Journal: bioRxiv

Article Title: Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

doi: 10.64898/2026.05.21.726951

Figure Lengend Snippet: ( A ) Overview of lineage tracing strategy to study Ebf3-lineage cells using Ebf3-CreERT2; Rosa26-LSL-tdTomato; Bglap-GFP mice. Tamoxifen was administered at 10 weeks of age and sacrificed after 2 days, 10 days, and 2 months of iPTH or vehicle treatment. ( B ) Representative fluorescent images of the proximal tibia showing Ebf3-CreERT2-labeled tdTomato+ cells accumulate on bone surfaces (outlined by Bglap-GFP+ osteoblasts) and differentiate into mature osteoblasts (tdTomato+GFP+) and matrix-embedded osteocytes under steady state condition, and this process is enhanced with iPTH treatment. Red = Ebf3-CreERT2-tdTomato; green = Bglap-GFP; blue = DAPI. Scale bar = 500 µm (lower magnification) and 50 µm (higher magnification). ( C ) Quantification of tdTomato+ cells and tdTomato+GFP+ osteoblasts on metaphyseal trabecular and ( D ) endocortical bone surfaces, ( E ) tdTomato+ osteocytes in the cortical bone, and ( F ) tdTomato+ cells in the central bone marrow. Distribution (area fraction) and cell morphology (aspect ratio) analysis of tdTomato+ cells relative to defined distances from the endocortical bone surface in the proximal tibia following ( G ) 2 days, ( H ) 10 days, and ( I ) 2 months of iPTH or vehicle treatment. Statistical test: Two-way ANOVA with ( C-E ) treatment and time, or ( G-I ) treatment and specified distance bins as factors followed by Šidák’s multiple comparisons test. Data is expressed as mean ± SD, n = 3-4 mice/group.

Article Snippet: For FACS isolation of mouse tdTomato+ stromal cells for single cell RNA-sequencing experiments, mouse bone single cell suspension collected after 7 rounds of serial enzymatic digestion underwent magnetic activated cell depletion of hematopoietic cells using mouse lineage depletion kit (Miltenyi Biotec, 130-110-470) combined with CD45 microbeads (Miltenyi Biotec, 130-052-301) according to the manufacturer’s instructions.

Techniques: Labeling

Journal: bioRxiv

Article Title: Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

doi: 10.64898/2026.05.21.726951

Figure Lengend Snippet: ( A) Representative fluorescent images of the humerus and ( B ) flow cytometry analysis shows tdTomato+ cells are not present in 10-week-old Ebf3-CreERT2; Rosa26-LSL-tdTomato mice when tamoxifen is not administered. (C) Region of interest (500 µm x 750 µm box; 400 µm below the growth plate) used to quantify tdTomato+ cells in the central marrow and cortical bone at the metaphyseal and diaphyseal region of the tibia. Scale bar = 500 µm.

Article Snippet: For FACS isolation of mouse tdTomato+ stromal cells for single cell RNA-sequencing experiments, mouse bone single cell suspension collected after 7 rounds of serial enzymatic digestion underwent magnetic activated cell depletion of hematopoietic cells using mouse lineage depletion kit (Miltenyi Biotec, 130-110-470) combined with CD45 microbeads (Miltenyi Biotec, 130-052-301) according to the manufacturer’s instructions.

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

doi: 10.64898/2026.05.21.726951

Figure Lengend Snippet: Fluorescent image quantification of Ebf3-CreERT2-labeled tdTomato+ and tdTomato+Bglap-GFP+ cells in the ( A ) diaphyseal endocortical, ( B) metaphyseal and diaphyseal periosteal, ( C ) diaphyseal cortical bone, and ( D ) diaphyseal central marrow skeletal regions following 2 days, 10 days, and 2 months of iPTH or vehicle treatment. (E) Fluorescent image quantification of Ebf3-CreERT2-labeled tdTomato+ cells from male and female mice at distinct skeletal sites of the humerus following 2 days and 2 months of iPTH or vehicle treatment. Statistical test: Two-way ANOVA with treatment and time ( A-E ) as factors followed by Šidák’s multiple comparisons test. Data is expressed as mean ± SD, n = 3-6 mice/group.

Article Snippet: For FACS isolation of mouse tdTomato+ stromal cells for single cell RNA-sequencing experiments, mouse bone single cell suspension collected after 7 rounds of serial enzymatic digestion underwent magnetic activated cell depletion of hematopoietic cells using mouse lineage depletion kit (Miltenyi Biotec, 130-110-470) combined with CD45 microbeads (Miltenyi Biotec, 130-052-301) according to the manufacturer’s instructions.

Techniques: Labeling

Journal: bioRxiv

Article Title: Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

doi: 10.64898/2026.05.21.726951

Figure Lengend Snippet: ( A ) Multicolor flow cytometry gating strategy used to analyze non-hematopoietic Ebf3-lineage tdTomato+ and tdTomato– skeletal stem and progenitor subsets. mSSC: Mouse skeletal stem cell; Pre-BCSP: Pre-bone/cartilage/stromal progenitor; BCSP: Bone/cartilage/stromal progenitor.

Article Snippet: For FACS isolation of mouse tdTomato+ stromal cells for single cell RNA-sequencing experiments, mouse bone single cell suspension collected after 7 rounds of serial enzymatic digestion underwent magnetic activated cell depletion of hematopoietic cells using mouse lineage depletion kit (Miltenyi Biotec, 130-110-470) combined with CD45 microbeads (Miltenyi Biotec, 130-052-301) according to the manufacturer’s instructions.

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

doi: 10.64898/2026.05.21.726951

Figure Lengend Snippet: ( A ) Experimental overview for labelling parental CAR cells using Ebf3-CreERT2; Rosa26-LSL-tdTomato; Cxcl12-GFP mice treated with tamoxifen at 10-weeks-old and sacrificed 2 days later. ( B ) Flow cytometry analysis showing Ebf3-CreERT2 labels approximately 85% of Cxcl12-GFP+ cells in enzymatically digested marrow plus bone fragments. ( C ) Flow cytometry analysis indicates parental CAR (tdTomato+Cxcl12-GFP+) cells predominately lie within the 6C3+ stromal progenitor subset. ( D ) RT-qPCR analysis of tdTomato mRNA of the remaining bone fragments post-serial digestion of tibia and femur from Ebf3-CreERT2; Rosa26-LSL-tdTomato mice following 2 weeks of iPTH or vehicle treatment. Statistical test: Student’s T-test ( D ). Data is expressed as mean ± SD, n = 5-6 mice/group.

Article Snippet: For FACS isolation of mouse tdTomato+ stromal cells for single cell RNA-sequencing experiments, mouse bone single cell suspension collected after 7 rounds of serial enzymatic digestion underwent magnetic activated cell depletion of hematopoietic cells using mouse lineage depletion kit (Miltenyi Biotec, 130-110-470) combined with CD45 microbeads (Miltenyi Biotec, 130-052-301) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

doi: 10.64898/2026.05.21.726951

Figure Lengend Snippet: Representative flow cytometry plots and analysis of non-hematopoietic Ebf3-CreERT2-labeled tdTomato+ cells and their skeletal stem and progenitor cell surface markers following 2 ( A-C) and 10 days ( D-F) of iPTH or vehicle treatment. Red box: CD45–Ter119–CD31–tdTomato+ fraction. Yellow Box: CD45–Ter119–CD31–tdTomato+Thy1–6C3– fraction. mSSC: Mouse skeletal stem cell; Pre-BCSP: Pre-bone/cartilage/stromal progenitor; BCSP: Bone/cartilage/stromal progenitor. ( G ) Experimental overview for in vitro culturing of FACS-isolated non-hematopoietic Ebf3-CreERT2-labeled tdTomato+ cells. ( H ) Cyclic adenosine monophosphate (cAMP) analysis, ( I ) immunocytochemical staining and ( J ) flow cytometry analysis for phospho-CREB, of in vitro cultured tdTomato+ cells thirty minutes after PTH, forskolin, or vehicle treatment. Statistical test: Student’s T-test ( B, E, H, and J ) and Two-way ANOVA with treatment and subpopulations ( C and F ) as factors followed by Šidák’s multiple comparisons test. Data is expressed as mean ± SD, n = 3-5 mice/group.

Article Snippet: For FACS isolation of mouse tdTomato+ stromal cells for single cell RNA-sequencing experiments, mouse bone single cell suspension collected after 7 rounds of serial enzymatic digestion underwent magnetic activated cell depletion of hematopoietic cells using mouse lineage depletion kit (Miltenyi Biotec, 130-110-470) combined with CD45 microbeads (Miltenyi Biotec, 130-052-301) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Labeling, In Vitro, Isolation, Staining, Cell Culture

Journal: bioRxiv

Article Title: Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

doi: 10.64898/2026.05.21.726951

Figure Lengend Snippet: (A) Harmony-integrated UMAP of single cell RNA-sequencing data from FACS-isolated CD45–Ter119–tdTomato+ cells labeled by Ebf3-CreERT2 following 2 and 10 days of iPTH or vehicle treatment (2 days: vehicle = 13,058 cells and PTH = 12,214 cells; 10 days: vehicle = 34,735 cells and PTH = 19,809 cells; n = 4 mice/group). ( B ) Dot plot summarizing the expression levels of cell-type marker genes across subclusters within Ebf3-lineage cells. Dot size represents the fraction of positive cells within the cluster expressing the gene of interest, and color scale denotes average gene expression level. Purple = higher expression and yellow = lower expression. ( C ) RNA velocity-based fate probability analysis using the CellRank to infer Ebf3-lineage CAR cell fates at steady state or in response to PTH. Color scale represents fate probability of each cell. Violin plots show the fate probability of Ebf3-lineage CAR subclusters transitioning toward the pre-osteoblast cluster under steady state or PTH-treated conditions. ( D ) Volcano plot showing the total number of significant differentially expressed genes (DEGs) of all Ebf3-lineage CAR subclusters regulated by PTH (adjusted P-value < 0.05 and |log2FC| > 0.5). ( E ) Gene set enrichment analysis of significantly up- and down-regulated DEGs by PTH using the WikiPathways 2024 Mouse database. Top 12 significantly enriched gene sets are shown (adjusted P-value < 0.05). ( F ) PTH-responsive module scores visualized by Feature plots of each Ebf3-lineage cell (top) and violin plots (bottom) across Ebf3-lineage CAR and osteoblast clusters in response to iPTH or vehicle treatment. Purple = higher expression and yellow = lower expression. Cliff’s delta (δ) = non-parametric effect size used to measure differences between module score distributions. ( G ) Violin plots showing the expression levels of canonical PTH target genes, ( H ) adipogenic genes, ( I ) blood supportive genes, and ( J ) CAR cell fate regulating transcription factor genes, across Ebf3-lineage CAR and osteoblast clusters in response to iPTH or vehicle treatment. Differentially expressed genes between PTH and control cells within each cluster was assessed using the MAST hurdle model . Asterisk (*) denotes significance (adjusted P-value < 0.05 and |log2FC| > 0.5).

Article Snippet: For FACS isolation of mouse tdTomato+ stromal cells for single cell RNA-sequencing experiments, mouse bone single cell suspension collected after 7 rounds of serial enzymatic digestion underwent magnetic activated cell depletion of hematopoietic cells using mouse lineage depletion kit (Miltenyi Biotec, 130-110-470) combined with CD45 microbeads (Miltenyi Biotec, 130-052-301) according to the manufacturer’s instructions.

Techniques: Single Cell, RNA Sequencing, Isolation, Labeling, Expressing, Marker, Gene Expression, Control

Journal: bioRxiv

Article Title: Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

doi: 10.64898/2026.05.21.726951

Figure Lengend Snippet: (A) Violin plots showing Sp7 expression level across Ebf3-lineage CAR and osteoblast clusters. ( B ) Overview of lineage tracing strategy to study the role of Sp7 in Ebf3-lineage cells following 10 days of iPTH or vehicle treatment. ( C ) Representative fluorescent images of the proximal tibia from wild type and Sp7 f/f mice following 10 days of iPTH or vehicle treatment. Red = Ebf3-CreERT2-tdTomato; green = Bglap-GFP; blue = DAPI. Scale bar = 500 µm (lower magnification) and 50 µm (higher magnification). ( D ) Quantification of tdTomato+ cells and tdTomato+GFP+ osteoblasts on metaphyseal trabecular and ( E ) endocortical bone surfaces of wild type and Sp7 f/f mice following 10 days of iPTH or vehicle treatment. ( F ) Distribution (area fraction) and ( G ) cell morphology (aspect ratio) analysis of tdTomato+ cells relative to defined distances from bone surfaces in the proximal tibia of wild type and Sp7 f/f mice following 10 days of iPTH or vehicle treatment. ( H ) Representative flow cytometry plots and analysis of non-hematopoietic Ebf3-CreERT2-labeled tdTomato+ cells ( I ) and their skeletal stem/progenitor cell surface markers ( J ) from wild type and Sp7 f/f mice following 10 days of iPTH or vehicle treatment. ( K and L) RT-qPCR analysis of FACS-isolated Lineage–tdTomato+ cells from wild type and Sp7 f/f mice following 10 days of iPTH and vehicle treatment. Statistical test: Two-way ANOVA with treatment and genotype ( D, E, I, and L ), or treatment and specified distance bins ( F and G ), or treatment and subpopulations ( J ) as factors followed by Šidák’s multiple comparisons test, and Student’s T-test ( K ). Data is expressed as mean ± SD, n = 4-5 mice/group.

Article Snippet: For FACS isolation of mouse tdTomato+ stromal cells for single cell RNA-sequencing experiments, mouse bone single cell suspension collected after 7 rounds of serial enzymatic digestion underwent magnetic activated cell depletion of hematopoietic cells using mouse lineage depletion kit (Miltenyi Biotec, 130-110-470) combined with CD45 microbeads (Miltenyi Biotec, 130-052-301) according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Labeling, Quantitative RT-PCR, Isolation

Journal: bioRxiv

Article Title: Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

doi: 10.64898/2026.05.21.726951

Figure Lengend Snippet: (A) Violin plots showing Pth1r expression level across Ebf3-lineage CAR and osteoblast clusters. ( B ) Overview of lineage tracing strategy to study the role of Pth1r in Ebf3-lineage cells following 10 days of iPTH or vehicle treatment. ( C ) Representative fluorescent images of the proximal tibia from wild type and Pth1r f/f mice following 10 days of iPTH or vehicle treatment. Red = Ebf3-CreERT2-tdTomato; green = Bglap-GFP; blue = DAPI. Scale bar = 500 µm (lower magnification) and 50 µm (higher magnification). ( D ) Quantification of tdTomato+ cells and tdTomato+GFP+ osteoblasts on metaphyseal trabecular and ( E ) endocortical bone surfaces of wild type and Pth1r f/f mice following 10 days of iPTH or vehicle treatment. ( F ) Distribution (area fraction) and ( G ) cell morphology (aspect ratio) analysis of tdTomato+ cells relative to defined distances from bone surfaces in the proximal tibia of wild type and Pth1r f/f mice following 10 days of iPTH or vehicle treatment. ( H ) Representative flow cytometry plots and analysis of non-hematopoietic Ebf3-CreERT2-tdTomato+ cells ( I ) and their skeletal stem/progenitor cell surface markers ( J ) from wild type and Sp7 f/f mice following 10 days of iPTH or vehicle treatment. ( K and L) RT-qPCR analysis of FACS-isolated Lineage–tdTomato+ cells from wild type and Sp7 f/f mice following 10 days of iPTH and vehicle treatment. Statistical test: Two-way ANOVA with treatment and genotype ( D, E, I, and L ), or treatment and specified distance bins ( F and G ), or treatment and subpopulations ( J ) as factors followed by Šidák’s multiple comparisons test, and Student’s T-test ( K ). Data is expressed as mean ± SD, n = 4-7 mice/group.

Article Snippet: For FACS isolation of mouse tdTomato+ stromal cells for single cell RNA-sequencing experiments, mouse bone single cell suspension collected after 7 rounds of serial enzymatic digestion underwent magnetic activated cell depletion of hematopoietic cells using mouse lineage depletion kit (Miltenyi Biotec, 130-110-470) combined with CD45 microbeads (Miltenyi Biotec, 130-052-301) according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Quantitative RT-PCR, Isolation

Journal: bioRxiv

Article Title: Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

doi: 10.64898/2026.05.21.726951

Figure Lengend Snippet: (A) Representative fluorescent images of TRAP-stained osteoclasts in proximal tibia tissue sections from 10-week-old Ebf3-CreERT2; Rosa26-LSL-tdTomato; Bglap-GFP mice following 2 and 10 days of iPTH or vehicle treatment. White = TRAP; Red = Ebf3-CreERT2-tdTomato; green = Bglap-GFP; blue = DRAQ5. Scale bar = 50 µm. (B) Quantification of Ebf3-CreERT2-labeled tdTomato+ cell distance relative to Bglap-GFP+ osteoblast or TRAP+ osteoclast following 10 days of vehicle treatment. ( C ) Quantification of Ebf3-CreERT2-labeled tdTomato+ cell numbers within 50 µm radius of Bglap-GFP+ osteoblast or TRAP+ osteoclast following 10 days of iPTH or vehicle treatment. ( D ) Experimental overview to selectively deplete osteoclasts by OPG-Fc in the Ebf3-CreERT2 lineage tracing iPTH system. ( E ) Representative fluorescent images of the proximal tibia from Ebf3-CreERT2; Rosa26-LSL-tdTomato; Bglap-GFP mice following 10 days of iPTH, OPG-Fc, combination or vehicle treatment. Red = Ebf3-CreERT2-tdTomato; green = Bglap-GFP; blue = DAPI. Scale bar = 500 µm (lower magnification) and 50 µm (higher magnification). ( F ) Quantification of TRAP+ cells on metaphyseal trabecular bone surfaces, and ( G ) tdTomato+ cells on metaphyseal trabecular and ( H ) endocortical bone surfaces following 10 days of iPTH, OPG-Fc, combination or vehicle treatment. ( I ) Distribution (area fraction) and ( J ) cell morphology (aspect ratio) analysis of tdTomato+ cells relative to defined distances from bone surfaces in the proximal tibia of Ebf3-CreERT2; Rosa26-LSL-tdTomato; Bglap-GFP mice following 10 days of iPTH, OPG-Fc, combination or vehicle treatment. ( K ) Representative flow cytometry plots and analysis of ( L ) non-hematopoietic Ebf3-CreERT2-labeled tdTomato+ cells and ( M ) their skeletal stem/progenitor cell surface markers following 10 days of iPTH, OPG-Fc, combination or vehicle treatment. Statistical test: Student’s T-test ( B ), Two-way ANOVA with treatment and cell-type ( C ), or treatment and specified distance bins ( I and J ), or treatment and subpopulations ( M ) as factors followed by Šidák’s multiple comparisons test, and one-way ANOVA followed by by Šidák’s multiple comparisons test ( F, G, H, and L ). Data is expressed as mean ± SD, n = 5-6 mice/group.

Article Snippet: For FACS isolation of mouse tdTomato+ stromal cells for single cell RNA-sequencing experiments, mouse bone single cell suspension collected after 7 rounds of serial enzymatic digestion underwent magnetic activated cell depletion of hematopoietic cells using mouse lineage depletion kit (Miltenyi Biotec, 130-110-470) combined with CD45 microbeads (Miltenyi Biotec, 130-052-301) according to the manufacturer’s instructions.

Techniques: Staining, Labeling, Flow Cytometry

Journal: bioRxiv

Article Title: Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

doi: 10.64898/2026.05.21.726951

Figure Lengend Snippet: (A) Harmony-integrated UMAP of single cell RNA-sequencing data from FACS-isolated CD45–Ter119–tdTomato+ cells labeled by Ebf3-CreERT2 following 10 days of iPTH, OPG-Fc, combination or vehicle treatment (Vehicle = 1,155 cells, PTH = 1,638 cells, OPG-Fc = 1,720, and OPG-Fc + PTH = 1,773; n = 5-6 mice/group). Inset shows feature plot of tdTomato mRNA expression across clusters. ( B ) Violin plots showing mRNA expression levels of CAR cell, pre-osteoblast, and mature osteoblast marker genes in Ebf3-lineage cell clusters. ( C ) Volcano plots showing the total number of significant differentially expressed genes (DEGs) of all Ebf3-lineage CAR subclusters regulated by PTH, OPG-Fc, or OPG-Fc + PTH (adjusted P-value < 0.05 and |log2FC| > 0.5). ( D ) PTH-responsive module scores visualized by Feature plots of each Ebf3-lineage CAR cell and pre-osteoblast (top) and violin plots (bottom) across Ebf3-lineage CAR and osteoblast clusters in response to iPTH, OPG-Fc, combination, or vehicle treatment. ( E ) Violin plots showing the expression levels of canonical PTH target genes and CAR cell fate regulating transcription factor genes across Ebf3-lineage CAR and pre-osteoblast clusters in response to iPTH, OPG-Fc, combination, or vehicle treatment. ( F ) Venn-diagrams comparing differentially expressed genes altered by PTH versus OPG-Fc + PTH treatment. ( G ) Gene set enrichment analysis of all significantly up- and down-regulated DEGs by PTH but not OPG-Fc + PTH in Ebf3-lineage CAR clusters using the MSigDB Hallmark 2020 database. The top 5 significantly enriched gene sets are shown (adjusted P-value < 0.05). ( H ) Violin plots showing the expression levels of TGFß-responsive genes across Ebf3-lineage CAR and pre-osteoblast clusters in response to iPTH, OPG-Fc, combination, or vehicle treatment. ( I ) TGFß-responsive module scores visualized by Feature plots of each Ebf3-lineage CAR cell and pre-osteoblast (top) and violin plots (bottom) across Ebf3-lineage CAR and osteoblast clusters in response to iPTH, OPG-Fc, combination, or vehicle treatment. Cliff’s delta (δ) = non-parametric effect size used to measure differences between module score distributions. Differential gene expression between treatment and control cells, and treatment interactions within each cluster was assessed using the MAST hurdle model. Black asterisks (*) denotes genes that are significantly differentially expressed in current dataset (adjusted P-value < 0.05 and |log2FC| > 0.5). Red asterisks indicate genes that were significantly regulated by iPTH in the single cell RNA-sequencing dataset from , but did not reach statistical significance in the current experiment, likely due to limited power. Blue hashtags (#) indicates a significant antagonistic treatment interaction between OPG-Fc and PTH. ( J ) Representative confocal images of immunohistochemical staining for phospho-SMAD2/3 in proximal tibia tissue sections from Ebf3-CreERT2; Rosa26-LSL-tdTomato; Bglap-GFP mice following 10 days of iPTH, OPG-Fc, combination or vehicle treatment. Red = Ebf3-CreERT2-tdTomato; green = Bglap-GFP; white = phospho-SMAD2/3; blue = DAPI. Scale bar = 50 µm.

Article Snippet: For FACS isolation of mouse tdTomato+ stromal cells for single cell RNA-sequencing experiments, mouse bone single cell suspension collected after 7 rounds of serial enzymatic digestion underwent magnetic activated cell depletion of hematopoietic cells using mouse lineage depletion kit (Miltenyi Biotec, 130-110-470) combined with CD45 microbeads (Miltenyi Biotec, 130-052-301) according to the manufacturer’s instructions.

Techniques: Single Cell, RNA Sequencing, Isolation, Labeling, Expressing, Marker, Gene Expression, Control, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

doi: 10.64898/2026.05.21.726951

Figure Lengend Snippet: ( A ) Overview of lineage tracing strategy to study the relative contribution of Ebf3-lineage cells following 1 month of iPTH or vehicle treatment in male mice. ( C ) Representative fluorescent images of the proximal tibia from wild type and Sp7 f/f mice following 1 month of iPTH or vehicle treatment. Red = Ebf3-CreERT2-tdTomato; green = Bglap-GFP; blue = DAPI. White arrow: tdTomato+ cell on bone surface; yellow arrow: tdTomato+ osteocyte. Scale bar = 500 µm (lower magnification) and 50 µm (higher magnification). ( C ) Quantification of tdTomato+ cells on metaphyseal trabecular and ( D ) endocortical bone surfaces, ( E ) tdTomato+ osteocytes in the cortical bone of wild type or Sp7 f/f mice following 1 month of iPTH or vehicle treatment. ( F ) Distribution (area fraction) and ( G ) cell morphology (aspect ratio) analysis of tdTomato+ cells relative to defined distances from endocortical bone surface in the proximal tibia of wild type and Sp7 f/f mice following 1 month of iPTH or vehicle treatment. Levels of serum bone formation marker ( H ) P1NP and resorption marker ( I ) CTX from wild type and Sp7 f/f mice following 1 month of iPTH or vehicle treatment. ( J ) Representative sagittal femur microCT images and quantification of ( K ) trabecular and ( L ) primary spongiosa bone volume/total volume (BV/TV) in wild type and Sp7 f/f mice following 1 month of iPTH or vehicle treatment. Scale bar = 1 mm.Statistical test: Two-way ANOVA with treatment and genotype ( C, D, E, H, I, K and L ), or treatment and specified distance bins ( F and G ) as factors followed by Šidák’s multiple comparisons test. Data is expressed as mean ± SD, n = 6-8 mice/group.

Article Snippet: For FACS isolation of mouse tdTomato+ stromal cells for single cell RNA-sequencing experiments, mouse bone single cell suspension collected after 7 rounds of serial enzymatic digestion underwent magnetic activated cell depletion of hematopoietic cells using mouse lineage depletion kit (Miltenyi Biotec, 130-110-470) combined with CD45 microbeads (Miltenyi Biotec, 130-052-301) according to the manufacturer’s instructions.

Techniques: Marker

Journal: bioRxiv

Article Title: Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

doi: 10.64898/2026.05.21.726951

Figure Lengend Snippet: ( A ) Fluorescent image quantification of Ebf3-CreERT2-labeled tdTomato+ cells from wild type and Sp7 f/f mice in the ( A ) diaphyseal endocortical bone surface, ( B ) diaphyseal cortical bone, ( C) metaphyseal and ( D ) diaphyseal periosteal bone surface, and ( E ) metaphyseal and ( F ) diaphyseal central marrow skeletal regions following 1 month of iPTH or vehicle treatment. ( G ) Flow cytometry analysis of non-hematopoietic Ebf3-CreERT2-tdTomato+ cells ( H ) and their skeletal stem/progenitor cell surface markers ( J ) from wild type and Sp7 f/f mice following 1 month of iPTH or vehicle treatment. Statistical test: Two-way ANOVA with treatment and genotype ( A-G ), or treatment and subpopulations ( H ) as factors followed by Šidák’s multiple comparisons test. Data is expressed as mean ± SD, n = 5-8 mice/group.

Article Snippet: For FACS isolation of mouse tdTomato+ stromal cells for single cell RNA-sequencing experiments, mouse bone single cell suspension collected after 7 rounds of serial enzymatic digestion underwent magnetic activated cell depletion of hematopoietic cells using mouse lineage depletion kit (Miltenyi Biotec, 130-110-470) combined with CD45 microbeads (Miltenyi Biotec, 130-052-301) according to the manufacturer’s instructions.

Techniques: Labeling, Flow Cytometry

Journal: bioRxiv

Article Title: Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

doi: 10.64898/2026.05.21.726951

Figure Lengend Snippet: ( A ) Violin plots showing Esr1 expression level across Ebf3-lineage CAR and osteoblast clusters. ( B ) Overview of lineage tracing strategy to study the effects of iPTH, OPG-Fc, or combination treatment on Ebf3-lineage cells under estrogen-deficient setting using female ovariectomized Ebf3-CreERT2; Rosa26-LSL-tdTomato mice. ( C ) Representative fluorescent images of the proximal tibia from ovariectomized Ebf3-CreERT2; Rosa26-LSL-tdTomato; Bglap-GFP mice following 2 weeks of iPTH, OPG-Fc, combination, or vehicle treatment. White = TRAP; Red = Ebf3-CreERT2-tdTomato; green = Bglap-GFP; blue = DAPI. Scale bar = 500 µm (lower magnification) and 50 µm (higher magnification). ( D ) Quantification of tdTomato+ cells on metaphyseal trabecular and ( E ) endocortical bone surfaces following 2 weeks of iPTH, OPG-Fc, combination or vehicle treatment. ( F ) Distribution (area fraction) and ( G ) cell morphology (aspect ratio) analysis of tdTomato+ cells relative to defined distances from bone surfaces in the proximal tibia. ( H ) Representative flow cytometry plots and analysis of ( I ) non-hematopoietic Ebf3-CreERT2-tdTomato+ cells and their ( J ) skeletal stem/progenitor cell surface markers from ovariectomized Ebf3-CreERT2; Rosa26-LSL-tdTomato; Bglap-GFP mice following 2 weeks of iPTH, OPG-Fc, combination, or vehicle treatment. Statistical test: One-way ANOVA followed by by Šidák’s multiple comparisons test ( D, E, and I ), and two-way ANOVA with treatment and specified distance bins ( F and G ), or treatment and subpopulations ( J ) as factors followed by Šidák’s multiple comparisons test. Data is expressed as mean ± SD, n = 5-6 mice/group.

Article Snippet: For FACS isolation of mouse tdTomato+ stromal cells for single cell RNA-sequencing experiments, mouse bone single cell suspension collected after 7 rounds of serial enzymatic digestion underwent magnetic activated cell depletion of hematopoietic cells using mouse lineage depletion kit (Miltenyi Biotec, 130-110-470) combined with CD45 microbeads (Miltenyi Biotec, 130-052-301) according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

doi: 10.64898/2026.05.21.726951

Figure Lengend Snippet: (A) UMAP visualization of human bone marrow single cell RNA-sequencing data following ( B ) Harmony-integration of bone marrow aspirate samples from postmenopausal women treated with teriparatide for 3 months (11,090 cells; n = 2 patient samples) and a naïve human bone marrow atlas. ( C ) Feature plots showing the expression CAR cell marker genes. ( D ) Violin plots showing mRNA expression levels of CAR cell and pre-osteoblast genes. ( E ) Volcano plot showing the total number of significant differentially expressed genes between each teriparatide-treated vs atlas CAR clusters (adjusted P-value < 0.05 and |log2FC| > 0.5). ( F ) Violin plots showing the expression levels of CAR cell genes, ( G ) adipogenic genes, ( H ) and osteogenic and bone matrix genes, across each teriparatide-treated versus atlas CAR clusters. ( I ) Gene set enrichment analysis of all up- and down-regulated differentially expressed genes identified in teriparatide-treated CAR clusters using the MSigDB Hallmark 2020 database. ( J ) Violin plots of TGFß-responsive module scores of teriparatide-treated versus atlas CAR clusters. Cliff’s delta (δ) = non-parametric effect size used to measure differences between module score distributions. (K) Violin plots showing the expression levels of TGFß-responsive genes between teriparatide-treated vs atlas CAR clusters. Differential gene expression between teriparatide-treated and atlas cells within each CAR cluster was assessed using the MAST hurdle model. Asterisk (*) denotes significance (adjusted P-value < 0.05 and |log2FC| > 0.5).

Article Snippet: For FACS isolation of mouse tdTomato+ stromal cells for single cell RNA-sequencing experiments, mouse bone single cell suspension collected after 7 rounds of serial enzymatic digestion underwent magnetic activated cell depletion of hematopoietic cells using mouse lineage depletion kit (Miltenyi Biotec, 130-110-470) combined with CD45 microbeads (Miltenyi Biotec, 130-052-301) according to the manufacturer’s instructions.

Techniques: Single Cell, RNA Sequencing, Expressing, Marker, Gene Expression