Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet: (A) Skeletal phenotypes of WT and Trim28 MKO mice at E19.5 visualized with Alcian blue and Alizarin red staining; arrowheads indicate thicker and shorter ribs and over-expanded hindlimb cartilage. Scale bar, 0.5 cm. (B) E17.5 mouse proximal tibial sections stained with Alcian blue Hematoxylin/Orange G. Gross image (left) and magnified views of each zone (right) are shown(RZ, resting zone; PZ, proliferating zone; HZ, hypertrophic zone). Scale bar, 200 μm. (C) Increased cell proliferation in the Trim 28 MKO proximal tibial sections (E17.5) as indicated by Ki67 immunohistochemistry (IHC). Bottom panel: area boxed in red is the magnified perichondrium (PC), while the yellow boxed area is the magnified periarticular resting zone (PRZ). Scale bars, 200 μm. (D) Type II collagen IHC of proximal tibial sections (E17.5). Area boxed in red is magnified in the bottom panels showing high levels of type II collagen retention inthe Trim28 MKO HZ. Scale bars, 200 μm. (E) Type X collagen IHC staining of distal femoral sections (E17.5). The red box area is magnified in the bottom panel, and red dotted lines define type X collagen-expressing areas. Scale bars, 200 μm. See also .

Article Snippet: Mouse: Trim28 f/f , Jackson Laboratories , Cat # 018552.

Techniques: Staining, Immunohistochemistry, Expressing

Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet: (A) Distribution of 13,683 qualified single cells from WT (6,235) and Trim28 MKO (7,448) hindlimb GPs in 8 clusters were visualized by uniform manifold approximation and projection (UMAP). (B) UMAP in (A) is divided into two genotype-specific UMAPs. (C) The proportion of each cell cluster in total WT or Trim28 MKO hindlimb GPs. (D) Enriched Wikipathways and KEGG pathways in Trim28 MKO -U cluster versus all cells, which includes total WT and Trim28 MKO cells. (E) Violin plots of the expression of previously reported mesenchymal progenitor/SSC marker genes in each of the eight cell clusters identified in (A). (F) Distribution of SSCs in scRNA-seq UMAPs (the same as B). The criteria for defining SSCs here are cells positive for Itgav and Cd200 and negative for Ptprc , Tek , Thy1 , Enpep , and Eng in mRNA expression. Dashed black line indicates the boundary of the Trim28 MKO -U cluster. (G) Colony-formation assay initiated by seeding 1,000 (top panels) or 100 (bottom panels) rib GP cells. Clones containing at least 50 cells from the 100-cell seeding group are quantified in the bottom (n = 6). p value = 0.0081. Scale bar, 1 cm. Comparisons are conducted by Student’s t test, two tailed. Data are presented as mean ± SEM. (H) Representative flow cytometric analysis of the proportion of SSCs (CD45 − Ter119 − TIE2 CD51 + 6C3 − THY1.2 − CD105 − CD200 + ) and pre-BCSPs (CD45 − Ter119 − TIE2 − CD51 + 6C3 THY1.2 − CD105 − CD200 − ) in hindlimb GPs (n = 3 for each genotype). The representative flow image in the bottom shows the Trim28 MKO sample has a population shift from pre-BCSP to SSC when gated by CD200 expression. p values are shown in the figure. All comparisons are conducted by Student’s t test, two tailed. Data are presented as mean ± SEM. and .

Article Snippet: Mouse: Trim28 f/f , Jackson Laboratories , Cat # 018552.

Techniques: Expressing, Marker, Colony Assay, Clone Assay, Two Tailed Test

Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet: (A) Dot plots comparing the expression of selected lineage/niche markers in total WT SSCs versus Trim28 MKO SSCs. (B) Ex vivo chondrogenesis (top panels) and osteogenesis (bottom panels) of FACS WT or Trim28 MKO GP SSCs. Representative staining is shown (n = 3 per genotype). Scale bar, 400 μm. (C) UMAP of 5 subclusters of SSCs from combined WT and Trim28 MKO hindlimb GP cells (top panel). Genotype-specific UMAPs of SSCs are shown in the bottom panels. (D) RNA velocity trajectory inference analysis of gross SSCs (left). The separated WT (top panel) and Trim28 MKO (bottom panel) velocity trajectories are shown on the right. (E) Proportion of each SSC subtype in WT or Trim28 MKO GP SSCs. (F) Enriched WiKiPathways and MSigDB_Hallmarks in Trim28 MKO -NSSCs versus all SSCs (WT plus Trim28 MKO ). (G) Dot plots comparing the expression of osteochondrogenic marker genes in WT and Trim28 MKO root-SSCs, Trim28 MKO -NSSCs, WT chondro- and osteo-SSCs. (H) Transcriptomic profiles of Trim28 MKO SSCs (MKO), but not WT SSCs (WT), show a strong similarity with neural crest cells (NCCs) according to Spearman correlation analysis. (I) Pseudo-bulk-level transcriptomic similarity analysis of NCC and SSC subtypes. The size of dots represents the cell number of each subcluster. (J) FACS Trim28 MKO GP SSCs exhibit more potent neurogenic differentiation than WT SSCs. Representative bright-field images are shown in the left panels, and percentages of neuron-like cell/total cell and neurite length/neuron-like cell are quantified in the right panel (n = 4 per genotype). Scale bar, 100 μm. p < 0.0001. Data comparisons are conducted by Student’s t test, two-tailed. Data are presented as mean ± SEM. (K) Trim28 MKO SSCs express higher levels of neurogenic markers than WT SSCs after 5 days of neurogenic induction (n = 3 per genotype). p values are shown in the figure. Data comparisons are conducted by Student’s t test, two-tailed. Data are presented as mean ± SEM. See also and and .

Article Snippet: Mouse: Trim28 f/f , Jackson Laboratories , Cat # 018552.

Techniques: Expressing, Ex Vivo, Staining, Marker, Two Tailed Test

Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet: (A) Volcano plot of RNA-seq data demonstrating significant changes in gene expression between WT and Trim28 MKO rib GP cells. Top 10 up- and down-regulated genes are shown. (B) mTORC1 and PI3K-AKT pathways are enriched in Trim28 MKO samples by gene set enrichment analysis (GSEA). (C) Global changes of H3K9me3 (left panel) and DNA methylation (right panel) caused by Trim28 deletion. (D) Trim28 MKO GP cells have decreased global H3K9me3 modifications at gene bodies (left panel) and enhancers (right panel). (E) Regions with down-regulated H3K9me3 (upper panel) and CpG DNA methylation (lower panel) show enriched binding motifs of TFs related to pluripotency and neurogenesis. (F) Chromatin accessibility status of SSCs and NCCs at the genomic regions that are silenced by TRIM28 through H3K9me3 (left panel) and CpG DNA methylation (right panel). (G) Proportion of TRIM28-silenced regions that contain TEs. The chromatin accessibility information is from (F). (H) Venn diagram of genes with upregulated mRNA expression (mRNA up), genes silenced by TRIM28 through H3K9me3 (H3K9me3 down), and genes silenced by TRIM28 through CpG DNA methylation (CpG DNA methylation down) identified 124 TRIM28-silenced genes. (I) DAVID tissue expression analysis of the 124 TRIM28-silenced genes. (J) DAVID GO biology process analysis of the 124 TRIM28-silenced genes. (K) TRIM28 silences the gamma-protocadherin gene cluster through H3K9me3. See also , , , , and .

Article Snippet: Mouse: Trim28 f/f , Jackson Laboratories , Cat # 018552.

Techniques: RNA Sequencing, Gene Expression, DNA Methylation Assay, Binding Assay, Expressing

Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet: (A) pS6 (an mTORC1 downstream effector) and pAKT (an mTORC1 upstream activator) levels are increased in E17.5 Trim28 MKO rib GPs (WT, n = 4; Trim28 MKO , n = 5). (B) pS6 level is increased in E17.5 Trim28 MKO mouse distal femur. GP areas are demarcated by white dashed lines. Red boxes are magnified in the bottom panel. Scale bars, 200 μm. (C) GREM1 and TRIM28 protein expressions are inversely related in the rib GP cells (WT, n = 3; Trim28 MKO , n = 2). (D) mTORC1 and AKT pathways are activated by recombinant GREM1 in ATDC5 cells in a time-dependent manner. (E) Levels of pAKT and pS6 were reduced by Grem1 gene knockdown with shRNA targeting Grem1 (sh Grem1) in cultured Trim28 MKO rib GP cells compared with control shRNA (shct). Densitometry quantification (n = 4) is shown on the right. p values are shown in the figure. Comparisons are conducted using one-way ANOVA analyses with Tukey’s post-hoc test. Data are presented as mean ± SEM. (F) H3K27ac and H3K9me3 marks at the promoter region of the Grem1 gene. (G) The occupancy status of CpG DNA methylation and H3K27ac at DMR1 and DMR2. The most distinctive changes are highlighted, and DMR regions are indicated with dark blue bars. (H) Luciferase reporter assays indicating the transcriptional activity of the Grem1 enhancers (DMR1 or DMR2) and promoter in ATDC5 cells. Schematics of the reporter constructs are in the top panel. Empty pGL3-basic vector was used as a negative control. Representative quantified results are shown in the bottom panel (n = 6). p values are shown in the figure. Comparisons are achieved using Student’s t test, two-tailed. Data are presented as mean ± SEM. (I) Grem1 mRNA levels in control (sg GFP ) and CRISPR-Cas9-mediated DMR1 (sgDMR1) or DMR2 (sgDMR2–1, sgDMR2–2) deleted WT and Trim28 MKO rib GP cells (n = 6). p values are shown in the figure. Comparisons are conducted using one-way ANOVA analyses with Tukey’s post-hoc test. Data are presented as mean ± SEM. See also .

Article Snippet: Mouse: Trim28 f/f , Jackson Laboratories , Cat # 018552.

Techniques: Recombinant, Knockdown, shRNA, Cell Culture, Control, DNA Methylation Assay, Luciferase, Activity Assay, Construct, Plasmid Preparation, Negative Control, Two Tailed Test, CRISPR

Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet: (A) Skeletal phenotypes of the E17.5 vector-, YL294002-, or rapamycin-treated mice visualized with Alcian blue and Alizarin red staining. Red arrows indicate reduced knee cartilage and black arrows indicate expanded rib cages in YL294002-and rapamycin-treated mice. Scale bars, 0.5 cm. (B) Representative Alcian blue Hematoxylin/Orange G stain of proximal tibial (right) and distal femoral (left) sections from mice treated with vector, YL294002, and rapamycin. Red arrows indicate rescued proliferating and hypertrophic zones. Scale bars, 200 μm. (C) Ex vivo neurogenesis of GP cells isolated from WT hindlimb treated with either GREM1 (300 ng/mL) or PBS in either neurogenic media or αMEM (n = 5 per group). p values are shown in the figure. Comparisons are achieved by one-way ANOVA analyses with Tukey’s post-hoc test. Data are presented as mean ± SEM. Scale bars, 100 μm. (D) Expression of key neurogenic markers (n = 3 per group) in cells cultured as described in (C). p values are shown in the figure. Comparisons are achieved by one-way ANOVA analyses with Tukey’s post-hoc test. Data are mean ± SEM. (E) Rapamycin (100 nM) treatment partially rescues the enhanced neurogenesis of hindlimb GP cells caused by Trim28 loss. DMSO was used as a negative control (n = 5 per group). p values are shown in the figure. Comparisons are achieved by one-way ANOVA analyses with Tukey’s post-hoc test. Data are presented as mean ± SEM. Scale bars, 100 μm. (F) mRNA expression of key neurogenic markers from cells treated as described in (E) (n = 3 per group). p values are shown in the figure. Comparisons are achieved by one-way ANOVA analyses with Tukey’s post-hoc test. Data are presented as mean ± SEM.

Article Snippet: Mouse: Trim28 f/f , Jackson Laboratories , Cat # 018552.

Techniques: Plasmid Preparation, Staining, Ex Vivo, Isolation, Expressing, Cell Culture, Negative Control

Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet:

Article Snippet: Mouse: Trim28 f/f , Jackson Laboratories , Cat # 018552.

Techniques: Virus, Recombinant, Plasmid Preparation, SYBR Green Assay, cDNA Synthesis, DNA HS Assay, Reporter Assay, Software, Imaging