury. Whole-mount muscles are shown. Uninjured contralateral muscles at 6 h are provided for comparison. Arrowheads mark examples of single cells that express the reporter. (C) Sectioned tissues from X-gal-stained, BaCl 2 -injured TA muscles from Enhancer 2, 3 and 2+3 reporter mice at 3-5 days. Uninjured muscles from Enhancer 2 and 3 reporters are shown. Arrows mark examples of single cells that express the reporter. Some variable and sporadic background staining is observed (asterisks). d, days post-injury. Scale bars: 20 µm.

Article Snippet: Probes utilized for assays were: Srp14 (Mm00726104_s1), Pax7 (Mm01354484_m1), Pdgfra (Mm00440701_m1), Cebpb (Mm00843434_s1), Wnt1 (Mm01300555_g1), Wnt10b (Mm00442104_m1), Wnt5a (Mm00437347_m1), Cebpa (Mm00514283_s1), Pparg (Mm00440940_m1), Axin2 (Mm_00443610).

Techniques: Labeling, Construct, Expressing, Muscles, Comparison, Staining

Journal: Development (Cambridge, England)

Article Title: Deletion of an enhancer that controls Wnt gene expression following tissue injury produces increased adipogenesis in regenerated muscle

doi: 10.1242/dev.204933

Figure Lengend Snippet: Loss of Enhancers 2 and 3 produces viable mice, but expression of Wnt1 and Wnt10b is reduced post-injury. (A) Schematic showing CRISPR-Cas9-mediated deletion of Enhancers 2 and 3. Guide RNAs (red arrowheads) and PCR genotyping primers (green arrowheads) are shown. Two independent mouse lines carrying Enhancer 2+3 deletions are labeled Δ1 and Δ2. Δ1 is 206 bp larger than Δ2. (B) PCR reactions from genotyping primers flanking Enh2+3. Product sizes are 1.6 kb (wt), 500 bp (Δ1) and 700 bp (Δ2). Δ1/Δ2 mice produce PCR bands that run as a doublet. (C) Δ1/Δ2 and wt mice display similar overall weights. Males (M) and females (F) were followed between 2.5 and 35 weeks. ( n =9 wt M, n =9 Δ1/Δ2 M; n =11 wt F, n =10 Δ1/Δ2 F) (D,E) Wnt1 , Wnt10b and Wnt5a expression assessed by mRNA in situ hybridization in adult TA muscles. Arrowheads mark examples of transcripts. (D) Staining at 3 days post-injury. n >3 mice. (E) Staining at 5 days post-injury. n >3 mice. (F-H) Quantification of Wnt-expressing nuclei at 3 and 5 days post-injury. Each dot on the box plots represents mean percentage of Wnt-positive nuclei normalized to total number of nuclei counted per field for each mouse. Violin plots display all quantified fields. (F) A slight decrease in Wnt1 -positive cells is detected at 3 days followed by significant reduction at d5. (G) A slight decrease in Wnt10b -positive cells is detected at d3, followed by significant reduction at d5. (H) Wnt5a expression shows no decline over time in Δ1/Δ2 muscles compared to wt. Box limits mark the 25th and 75th percentiles, whiskers mark minimum to maximum values, and horizontal line marks the median; red lines mark the median in violin plots. n =5 wt, n =5 del for Wnt1 , Wnt10b and Wnt5a at d3; n =5 wt, n =4 del for Wnt1 , Wnt10b at d5; n =4 wt, n =4 del for Wnt5a at d5. Wnt1 d3, Mann–Whitney test; Wnt1 d5, * P =0.0159 Mann–Whitney test; Wnt10b d3, unpaired t -test; Wnt10b d5, * P =0.0157 unpaired t -test; Wnt5a d3, Welch's t -test; Wnt5a d5, unpaired t -test. d, days post-injury; del, Δ1/Δ2; ns, not significant. Scale bars: 20 µm.

Article Snippet: Probes utilized for assays were: Srp14 (Mm00726104_s1), Pax7 (Mm01354484_m1), Pdgfra (Mm00440701_m1), Cebpb (Mm00843434_s1), Wnt1 (Mm01300555_g1), Wnt10b (Mm00442104_m1), Wnt5a (Mm00437347_m1), Cebpa (Mm00514283_s1), Pparg (Mm00440940_m1), Axin2 (Mm_00443610).

Techniques: Expressing, CRISPR, Labeling, In Situ Hybridization, Muscles, Staining, MANN-WHITNEY

Journal: Animal Bioscience

Article Title: Transcriptomic analysis identifies CXCL12 as a novel candidate gene for litter size in rabbits

doi: 10.5713/ab.24.0640

Figure Lengend Snippet: CXCL12 regulated the expression of genes and proteins related to litter size and ovarian functions. The mRNA expression level of TAF4B , CITED1 , WNT2 , WNT10B and HSD17B1 were detected after overexpression (A) and knockdown (B) of CXCL12 in GCs. (C) The protein expression levels of CITED1 and WNT10B were detected after overexpression and knockdown of CXCL12 in GCs (The t test was used for the above analyses comparing two individual samples. * p<0.05; ** p<0.01; *** p<0.001). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, granulosa cells.

Article Snippet: Protein detection was achieved using the following antibodies: anti-CCND1 mouse monoclonal antibody (1:250, Proteintech), anti-PCNA rabbit polyclonal antibody (1:250, Proteintech), anti-Bcl2 rabbit polyclonal antibody (1:250, Proteintech), anti-Bax rabbit polyclonal antibody (1:250, Proteintech), anti-CITED1 rabbit polyclonal antibody (1:50, Proteintech), anti-WNT10B mouse monoclonal polyclonal antibody (1:250, Proteintech), anti-CXCR4 mouse monoclonal polyclonal antibody (1:250, Proteintech), anti-phospho-JAK2 rabbit monoclonal polyclonal antibody (1:250, Abcam, Cambridge, UK), anti-JAK2 rabbit monoclonal polyclonal antibody (1:250, Abcam), anti-phospho-STAT1 rabbit polyclonal antibody (1:250, Proteintech), anti-STAT1 rabbit polyclonal antibody (1:250, Proteintech), anti-GAPDH mouse monoclonal antibody (1:2,500, Proteintech), 1:1,000 goat anti-rabbit secondary antibody IgG (Proteintech) and 1:1,000 goat anti-mouse secondary antibody IgG (Proteintech).

Techniques: Expressing, Over Expression, Knockdown

Journal: Diabetes/Metabolism Research and Reviews

Article Title: Fibre‐Enriched High‐Carbohydrate (FEHC) Diet Modulates Inflammation Without Affecting Bone Health in Older Women With Obesity: A Randomised Clinical Trial

doi: 10.1002/dmrr.70089

Figure Lengend Snippet: Gene expression analysis by Real‐time PCR of Wnt signaling and bone metabolism–related genes in bone tissue at T1 in a fiber‐enriched high‐carbohydrate diet (FEHC, green bars) and a control diet (CD, pink bars) group. (A) Wnt family member 5A (WNT5A). (B) Wnt family member 10B (WNT10B). (C) Sclerostin (SOST). (D) Dickkopf WNT signaling pathway inhibitor 1 (DKK1). (E) Lymphoid enhancer‐binding factor 1 (LEF1). (F) Osteocalcin (OCN). (G) Runt‐related transcription factor 2 (RUNX2). (H) Collagen type I alpha 1 chain (COL1A1). Data are presented as median values with interquartile ranges (IQR). Statistical analysis was performed using two‐way ANOVA followed by Tukey’s multiple comparisons test. Comparisons were considered statistically significant at p < 0.05.

Article Snippet: Gene expression of WNT5A (Hs00998537_m1), WNT10B (Hs00928823_m1), SOST (Hs00228830_m1), IGF‐1 (Hs01547656_m1), IL‐6 (Hs00174131_m1), IL‐8 (Hs00174103_m1), IL‐10 (Hs00961622_m1), TNFα (Hs00174128_m1), ADIPOQ (Hs00605917_m1), OCN (Hs01587814_g1), RUNX2 (Hs00231692_m1), DKK‐1 (Hs00183740_m1), LEF‐1 (Hs01547250_m1), COL1A1 (Hs00164004_m1) were evaluated in bone and muscle tissue biopsies.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Control, Binding Assay

Journal: Diabetes/Metabolism Research and Reviews

Article Title: Fibre‐Enriched High‐Carbohydrate (FEHC) Diet Modulates Inflammation Without Affecting Bone Health in Older Women With Obesity: A Randomised Clinical Trial

doi: 10.1002/dmrr.70089

Figure Lengend Snippet: Gene expression analysis by Real‐time PCR of Wnt signaling and bone metabolism–related genes in muscle tissue at T1 in a fiber‐enriched high‐carbohydrate diet (FEHC, green bars) and a control diet (CD, pink bars) group. (A) Wnt family member 5A (WNT5A). (B) Wnt family member 10B (WNT10B). (C) Sclerostin (SOST). (D) Dickkopf WNT signaling pathway inhibitor 1 (DKK1). (E) Lymphoid enhancer‐binding factor 1 (LEF1). (F) Osteocalcin (OCN). (G) Runt‐related transcription factor 2 (RUNX2). (H) Collagen type I alpha 1 chain (COL1A1). Data are presented as median values with interquartile ranges (IQR). Statistical analysis was performed using two‐way ANOVA followed by Tukey’s multiple comparisons test. Comparisons were considered statistically significant at p < 0.05.

Article Snippet: Gene expression of WNT5A (Hs00998537_m1), WNT10B (Hs00928823_m1), SOST (Hs00228830_m1), IGF‐1 (Hs01547656_m1), IL‐6 (Hs00174131_m1), IL‐8 (Hs00174103_m1), IL‐10 (Hs00961622_m1), TNFα (Hs00174128_m1), ADIPOQ (Hs00605917_m1), OCN (Hs01587814_g1), RUNX2 (Hs00231692_m1), DKK‐1 (Hs00183740_m1), LEF‐1 (Hs01547250_m1), COL1A1 (Hs00164004_m1) were evaluated in bone and muscle tissue biopsies.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Control, Binding Assay