Journal: Signal Transduction and Targeted Therapy

Article Title: EphrinB2-mediated CDK5/ISL1 pathway enhances cardiac lymphangiogenesis and alleviates ischemic injury by resolving post-MI inflammation

doi: 10.1038/s41392-024-02019-4

Figure Lengend Snippet: A key role of EphrinB2 in cardiac lymphangiogenesis after acute myocardial infarction (AMI). a Representative immunoblotting images showing EphrinB2 protein levels in the hearts of wild-type (WT) mice subjected to sham or MI operation (at 3, 7, and 14 days after operation). b Quantification of a normalized to Actin and presented relative to the sham group (n = 4 per group). c t-distributed stochastic neighbor embedding (t-SNE) plot showing the cardiac cells isolated from murine hearts that were clustered into four cell populations using the single-cell RNA sequencing data (GSE120064). Colors indicate different cell populations. d Dot plot showing feature genes for the four cell populations. e Feature plot showing the transcriptional expression of Efnb2 in each cell. Colors denote the relative expression of Efnb2 . f Violin plot showing the transcriptional expression of Efnb2 across the four cell populations. g Representative immunofluorescence staining images showing myocardium co-stained by EphrinB2 (green) and DAPI (blue) with CD31 (red), cTnT (red), αSMA (red) and CD68 (red), respectively. Scar bar: 20 μm. h Representative M-mode echocardiographic images showing the cardiac function of Efnb2 +/− mice and their WT littermates after sham or MI operation. The yellow lines indicate the endocardium of the anterior and posterior walls at mid-papillary muscle level. i – k Quantification of echocardiographic parameters in h (LVEF, LVIDs, and LVIDd, n = 6 per group). l Representative histological images showing infarct and fibrosis area of Efnb2 +/− mice and their WT littermates after MI operation assessed by Masson Trichrome staining. Magnified views of black boxes are shown in the right lane. Scar bar: 1 mm. m , n Quantification of infarct size of myocardium and wall thickness of the infarct area (n = 5 per group). o Quantification of p (n = 5 per group). (p) Representative immunofluorescence images showing myocardium co-stained by CD31 (red), VEGFR3 (green), and DAPI (blue) of Efnb2 +/− mice and their WT littermates after MI operation. Scar bar: 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001, *** P < 0.0001. b by Kruskal–Wallis with Dunn test, i – k by one-way ANOVA with Tukey posthoc test, and m – o by unpaired Student’s test. D days, ave. exp. average expression, per. exp., percent expresse, LVEF left ventricularejection fraction, LVIDs left ventricular internal dimension during systole, LVIDd left ventricular internal dimension during diastole, DAPI 4’,6-diamidino-2-phenylindole, and TUNEL terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling

Article Snippet: The antibodies utilized were as follows: EphrinB2 (10 μg/ml;# AF496, R&D system), VEGFR3/FLT4 (2 μg/ml; #AF743, R&D system), LYVE-1 (1:100; #67538, Cell Signal Technology), CD31 (1:100; #ab9498, Abcam), CD68 (1:100; #25747, Proteintech), cTnT (1:100; #ab8295 Abcam), αSMA (1:500; #ab7817, Abcam), ISL1(1:200; #ab86501, Abcam), CDK5 (1:200; #10430-1-AP, Proteintech).

Techniques: Western Blot, Isolation, RNA Sequencing Assay, Expressing, Immunofluorescence, Staining, TUNEL Assay, End Labeling

Journal: Signal Transduction and Targeted Therapy

Article Title: EphrinB2-mediated CDK5/ISL1 pathway enhances cardiac lymphangiogenesis and alleviates ischemic injury by resolving post-MI inflammation

doi: 10.1038/s41392-024-02019-4

Figure Lengend Snippet: Overexpression of EphrinB2 promotes cardiac lymphangiogenesis and reduces cardiac inflammation post-MI. a Whole mount imaging showing endogenous tdTomato (white) fluorescence in the hearts from Lyve1 -Cre; Rosa26 -tdTomato mice after sham or MI operation. Magnified views of yellow dashed boxes are shown in the right lane. The yellow arrows highlight the lymphatics in the infarct area and border zone. Scar bar: 1 mm. b (Left) Representative immunofluorescence staining images showing myocardium co-stained by CD31 (red), VEGFR3 (green), and DAPI (blue) of mice injected with AAV- Efnb2 or AAV-NC after MI operation. Scar bar: 50 μm. (Right) Quantification of VEGFR3 + lymphatics (n = 5 per group). c (Left) Representative immunoblotting images showing the protein levels of VEGFR3 and LYVE1 in the hearts from indicated groups. (Right) Quantification of immunoblotting results normalized to Actin and presented relative to the AAV-NC group (n = 5 per group). d Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis determining the mRNA expressions of pro-inflammatory genes in the hearts of mice that were injected with AAV-NC or AAV- Efnb2 at day 7 post-operation (n = 5 per group). e (Left) Fluorescence-activated cell sorter (FACS) analysis of CD45 + CD11b + Ly6G − F4/80 + Ly6C high macrophages in the murine hearts at day 7 post-MI. (Right) Quantification of CD45 + CD11b + Ly6G − F4/80 + Ly6C high macrophages as the percentage of CD45 + cells (n = 5 per group). f Representative immunofluorescence staining images showing the myocardium co-stained by CD68 (red), VEGFR3 (green), and DAPI (blue) in indicated groups. Scar bar: 20 μm. g Schematic diagram depicting the experimental strategy for Evans blue injection from cardiac apex to lymph nodes through the lymphatic system. h Representative heart images of Evans blue in the mediastinal lymph nodes and lymphatics. Scar bar: 1 mm. i (Left) Representative immunofluorescence staining images showing mediastinal lymph node co-stained by CD68 (red) and DAPI (blue) in indicated groups. Scar bar: 50 μm. (Right) Quantification of CD68 + macrophages as the percentage of cells in MLNs. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. c , d by one-way ANOVA with Tukey post-hoc test, b by unpaired Student’s test, and e and i by Mann–Whitney U test. MLN mediastinal lymphatic node, and LV lymphatic vessel

Article Snippet: The antibodies utilized were as follows: EphrinB2 (10 μg/ml;# AF496, R&D system), VEGFR3/FLT4 (2 μg/ml; #AF743, R&D system), LYVE-1 (1:100; #67538, Cell Signal Technology), CD31 (1:100; #ab9498, Abcam), CD68 (1:100; #25747, Proteintech), cTnT (1:100; #ab8295 Abcam), αSMA (1:500; #ab7817, Abcam), ISL1(1:200; #ab86501, Abcam), CDK5 (1:200; #10430-1-AP, Proteintech).

Techniques: Over Expression, Imaging, Fluorescence, Immunofluorescence, Staining, Injection, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, MANN-WHITNEY

Journal: Signal Transduction and Targeted Therapy

Article Title: EphrinB2-mediated CDK5/ISL1 pathway enhances cardiac lymphangiogenesis and alleviates ischemic injury by resolving post-MI inflammation

doi: 10.1038/s41392-024-02019-4

Figure Lengend Snippet: Lyve1 deficiency abrogates the anti-inflammatory effects of EphrinB2 post-MI. a Schematic diagram depicting the experimental strategy for EphrinB2 overexpression in Lyve1 −/− mice and their WT littermates. b Representative histological images from Lyve1 −/− mice and their WT littermates that were injected with AAV-NC or AAV- Efnb2 assessed by Masson Trichrome staining. Magnified views of black boxes are shown in the bottom lane. Scar bar: 1 mm. c Representative M-mode echocardiographic images showing the cardiac function of Lyve1 −/− mice and their WT littermates that were injected with AAV-NC or AAV- Efnb2 . The yellow lines indicate the endocardium of the anterior and posterior walls at mid-papillary muscle level. d – f Quantification of echocardiographic parameters in c (LVEF, LVIDs, and LVIDd, n = 6 per group). g Quantification of infarct size of myocardium in b (n = 5 per group). h Quantification of apoptotic cells as percentage of all cells in i . i Representative TUNEL staining images showing cell apoptosis in indicated groups. Scar bar: 50 μm. j Representative immunofluorescence staining images showing the myocardium co-stained by CD31 (red), VEGFR3 (green), and DAPI (blue) in indicated groups. Scar bar: 50 μm. k Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis determining the mRNA expressions of pro-inflammatory genes in the hearts in indicated groups at day 7 post-MI (n = 5 per group). l (Left) Fluorescence-activated cell sorter (FACS) analysis of CD45 + CD11b + Ly6G − F4/80 + Ly6C high macrophages in the murine hearts at day 7 post-MI. m Quantification of the density of VEGFR3 + lymphatics in j . n Quantification of CD45 + CD11b + Ly6G − F4/80 + Ly6C high macrophages as the percentage of CD45 + cells in l (n = 4 per group). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. d–h , k , and m by one-way ANOVA with Tukey post-hoc test, and n by Mann-Whitney U test. LVEF left ventricular ejection fraction, LVIDs left ventricular internal dimension during systole, LVIDd left ventricular internal dimension during diastole, DAPI 4’,6-diamidino-2-phenylindole, HE hematoxylin and eosin, and TUNEL terminal deoxynucleotidyltransferase-mediated dUTPbiotin nick end labeling

Article Snippet: The antibodies utilized were as follows: EphrinB2 (10 μg/ml;# AF496, R&D system), VEGFR3/FLT4 (2 μg/ml; #AF743, R&D system), LYVE-1 (1:100; #67538, Cell Signal Technology), CD31 (1:100; #ab9498, Abcam), CD68 (1:100; #25747, Proteintech), cTnT (1:100; #ab8295 Abcam), αSMA (1:500; #ab7817, Abcam), ISL1(1:200; #ab86501, Abcam), CDK5 (1:200; #10430-1-AP, Proteintech).

Techniques: Over Expression, Injection, Staining, TUNEL Assay, Immunofluorescence, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Fluorescence, MANN-WHITNEY, End Labeling

Journal: Signal Transduction and Targeted Therapy

Article Title: EphrinB2-mediated CDK5/ISL1 pathway enhances cardiac lymphangiogenesis and alleviates ischemic injury by resolving post-MI inflammation

doi: 10.1038/s41392-024-02019-4

Figure Lengend Snippet: EphrinB2 promoted lymphatic proliferation and migration via the upregulation and nuclear translocation of ISL1. a Schematic diagram depicting the experimental strategy for RNA sequencing. b Volcano plot showing significantly differentially expressed genes (DEGs) in lymphatic endothelial cells (LECs) transfected with Adv-NC or Adv- Efnb2 following RNA sequencing. Genes with log 2 fold-change ≥2.0 and adjusted P < 0.05 were considered DEGs. c Barplot showing significantly enriched gene ontology (GO) pathways. d Heat map showing DEGs involed in endothelial cell proliferation. e RT-qPCR analysis determining the mRNA expressions of genes in d from LECs transfected with Adv-NC and Adv- Efnb2 in hypoxic conditions (n = 6 per group). f (Top) Representative immunoblotting images showing ISL1 protein levels in LECs treated with Adv-NC or Adv- Efnb2 under hypoxia. (Bottom) Quantification of the immunoblotting results normalized to Actin (n = 5 per group). g Cell proliferation activity of LECs transfected with Adv-NC or Adv- Efnb2 and Adv-shScram or Adv-sh ISL1 under hypoxic conditions for indicated times (0, 6, 12, 24 hours) assessed by CCK-8 assay. *(yellow) P < 0.05, **(yellow) P < 0.01 for Adv- Efnb2 + Adv-shScram vs Adv-NC + Adv-shScram; # (red) P < 0.05, ## (red) P < 0.01 for Adv- Efnb2 +Adv-sh ISL1 vs Adv- Efnb2 + Adv-shScram. h (Left) Wound healing assay determing the effect of ISL1 knockdown under normoxic or hypoxic conditions for 24 hours. The white dashed lines indicate the terminals of the scratch. Scar bar: 200 μm. (Right) Quantification of results presented relative to normoxia+Adv-shScram group (n = 5 per group). i (Left) Representative immunoblotting images showing the nuclear and cytoplasmic extracts of ISL1 in LECs transfected with Adv-NC and Adv- Efnb2 under normoxic or hypoxic conditions. (Right) Quantification of the immunoblotting results normalized to Actin and presented relative to normoxia + Adv-NC group (n = 6 per group). j (Left) Representative immunofluorescence staining images showing LECs co-stained by ISL1 (green) and DAPI (blue) in indicated groups. Scar bar: 20 μm. (Right) Quantification of the immunofluorescence staining intensity in the LEC nuclei presented relative to the Adv-NC group (n = 5 per group). k Schematic illustration of conserved ISL1 consensus located at FLT4 promoter region across species. l Representative ChIP-seq track of ISL1 peaks across the FLT4 gene. The grey interval indicates the promoter region of FLT4 gene. (m) Chromatin immunoprecipitation (ChIP) assay showing the relative recruitment of ISL1 at FLT4 promoter region in indicated groups (n = 5 per group). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. e , f by unpaired Student’s test. h – j, and m by one-way ANOVA with Tukey post-hoc test, and g by two-way repeated measurement ANOVA with Bonferroni’s multiple comparisons test. BP biological process, MF molecular function, false discovery rate, and FC fodl change, and hg human genome

Article Snippet: The antibodies utilized were as follows: EphrinB2 (10 μg/ml;# AF496, R&D system), VEGFR3/FLT4 (2 μg/ml; #AF743, R&D system), LYVE-1 (1:100; #67538, Cell Signal Technology), CD31 (1:100; #ab9498, Abcam), CD68 (1:100; #25747, Proteintech), cTnT (1:100; #ab8295 Abcam), αSMA (1:500; #ab7817, Abcam), ISL1(1:200; #ab86501, Abcam), CDK5 (1:200; #10430-1-AP, Proteintech).

Techniques: Migration, Translocation Assay, RNA Sequencing Assay, Transfection, Quantitative RT-PCR, Western Blot, Activity Assay, CCK-8 Assay, Wound Healing Assay, Knockdown, Immunofluorescence, Staining, ChIP-sequencing, Chromatin Immunoprecipitation

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a . Representative FACS plots showing gating strategies for the isolation of ECs from Cdh5-mTnG reporter bone. b . Tile scan confocal images showing sections of 6, 22 or 55-week-old Cdh5-mTnG reporter bone co-stained for EMCN (red) and VEGFR3 (blue). The avascular growth plate (GP) and regions containing bmECs, mpECs and rECs are indicated. Small panels show higher magnifications of these areas. Arrowheads mark type R capillaries. Scale bars, 100μm (overview images) and 500μm (small panels). c . Heatmap showing the expression of selected marker genes for each EC subcluster (based on integrated scRNA-seq data for all age groups). d . Representative confocal images showing immunostaining for EMCN (red) and SOX11 (green, white arrowheads), marking pECs in the young but not adult metaphysis. e . CXADR (green) staining of bmECs in young, adult, and aging femur. Growth plate (GP) and epiphysis are indicated. f . MAdCAM1 (green, white arrowheads) expression in diaphyseal bmECs in young and adult femur. Scale bars, 50μm ( d ), 500μm ( e ), 100μm ( f ).

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Isolation, Staining, Expressing, Marker, Immunostaining

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a, b , Freshly isolated wild-type femurs at the age of 3, 6, 8 or 12 weeks (W), as indicated. Tile scan confocal images of EMCN + VEGFR3 − ECs (arrowheads) in sectioned femur at the indicated ages ( b ). Epiphysis and growth plate are labelled. Scale bars, 1000μm ( a ), 500μm ( b ). c . The UMAP plot (integrated scRNA-seq data of all age groups) showing Bmx expression in arterial endothelial cells (aECs), marked by arrowhead. Scheme of Bmx-CreERT2 genetic fate mapping. 4-hydroxy tamoxifen (4-OHT) administration (1mg/mouse) is indicated by black arrow and red arrows mark time points of analysis. d, e , High-resolution confocal images and tile scan overview images of fate-tracked Bmx-CreERT2 R26-mTmG (GFP, green) in femur at the indicated time points after 4-OHT administration. Insets in ( e ) show metaphysis (i) and diaphysis (ii), respectively. White arrowheads mark CAV1 + EMCN − aECs and yellow arrowheads GFP + CAV1 + EMCN − aECs. Green arrowheads indicate EMCN + CAV1 + rECs, which are devoid of GFP signal. Scale bars, 100μm ( d ) and 500μm or 100μm ( e ).

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Isolation, Expressing

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , UMAP plots showing colour-coded subclusters in juvenile and adult bone ECs. Dashed black line encompasses rEC clusters. Red arrowhead indicates expansion of type R ECs in adult. b , Bar plots showing colour-coded subclusters in juvenile and adult bone ECs. Relative representation (in %) of rECs and aECs is indicated. c , High-magnification images of femurs immunostained for EMCN (red), VEGFR3 (blue) and CAV1 (green) showing the emergence of EMCN + VEGFR3 − CAV1 + (yellow) rECs (white arrowheads) around trabecular bone (TB). d , Two-photon microscopic image of immunostained EMCN + VEGFR3 − rECs (white arrowheads) around TB visualized by second-harmonic generation. e , Representative confocal images of 3-, 6-, 8- and 12-week-old femurs immunostained for EMCN (red) and VEGFR3 (green). White arrowheads indicate increasing age-dependent abundance of EMCN + VEGFR3 − rECs around TB. f , Quantitation of rECs (EMCN + VEGFR3 − area) showing the age-dependent increase in the 3-, 6-, 8- and 12-week-old TB relative to samples from 3 weeks. n = 3 mice per group. Mean ± s.e.m. P values, one-way analysis of variance (ANOVA). g , Schematic representation of type R capillary expansion in postnatal and adult long bone. Data in a show individual samples from different age groups, whereas b is based on integrated scRNA-seq data.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Quantitation Assay

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , Perfusion of EMCN + VEGFR3 − type R capillaries (arrowheads) near TB demonstrated by injected 2,000 kDa TRITC dextran (yellow) in 12-week-old wild-type femur. b , Representative confocal images of 12-week-old Efnb2-H2B-GFP (green) femur section co-stained for EMCN (red) and VEGFR3 (blue). Efnb2 + EMCN + rECs (white arrowheads) are connected to Efnb2 + EMCN − arterioles and arteries (green arrowheads). c , d , Type III collagen (COL3A1) ( c ) and type IV collagen (COL4A1) ( d ) are tightly associated with EMCN + VEGFR3 − type R capillaries (arrowheads) in 12-week-old wild-type femur, whereas the surrounding sinusoidal vessels show a loose reticular fibre network. e , High-magnification images showing filopodia (arrowheads) extending from EMCN + VEGFR3 − rECs around 12-week-old TB. f , Proliferating rECs (white arrowheads) near 6-week-old TB. Cdh5-mTnG reporter (nGFP, red) shows EC nuclei co-stained with KI-67 (green). g , Scheme of genetic fate-mapping strategy. 4-OHT administration (1 mg per mouse) is indicated by black arrow and red arrows mark time points of analysis. h , i , Tile-scan confocal images ( h ) and higher magnification of insets ( i ) showing fate-tracked Flt4-CreERT2 R26-mTmG (GFP, green)-labelled ECs in femur at 8 weeks (48 h after Cre induction) and 12 weeks (4 weeks after Cre induction). White arrowheads indicate EMCN + CAV1 + rECs and yellow arrowheads mark GFP + traced rECs. j , Quantitative analysis of GFP + rECs (EMCN + CAV1 + FLT4-GFP + ) at 48 h and 4 weeks post-induction, respectively. n = 3 mice per group. Mean ± s.e.m. P values were obtained using an unpaired two-tailed t -test. k , Schematic illustration of genetic fate mapping of rECs in Flt4-CreERT2 R26-mTmG femur.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Injection, Staining, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , b , High-resolution confocal images showing EMCN + VEGFR3 − rECs (arrowheads) around TB in relation to RUNX2 + osteoprogenitors (green) ( a ) and ATP6V1B1/B2 + osteoclasts (green) ( b ). c , d , Maximum-intensity projection showing Sp7-mCherry + osteoblasts (yellow) and ATP6V1B1/B2 + osteoclasts (green) in in relation to EMCN + VEGFR3 − rECs around 6-week-old femoral TB ( c ). Single xy plane ( d ). Green arrowheads mark rECs near osteoclasts and yellow arrowheads near osteoblasts. e , f , Distribution of EMCN + VEGFR3 − type R vessels in relation to Sp7-mCherry + osteoblasts (yellow arrowheads) and ATP6V1B1/B2 + osteoclasts (green arrowheads) around 6-week-old and 12-week-old TB ( e ). Isolated xy plane ( f ). g , Confocal images showing VEGFR3 − VEGFR2 + ECs (white arrowheads) around TB in young and aged patient samples. VEGFR3 (yellow), VEGFR2 (red) and nuclei (DAPI, blue). DAPI, 4,6-diamidino-2-phenylindole.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Isolation

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , High-magnification two-photon microscopy images of compact bone immunostained for EMCN (red) and CAV1 (blue) together with second-harmonic generation (SHG, white). Type R capillaries (arrowheads) and trabecular bone (TB) are indicated. Scale bars, 50μm. b, c , Maximum-intensity projections of sections from 3, 6, 8 or 12-week-old femur immunostained for EMCN (red) and OSTERIX (green) ( b ) or EMCN (red), VEGFR3 (blue) and ATP6V1B1/B2 (green) ( c ). Arrowheads mark type R capillaries near trabecular bone (TB). Scale bars, 50μm. Diagram on the right depicts association of rECs with osteoblast-lineage cells and osteoclasts. d . Injection scheme and time points of imaging after labelling of wild-type mice with Calcein Green (CG) and Alizarin Red (AZ). Tile scan imaging of sectioned femur from 8-week-old wild-type after double labelling. Insets depict active resorption in the metaphysis with CG − AR high labelling (top row), active osteogenesis (CG high AR high ) in compact bone (middle row), and active remodelling (CG low AR high ) of trabecular bone (bottom row). n=3 mice. Scale bars, 500μm (overview images) and 50μm (insets).

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Microscopy, Injection, Imaging

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a, b . High-resolution confocal images showing VEGFR3 (yellow) and VEGFR2 (red) immunostaining together with DAPI (blue). VEGFR3 − VEGFR2 + capillaries (white arrowheads) around trabecular bone (TB) and nearby VEGFR3 + VEGFR2 + vessels (yellow arrowheads) in samples from young ( a ) and aged patients ( b ) are indicated. Scale bars, 200μm. c . Tile scan confocal images of EMCN + VEGFR3 − ECs in 12-week-old female and male murine femur. Trabecular bone (TB) is indicated. Scale bars, 500μm. d . Quantitation of EMCN + VEGFR3 − vessel density in 12-week-old female and male femur (n =3 mice per group). Mean ± SEM. P values, unpaired two-tailed t-test. e . EMCN (red) and VEGFR3 (blue) immunostaining showing the presence of VEGFR3 − EMCN + vessels (white arrowheads) around trabecular bone (TB) in female and male femur. Scale bars, 100μm.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Immunostaining, Quantitation Assay, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , UMAP plot (integrated scRNA-seq dataset of all age groups) showing Dach1 expression in rECs (arrow). Colour bar illustrates the expression level. b , Representative confocal images of DACH1 immunostaining (green) in 6-week-old Cdh5-mTnG (red) reporter femur. DACH1 + rECs near TB (green) are marked by white arrowheads. c , Scheme of tamoxifen-induced EC-specific Dach1 inactivation. d , Tile-scan confocal images of EMCN + VEGFR3 − CAV1 + rECs (white arrowheads) and EMCN − VEGFR3 − CAV1 + aECs (yellow arrowheads) in 12-week-old Dach1 iΔEC loss-of-function and littermate control femur. Growth plate (GP) is indicated. e , Quantitation of EMCN + vessel density, EMCN + VEGFR3 − vessel density, and number of CAV1 + arteries in 12-week-old Dach1 iΔEC and control femur ( n = 3–4 female mice per group). Mean ± s.e.m. P values, unpaired two-tailed t -test. Emcn + vessel density plotted with Welch’s correction. f , High-magnification images of metaphysis near GP (left) and of EMCN + VEGFR3 − vessels (white arrowheads; right) around TB in 12-week-old male Dach1 iΔEC and control femurs. White dashed lines in d and f indicate type H area. g , Representative 3D reconstruction of µCT measurements of 12-week-old Dach1 iΔEC and control femoral bone. Dashed yellow lines indicate area analysed. h , Quantitation shows relative bone volume, represented as bone volume/tissue volume (BV/TV) and trabecular thickness (per mm) ( n = 3–4 female mice per group and n = 3 male mice per group). Mean ± s.e.m. P values were plotted using an unpaired two-tailed t -test.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Expressing, Immunostaining, Control, Quantitation Assay, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , Schematic representation of tamoxifen-inducible Dach1 overexpression in Aplnr + ECs. b , Tile-scan confocal images showing expansion of CAV1 (green, white arrowheads) immunostained arteries and type R capillaries in the Dach1 gain-of-function ( Dach1 OE ) femur relative to littermate control at postnatal day 30 (P30). c , High-magnification confocal images of EMCN (red) and VEGFR3 (green) immunostained femurs showing the expansion of type R capillaries near TB in Dach1 OE mutants relative to littermate control. White arrowheads indicate type R capillaries. d , Quantitation of arteries/arterioles and CAV1 + area in control and Dach1 OE mutants. n = 3 mice per group. Mean ± s.e.m. P values were obtained by unpaired two-tailed t -test. e , Representative three-dimensional (3D) reconstruction of µCT measurements of P30 Dach1 OE and control femoral TB. f , Quantitation of parameters: bone volume/tissue volume (BV/TV), trabecular number represented by number of trabeculae per millimetre, connectivity density (connectivity density per cubic millimetre) and cortical thickness (mm). n = 3 mice per group. Mean ± s.e.m. P values were obtained by an unpaired two-tailed t -test. g , Schematic overview of scRNA-seq workflow for non-haematopoietic cells from Dach1 OE and littermate control long bone. h , i , UMAP plots of BMSCs ( n = 10,315) with colour-coded subclusters, namely osteoblasts (OBs), osteocytes (OCYs), septoclasts (SCs), proliferating BMSCs (pBMSCs), mpMSCs and dpMSCs (dpMSCs1 and dpMSCs2) ( h ). UMAP distribution of Dach1 OE and control BMSCs, as indicated by colour ( i ). j , Bar plots showing proportion of cells in Dach1 OE and control BMSC subclusters. k , Heatmap showing the top three marker genes for each BMSC subcluster. l , Heatmap illustrating differentially expressed genes in Dach1 OE and control BMSCs related to hypoxia, growth factors, adipogenic and osteogenic transcription factors, and regulators of osteogenesis. Texts in red are the areas of interest. Colour bars in k and l illustrate the expression level (log 2 fold change). Data in h – l are derived from an integrated scRNA-seq dataset of two conditions.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Over Expression, Control, Quantitation Assay, Two Tailed Test, Marker, Expressing, Derivative Assay

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a, b . Confocal images of femur sections immunostained for EMCN (red) together with OSTERIX (OSX, green) ( a ) or ATP6V1B1/B2 (green) ( b ). Scale bars, 100μm. c . Quantitation of OSX + osteoblast-lineage cells and ATP6V1B1/B2 + osteoclasts in Dach1 OE and control femur (n = 3 in each group). Mean ± SEM. P values, unpaired two-tailed test with Welch’s correction. d-f . Injection scheme and time points of imaging after labelling of Dach1 OE and control with Calcein Green (CG) and Alizarin Red (AZ) ( d ) and tile scan imaging of sectioned femur from P30 Dach1 OE and control after double labelling ( e ). Scale bars, 500μm. Bottom panels in ( e ) show stained trabecular bone. Scale bars, 20μm. f . Quantitation showing extent of bone surface actively mineralizing as mineralizing surface/bone surface (MS/BS) in percentage, mineral apposition rate (MAR) as per micrometre/day, and bone formation rate (BFR) (n=3 mice per group). Mean ± SEM. P values, unpaired two-tailed t-test with Welch’s correction for MS/BS, MAR-trabecular bone and BFR. g . Maximum-intensity projections showing prominent HIF1α + immunostaining (yellow arrowheads) in 6-week-old Sp7-mCherry + trabecular osteoblasts, whereas HIF1α + is strongly decreased after emergence of EMCN + VEGFR3 − type R vessels at 12 weeks. Scale bars, 50 μm. h . Maximum-intensity projections of 12-week-old femurs showing immunostaining for hypoxia-related markers in relation to EMCN + VEGFR3 − type R vessels. Shown are immunostaining for Glucose-6-Phosphate Isomerase (GPI), 5′-nucleotidase/CD73 and Haem Oxygenase-1 (HMOX1)-expressing macrophages (yellow arrowheads), all of which are elevated near juvenile trabecular bone relative to the equivalent region in adult. White arrowheads mark EMCN + VEGFR3 − type R vessels. i, j . UMAP plots of bone ECs (n=18383) with colour-coded subclusters ( f ), namely sinusoidal bmECs, metaphyseal mpECs, arterial aECs, remodelling rECs, and proliferating pECs. UMAP distribution of Dach1 OE and control ECs, as indicated by colour ( g ). k . Bar plots showing proportion of cells in Dach1 OE and control samples. l . UMAP plots comparing Dach1 expression in Dach1 OE (bottom) and control (top) EC subclusters. m . Heatmap of differentially expressed genes between EC subclusters. n . Heatmap of differentially expressed genes related to hypoxia and, tissue oxygenation in Dach1 OE and control bone ECs. o . Confocal images showing reduced HIF1α immunostaining (white arrowheads) in Dach1 OE metaphysis and around trabecular bone relative to littermate control. Scale bars, 50μm. Results in panels i, j, m , and n show the integrated mutant and control scRNA-seq data, whereas panels k and l show separated samples derived from the integrated data.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Quantitation Assay, Control, Two Tailed Test, Injection, Imaging, Staining, Immunostaining, Expressing, Mutagenesis, Derivative Assay

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a . Violin plots showing selected genes related to reduction of bmEC markers and increased arterialization in Dach1 OE and control. b . Heatmap of differentially expressed genes related to Notch signalling in Dach1 OE and control bone ECs. c . Representative confocal images showing increased Delta-like 4 (DLL4) (green, white arrowheads) expression in vessels around Dach1 OE trabecular bone (TB). Scale bars, 100μm. d . Scheme of tamoxifen-induced (TMX) EC-specific Dll4 inactivation with Cdh5-CreERT2 line. e, f , Tile scan confocal images of EMCN + VEGFR3 − ECs (white arrowheads) (e) and CAV1 + aECs (green arrowheads) (f) in 12-week-old Dll4 iΔEC loss-of-function and control femur. Trabecular bone (TB) is indicated. Scale bars, 500μm. g , Quantitation of EMCN + VEGFR3 − vessel density (left), and number of CAV1 + arteries (right) in 12-week-old Dll4 iΔEC and control femur (n =3 female mice per group). Mean ± SEM. P values, unpaired two-tailed t-test. h . Heatmap showing differentially expressed transcripts for secreted factors in bone EC subpopulations. i, j , Representative images (i) and quantitation (j) of calcified nodules in human mesenchymal stem cells (HMSC) cultured in osteogenic differentiation medium (ODM) supplemented with 100ng/ml Complement C1q and Tumour Necrosis Factor-Related Protein 9 (CTRP9), Neurotrophin 3 (NTF3), Platelet-Derived Growth Factor D (PDGFD), or Semaphorin 7A (SEMA7A) (n=3 independent experiments for each group). Scale bars, 100μm. Graph shows quantitation of absorbance at 405nm. Mean ± SEM. P values, ordinary one-way ANOVA and plotted with Dunnett’s multiple comparisons test. k, l , Representative images (k) and quantitation (m) of multinucleated TRAP+ osteoclasts generated from bone marrow-derived monocytes/macrophages treated with RANKL in osteoclast medium (OCM) supplemented with 100ng/ml CTRP9, NTF3, PDGFD, or SEMA7A n=8 (4 independent experiments) for each group. Scale bars, 500μm. Graph shows quantitation of TRAP + multinucleated cells between OCM and treated conditions. Mean ± SEM. P values, ordinary one-way ANOVA and plotted with Dunnett’s multiple comparisons test. Panels a and b show separated conditions derived from the integrated mutant and control data, whereas panel h is based on the integrated scRNA-seq data.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Control, Expressing, Quantitation Assay, Two Tailed Test, Cell Culture, Derivative Assay, Generated, Mutagenesis

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , UMAP visualization of bone ECs from 75-week-old bone with colour-coded subclusters. b , Bar plot showing the proportion of cells from each EC subcluster in adult and aged bone. Percentage (%) differences are indicated for rECs and aECs. Data are representative of an individual sample ( a ) and integrated scRNA-seq data from all age groups ( b ). c , d , High-magnification confocal images of TB ( c ) and compact bone (CB) ( d ) immunostained for EMCN (red), VEGFR3 (blue) RUNX2 (yellow arrowheads, marking osteoprogenitors) and ATP6V1B1B2 (green arrowheads, marking osteoclasts). e , High-magnification two-photon microscopy images of CB immunostained for EMCN (red) and VEGFR3 (blue) together with second-harmonic generation (white) showing the changes during ageing. f , Confocal tile-scan images showing EMCN + VEGFR3 − rECs in untreated 12-week-old and 75-week-old mice femur and in response to treatment with alendronate (ALN) or PTH, as indicated. Note expansion of EMCN + VEGFR3 − vessels in CB in response to ageing or treatments (arrowheads). g , Representative confocal images showing EMCN + VEGFR3 − rECs and OSTERIX (green) immunostaining in 75-week-old control, ALN-treated or PTH-treated CB (white arrowheads, marking type R capillaries during ageing). White dashed lines in d – g indicate compact bone (CB) area. h , i , Quantitation of EMCN + CAV1 + vessel density across age groups ( h ), EMCN + VEGFR3 − vessel density in 75-week-old control, ALN-treated or PTH-treated groups, and number of OSTERIX + cells in CB after ALN and PTH treatment compared with respective controls ( i ). n = 3 mice per group. Mean ± s.e.m. P values were obtained using ordinary ANOVA.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Microscopy, Immunostaining, Control, Quantitation Assay

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , Scheme for the treatment of adult mice with parathyroid hormone (PTH) or Alendronate. Black arrows indicate onset of treatment, red arrows time points of imaging. b , Confocal images of OSTEOPONTIN (blue) immunostaining of the metaphysis near the growth plate (GP) together with EMCN (red), CAV1 (green) and DAPI (white) to show the response to treatments. Scale bars, 100μm. c , UMAP visualization of sorted bone ECs from control and treatment groups with colour-coded subclusters. d , Bar plots showing the proportions of EC subpopulations in 12-week-old mice. Differences in percentage of rECs and aECs compared to control are indicated. Panels c and d show the individual samples from an integrated scRNA-seq dataset of three conditions. e, f , Confocal images showing EMCN + VEGFR3 − CAV1 + rECs (white arrowheads) around trabecular bone (TB) ( e ) and association of ATP6V1B1/B2 + (green) osteoclasts with EMCN + VEGFR3 − type R capillaries ( f ) in 12-week-old control and indicated treatment conditions. Scale bars, 50μm. g , Effect of treatments on EMCN + VEGFR3 − rECs (white arrowheads) and RUNX2 + (green) osteoprogenitors around trabecular bone (TB). Scale bars, 100μm.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Imaging, Immunostaining, Control

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a . Representative FACS plots showing gating strategies for the isolation of ECs from Cdh5-mTnG reporter bone. b . Tile scan confocal images showing sections of 6, 22 or 55-week-old Cdh5-mTnG reporter bone co-stained for EMCN (red) and VEGFR3 (blue). The avascular growth plate (GP) and regions containing bmECs, mpECs and rECs are indicated. Small panels show higher magnifications of these areas. Arrowheads mark type R capillaries. Scale bars, 100μm (overview images) and 500μm (small panels). c . Heatmap showing the expression of selected marker genes for each EC subcluster (based on integrated scRNA-seq data for all age groups). d . Representative confocal images showing immunostaining for EMCN (red) and SOX11 (green, white arrowheads), marking pECs in the young but not adult metaphysis. e . CXADR (green) staining of bmECs in young, adult, and aging femur. Growth plate (GP) and epiphysis are indicated. f . MAdCAM1 (green, white arrowheads) expression in diaphyseal bmECs in young and adult femur. Scale bars, 50μm ( d ), 500μm ( e ), 100μm ( f ).

Article Snippet: Primary antibodies were: rat monoclonal anti-endomucin (V.7C7) (Santa Cruz, sc-65495, 1:100 dilution), goat polyclonal anti-Vegfr3 (R&D Systems, AF743, 1:50 dilution), rabbit polyclonal anti-Cav1 (Cell Signalling, 3238, 1:50 dilution), chicken polyclonal anti-GFP (Abcam, ab13970, 1:200 dilution), rabbit polyclonal anti-OSTERIX (Abcam, ab22552, 1:300 dilution), rabbit monoclonal anti-Runx2 (Abcam, ab192256, 1:200 dilution), rabbit monoclonal anti-Ki-67 (Cell Signalling, 12202, 1:200 dilution), rabbit monoclonal anti-vATPase (Abcam, ab200839, 1:200 dilution), rabbit polyclonal anti-collagen IV (AbD Serotec, 2150-1470, 1:100 dilution), rabbit polyclonal anti-collagen IIIA1 (Abcam, ab7778, 1:100 dilution), goat polyclonal anti-Hif1a (R&D Systems, AF1935, 1:100 dilution), rabbit polyclonal anti-Hif1a (Santa Cruz, sc-10790, 1:100 dilution), rabbit monoclonal anti-GPI (Cell Signalling, 94068, 1:200 dilution), rabbit monoclonal anti-CD73 (Cell Signalling, 13160, 1:200 dilution), rabbit monoclonal anti-Hmox1 (Cell Signalling, 86806, 1:200 dilution), rabbit polyclonal anti-Dach1 (Proteintech, 10914-1-AP, 1:200 dilution), rabbit polyclonal anti-NG2 (Millipore, AB5320, 1:200 dilution), goat polyclonal anti-Cxadr (R&D Systems, AF2654, 1:50 dilution), rabbit monoclonal anti-Sox11 (Abcam, ab134107, 1:800 dilution), rat monoclonal anti-Madcam1 (Abcam, ab80680, 1:100 dilution), rabbit polyclonal anti-Fabp4 (Abcam, ab13979, 1:100 dilution), mouse monoclonal anti-Mct4 (Santa Cruz, AF647, 1:100 dilution), rabbit monoclonal anti-Glut1 (Cell Signalling, 12939, 1:100 dilution), goat polyclonal anti-Dll4 (R&D Systems, AF1389, 1:50 dilution) and chicken polyclonal anti-Mct1 (Millipore, AB1286-I, 1:100 dilution).

Techniques: Isolation, Staining, Expressing, Marker, Immunostaining

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a, b , Freshly isolated wild-type femurs at the age of 3, 6, 8 or 12 weeks (W), as indicated. Tile scan confocal images of EMCN + VEGFR3 − ECs (arrowheads) in sectioned femur at the indicated ages ( b ). Epiphysis and growth plate are labelled. Scale bars, 1000μm ( a ), 500μm ( b ). c . The UMAP plot (integrated scRNA-seq data of all age groups) showing Bmx expression in arterial endothelial cells (aECs), marked by arrowhead. Scheme of Bmx-CreERT2 genetic fate mapping. 4-hydroxy tamoxifen (4-OHT) administration (1mg/mouse) is indicated by black arrow and red arrows mark time points of analysis. d, e , High-resolution confocal images and tile scan overview images of fate-tracked Bmx-CreERT2 R26-mTmG (GFP, green) in femur at the indicated time points after 4-OHT administration. Insets in ( e ) show metaphysis (i) and diaphysis (ii), respectively. White arrowheads mark CAV1 + EMCN − aECs and yellow arrowheads GFP + CAV1 + EMCN − aECs. Green arrowheads indicate EMCN + CAV1 + rECs, which are devoid of GFP signal. Scale bars, 100μm ( d ) and 500μm or 100μm ( e ).

Article Snippet: Primary antibodies were: rat monoclonal anti-endomucin (V.7C7) (Santa Cruz, sc-65495, 1:100 dilution), goat polyclonal anti-Vegfr3 (R&D Systems, AF743, 1:50 dilution), rabbit polyclonal anti-Cav1 (Cell Signalling, 3238, 1:50 dilution), chicken polyclonal anti-GFP (Abcam, ab13970, 1:200 dilution), rabbit polyclonal anti-OSTERIX (Abcam, ab22552, 1:300 dilution), rabbit monoclonal anti-Runx2 (Abcam, ab192256, 1:200 dilution), rabbit monoclonal anti-Ki-67 (Cell Signalling, 12202, 1:200 dilution), rabbit monoclonal anti-vATPase (Abcam, ab200839, 1:200 dilution), rabbit polyclonal anti-collagen IV (AbD Serotec, 2150-1470, 1:100 dilution), rabbit polyclonal anti-collagen IIIA1 (Abcam, ab7778, 1:100 dilution), goat polyclonal anti-Hif1a (R&D Systems, AF1935, 1:100 dilution), rabbit polyclonal anti-Hif1a (Santa Cruz, sc-10790, 1:100 dilution), rabbit monoclonal anti-GPI (Cell Signalling, 94068, 1:200 dilution), rabbit monoclonal anti-CD73 (Cell Signalling, 13160, 1:200 dilution), rabbit monoclonal anti-Hmox1 (Cell Signalling, 86806, 1:200 dilution), rabbit polyclonal anti-Dach1 (Proteintech, 10914-1-AP, 1:200 dilution), rabbit polyclonal anti-NG2 (Millipore, AB5320, 1:200 dilution), goat polyclonal anti-Cxadr (R&D Systems, AF2654, 1:50 dilution), rabbit monoclonal anti-Sox11 (Abcam, ab134107, 1:800 dilution), rat monoclonal anti-Madcam1 (Abcam, ab80680, 1:100 dilution), rabbit polyclonal anti-Fabp4 (Abcam, ab13979, 1:100 dilution), mouse monoclonal anti-Mct4 (Santa Cruz, AF647, 1:100 dilution), rabbit monoclonal anti-Glut1 (Cell Signalling, 12939, 1:100 dilution), goat polyclonal anti-Dll4 (R&D Systems, AF1389, 1:50 dilution) and chicken polyclonal anti-Mct1 (Millipore, AB1286-I, 1:100 dilution).

Techniques: Isolation, Expressing

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , UMAP plots showing colour-coded subclusters in juvenile and adult bone ECs. Dashed black line encompasses rEC clusters. Red arrowhead indicates expansion of type R ECs in adult. b , Bar plots showing colour-coded subclusters in juvenile and adult bone ECs. Relative representation (in %) of rECs and aECs is indicated. c , High-magnification images of femurs immunostained for EMCN (red), VEGFR3 (blue) and CAV1 (green) showing the emergence of EMCN + VEGFR3 − CAV1 + (yellow) rECs (white arrowheads) around trabecular bone (TB). d , Two-photon microscopic image of immunostained EMCN + VEGFR3 − rECs (white arrowheads) around TB visualized by second-harmonic generation. e , Representative confocal images of 3-, 6-, 8- and 12-week-old femurs immunostained for EMCN (red) and VEGFR3 (green). White arrowheads indicate increasing age-dependent abundance of EMCN + VEGFR3 − rECs around TB. f , Quantitation of rECs (EMCN + VEGFR3 − area) showing the age-dependent increase in the 3-, 6-, 8- and 12-week-old TB relative to samples from 3 weeks. n = 3 mice per group. Mean ± s.e.m. P values, one-way analysis of variance (ANOVA). g , Schematic representation of type R capillary expansion in postnatal and adult long bone. Data in a show individual samples from different age groups, whereas b is based on integrated scRNA-seq data.

Article Snippet: Primary antibodies were: rat monoclonal anti-endomucin (V.7C7) (Santa Cruz, sc-65495, 1:100 dilution), goat polyclonal anti-Vegfr3 (R&D Systems, AF743, 1:50 dilution), rabbit polyclonal anti-Cav1 (Cell Signalling, 3238, 1:50 dilution), chicken polyclonal anti-GFP (Abcam, ab13970, 1:200 dilution), rabbit polyclonal anti-OSTERIX (Abcam, ab22552, 1:300 dilution), rabbit monoclonal anti-Runx2 (Abcam, ab192256, 1:200 dilution), rabbit monoclonal anti-Ki-67 (Cell Signalling, 12202, 1:200 dilution), rabbit monoclonal anti-vATPase (Abcam, ab200839, 1:200 dilution), rabbit polyclonal anti-collagen IV (AbD Serotec, 2150-1470, 1:100 dilution), rabbit polyclonal anti-collagen IIIA1 (Abcam, ab7778, 1:100 dilution), goat polyclonal anti-Hif1a (R&D Systems, AF1935, 1:100 dilution), rabbit polyclonal anti-Hif1a (Santa Cruz, sc-10790, 1:100 dilution), rabbit monoclonal anti-GPI (Cell Signalling, 94068, 1:200 dilution), rabbit monoclonal anti-CD73 (Cell Signalling, 13160, 1:200 dilution), rabbit monoclonal anti-Hmox1 (Cell Signalling, 86806, 1:200 dilution), rabbit polyclonal anti-Dach1 (Proteintech, 10914-1-AP, 1:200 dilution), rabbit polyclonal anti-NG2 (Millipore, AB5320, 1:200 dilution), goat polyclonal anti-Cxadr (R&D Systems, AF2654, 1:50 dilution), rabbit monoclonal anti-Sox11 (Abcam, ab134107, 1:800 dilution), rat monoclonal anti-Madcam1 (Abcam, ab80680, 1:100 dilution), rabbit polyclonal anti-Fabp4 (Abcam, ab13979, 1:100 dilution), mouse monoclonal anti-Mct4 (Santa Cruz, AF647, 1:100 dilution), rabbit monoclonal anti-Glut1 (Cell Signalling, 12939, 1:100 dilution), goat polyclonal anti-Dll4 (R&D Systems, AF1389, 1:50 dilution) and chicken polyclonal anti-Mct1 (Millipore, AB1286-I, 1:100 dilution).

Techniques: Quantitation Assay

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , Perfusion of EMCN + VEGFR3 − type R capillaries (arrowheads) near TB demonstrated by injected 2,000 kDa TRITC dextran (yellow) in 12-week-old wild-type femur. b , Representative confocal images of 12-week-old Efnb2-H2B-GFP (green) femur section co-stained for EMCN (red) and VEGFR3 (blue). Efnb2 + EMCN + rECs (white arrowheads) are connected to Efnb2 + EMCN − arterioles and arteries (green arrowheads). c , d , Type III collagen (COL3A1) ( c ) and type IV collagen (COL4A1) ( d ) are tightly associated with EMCN + VEGFR3 − type R capillaries (arrowheads) in 12-week-old wild-type femur, whereas the surrounding sinusoidal vessels show a loose reticular fibre network. e , High-magnification images showing filopodia (arrowheads) extending from EMCN + VEGFR3 − rECs around 12-week-old TB. f , Proliferating rECs (white arrowheads) near 6-week-old TB. Cdh5-mTnG reporter (nGFP, red) shows EC nuclei co-stained with KI-67 (green). g , Scheme of genetic fate-mapping strategy. 4-OHT administration (1 mg per mouse) is indicated by black arrow and red arrows mark time points of analysis. h , i , Tile-scan confocal images ( h ) and higher magnification of insets ( i ) showing fate-tracked Flt4-CreERT2 R26-mTmG (GFP, green)-labelled ECs in femur at 8 weeks (48 h after Cre induction) and 12 weeks (4 weeks after Cre induction). White arrowheads indicate EMCN + CAV1 + rECs and yellow arrowheads mark GFP + traced rECs. j , Quantitative analysis of GFP + rECs (EMCN + CAV1 + FLT4-GFP + ) at 48 h and 4 weeks post-induction, respectively. n = 3 mice per group. Mean ± s.e.m. P values were obtained using an unpaired two-tailed t -test. k , Schematic illustration of genetic fate mapping of rECs in Flt4-CreERT2 R26-mTmG femur.

Article Snippet: Primary antibodies were: rat monoclonal anti-endomucin (V.7C7) (Santa Cruz, sc-65495, 1:100 dilution), goat polyclonal anti-Vegfr3 (R&D Systems, AF743, 1:50 dilution), rabbit polyclonal anti-Cav1 (Cell Signalling, 3238, 1:50 dilution), chicken polyclonal anti-GFP (Abcam, ab13970, 1:200 dilution), rabbit polyclonal anti-OSTERIX (Abcam, ab22552, 1:300 dilution), rabbit monoclonal anti-Runx2 (Abcam, ab192256, 1:200 dilution), rabbit monoclonal anti-Ki-67 (Cell Signalling, 12202, 1:200 dilution), rabbit monoclonal anti-vATPase (Abcam, ab200839, 1:200 dilution), rabbit polyclonal anti-collagen IV (AbD Serotec, 2150-1470, 1:100 dilution), rabbit polyclonal anti-collagen IIIA1 (Abcam, ab7778, 1:100 dilution), goat polyclonal anti-Hif1a (R&D Systems, AF1935, 1:100 dilution), rabbit polyclonal anti-Hif1a (Santa Cruz, sc-10790, 1:100 dilution), rabbit monoclonal anti-GPI (Cell Signalling, 94068, 1:200 dilution), rabbit monoclonal anti-CD73 (Cell Signalling, 13160, 1:200 dilution), rabbit monoclonal anti-Hmox1 (Cell Signalling, 86806, 1:200 dilution), rabbit polyclonal anti-Dach1 (Proteintech, 10914-1-AP, 1:200 dilution), rabbit polyclonal anti-NG2 (Millipore, AB5320, 1:200 dilution), goat polyclonal anti-Cxadr (R&D Systems, AF2654, 1:50 dilution), rabbit monoclonal anti-Sox11 (Abcam, ab134107, 1:800 dilution), rat monoclonal anti-Madcam1 (Abcam, ab80680, 1:100 dilution), rabbit polyclonal anti-Fabp4 (Abcam, ab13979, 1:100 dilution), mouse monoclonal anti-Mct4 (Santa Cruz, AF647, 1:100 dilution), rabbit monoclonal anti-Glut1 (Cell Signalling, 12939, 1:100 dilution), goat polyclonal anti-Dll4 (R&D Systems, AF1389, 1:50 dilution) and chicken polyclonal anti-Mct1 (Millipore, AB1286-I, 1:100 dilution).

Techniques: Injection, Staining, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , b , High-resolution confocal images showing EMCN + VEGFR3 − rECs (arrowheads) around TB in relation to RUNX2 + osteoprogenitors (green) ( a ) and ATP6V1B1/B2 + osteoclasts (green) ( b ). c , d , Maximum-intensity projection showing Sp7-mCherry + osteoblasts (yellow) and ATP6V1B1/B2 + osteoclasts (green) in in relation to EMCN + VEGFR3 − rECs around 6-week-old femoral TB ( c ). Single xy plane ( d ). Green arrowheads mark rECs near osteoclasts and yellow arrowheads near osteoblasts. e , f , Distribution of EMCN + VEGFR3 − type R vessels in relation to Sp7-mCherry + osteoblasts (yellow arrowheads) and ATP6V1B1/B2 + osteoclasts (green arrowheads) around 6-week-old and 12-week-old TB ( e ). Isolated xy plane ( f ). g , Confocal images showing VEGFR3 − VEGFR2 + ECs (white arrowheads) around TB in young and aged patient samples. VEGFR3 (yellow), VEGFR2 (red) and nuclei (DAPI, blue). DAPI, 4,6-diamidino-2-phenylindole.

Article Snippet: Primary antibodies were: rat monoclonal anti-endomucin (V.7C7) (Santa Cruz, sc-65495, 1:100 dilution), goat polyclonal anti-Vegfr3 (R&D Systems, AF743, 1:50 dilution), rabbit polyclonal anti-Cav1 (Cell Signalling, 3238, 1:50 dilution), chicken polyclonal anti-GFP (Abcam, ab13970, 1:200 dilution), rabbit polyclonal anti-OSTERIX (Abcam, ab22552, 1:300 dilution), rabbit monoclonal anti-Runx2 (Abcam, ab192256, 1:200 dilution), rabbit monoclonal anti-Ki-67 (Cell Signalling, 12202, 1:200 dilution), rabbit monoclonal anti-vATPase (Abcam, ab200839, 1:200 dilution), rabbit polyclonal anti-collagen IV (AbD Serotec, 2150-1470, 1:100 dilution), rabbit polyclonal anti-collagen IIIA1 (Abcam, ab7778, 1:100 dilution), goat polyclonal anti-Hif1a (R&D Systems, AF1935, 1:100 dilution), rabbit polyclonal anti-Hif1a (Santa Cruz, sc-10790, 1:100 dilution), rabbit monoclonal anti-GPI (Cell Signalling, 94068, 1:200 dilution), rabbit monoclonal anti-CD73 (Cell Signalling, 13160, 1:200 dilution), rabbit monoclonal anti-Hmox1 (Cell Signalling, 86806, 1:200 dilution), rabbit polyclonal anti-Dach1 (Proteintech, 10914-1-AP, 1:200 dilution), rabbit polyclonal anti-NG2 (Millipore, AB5320, 1:200 dilution), goat polyclonal anti-Cxadr (R&D Systems, AF2654, 1:50 dilution), rabbit monoclonal anti-Sox11 (Abcam, ab134107, 1:800 dilution), rat monoclonal anti-Madcam1 (Abcam, ab80680, 1:100 dilution), rabbit polyclonal anti-Fabp4 (Abcam, ab13979, 1:100 dilution), mouse monoclonal anti-Mct4 (Santa Cruz, AF647, 1:100 dilution), rabbit monoclonal anti-Glut1 (Cell Signalling, 12939, 1:100 dilution), goat polyclonal anti-Dll4 (R&D Systems, AF1389, 1:50 dilution) and chicken polyclonal anti-Mct1 (Millipore, AB1286-I, 1:100 dilution).

Techniques: Isolation

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , High-magnification two-photon microscopy images of compact bone immunostained for EMCN (red) and CAV1 (blue) together with second-harmonic generation (SHG, white). Type R capillaries (arrowheads) and trabecular bone (TB) are indicated. Scale bars, 50μm. b, c , Maximum-intensity projections of sections from 3, 6, 8 or 12-week-old femur immunostained for EMCN (red) and OSTERIX (green) ( b ) or EMCN (red), VEGFR3 (blue) and ATP6V1B1/B2 (green) ( c ). Arrowheads mark type R capillaries near trabecular bone (TB). Scale bars, 50μm. Diagram on the right depicts association of rECs with osteoblast-lineage cells and osteoclasts. d . Injection scheme and time points of imaging after labelling of wild-type mice with Calcein Green (CG) and Alizarin Red (AZ). Tile scan imaging of sectioned femur from 8-week-old wild-type after double labelling. Insets depict active resorption in the metaphysis with CG − AR high labelling (top row), active osteogenesis (CG high AR high ) in compact bone (middle row), and active remodelling (CG low AR high ) of trabecular bone (bottom row). n=3 mice. Scale bars, 500μm (overview images) and 50μm (insets).

Article Snippet: Primary antibodies were: rat monoclonal anti-endomucin (V.7C7) (Santa Cruz, sc-65495, 1:100 dilution), goat polyclonal anti-Vegfr3 (R&D Systems, AF743, 1:50 dilution), rabbit polyclonal anti-Cav1 (Cell Signalling, 3238, 1:50 dilution), chicken polyclonal anti-GFP (Abcam, ab13970, 1:200 dilution), rabbit polyclonal anti-OSTERIX (Abcam, ab22552, 1:300 dilution), rabbit monoclonal anti-Runx2 (Abcam, ab192256, 1:200 dilution), rabbit monoclonal anti-Ki-67 (Cell Signalling, 12202, 1:200 dilution), rabbit monoclonal anti-vATPase (Abcam, ab200839, 1:200 dilution), rabbit polyclonal anti-collagen IV (AbD Serotec, 2150-1470, 1:100 dilution), rabbit polyclonal anti-collagen IIIA1 (Abcam, ab7778, 1:100 dilution), goat polyclonal anti-Hif1a (R&D Systems, AF1935, 1:100 dilution), rabbit polyclonal anti-Hif1a (Santa Cruz, sc-10790, 1:100 dilution), rabbit monoclonal anti-GPI (Cell Signalling, 94068, 1:200 dilution), rabbit monoclonal anti-CD73 (Cell Signalling, 13160, 1:200 dilution), rabbit monoclonal anti-Hmox1 (Cell Signalling, 86806, 1:200 dilution), rabbit polyclonal anti-Dach1 (Proteintech, 10914-1-AP, 1:200 dilution), rabbit polyclonal anti-NG2 (Millipore, AB5320, 1:200 dilution), goat polyclonal anti-Cxadr (R&D Systems, AF2654, 1:50 dilution), rabbit monoclonal anti-Sox11 (Abcam, ab134107, 1:800 dilution), rat monoclonal anti-Madcam1 (Abcam, ab80680, 1:100 dilution), rabbit polyclonal anti-Fabp4 (Abcam, ab13979, 1:100 dilution), mouse monoclonal anti-Mct4 (Santa Cruz, AF647, 1:100 dilution), rabbit monoclonal anti-Glut1 (Cell Signalling, 12939, 1:100 dilution), goat polyclonal anti-Dll4 (R&D Systems, AF1389, 1:50 dilution) and chicken polyclonal anti-Mct1 (Millipore, AB1286-I, 1:100 dilution).

Techniques: Microscopy, Injection, Imaging

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a, b . High-resolution confocal images showing VEGFR3 (yellow) and VEGFR2 (red) immunostaining together with DAPI (blue). VEGFR3 − VEGFR2 + capillaries (white arrowheads) around trabecular bone (TB) and nearby VEGFR3 + VEGFR2 + vessels (yellow arrowheads) in samples from young ( a ) and aged patients ( b ) are indicated. Scale bars, 200μm. c . Tile scan confocal images of EMCN + VEGFR3 − ECs in 12-week-old female and male murine femur. Trabecular bone (TB) is indicated. Scale bars, 500μm. d . Quantitation of EMCN + VEGFR3 − vessel density in 12-week-old female and male femur (n =3 mice per group). Mean ± SEM. P values, unpaired two-tailed t-test. e . EMCN (red) and VEGFR3 (blue) immunostaining showing the presence of VEGFR3 − EMCN + vessels (white arrowheads) around trabecular bone (TB) in female and male femur. Scale bars, 100μm.

Article Snippet: Primary antibodies were: rat monoclonal anti-endomucin (V.7C7) (Santa Cruz, sc-65495, 1:100 dilution), goat polyclonal anti-Vegfr3 (R&D Systems, AF743, 1:50 dilution), rabbit polyclonal anti-Cav1 (Cell Signalling, 3238, 1:50 dilution), chicken polyclonal anti-GFP (Abcam, ab13970, 1:200 dilution), rabbit polyclonal anti-OSTERIX (Abcam, ab22552, 1:300 dilution), rabbit monoclonal anti-Runx2 (Abcam, ab192256, 1:200 dilution), rabbit monoclonal anti-Ki-67 (Cell Signalling, 12202, 1:200 dilution), rabbit monoclonal anti-vATPase (Abcam, ab200839, 1:200 dilution), rabbit polyclonal anti-collagen IV (AbD Serotec, 2150-1470, 1:100 dilution), rabbit polyclonal anti-collagen IIIA1 (Abcam, ab7778, 1:100 dilution), goat polyclonal anti-Hif1a (R&D Systems, AF1935, 1:100 dilution), rabbit polyclonal anti-Hif1a (Santa Cruz, sc-10790, 1:100 dilution), rabbit monoclonal anti-GPI (Cell Signalling, 94068, 1:200 dilution), rabbit monoclonal anti-CD73 (Cell Signalling, 13160, 1:200 dilution), rabbit monoclonal anti-Hmox1 (Cell Signalling, 86806, 1:200 dilution), rabbit polyclonal anti-Dach1 (Proteintech, 10914-1-AP, 1:200 dilution), rabbit polyclonal anti-NG2 (Millipore, AB5320, 1:200 dilution), goat polyclonal anti-Cxadr (R&D Systems, AF2654, 1:50 dilution), rabbit monoclonal anti-Sox11 (Abcam, ab134107, 1:800 dilution), rat monoclonal anti-Madcam1 (Abcam, ab80680, 1:100 dilution), rabbit polyclonal anti-Fabp4 (Abcam, ab13979, 1:100 dilution), mouse monoclonal anti-Mct4 (Santa Cruz, AF647, 1:100 dilution), rabbit monoclonal anti-Glut1 (Cell Signalling, 12939, 1:100 dilution), goat polyclonal anti-Dll4 (R&D Systems, AF1389, 1:50 dilution) and chicken polyclonal anti-Mct1 (Millipore, AB1286-I, 1:100 dilution).

Techniques: Immunostaining, Quantitation Assay, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , UMAP plot (integrated scRNA-seq dataset of all age groups) showing Dach1 expression in rECs (arrow). Colour bar illustrates the expression level. b , Representative confocal images of DACH1 immunostaining (green) in 6-week-old Cdh5-mTnG (red) reporter femur. DACH1 + rECs near TB (green) are marked by white arrowheads. c , Scheme of tamoxifen-induced EC-specific Dach1 inactivation. d , Tile-scan confocal images of EMCN + VEGFR3 − CAV1 + rECs (white arrowheads) and EMCN − VEGFR3 − CAV1 + aECs (yellow arrowheads) in 12-week-old Dach1 iΔEC loss-of-function and littermate control femur. Growth plate (GP) is indicated. e , Quantitation of EMCN + vessel density, EMCN + VEGFR3 − vessel density, and number of CAV1 + arteries in 12-week-old Dach1 iΔEC and control femur ( n = 3–4 female mice per group). Mean ± s.e.m. P values, unpaired two-tailed t -test. Emcn + vessel density plotted with Welch’s correction. f , High-magnification images of metaphysis near GP (left) and of EMCN + VEGFR3 − vessels (white arrowheads; right) around TB in 12-week-old male Dach1 iΔEC and control femurs. White dashed lines in d and f indicate type H area. g , Representative 3D reconstruction of µCT measurements of 12-week-old Dach1 iΔEC and control femoral bone. Dashed yellow lines indicate area analysed. h , Quantitation shows relative bone volume, represented as bone volume/tissue volume (BV/TV) and trabecular thickness (per mm) ( n = 3–4 female mice per group and n = 3 male mice per group). Mean ± s.e.m. P values were plotted using an unpaired two-tailed t -test.

Article Snippet: Primary antibodies were: rat monoclonal anti-endomucin (V.7C7) (Santa Cruz, sc-65495, 1:100 dilution), goat polyclonal anti-Vegfr3 (R&D Systems, AF743, 1:50 dilution), rabbit polyclonal anti-Cav1 (Cell Signalling, 3238, 1:50 dilution), chicken polyclonal anti-GFP (Abcam, ab13970, 1:200 dilution), rabbit polyclonal anti-OSTERIX (Abcam, ab22552, 1:300 dilution), rabbit monoclonal anti-Runx2 (Abcam, ab192256, 1:200 dilution), rabbit monoclonal anti-Ki-67 (Cell Signalling, 12202, 1:200 dilution), rabbit monoclonal anti-vATPase (Abcam, ab200839, 1:200 dilution), rabbit polyclonal anti-collagen IV (AbD Serotec, 2150-1470, 1:100 dilution), rabbit polyclonal anti-collagen IIIA1 (Abcam, ab7778, 1:100 dilution), goat polyclonal anti-Hif1a (R&D Systems, AF1935, 1:100 dilution), rabbit polyclonal anti-Hif1a (Santa Cruz, sc-10790, 1:100 dilution), rabbit monoclonal anti-GPI (Cell Signalling, 94068, 1:200 dilution), rabbit monoclonal anti-CD73 (Cell Signalling, 13160, 1:200 dilution), rabbit monoclonal anti-Hmox1 (Cell Signalling, 86806, 1:200 dilution), rabbit polyclonal anti-Dach1 (Proteintech, 10914-1-AP, 1:200 dilution), rabbit polyclonal anti-NG2 (Millipore, AB5320, 1:200 dilution), goat polyclonal anti-Cxadr (R&D Systems, AF2654, 1:50 dilution), rabbit monoclonal anti-Sox11 (Abcam, ab134107, 1:800 dilution), rat monoclonal anti-Madcam1 (Abcam, ab80680, 1:100 dilution), rabbit polyclonal anti-Fabp4 (Abcam, ab13979, 1:100 dilution), mouse monoclonal anti-Mct4 (Santa Cruz, AF647, 1:100 dilution), rabbit monoclonal anti-Glut1 (Cell Signalling, 12939, 1:100 dilution), goat polyclonal anti-Dll4 (R&D Systems, AF1389, 1:50 dilution) and chicken polyclonal anti-Mct1 (Millipore, AB1286-I, 1:100 dilution).

Techniques: Expressing, Immunostaining, Control, Quantitation Assay, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , Schematic representation of tamoxifen-inducible Dach1 overexpression in Aplnr + ECs. b , Tile-scan confocal images showing expansion of CAV1 (green, white arrowheads) immunostained arteries and type R capillaries in the Dach1 gain-of-function ( Dach1 OE ) femur relative to littermate control at postnatal day 30 (P30). c , High-magnification confocal images of EMCN (red) and VEGFR3 (green) immunostained femurs showing the expansion of type R capillaries near TB in Dach1 OE mutants relative to littermate control. White arrowheads indicate type R capillaries. d , Quantitation of arteries/arterioles and CAV1 + area in control and Dach1 OE mutants. n = 3 mice per group. Mean ± s.e.m. P values were obtained by unpaired two-tailed t -test. e , Representative three-dimensional (3D) reconstruction of µCT measurements of P30 Dach1 OE and control femoral TB. f , Quantitation of parameters: bone volume/tissue volume (BV/TV), trabecular number represented by number of trabeculae per millimetre, connectivity density (connectivity density per cubic millimetre) and cortical thickness (mm). n = 3 mice per group. Mean ± s.e.m. P values were obtained by an unpaired two-tailed t -test. g , Schematic overview of scRNA-seq workflow for non-haematopoietic cells from Dach1 OE and littermate control long bone. h , i , UMAP plots of BMSCs ( n = 10,315) with colour-coded subclusters, namely osteoblasts (OBs), osteocytes (OCYs), septoclasts (SCs), proliferating BMSCs (pBMSCs), mpMSCs and dpMSCs (dpMSCs1 and dpMSCs2) ( h ). UMAP distribution of Dach1 OE and control BMSCs, as indicated by colour ( i ). j , Bar plots showing proportion of cells in Dach1 OE and control BMSC subclusters. k , Heatmap showing the top three marker genes for each BMSC subcluster. l , Heatmap illustrating differentially expressed genes in Dach1 OE and control BMSCs related to hypoxia, growth factors, adipogenic and osteogenic transcription factors, and regulators of osteogenesis. Texts in red are the areas of interest. Colour bars in k and l illustrate the expression level (log 2 fold change). Data in h – l are derived from an integrated scRNA-seq dataset of two conditions.

Article Snippet: Primary antibodies were: rat monoclonal anti-endomucin (V.7C7) (Santa Cruz, sc-65495, 1:100 dilution), goat polyclonal anti-Vegfr3 (R&D Systems, AF743, 1:50 dilution), rabbit polyclonal anti-Cav1 (Cell Signalling, 3238, 1:50 dilution), chicken polyclonal anti-GFP (Abcam, ab13970, 1:200 dilution), rabbit polyclonal anti-OSTERIX (Abcam, ab22552, 1:300 dilution), rabbit monoclonal anti-Runx2 (Abcam, ab192256, 1:200 dilution), rabbit monoclonal anti-Ki-67 (Cell Signalling, 12202, 1:200 dilution), rabbit monoclonal anti-vATPase (Abcam, ab200839, 1:200 dilution), rabbit polyclonal anti-collagen IV (AbD Serotec, 2150-1470, 1:100 dilution), rabbit polyclonal anti-collagen IIIA1 (Abcam, ab7778, 1:100 dilution), goat polyclonal anti-Hif1a (R&D Systems, AF1935, 1:100 dilution), rabbit polyclonal anti-Hif1a (Santa Cruz, sc-10790, 1:100 dilution), rabbit monoclonal anti-GPI (Cell Signalling, 94068, 1:200 dilution), rabbit monoclonal anti-CD73 (Cell Signalling, 13160, 1:200 dilution), rabbit monoclonal anti-Hmox1 (Cell Signalling, 86806, 1:200 dilution), rabbit polyclonal anti-Dach1 (Proteintech, 10914-1-AP, 1:200 dilution), rabbit polyclonal anti-NG2 (Millipore, AB5320, 1:200 dilution), goat polyclonal anti-Cxadr (R&D Systems, AF2654, 1:50 dilution), rabbit monoclonal anti-Sox11 (Abcam, ab134107, 1:800 dilution), rat monoclonal anti-Madcam1 (Abcam, ab80680, 1:100 dilution), rabbit polyclonal anti-Fabp4 (Abcam, ab13979, 1:100 dilution), mouse monoclonal anti-Mct4 (Santa Cruz, AF647, 1:100 dilution), rabbit monoclonal anti-Glut1 (Cell Signalling, 12939, 1:100 dilution), goat polyclonal anti-Dll4 (R&D Systems, AF1389, 1:50 dilution) and chicken polyclonal anti-Mct1 (Millipore, AB1286-I, 1:100 dilution).

Techniques: Over Expression, Control, Quantitation Assay, Two Tailed Test, Marker, Expressing, Derivative Assay

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a, b . Confocal images of femur sections immunostained for EMCN (red) together with OSTERIX (OSX, green) ( a ) or ATP6V1B1/B2 (green) ( b ). Scale bars, 100μm. c . Quantitation of OSX + osteoblast-lineage cells and ATP6V1B1/B2 + osteoclasts in Dach1 OE and control femur (n = 3 in each group). Mean ± SEM. P values, unpaired two-tailed test with Welch’s correction. d-f . Injection scheme and time points of imaging after labelling of Dach1 OE and control with Calcein Green (CG) and Alizarin Red (AZ) ( d ) and tile scan imaging of sectioned femur from P30 Dach1 OE and control after double labelling ( e ). Scale bars, 500μm. Bottom panels in ( e ) show stained trabecular bone. Scale bars, 20μm. f . Quantitation showing extent of bone surface actively mineralizing as mineralizing surface/bone surface (MS/BS) in percentage, mineral apposition rate (MAR) as per micrometre/day, and bone formation rate (BFR) (n=3 mice per group). Mean ± SEM. P values, unpaired two-tailed t-test with Welch’s correction for MS/BS, MAR-trabecular bone and BFR. g . Maximum-intensity projections showing prominent HIF1α + immunostaining (yellow arrowheads) in 6-week-old Sp7-mCherry + trabecular osteoblasts, whereas HIF1α + is strongly decreased after emergence of EMCN + VEGFR3 − type R vessels at 12 weeks. Scale bars, 50 μm. h . Maximum-intensity projections of 12-week-old femurs showing immunostaining for hypoxia-related markers in relation to EMCN + VEGFR3 − type R vessels. Shown are immunostaining for Glucose-6-Phosphate Isomerase (GPI), 5′-nucleotidase/CD73 and Haem Oxygenase-1 (HMOX1)-expressing macrophages (yellow arrowheads), all of which are elevated near juvenile trabecular bone relative to the equivalent region in adult. White arrowheads mark EMCN + VEGFR3 − type R vessels. i, j . UMAP plots of bone ECs (n=18383) with colour-coded subclusters ( f ), namely sinusoidal bmECs, metaphyseal mpECs, arterial aECs, remodelling rECs, and proliferating pECs. UMAP distribution of Dach1 OE and control ECs, as indicated by colour ( g ). k . Bar plots showing proportion of cells in Dach1 OE and control samples. l . UMAP plots comparing Dach1 expression in Dach1 OE (bottom) and control (top) EC subclusters. m . Heatmap of differentially expressed genes between EC subclusters. n . Heatmap of differentially expressed genes related to hypoxia and, tissue oxygenation in Dach1 OE and control bone ECs. o . Confocal images showing reduced HIF1α immunostaining (white arrowheads) in Dach1 OE metaphysis and around trabecular bone relative to littermate control. Scale bars, 50μm. Results in panels i, j, m , and n show the integrated mutant and control scRNA-seq data, whereas panels k and l show separated samples derived from the integrated data.

Article Snippet: Primary antibodies were: rat monoclonal anti-endomucin (V.7C7) (Santa Cruz, sc-65495, 1:100 dilution), goat polyclonal anti-Vegfr3 (R&D Systems, AF743, 1:50 dilution), rabbit polyclonal anti-Cav1 (Cell Signalling, 3238, 1:50 dilution), chicken polyclonal anti-GFP (Abcam, ab13970, 1:200 dilution), rabbit polyclonal anti-OSTERIX (Abcam, ab22552, 1:300 dilution), rabbit monoclonal anti-Runx2 (Abcam, ab192256, 1:200 dilution), rabbit monoclonal anti-Ki-67 (Cell Signalling, 12202, 1:200 dilution), rabbit monoclonal anti-vATPase (Abcam, ab200839, 1:200 dilution), rabbit polyclonal anti-collagen IV (AbD Serotec, 2150-1470, 1:100 dilution), rabbit polyclonal anti-collagen IIIA1 (Abcam, ab7778, 1:100 dilution), goat polyclonal anti-Hif1a (R&D Systems, AF1935, 1:100 dilution), rabbit polyclonal anti-Hif1a (Santa Cruz, sc-10790, 1:100 dilution), rabbit monoclonal anti-GPI (Cell Signalling, 94068, 1:200 dilution), rabbit monoclonal anti-CD73 (Cell Signalling, 13160, 1:200 dilution), rabbit monoclonal anti-Hmox1 (Cell Signalling, 86806, 1:200 dilution), rabbit polyclonal anti-Dach1 (Proteintech, 10914-1-AP, 1:200 dilution), rabbit polyclonal anti-NG2 (Millipore, AB5320, 1:200 dilution), goat polyclonal anti-Cxadr (R&D Systems, AF2654, 1:50 dilution), rabbit monoclonal anti-Sox11 (Abcam, ab134107, 1:800 dilution), rat monoclonal anti-Madcam1 (Abcam, ab80680, 1:100 dilution), rabbit polyclonal anti-Fabp4 (Abcam, ab13979, 1:100 dilution), mouse monoclonal anti-Mct4 (Santa Cruz, AF647, 1:100 dilution), rabbit monoclonal anti-Glut1 (Cell Signalling, 12939, 1:100 dilution), goat polyclonal anti-Dll4 (R&D Systems, AF1389, 1:50 dilution) and chicken polyclonal anti-Mct1 (Millipore, AB1286-I, 1:100 dilution).

Techniques: Quantitation Assay, Control, Two Tailed Test, Injection, Imaging, Staining, Immunostaining, Expressing, Mutagenesis, Derivative Assay

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a . Violin plots showing selected genes related to reduction of bmEC markers and increased arterialization in Dach1 OE and control. b . Heatmap of differentially expressed genes related to Notch signalling in Dach1 OE and control bone ECs. c . Representative confocal images showing increased Delta-like 4 (DLL4) (green, white arrowheads) expression in vessels around Dach1 OE trabecular bone (TB). Scale bars, 100μm. d . Scheme of tamoxifen-induced (TMX) EC-specific Dll4 inactivation with Cdh5-CreERT2 line. e, f , Tile scan confocal images of EMCN + VEGFR3 − ECs (white arrowheads) (e) and CAV1 + aECs (green arrowheads) (f) in 12-week-old Dll4 iΔEC loss-of-function and control femur. Trabecular bone (TB) is indicated. Scale bars, 500μm. g , Quantitation of EMCN + VEGFR3 − vessel density (left), and number of CAV1 + arteries (right) in 12-week-old Dll4 iΔEC and control femur (n =3 female mice per group). Mean ± SEM. P values, unpaired two-tailed t-test. h . Heatmap showing differentially expressed transcripts for secreted factors in bone EC subpopulations. i, j , Representative images (i) and quantitation (j) of calcified nodules in human mesenchymal stem cells (HMSC) cultured in osteogenic differentiation medium (ODM) supplemented with 100ng/ml Complement C1q and Tumour Necrosis Factor-Related Protein 9 (CTRP9), Neurotrophin 3 (NTF3), Platelet-Derived Growth Factor D (PDGFD), or Semaphorin 7A (SEMA7A) (n=3 independent experiments for each group). Scale bars, 100μm. Graph shows quantitation of absorbance at 405nm. Mean ± SEM. P values, ordinary one-way ANOVA and plotted with Dunnett’s multiple comparisons test. k, l , Representative images (k) and quantitation (m) of multinucleated TRAP+ osteoclasts generated from bone marrow-derived monocytes/macrophages treated with RANKL in osteoclast medium (OCM) supplemented with 100ng/ml CTRP9, NTF3, PDGFD, or SEMA7A n=8 (4 independent experiments) for each group. Scale bars, 500μm. Graph shows quantitation of TRAP + multinucleated cells between OCM and treated conditions. Mean ± SEM. P values, ordinary one-way ANOVA and plotted with Dunnett’s multiple comparisons test. Panels a and b show separated conditions derived from the integrated mutant and control data, whereas panel h is based on the integrated scRNA-seq data.

Article Snippet: Primary antibodies were: rat monoclonal anti-endomucin (V.7C7) (Santa Cruz, sc-65495, 1:100 dilution), goat polyclonal anti-Vegfr3 (R&D Systems, AF743, 1:50 dilution), rabbit polyclonal anti-Cav1 (Cell Signalling, 3238, 1:50 dilution), chicken polyclonal anti-GFP (Abcam, ab13970, 1:200 dilution), rabbit polyclonal anti-OSTERIX (Abcam, ab22552, 1:300 dilution), rabbit monoclonal anti-Runx2 (Abcam, ab192256, 1:200 dilution), rabbit monoclonal anti-Ki-67 (Cell Signalling, 12202, 1:200 dilution), rabbit monoclonal anti-vATPase (Abcam, ab200839, 1:200 dilution), rabbit polyclonal anti-collagen IV (AbD Serotec, 2150-1470, 1:100 dilution), rabbit polyclonal anti-collagen IIIA1 (Abcam, ab7778, 1:100 dilution), goat polyclonal anti-Hif1a (R&D Systems, AF1935, 1:100 dilution), rabbit polyclonal anti-Hif1a (Santa Cruz, sc-10790, 1:100 dilution), rabbit monoclonal anti-GPI (Cell Signalling, 94068, 1:200 dilution), rabbit monoclonal anti-CD73 (Cell Signalling, 13160, 1:200 dilution), rabbit monoclonal anti-Hmox1 (Cell Signalling, 86806, 1:200 dilution), rabbit polyclonal anti-Dach1 (Proteintech, 10914-1-AP, 1:200 dilution), rabbit polyclonal anti-NG2 (Millipore, AB5320, 1:200 dilution), goat polyclonal anti-Cxadr (R&D Systems, AF2654, 1:50 dilution), rabbit monoclonal anti-Sox11 (Abcam, ab134107, 1:800 dilution), rat monoclonal anti-Madcam1 (Abcam, ab80680, 1:100 dilution), rabbit polyclonal anti-Fabp4 (Abcam, ab13979, 1:100 dilution), mouse monoclonal anti-Mct4 (Santa Cruz, AF647, 1:100 dilution), rabbit monoclonal anti-Glut1 (Cell Signalling, 12939, 1:100 dilution), goat polyclonal anti-Dll4 (R&D Systems, AF1389, 1:50 dilution) and chicken polyclonal anti-Mct1 (Millipore, AB1286-I, 1:100 dilution).

Techniques: Control, Expressing, Quantitation Assay, Two Tailed Test, Cell Culture, Derivative Assay, Generated, Mutagenesis

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , UMAP visualization of bone ECs from 75-week-old bone with colour-coded subclusters. b , Bar plot showing the proportion of cells from each EC subcluster in adult and aged bone. Percentage (%) differences are indicated for rECs and aECs. Data are representative of an individual sample ( a ) and integrated scRNA-seq data from all age groups ( b ). c , d , High-magnification confocal images of TB ( c ) and compact bone (CB) ( d ) immunostained for EMCN (red), VEGFR3 (blue) RUNX2 (yellow arrowheads, marking osteoprogenitors) and ATP6V1B1B2 (green arrowheads, marking osteoclasts). e , High-magnification two-photon microscopy images of CB immunostained for EMCN (red) and VEGFR3 (blue) together with second-harmonic generation (white) showing the changes during ageing. f , Confocal tile-scan images showing EMCN + VEGFR3 − rECs in untreated 12-week-old and 75-week-old mice femur and in response to treatment with alendronate (ALN) or PTH, as indicated. Note expansion of EMCN + VEGFR3 − vessels in CB in response to ageing or treatments (arrowheads). g , Representative confocal images showing EMCN + VEGFR3 − rECs and OSTERIX (green) immunostaining in 75-week-old control, ALN-treated or PTH-treated CB (white arrowheads, marking type R capillaries during ageing). White dashed lines in d – g indicate compact bone (CB) area. h , i , Quantitation of EMCN + CAV1 + vessel density across age groups ( h ), EMCN + VEGFR3 − vessel density in 75-week-old control, ALN-treated or PTH-treated groups, and number of OSTERIX + cells in CB after ALN and PTH treatment compared with respective controls ( i ). n = 3 mice per group. Mean ± s.e.m. P values were obtained using ordinary ANOVA.

Article Snippet: Primary antibodies were: rat monoclonal anti-endomucin (V.7C7) (Santa Cruz, sc-65495, 1:100 dilution), goat polyclonal anti-Vegfr3 (R&D Systems, AF743, 1:50 dilution), rabbit polyclonal anti-Cav1 (Cell Signalling, 3238, 1:50 dilution), chicken polyclonal anti-GFP (Abcam, ab13970, 1:200 dilution), rabbit polyclonal anti-OSTERIX (Abcam, ab22552, 1:300 dilution), rabbit monoclonal anti-Runx2 (Abcam, ab192256, 1:200 dilution), rabbit monoclonal anti-Ki-67 (Cell Signalling, 12202, 1:200 dilution), rabbit monoclonal anti-vATPase (Abcam, ab200839, 1:200 dilution), rabbit polyclonal anti-collagen IV (AbD Serotec, 2150-1470, 1:100 dilution), rabbit polyclonal anti-collagen IIIA1 (Abcam, ab7778, 1:100 dilution), goat polyclonal anti-Hif1a (R&D Systems, AF1935, 1:100 dilution), rabbit polyclonal anti-Hif1a (Santa Cruz, sc-10790, 1:100 dilution), rabbit monoclonal anti-GPI (Cell Signalling, 94068, 1:200 dilution), rabbit monoclonal anti-CD73 (Cell Signalling, 13160, 1:200 dilution), rabbit monoclonal anti-Hmox1 (Cell Signalling, 86806, 1:200 dilution), rabbit polyclonal anti-Dach1 (Proteintech, 10914-1-AP, 1:200 dilution), rabbit polyclonal anti-NG2 (Millipore, AB5320, 1:200 dilution), goat polyclonal anti-Cxadr (R&D Systems, AF2654, 1:50 dilution), rabbit monoclonal anti-Sox11 (Abcam, ab134107, 1:800 dilution), rat monoclonal anti-Madcam1 (Abcam, ab80680, 1:100 dilution), rabbit polyclonal anti-Fabp4 (Abcam, ab13979, 1:100 dilution), mouse monoclonal anti-Mct4 (Santa Cruz, AF647, 1:100 dilution), rabbit monoclonal anti-Glut1 (Cell Signalling, 12939, 1:100 dilution), goat polyclonal anti-Dll4 (R&D Systems, AF1389, 1:50 dilution) and chicken polyclonal anti-Mct1 (Millipore, AB1286-I, 1:100 dilution).

Techniques: Microscopy, Immunostaining, Control, Quantitation Assay

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , Scheme for the treatment of adult mice with parathyroid hormone (PTH) or Alendronate. Black arrows indicate onset of treatment, red arrows time points of imaging. b , Confocal images of OSTEOPONTIN (blue) immunostaining of the metaphysis near the growth plate (GP) together with EMCN (red), CAV1 (green) and DAPI (white) to show the response to treatments. Scale bars, 100μm. c , UMAP visualization of sorted bone ECs from control and treatment groups with colour-coded subclusters. d , Bar plots showing the proportions of EC subpopulations in 12-week-old mice. Differences in percentage of rECs and aECs compared to control are indicated. Panels c and d show the individual samples from an integrated scRNA-seq dataset of three conditions. e, f , Confocal images showing EMCN + VEGFR3 − CAV1 + rECs (white arrowheads) around trabecular bone (TB) ( e ) and association of ATP6V1B1/B2 + (green) osteoclasts with EMCN + VEGFR3 − type R capillaries ( f ) in 12-week-old control and indicated treatment conditions. Scale bars, 50μm. g , Effect of treatments on EMCN + VEGFR3 − rECs (white arrowheads) and RUNX2 + (green) osteoprogenitors around trabecular bone (TB). Scale bars, 100μm.

Article Snippet: Primary antibodies were: rat monoclonal anti-endomucin (V.7C7) (Santa Cruz, sc-65495, 1:100 dilution), goat polyclonal anti-Vegfr3 (R&D Systems, AF743, 1:50 dilution), rabbit polyclonal anti-Cav1 (Cell Signalling, 3238, 1:50 dilution), chicken polyclonal anti-GFP (Abcam, ab13970, 1:200 dilution), rabbit polyclonal anti-OSTERIX (Abcam, ab22552, 1:300 dilution), rabbit monoclonal anti-Runx2 (Abcam, ab192256, 1:200 dilution), rabbit monoclonal anti-Ki-67 (Cell Signalling, 12202, 1:200 dilution), rabbit monoclonal anti-vATPase (Abcam, ab200839, 1:200 dilution), rabbit polyclonal anti-collagen IV (AbD Serotec, 2150-1470, 1:100 dilution), rabbit polyclonal anti-collagen IIIA1 (Abcam, ab7778, 1:100 dilution), goat polyclonal anti-Hif1a (R&D Systems, AF1935, 1:100 dilution), rabbit polyclonal anti-Hif1a (Santa Cruz, sc-10790, 1:100 dilution), rabbit monoclonal anti-GPI (Cell Signalling, 94068, 1:200 dilution), rabbit monoclonal anti-CD73 (Cell Signalling, 13160, 1:200 dilution), rabbit monoclonal anti-Hmox1 (Cell Signalling, 86806, 1:200 dilution), rabbit polyclonal anti-Dach1 (Proteintech, 10914-1-AP, 1:200 dilution), rabbit polyclonal anti-NG2 (Millipore, AB5320, 1:200 dilution), goat polyclonal anti-Cxadr (R&D Systems, AF2654, 1:50 dilution), rabbit monoclonal anti-Sox11 (Abcam, ab134107, 1:800 dilution), rat monoclonal anti-Madcam1 (Abcam, ab80680, 1:100 dilution), rabbit polyclonal anti-Fabp4 (Abcam, ab13979, 1:100 dilution), mouse monoclonal anti-Mct4 (Santa Cruz, AF647, 1:100 dilution), rabbit monoclonal anti-Glut1 (Cell Signalling, 12939, 1:100 dilution), goat polyclonal anti-Dll4 (R&D Systems, AF1389, 1:50 dilution) and chicken polyclonal anti-Mct1 (Millipore, AB1286-I, 1:100 dilution).

Techniques: Imaging, Immunostaining, Control

Journal: bioRxiv

Article Title: Embryological cellular origins and hypoxia-mediated mechanisms in PIK3CA -Driven refractory vascular malformations

doi: 10.1101/2024.10.16.618777

Figure Lengend Snippet: Tie2-Cre; R26R-Pik3ca H1047R embryos mimic human venous and lymphatic malformations. (A) Gross morphology of control and Tie2-Cre; R26R-Pik3ca H1047R mutant embryos at E13.5. (B, C-D’’”, G-H’’”, K-L’’”, O-P’’”) Immunostaining of sagittal sections with the indicated antibodies. (B-F) In the mutants, the cardinal vein (CV) is dilated (E) , and there is an increase in blood-filled PRECAM + / Prox1 + /VEGFR3 + lymphatic vessels surrounding the CV (white arrows) (D) . (B, G-J) In the mandible-tongue region, there is an expansion of blood-filled PECAM + /Prox1 - /partially VEGFR3 + blood vessels with an area greater than 5000 μm 2 (white dotted lines) (H) . No significant increase in the total number of PECAM + vessels was observed (J) . (B, K-N) In the liver, irregularly shaped, dilated PECAM + /Prox1 - /partially VEGFR3 + blood vessels with an area greater than 5000 μm 2 (white dotted lines) were observed (M, N) . (O, P) Similarly, in the brain, irregularly shaped and dilated PECAM + /Prox1 - /VEGFR3 + blood vessels with an area greater than 2000 μm 2 were observed (Q, R) . Each dot represents a value obtained from one sample. Scale bars: 100 μm (C-D””, G-H””, K-L””, O-P””) and 1 mm (A, B) . The nonparametric Mann–Whitney U test was used for statistical analysis, with exact p- values indicated. ns ≥ 0.05.

Article Snippet: The sections were immunostained using primary antibodies against PECAM (DIA-310, Dianova, 1:100, RRID:AB_2631039; M0823, DAKO, 1:100, RRID:AB_2114471), Prox1 (11-002, AngioBio, 1:100, RRID:AB_10013720; AF2727, R&D Systems, 1:100, RRID:AB_2170716), VEGFR3 (AF743, R&D Systems, 1:100, RRID:AB_355563), GFP (ab290, Abcam, 1:250, RRID:AB_303395), phospho-S6 (#2215, CST, 1:100, RRID:AB_331682), Ki67 (ab15580, Abcam, 1:250, RRID:AB_443209), D2-40 (anti-PDPN) (413151, Nichirei Biosciences, ready to use), HIF-1α (ab114977, Abcam, 1:100, RRID:AB_10900336), and VEGF-A (ab52917, Abcam, 1:100, RRID:AB_883427; ab51745, Abcam, 1:100, RRID:AB_2256948).

Techniques: Control, Mutagenesis, Immunostaining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Embryological cellular origins and hypoxia-mediated mechanisms in PIK3CA -Driven refractory vascular malformations

doi: 10.1101/2024.10.16.618777

Figure Lengend Snippet: Pik3ca H1047R expression timing influences the location of vascular malformations but does not fully recapitulate human pathology. (A, H) Gross morphology of the control and CDH5-CreERT2; R26R-Pik3ca H1047R mutant embryos at E16.5, after tamoxifen administration at E9.5. (B-G’, I-N’) Immunostaining of sagittal sections with the indicated antibodies. (B, B’, I, I’) DAPI-stained sections of the same specimen. (D, D’, K, K’) In the neck region of mutant embryos, dilated jugular veins are visible (K, white dotted lines) . Compared with controls, smaller blood-filled PECAM + /Prox1 + /VEGFR3 + lymphatic vessels (K’, white arrows) and PECAM + /Prox1 - /VEGFR3 - blood vessels (K’, red arrows) are also seen. (E, E’, L, L’) In the liver, dilated PECAM + /Prox1 - /partially VEGFR3 + blood vessels are observed ( L, white dotted lines ). The number of PECAM + /Prox1 - /partially VEGFR3 + blood vessels with an area ≥ 20,000 μm 2 in the liver is 0 ± 0 (median ± SEM) (n = 5) in controls and 3 ± 0.33 (median ± SEM) (n = 3) in mutants (p = 0.0179). (F-G’, M-N’) Similar observations are made in the skin and mesentery, where blood-filled PECAM + /Prox1 + /VEGFR3 + lymphatic vessels ( M’, N’, white arrows ) and dilated PECAM + /Prox1 - /VEGFR3 - blood vessels (M’, N’, red arrows) are seen. (O, V) Gross morphology of the control and CDH5-CreERT2; R26R-Pik3ca H1047R mutant embryos at E16.5, after tamoxifen administration at E12.5. Compared with embryos treated at E9.5, blood-filled dilated vessels are less prominent, but generalized edema is more severe. (P, P’, W, W’) DAPI- stained sections of the same specimen. (P-U’, W-AB’) Immunostaining of sagittal sections with the indicated antibodies. (S, S’, Z, Z’) In the liver, dilated PECAM + /Prox1 - /partially VEGFR3 + blood vessels are observed (Z, Z’, white dotted lines) . The number of PECAM + vessels with an area ≥ 20,000 μm 2 in the liver is 0 ± 0 (median ± SEM) (n = 5) in controls and 3 ± 0.29 (median ± SEM) (n = 3) in mutants (p = 0.0179). (T-U’, AA-AB’) Dilated, blood- filled PECAM + /weak Prox1 + /VEGFR3 + lymphatic vessels (AA’, AB’, white arrows) are observed in the skin and mesentery. (AC, AD) Statistical analysis of the tongue, neck, skin, and mesentery. Scale bars: 100 μm (C-G’, J-N’, Q-U’, X-AB’) , 1 mm (B, B’, I, I’, P, P’, W, W’) , and 2 mm (A, H, O, V) . The nonparametric Mann–Whitney U test was used for statistical analysis, with exact p-values indicated. ns ≥ 0.05.

Article Snippet: The sections were immunostained using primary antibodies against PECAM (DIA-310, Dianova, 1:100, RRID:AB_2631039; M0823, DAKO, 1:100, RRID:AB_2114471), Prox1 (11-002, AngioBio, 1:100, RRID:AB_10013720; AF2727, R&D Systems, 1:100, RRID:AB_2170716), VEGFR3 (AF743, R&D Systems, 1:100, RRID:AB_355563), GFP (ab290, Abcam, 1:250, RRID:AB_303395), phospho-S6 (#2215, CST, 1:100, RRID:AB_331682), Ki67 (ab15580, Abcam, 1:250, RRID:AB_443209), D2-40 (anti-PDPN) (413151, Nichirei Biosciences, ready to use), HIF-1α (ab114977, Abcam, 1:100, RRID:AB_10900336), and VEGF-A (ab52917, Abcam, 1:100, RRID:AB_883427; ab51745, Abcam, 1:100, RRID:AB_2256948).

Techniques: Expressing, Control, Mutagenesis, Immunostaining, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Embryological cellular origins and hypoxia-mediated mechanisms in PIK3CA -Driven refractory vascular malformations

doi: 10.1101/2024.10.16.618777

Figure Lengend Snippet: Pik3ca H1047R expression in Isl1+ CPM leads to vascular malformations confined to the head and neck region. (A) Gross morphology of the control and Isl1-Cre;R26R-Pik3ca H1047R ;R26R-eYFP mutant embryos at E11.5. Mutant mice show widespread blood-filled, dilated vessels in the first and second pharyngeal arches, along with eYFP expression in the pharyngeal mesoderm (red arrows) (B-C”’) Immunostaining of sagittal sections with the indicated antibodies. Mutant mice exhibit dilated, blood-filled PECAM + /Prox1 - /partially VEGFR3 + blood vessels in the second pharyngeal arch ( B-B’, C-C’ , white dotted lines). (B”, B”’, C”, C”’) Enzyme-antibody staining reveals pS6 expression in both endothelial and mesenchymal cells. eYFP, detected by GFP antibody, is expressed in ECs of dilated vessels (red arrows). (D, D’) Gross morphology of the control and Isl1-Cre;R26R-Pik3ca H1047R mutant embryos at E13.5. Mutant embryos display extensive blood-filled, dilated vessels on both the left and right sides of the head and neck region (white arrows). (E-J”) Enzyme-antibody staining of sagittal sections. In mutants, large PECAM + vessels extend from the lower jaw to the jugular vein ( E-H’ , red dotted lines). Controls show small partially PECAM + /Prox1 + /VEGFR3 + lymphatic vessels in the lower jaw, and mutants exhibit enlarged PECAM + /Prox1 + /VEGFR3 + lymphatic vessels and blood-filled lymph sacs ( F-J” , red arrows). (K-M) Statistical analysis. Each dot represents a value from one sample. Scale bars: 100 μm (B’-C”’, F-J”) and 1 mm (A-C, D, D’, E, E’, H, H’) . The nonparametric Mann–Whitney U test was used for statistical analysis.

Article Snippet: The sections were immunostained using primary antibodies against PECAM (DIA-310, Dianova, 1:100, RRID:AB_2631039; M0823, DAKO, 1:100, RRID:AB_2114471), Prox1 (11-002, AngioBio, 1:100, RRID:AB_10013720; AF2727, R&D Systems, 1:100, RRID:AB_2170716), VEGFR3 (AF743, R&D Systems, 1:100, RRID:AB_355563), GFP (ab290, Abcam, 1:250, RRID:AB_303395), phospho-S6 (#2215, CST, 1:100, RRID:AB_331682), Ki67 (ab15580, Abcam, 1:250, RRID:AB_443209), D2-40 (anti-PDPN) (413151, Nichirei Biosciences, ready to use), HIF-1α (ab114977, Abcam, 1:100, RRID:AB_10900336), and VEGF-A (ab52917, Abcam, 1:100, RRID:AB_883427; ab51745, Abcam, 1:100, RRID:AB_2256948).

Techniques: Expressing, Control, Mutagenesis, Immunostaining, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Embryological cellular origins and hypoxia-mediated mechanisms in PIK3CA -Driven refractory vascular malformations

doi: 10.1101/2024.10.16.618777

Figure Lengend Snippet: HIF-1α and VEGF-A inhibitors significantly suppress vascular malformations in the dorsal skin of mice. ( A ) Experimental design for inducing and treating progressive vascular malformations with bevacizumab, LW6, rapamycin, or control (DMSO for LW6 and rapamycin; PBS for bevacizumab). ( B ) Macroscopic effects of treatment: the left panel shows the control group (red dotted line), and the right panel shows the treatment group (blue dotted line). ( C ) Normal dorsal skin, displaying PECAM + blood vessels and VEGFR3 + lymphatic vessels (blue arrows). ( D, E ) Sections were prepared vertically from the epidermis to the dermis. Immunohistochemistry was performed using the indicated antibodies. The treatment groups exhibited a reduction in PECAM + vessels (red arrows). VEGFR3 + lymphatic vessels were markedly reduced in the LW6 group, with a similar reduction observed in the bevacizumab group. However, the rapamycin-treated group retained noticeably enlarged blood vessels (green arrows). Each dot represents data from an individual sample. The white, vacuole-like structures in the image are oil droplets from the tamoxifen injection. Scale bars: 100 μm (C, D, E) . Statistical analysis was performed using the nonparametric Mann–Whitney U test. ns ≥ 0.05.

Article Snippet: The sections were immunostained using primary antibodies against PECAM (DIA-310, Dianova, 1:100, RRID:AB_2631039; M0823, DAKO, 1:100, RRID:AB_2114471), Prox1 (11-002, AngioBio, 1:100, RRID:AB_10013720; AF2727, R&D Systems, 1:100, RRID:AB_2170716), VEGFR3 (AF743, R&D Systems, 1:100, RRID:AB_355563), GFP (ab290, Abcam, 1:250, RRID:AB_303395), phospho-S6 (#2215, CST, 1:100, RRID:AB_331682), Ki67 (ab15580, Abcam, 1:250, RRID:AB_443209), D2-40 (anti-PDPN) (413151, Nichirei Biosciences, ready to use), HIF-1α (ab114977, Abcam, 1:100, RRID:AB_10900336), and VEGF-A (ab52917, Abcam, 1:100, RRID:AB_883427; ab51745, Abcam, 1:100, RRID:AB_2256948).

Techniques: Control, Immunohistochemistry, Injection, MANN-WHITNEY