Journal: bioRxiv

Article Title: Regulation of hematopoietic stem cell (HSC) proliferation by Epithelial Growth Factor Like-7 (EGFL7)

doi: 10.1101/2025.01.21.634107

Figure Lengend Snippet: (A) 12-16 weeks old WT or Egfl7 KO mice were sacrificed, and BM cell populations were analyzed by flow cytometry, and LTC-IC assays. Total number of BM cells in Egfl7 KO mice compared to WT. (B) Number of BM LSK (Lin-Sca1+ cKit+ cells), (C) number of BM HSCs (Lin-Sca1+ cKit+ CD150+ CD48-cells) and (D) number of BM Long-Term (LT) HSCs (Lin-Sca1+ cKit+ CD150+ CD48-CD34-cells) and Short-Term (ST) HSCs (Lin-Sca1+ cKit+ CD150+ CD48-CD34-cells) in Egfl7 KO mice compared to WT. (E) Representative Z-stack immunofluorescence images of sterna from Egfl7 KO mice stained with Lin (green), CD41 (green), CD48 (green), and CD150 (red) compared to WT. (F) Quantification of number of HSCs per sternum segment in Egfl7 KO mice compared to WT. (G) Total number of LTC-IC colonies from BM of Egfl7 KO mice compared to WT. (H) Lethally irradiated WT BoyJ mice were competitively transplanted at 1:1 ratio with sort-purified CD45.2 + LSK cells from BM of Egfl7 KO or WT mice and sort-purified WT CD45.1 + LSK cells. Mice were bled every 4 weeks and peripheral blood chimerism was analyzed by flow cytometry. At 16 weeks post transplantation, mice were sacrificed, and BM cells were analyzed by flow cytometry. Percentage of CD45.2 + cells in peripheral blood of transplanted mice at 4-16 weeks post transplantation. (I) Percentage of CD45.2 + BM cells and (J) total number of BM cells in transplanted mice at 16 weeks post-transplantation. These data are representative of 3 or more experiments done with N= 4-6 mice per group. P = *<0.05, **<0.01, ***<0.001.

Article Snippet: 12-16 weeks old C57/Bl6 mice were injected i.p with 10 µg/mouse of recombinant EGFL7 protein (rEGFL7) (Peprotech) daily for 10 days.

Techniques: Flow Cytometry, Immunofluorescence, Staining, Irradiation, Purification, Transplantation Assay

Journal: bioRxiv

Article Title: Regulation of hematopoietic stem cell (HSC) proliferation by Epithelial Growth Factor Like-7 (EGFL7)

doi: 10.1101/2025.01.21.634107

Figure Lengend Snippet: (A) Lethally irradiated WT BoyJ mice were transplanted with BM from 12-16 weeks old WT or Egfl7 KO mice mixed with BM from WT BoyJ mice at 10:1 ratio. Mice were bled every 4 weeks and peripheral blood chimerism was analyzed by flow cytometry. At 16 weeks post transplantation, mice were sacrificed, and BM cells were analyzed by flow cytometry. Percentage of CD45.2 + cells in peripheral blood of transplanted mice at 4-16 weeks post-transplantation. (B) Percentage of BM CD45.2 + cells, (C) percentage of CD45.2 + BM LSK cells, and (D) percentage of CD45.2 + BM HSCs in transplanted mice at 16 weeks post-transplantation. (E) BM from above primary transplanted mice were transplanted into lethally irradiated WT BoyJ mice. Recipient secondary transplanted mice were bled every 4 weeks and peripheral blood chimerism was analyzed by flow cytometry. At 16 weeks post transplantation, secondary transplanted mice were sacrificed, and BM cells were analyzed by flow cytometry. Percentage of CD45.2 + cells in peripheral blood of secondary transplanted mice. (F) Percentage of BM CD45.2 + cells, (G) total number of BM cells, (H) percentage of CD45.2 + BM LSK cells, and (I) percentage of CD45.2 + BM HSCs in secondary transplanted mice. These data are representative of 3 or more experiments done with N= 3-6 mice per group. P = *<0.05, **<0.01, ***<0.001.

Article Snippet: 12-16 weeks old C57/Bl6 mice were injected i.p with 10 µg/mouse of recombinant EGFL7 protein (rEGFL7) (Peprotech) daily for 10 days.

Techniques: Irradiation, Flow Cytometry, Transplantation Assay

Journal: bioRxiv

Article Title: Regulation of hematopoietic stem cell (HSC) proliferation by Epithelial Growth Factor Like-7 (EGFL7)

doi: 10.1101/2025.01.21.634107

Figure Lengend Snippet: 12-16 weeks old WT or Egfl7 KO mice were injected with BrdU (50µg/gm, i.p), followed by daily BrdU treatment through drinking water (0.8 mg/ml) until mice were sacrificed at 1-, 3-, 7-, and 30-days post BrdU injection. BM populations were analyzed for BrdU incorporation by flow cytometry. (A) Percentage of HSCs in the G 0 -G 1 , S, and G 2 -M cell cycle phases in Egfl7 KO mice compared to control on day 1, (B) 3, and (C) 7 post BrdU treatment. Mice were sacrificed 30 days post BrdU treatment and BM was analyzed for BrdU incorporation by flow cytometry. (D) (left) Representative flow plots of BrdU incorporation assay at 30 days in BM HSCs from Egfl7 KO mice compared to WT. (right) Percentage of cells in G 0 -G 1 , S, and G 2 M phases in Egfl7 KO mice compared to WT. (E) BM HSCs were sorted from 12-16 weeks old WT or Egfl7 KO mice and analyzed for EGFL7 expression by immunofluorescence. Representative immunofluorescence images of BM HSCs stained for EGFL7 (red) and Ki67 (green). Nuclei are counterstained with DAPI. (F) Mean fluorescence intensity of EGFL7 in Ki67+ BM HSCs compared to Ki67-HSCs from WT mice. (G) Quantification of percentage of Ki67+ BM HSCs in Egfl7 KO mice compared to WT. These data are representative of 3 or more experiments with N=4-7 mice per group. P = *<0.05, **<0.01, ***<0.001.

Article Snippet: 12-16 weeks old C57/Bl6 mice were injected i.p with 10 µg/mouse of recombinant EGFL7 protein (rEGFL7) (Peprotech) daily for 10 days.

Techniques: Injection, BrdU Incorporation Assay, Flow Cytometry, Control, Expressing, Immunofluorescence, Staining, Fluorescence

Journal: bioRxiv

Article Title: Regulation of hematopoietic stem cell (HSC) proliferation by Epithelial Growth Factor Like-7 (EGFL7)

doi: 10.1101/2025.01.21.634107

Figure Lengend Snippet: (A) Schematic representation of experiment. WT mice were treated with rEGFL7 or PBS (control) for 10 days after which mice were sacrificed, and BM was analyzed by flow cytometry, and LTC-IC assays. (B) Total BM cells, (C) percentage of BM LSKs, (D) total BM LSKs, (E) total HSCs, (F) total BM LT-HSCs, (G) percentage of BM HSCs, and (H) percentage of BM LT-HSCs in WT mice 10 days after treatment with rEGFL7 compared to control. (I) Total number of LTC-IC colonies in BM from WT mice after treatment with rEGFL7 compared to control for 10 days. (J) BM from rEGFL7-treated or control mice was transplanted into lethally irradiated WT BoyJ mice. Mice were bled every 4 weeks and BM was analyzed at 20 weeks post-transplantation. Percentage of CD45.2 + cells in peripheral blood of transplanted mice at 4-20 weeks post-transplantation. (K) Percentage of CD45.2 + cells in BM of transplanted mice at 20 weeks post-transplantation. These data are representative of 3 or more experiments performed in triplicates with N= 3-6 mice per group. P = *<0.05, **<0.01, ***<0.001.

Article Snippet: 12-16 weeks old C57/Bl6 mice were injected i.p with 10 µg/mouse of recombinant EGFL7 protein (rEGFL7) (Peprotech) daily for 10 days.

Techniques: Control, Flow Cytometry, Irradiation, Transplantation Assay

Journal: bioRxiv

Article Title: Regulation of hematopoietic stem cell (HSC) proliferation by Epithelial Growth Factor Like-7 (EGFL7)

doi: 10.1101/2025.01.21.634107

Figure Lengend Snippet: (A) 12-16 weeks old Egfl7 KO mice were treated with rEGFL7 (Egfl7 KO – rE7) or PBS (Egfl7 KO – CNT) for 10 days after which BM was analyzed by flow cytometry. WT mice treated with PBS were used as controls (WT – CNT). Total number of BM cells, (B) total number of BM LSKs, (C) total number of BM HSCs, (D) total number of BM LT-HSCs in Egfl7 KO – rE7 and Egfl7 KO – CNT mice compared to WT – CNT. (E) 12-16 weeks old Egfl7 KO mice were treated with rEGFL7 (Egfl7 KO – rE7) or PBS (Egfl7 KO – CNT) for 10 days. For the BrdU assay, mice were injected with BrdU (50µg/gm, i.p) on day 0, followed by daily BrdU treatment through drinking water (0.8 mg/ml) until mice were sacrificed on day 11 and BM was analyzed for BrdU incorporation by flow cytometry. Percentage of BM LSK cells in G 0 -G 1 , S, and G 2 M phases in in Egfl7 KO – rE7 and Egfl7 KO – CNT mice compared to WT – CNT. (F) Schematic representation of 5-FU survival assay. 12-16 weeks old WT or Egfl7 KO mice were injected with 5-FU (150 mg/kg, i.p.) once a week for 4 weeks. Mice were monitored daily for survival. (G) Kaplan-Meier survival curve of WT and Egfl7 KO mice after 5-FU treatment. (H) 12-16 weeks old WT or Egfl7 KO mice injected with 5-FU (150 mg/kg, i.p.) were sacrificed 3 days post 5-FU injection and peripheral blood and BM was analyzed by an automated hematology analyzer. BM cells were stained with Ki67 and DAPI to analyze proliferating LSK cells, or with Annexin V to analyze apoptotic cells by flow cytometry. Number of peripheral blood WBCs in peripheral blood, (I) number of BM cells, (J) number of BM LSK cells, (K) number of BM HSCs in WT or Egfl7 KO mice on day 3 after 5-FU treatment. (L) Percentage of BM LSK cells in G 0 -G 1 , S, and G 2 M phases in WT or Egfl7 KO mice. (M) Percentage of apoptotic BM HSCs in WT or Egfl7 KO mice. These data represent 3 or more experiments performed in triplicates with N= 3-6 mice per group. P = *<0.05, **<0.01, ***<0.001.

Article Snippet: 12-16 weeks old C57/Bl6 mice were injected i.p with 10 µg/mouse of recombinant EGFL7 protein (rEGFL7) (Peprotech) daily for 10 days.

Techniques: Flow Cytometry, BrdU Staining, Injection, BrdU Incorporation Assay, Clonogenic Cell Survival Assay, Staining

Journal: bioRxiv

Article Title: Regulation of hematopoietic stem cell (HSC) proliferation by Epithelial Growth Factor Like-7 (EGFL7)

doi: 10.1101/2025.01.21.634107

Figure Lengend Snippet: (A) BM cKit + cells (HSPCs) from 12-16 weeks old WT and Egfl7 KO mice were subjected to scRNA-sequencing. Uniform Manifold Approximation and Projection (UMAP) of HSPCs from BM of Egfl7 KO and WT mice, colored by clustering and annotated based on gene signature. (B) UMAP of HSPCs colored by expression of key cell-type marker genes Hlf (HSCs), Pf4 (MPP2), Ctsg (MPP3), and Dntt (MPP4). (C) Violin plot distribution of Egfl7 expression levels across all HSPC clusters. (D) Heatmap of differentially expressed genes in HSC cluster from Egfl7 KO mice compared to WT. (E) Relative mRNA expression of Egr1 , Klf2 , Klf4 , and Mid1 in HSCs sorted from Egfl7 KO mice compared to WT, as determined by qRT-PCR. mRNA expression is calculated relative to Gapdh . N= 5 mice per group. (F) (left) Percentage of HSCs (cluster 3) in G1, S, and G2M cell cycle phases in Egfl7 KO mice compared to WT. (right) Violin plots of cell cycle scores in HSCs (cluster 3) from Egfl7 KO mice compared to WT. (G) UMAP of BM HSCs (LSK CD150+ cells) from Egfl7 KO and WT mice, colored by clustering. (H) Violin plot distribution of Hlf, (I) Mki67 and Egfl7 expression levels across all HSC clusters. (J) UMAP of HSCs colored by cell cycle scores in WT (top) and Egfl7 KO (bottom) mice. (K) Top differentially up– and down-regulated signaling pathways in Egfl7 KO HSCs compared to WT. P = *<0.05, **<0.01, ***<0.001.

Article Snippet: 12-16 weeks old C57/Bl6 mice were injected i.p with 10 µg/mouse of recombinant EGFL7 protein (rEGFL7) (Peprotech) daily for 10 days.

Techniques: Sequencing, Expressing, Marker, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Regulation of hematopoietic stem cell (HSC) proliferation by Epithelial Growth Factor Like-7 (EGFL7)

doi: 10.1101/2025.01.21.634107

Figure Lengend Snippet: (A) WT mice were treated with rEGFL7 or PBS (control) for 10 days after which BM cKit+ cells (HSPCs) were subjected to scRNA-seq. UMAP of HSPCs from BM of rEGFL7-treated and control mice, colored by clustering and annotated based on gene signature. (B) UMAP of HSPCs colored by treatment group-rEGFL7-treated (red) and control (black). (C) UMAP of HSPCs colored by expression of key cell-type marker genes Hlf (HSCs), Pf4 (MPP2), Ctsg (MPP3), and Dntt (MPP4). (D) Violin plot distribution of Hlf expression levels across all HSPC clusters. (E) Heatmap of differentially expressed genes in HSC cluster from rEGFL7-treated mice compared to control. (F) Relative mRNA expression of S100a8 , S100a9 , Vegfa , Nr4a1 , Nr4a2 , and Nfkbiz in HSCs sorted from rEGFL7-treated mice compared to control, as determined by qRT-PCR. mRNA expression is calculated relative to Gapdh . N=5 mice per group. P = *<0.05, **<0.01, ***<0.001.

Article Snippet: 12-16 weeks old C57/Bl6 mice were injected i.p with 10 µg/mouse of recombinant EGFL7 protein (rEGFL7) (Peprotech) daily for 10 days.

Techniques: Control, Expressing, Marker, Quantitative RT-PCR

Journal: Biomedicines

Article Title: Effect of Pravastatin on Placental Expression of Epidermal Growth Factor-like Domain 7 in Early-Onset Pre-Eclampsia: A New Potential Mechanism of Action

doi: 10.3390/biomedicines12081929

Figure Lengend Snippet: Primer sequences, amplicon size, and gene accession number.

Article Snippet: Membranes were then incubated overnight at 4 °C with rabbit anti-EGFL7 (Abcam, Cambridge, UK, cat. ab256451, 1:1000) or mouse anti-GAPDH (clone 6C5, Santa Cruz, CA, USA, cat. sc-32233, 1:2000), all diluted in TBS/T with 5% ( w / v ) bovine serum albumin (BSA).

Techniques: Amplification, Sequencing

Journal: Biomedicines

Article Title: Effect of Pravastatin on Placental Expression of Epidermal Growth Factor-like Domain 7 in Early-Onset Pre-Eclampsia: A New Potential Mechanism of Action

doi: 10.3390/biomedicines12081929

Figure Lengend Snippet: EGFL7 gene expression levels in chorionic villous explants immediately after dissection. ( A ): Representative phase-contrast images of villous explant samples from control and PE placenta. ( B ): qRT-PCR analysis demonstrating reduced expression of EGFL7 in PE villous explant samples compared to the healthy controls. Scale bar in panel images = 250 μM. Statistical analysis was performed using Mann–Whitney test (** p = 0.0044).

Article Snippet: Membranes were then incubated overnight at 4 °C with rabbit anti-EGFL7 (Abcam, Cambridge, UK, cat. ab256451, 1:1000) or mouse anti-GAPDH (clone 6C5, Santa Cruz, CA, USA, cat. sc-32233, 1:2000), all diluted in TBS/T with 5% ( w / v ) bovine serum albumin (BSA).

Techniques: Expressing, Dissection, Control, Quantitative RT-PCR, MANN-WHITNEY

Journal: Biomedicines

Article Title: Effect of Pravastatin on Placental Expression of Epidermal Growth Factor-like Domain 7 in Early-Onset Pre-Eclampsia: A New Potential Mechanism of Action

doi: 10.3390/biomedicines12081929

Figure Lengend Snippet: Effect of pravastatin administration to ex vivo chorionic villous explant cultures on EGFL7 expression. ( A ): Representative phase-contrast images of villous explants from healthy control and PE placenta after 24 h of culture in the presence or absence of 10 μM pravastatin (PRA). ( B ): qRT-PCR analysis showing that PRA treatment did not affect EGFL7 expression in both healthy control and PE villous cultures. Scale bar in panel images = 250 μM. Statistical analysis was performed using Student’s t test. − and + PRA: without or with 10 μM pravastatin.

Article Snippet: Membranes were then incubated overnight at 4 °C with rabbit anti-EGFL7 (Abcam, Cambridge, UK, cat. ab256451, 1:1000) or mouse anti-GAPDH (clone 6C5, Santa Cruz, CA, USA, cat. sc-32233, 1:2000), all diluted in TBS/T with 5% ( w / v ) bovine serum albumin (BSA).

Techniques: Ex Vivo, Expressing, Control, Quantitative RT-PCR

Journal: Biomedicines

Article Title: Effect of Pravastatin on Placental Expression of Epidermal Growth Factor-like Domain 7 in Early-Onset Pre-Eclampsia: A New Potential Mechanism of Action

doi: 10.3390/biomedicines12081929

Figure Lengend Snippet: Identification of high-responder and low-responder PE pregnancies following treatment of chorionic villous explant cultures with 10 μM pravastatin (PRA) for 24 h. ( A , B ): qRT-PCR analysis demonstrating increased expression of EGFL7 in high-responder PE villous explant cultures following PRA treatment ( A ) and no changes in low-responder PE villous cultures ( B ) when compared to villi cultured without PRA. ( C ): Western blot analysis of villous explant cultures with or without PRA, indicating increased expression of EGFL7 in high-responder PE villous cultures. ( D ): Quantification of Western blot analysis ( n = 3). Statistical analysis was performed using Student’s t test (* p = 0.0467 for qRT-PCR; * p = 0.028 for western blot). − and + PRA: without or with 10 μM pravastatin.

Article Snippet: Membranes were then incubated overnight at 4 °C with rabbit anti-EGFL7 (Abcam, Cambridge, UK, cat. ab256451, 1:1000) or mouse anti-GAPDH (clone 6C5, Santa Cruz, CA, USA, cat. sc-32233, 1:2000), all diluted in TBS/T with 5% ( w / v ) bovine serum albumin (BSA).

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Western Blot

Journal: Biomedicines

Article Title: Effect of Pravastatin on Placental Expression of Epidermal Growth Factor-like Domain 7 in Early-Onset Pre-Eclampsia: A New Potential Mechanism of Action

doi: 10.3390/biomedicines12081929

Figure Lengend Snippet: Maternal socio-demographic characteristics and biochemical findings of women with pre-eclampsia according to EGFL7 expression in response to pravastatin treatment.

Article Snippet: Membranes were then incubated overnight at 4 °C with rabbit anti-EGFL7 (Abcam, Cambridge, UK, cat. ab256451, 1:1000) or mouse anti-GAPDH (clone 6C5, Santa Cruz, CA, USA, cat. sc-32233, 1:2000), all diluted in TBS/T with 5% ( w / v ) bovine serum albumin (BSA).

Techniques: Expressing

Journal: Biomedicines

Article Title: Effect of Pravastatin on Placental Expression of Epidermal Growth Factor-like Domain 7 in Early-Onset Pre-Eclampsia: A New Potential Mechanism of Action

doi: 10.3390/biomedicines12081929

Figure Lengend Snippet: Perinatal and neonatal outcome of women with pre-eclampsia according to EGFL7 expression in response to pravastatin treatment.

Article Snippet: Membranes were then incubated overnight at 4 °C with rabbit anti-EGFL7 (Abcam, Cambridge, UK, cat. ab256451, 1:1000) or mouse anti-GAPDH (clone 6C5, Santa Cruz, CA, USA, cat. sc-32233, 1:2000), all diluted in TBS/T with 5% ( w / v ) bovine serum albumin (BSA).

Techniques: Expressing

Journal: Nature Communications

Article Title: Epidermal growth factor-like domain 7 drives brain lymphatic endothelial cell development through integrin αvβ3

doi: 10.1038/s41467-024-50389-8

Figure Lengend Snippet: a – d Tg(egfl7:YFP) was generated showing YFP in blood vessels and lymphatics. The distribution of YFP is similar with CFP and DsRed in the triple transgenic line Tg(egfl7:YFP; lyve1b: DsRed; kdrl: CFP-NTR) at 5 dpf. n = 25/25 embryos. The low-magnified image shows the dorsal view of the head ( a ), the high-magnified image in another embryo shows the magnified BLECs loop in the dorsal brain ( b ). The lateral facial lymphatics of the head shows in c . The trunk lymphatics shows in d . BLECs, brain lymphatic endothelial cells, MsV, mesencephalic vein, OLV, otolithic lymphatic vessel, FL, facial lymphatics, TD, thoracic duct. Scale bar, 50 μm. e Schematic diagram showing the dorsal view of a dissected adult zebrafish brain. f egfl7 promoter-driven YFP is expressed in all BLECs and blood vessels. n = 6/8 adults. g High-magnification inset showing the YFP overlap with DsRed in the double transgenic line Tg(egfl7:YFP; lyve1b: DsRed) . Scale bars, 200 μm in f and 50 μm in g .

Article Snippet: The pT2KXIGD-lyve1b-egfl7-p2A-GFP plasmid was created using the In-Fusion cloning method (Clontech, #639619).

Techniques: Generated, Transgenic Assay

Journal: Nature Communications

Article Title: Epidermal growth factor-like domain 7 drives brain lymphatic endothelial cell development through integrin αvβ3

doi: 10.1038/s41467-024-50389-8

Figure Lengend Snippet: a Confocal images of the BLECs, facial lymphatics, and trunk lymphatics in Tg(kdrl: DenNTR;lyve1b: DsRed) transgenic lines from 4 dpf to 6 dpf in WT. b Loss of egfl7 prevents the formation of the BLECs from 4 dpf to 6 dpf, but the facial lymphatics of the lateral head and trunk lymphatics are normal in egfl7 mutant embryos at 6 dpf. c Percentage of embryos that have double lymphatic loops, single loops, and none of the loops in the brain (WT, n = 60 embryos, egfl7-/- , n = 75 embryos, χ 2 test). d The statistics show the percentage of TD formation per 6 somites ( n = 10 embryos, two-tailed unpaired t -test; ns, no significance. Data are represented as mean ± SD). e The statistics show the total lengths of LFLs, OLVs, and MFLs at 6 dpf ( n = 8 embryos, 2way ANOVA multiple comparisons test; ns, no significance. Box plots show the five-number summary of a set of data: including the minimum score (shown at the end of the lower whisker), first (lower) quartile, median, third (upper) quartile, and maximum score (shown at the end of the upper whisker)). BLECs, brain lymphatic endothelial cells; LFL lateral facial lymphatic, MFL medial facial lymphatic, LAA lymphatic branchial arches, OLV otolithic lymphatic vessel, ISLV intersegmental lymphatic vessels, DLLV dorsal longitudinal lymphatic vessel, TD thoracic duct. f , g Double labeling of FISH- vegfr3 and anti-DsRed in Tg(lyve1b:DsRed) transgenic background at 6 dpf. Arrowheads point to the BLECs in the top layer of the brain. h , i Double labeling of FISH- mrc1a and anti-Dendra2 in Tg(kdrl:DenNTR) transgenic background at 5 dpf. j The statistics show the number of vegfr3 + and mrc1a + BLECs in WT and egfl7 mutant ( n = 9 embryos, 2-way ANOVA multiple comparisons test. Box plots show the five-number summary of a set of data: including the minimum score (shown at the end of the lower whisker), first (lower) quartile, median, third (upper) quartile, and maximum score (shown at the end of the upper whisker). k – n Dissection and whole-mount images of BLECs and meningeal vascular in Tg(kdrl: DenNTR;lyve1b: DsRed) adult brain at 6 mpf (months post-fertilization) in WT ( k , l ) and egfl7 mutant ( m , n ). o , p The statistics show the number of lyve1b + BLECs in the adult brain ( o , n = 6 brains; two-tailed unpaired t test) and the number of BV junctions per 0.16 mm 2 of a brain ( p , n = 6 brain areas; two-tailed unpaired t test). Data are represented as mean ± SD. Scale bar, 50 μm.

Article Snippet: The pT2KXIGD-lyve1b-egfl7-p2A-GFP plasmid was created using the In-Fusion cloning method (Clontech, #639619).

Techniques: Transgenic Assay, Mutagenesis, Two Tailed Test, Whisker Assay, Labeling, Dissection

Journal: Nature Communications

Article Title: Epidermal growth factor-like domain 7 drives brain lymphatic endothelial cell development through integrin αvβ3

doi: 10.1038/s41467-024-50389-8

Figure Lengend Snippet: a – d In the stable transgenic line Tg(kdrl:egfl7-p2A-GFP) , the replenishment of Egfl7 in BVs can rescue the absence of BLECs in the egfl7 mutant ( d ) in contrast to the mutant under the Tg(kdrl: GFP) transgenic line ( b ). For WT, overexpression of Egfl7 in the BVs results in increased BLECs emerge in the bilateral loop over the brain ( c ) compared to the WT under the Tg(kdrl:GFP) ( a ). e The statistics show the number of BLECs per double loops in WT and egfl7 mutant under Tg(kdrl: GFP) and Tg(kdrl:egfl7-p2A-GFP) transgenic lines ( n = 24 embryos, 2-way ANOVA. Box plots show the five-number summary of a set of data: including the minimum score (shown at the end of the lower whisker), first (lower) quartile, median, third (upper) quartile, and maximum score (shown at the end of the upper whisker)). f – i In contrast to the mutant under Tg(lyve1b: GFP) transgenic line ( g ), the replenishment of Egfl7 in LECs under Tg(lyve1b:egfl7-p2A-GFP) is able to re-form the BLECs bilateral loop in the egfl7 mutant ( i ). And overexpression of Egfl7 in WT lymphatics shows the BLECs are unaltered ( f , h ). j The statistics show the number of BLECs per double loops in WT and egfl7 mutant under Tg(lyve1b: GFP) and Tg(lyve1b:egfl7-p2A-GFP) transgenic lines ( n = 24 embryos, 2-way ANOVA. Box plots show the five-number summary of a set of data: including the minimum score (shown at the end of the lower whisker), first (lower) quartile, median, third (upper) quartile, and maximum score (shown at the end of the upper whisker)). Scale bar, 50 μm.

Article Snippet: The pT2KXIGD-lyve1b-egfl7-p2A-GFP plasmid was created using the In-Fusion cloning method (Clontech, #639619).

Techniques: Transgenic Assay, Mutagenesis, Over Expression, Whisker Assay

Journal: Nature Communications

Article Title: Epidermal growth factor-like domain 7 drives brain lymphatic endothelial cell development through integrin αvβ3

doi: 10.1038/s41467-024-50389-8

Figure Lengend Snippet: a Schematic diagram showing the lateral view and the lower middle layer of the brain, respectively. Black frames indicate the image area of corresponding panels. CVP, choroidal vascular plexus. b , c Immunofluorescence staining for blood endothelial cells (Anti-Dendra2), BLECs (Anti-DsRed), and Anti-Prox1 (blue nuclei) in the WT ( b ) and egfl7 mutant ( c ) at 3 dpf. Arrowheads of the magnified 2D single slice image point to the Prox1+ BLECs progenitors sprout from CVP. d The statistics show the ratio of BLECs progenitors positive for Prox1 in WT and egfl7 mutant ( n = 9 embryos; two-tailed unpaired t test; ns, no significance. Data are represented as mean ± SD). e – h Confocal image showing the triple labeling of FISH- vegfr3 or FISH -mrc1a , anti-DsRed, and Dendra2 in Tg(kdrl:DenNTR; lyve1b:DsRed) transgenic background in WT ( e , g ) and egfl7 mutant ( f , h ). Arrowheads in the magnified image shows vegfr3 mRNA and mrc1a mRNA are expressed in the lyve1b positive and low-level kdrl expressing BLECs progenitors departing the CVP. i The statistics show the ratio of BLECs positive for vegfr3 and mrc1a in WT and egfl7 mutant ( n = 8 embryos; 2way ANOVA multiple comparisons test; ns, no significance. Box plots show the five-number summary of a set of data: including the minimum score (shown at the end of the lower whisker), first (lower) quartile, median, third (upper) quartile, and maximum score (shown at the end of the upper whisker)). j The statistics show the number of vegfr3 + and mrc1a + BLECs in WT and egfl7 mutant ( n = 8 embryos; 2way ANOVA multiple comparisons test. Box plots show the five-number summary of a set of data: including the minimum score (shown at the end of the lower whisker), first (lower) quartile, median, third (upper) quartile, and maximum score (shown at the end of the upper whisker)). Scale bar, 50 μm.

Article Snippet: The pT2KXIGD-lyve1b-egfl7-p2A-GFP plasmid was created using the In-Fusion cloning method (Clontech, #639619).

Techniques: Immunofluorescence, Staining, Mutagenesis, Two Tailed Test, Labeling, Transgenic Assay, Expressing, Whisker Assay

Journal: Nature Communications

Article Title: Epidermal growth factor-like domain 7 drives brain lymphatic endothelial cell development through integrin αvβ3

doi: 10.1038/s41467-024-50389-8

Figure Lengend Snippet: a Schematic diagram showing the lower middle layer and top layer of the vessels in the brain, respectively. Black frames indicate the image area of corresponding panels. CVP (choroidal vascular plexus) is observed in the lower middle layer; MsV (mesencephalic vein) is in the top layer. b Time-lapse image of Tg(lyve1b: DsRed; kdrl: DenNTR) from 66 hpf to 122 hpf show the BLECs (arrows) sprout from CVP (arrowhead at 66 hpf) and migrate along the MsV (arrowhead) to form the lymphatic loop in WT at 122 hpf. n = 15 embryos. c The BLECs (arrows) of egfl7 mutant shows a delayed sprouting from CVP (arrowhead at 66 hpf), fail to migrate along the MsV (arrowhead), and is unable to form the loop at 122 hpf. n = 15 embryos. d – f EdU staining shows the proliferation of BLECs in WT and egfl7 mutant at 6 dpf, the EdU is injected at 56 hpf. Note the EdU+ BLECs is significantly decreased in the mutant ( d, e ). Arrowheads indicate the EdU+ BLECs in WT. The statistics show the number of EdU+ BLECs in double loops at 6 dpf in WT and egfl7 mutant ( f , n = 18 embryos; two-tailed unpaired t -test). Data are represented as mean ± SD. Scale bar, 50 μm.

Article Snippet: The pT2KXIGD-lyve1b-egfl7-p2A-GFP plasmid was created using the In-Fusion cloning method (Clontech, #639619).

Techniques: Mutagenesis, Staining, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Epidermal growth factor-like domain 7 drives brain lymphatic endothelial cell development through integrin αvβ3

doi: 10.1038/s41467-024-50389-8

Figure Lengend Snippet: a Schematic diagram showing the experimental design for detecting the development of LECs after the ectopic, mosaic expression of Vegfc and Venus. Illustrations of the method, transgenic lines, plasmids, and time points of heat shock, and imaging. b , d Compared to the heatshock of Venus, overexpression of Vegfc, causing a hyperbranching in adjacent PLs (arrows) and hyperbranched ISVs in the horizontal myoseptum (arrowheads) of WT ( b ) and egfl7 mutant ( d ). f , g The statistics shows the number of PLs per 6 somites ( f ) and the number of hyperbranched ISVs ( g ) after ectopic expression of Vegfc and Venus in the trunk ( n = 10 embryos; two-tailed unpaired t -test). c , e Compared to the heatshock of Venus, overexpression of Vegfc in the brain does not rescue the devoid of BLECs in the egfl7 mutant. h Quantification of the number of BLECs in the brain after ectopic expression of Vegfc and Venus in the brain ( n = 10 embryos; two-tailed unpaired t-test). Data are represented as mean ± SD. Scale bar, 50 μm.

Article Snippet: The pT2KXIGD-lyve1b-egfl7-p2A-GFP plasmid was created using the In-Fusion cloning method (Clontech, #639619).

Techniques: Expressing, Transgenic Assay, Imaging, Over Expression, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: Epidermal growth factor-like domain 7 drives brain lymphatic endothelial cell development through integrin αvβ3

doi: 10.1038/s41467-024-50389-8

Figure Lengend Snippet: a , b , g Schematic diagram showing the lateral view, the lower middle layer, and the top layer of the vessels in the dorsal view, respectively. Black frames indicate the orientation and the image area of corresponding panels. c – f In Tg(lyve1b: GFP; kdrl: DenNTR) transgenic background, FISH and antibody staining show itgb3a and itgb3b are expressed in CVPs at 54 hpf ( c , n = 20/21 embryos, e n = 20/20 embryos, arrowheads), and expressed in BVs and departed BLECs at 3 dpf ( d , n = 20/20 embryos, f , n = 19/20 embryos). h Double labeling of FISH- itgav and anti-GFP in Tg(lyve1b: GFP) transgenic background at 4 dpf, arrowheads points the itgav expressed in BLECs. n = 15/16 embryos. i HEK293T cells were transfected with Egfl7-Myc and Itgb3b-Flag or Itgav-HA. Egfl7 was immunoprecipitated using anti-Myc antibody and the immunoprecipitants were analyzed for the presence of Itgav-HA or Itgb3b-Flag by Western blot. j – l Inhibition of integrin αvβ3 by Cilengitide treatment from 54 hpf to 5 dpf phenocopy the devoid of the lymphatic loop in the brain ( k ). Arrows point to the BLECs in the control group ( j ). The statistics show the number of BLECs in the double loops after Cilengitide treatment ( l , n = 15 embryos; two-tailed unpaired t -test. Data are represented as mean ± SD). m – p The itgb3b transcript elongation inhibition causes BLECs reduction in the brain. Dorsal view of BLECs loop-like structure of uninjected siblings and samples co-injected with dCas9-KRAB mRNA and itgb3b gRNAs ( m, n ). The co-injection of gRNA and CRISPRi results in decreased expression of itgb3b and the number of BLECs ( o , n = 3 replicants; p , n = 23 embryos; two-tailed unpaired t -test. Data are represented as mean ± SD). Scale bar, 50 μm.

Article Snippet: The pT2KXIGD-lyve1b-egfl7-p2A-GFP plasmid was created using the In-Fusion cloning method (Clontech, #639619).

Techniques: Transgenic Assay, Staining, Labeling, Transfection, Immunoprecipitation, Western Blot, Inhibition, Control, Two Tailed Test, Injection, Expressing

Journal: Nature Communications

Article Title: Epidermal growth factor-like domain 7 drives brain lymphatic endothelial cell development through integrin αvβ3

doi: 10.1038/s41467-024-50389-8

Figure Lengend Snippet: a FISH and antibody staining shows ilk is expressed in GFP+ BLECs at 4 dpf (arrowheads). n = 17/18 embryos. b – g Inhibition of ILK by cpd22 can partially rescue the absence of BLECs in the egfl7 mutant at 6 dpf ( b – e ). Quantification of the number of BLECs in the double loops of brain in different treatment groups ( f , n = 10 embryos, two-tailed unpaired t test. Data are represented as mean ± SD). The statistics show the percentage of embryos that have double lymphatic loops, single loops, and none of the loops in the brain ( g , WT, n = 51 embryos, WT + cpd22, n = 67 embryos, egfl7-/- , n = 116 embryos, egfl7-/- + cpd22, n = 127 embryos, χ 2 test).). h – k An egfl7 mutant was crossed with an ilk heterozygote to generate two types of larvae: ilk +/+; egfl7 -/- and ilk +/-; egfl7 -/-, which were then studied for BLECs loop-structures formation. The larvae were classified into three categories based on their phenotype: Double loops, single loops, and none of the loops ( h – j ). The results showed that compared to ilk +/+; egfl7 -/-, the ilk +/-; egfl7 -/- larvae had a decreased percentage of the none of the loops phenotype, and a partially rescued percentage of double loops and single loops phenotype ( k , ilk +/+; egfl7 -/-, n = 49 embryos, ilk +/-; egfl7 -/-, n = 47 embryos, χ2 test). Scale bar, 50 μm.

Article Snippet: The pT2KXIGD-lyve1b-egfl7-p2A-GFP plasmid was created using the In-Fusion cloning method (Clontech, #639619).

Techniques: Staining, Inhibition, Mutagenesis, Two Tailed Test

Journal: Heliyon

Article Title: Tetramethylpyrazine promotes angiogenesis and nerve regeneration and nerve defect repair in rats with spinal cord injury

doi: 10.1016/j.heliyon.2023.e21549

Figure Lengend Snippet: Primer sequences used in PCR.

Article Snippet: After being separated by 12 % SDS-PAGE, the samples were loaded onto polyvinylidene fluoride membranes (Bio-Rad, USA) which were then counteracted with primary antibodies EGFL7 (19291-1-AP, 1:500, Proteintech), CD31 (ab28364, 1:500, Abcam), and GAPDH (2118, 1:1000, CST) and co-incubated with the secondary antibody (ab6734, 1:3000, Abcam).

Techniques:

Journal: Heliyon

Article Title: Tetramethylpyrazine promotes angiogenesis and nerve regeneration and nerve defect repair in rats with spinal cord injury

doi: 10.1016/j.heliyon.2023.e21549

Figure Lengend Snippet: TMP promotes angiogenesis, nerve regeneration and nerve defect repair in rats with SCI. A: BBB score to test the motor behavior of rats; B–C: HE staining and TUNEL staining to assess the lesions of rat spinal cord; D: Western blot to detect CD31 expression to assess angiogenesis; E–G: ELISA to detect inflammatory factors; H–I: RT-qPCR or Western blot to detect miR-497-5p and EGFL7 expression; the values are expressed by the mean standard deviation. * P < 0.05 vs. Sham; # P < 0.05 vs. Model.

Article Snippet: After being separated by 12 % SDS-PAGE, the samples were loaded onto polyvinylidene fluoride membranes (Bio-Rad, USA) which were then counteracted with primary antibodies EGFL7 (19291-1-AP, 1:500, Proteintech), CD31 (ab28364, 1:500, Abcam), and GAPDH (2118, 1:1000, CST) and co-incubated with the secondary antibody (ab6734, 1:3000, Abcam).

Techniques: Staining, TUNEL Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Standard Deviation

Journal: Heliyon

Article Title: Tetramethylpyrazine promotes angiogenesis and nerve regeneration and nerve defect repair in rats with spinal cord injury

doi: 10.1016/j.heliyon.2023.e21549

Figure Lengend Snippet: MiR-497-5p inhibits EGFL7 expression. A: Bioinformatics website to predict that miR-497-5p and EGFL7 have targeted binding sites; B: Dual-luciferase reporter gene detection to confirm the targeting relationship between miR-497-5p and EGFL7; C –D: RT-qPCR and Western blot to detect EGFL7 expression; the values were expressed by the mean standard deviation. * P < 0.05 vs. TMP + antagomir NC; # P < 0.05 vs. TMP + agomir NC.

Article Snippet: After being separated by 12 % SDS-PAGE, the samples were loaded onto polyvinylidene fluoride membranes (Bio-Rad, USA) which were then counteracted with primary antibodies EGFL7 (19291-1-AP, 1:500, Proteintech), CD31 (ab28364, 1:500, Abcam), and GAPDH (2118, 1:1000, CST) and co-incubated with the secondary antibody (ab6734, 1:3000, Abcam).

Techniques: Expressing, Binding Assay, Luciferase, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Heliyon

Article Title: Tetramethylpyrazine promotes angiogenesis and nerve regeneration and nerve defect repair in rats with spinal cord injury

doi: 10.1016/j.heliyon.2023.e21549

Figure Lengend Snippet: EGFL7 upregulation promotes TMP-mediated protection on SCI rats. A: RT-qPCR or Western blot to detect EGFL7 expression; B: BBB score to test the motor behavior of rats; C –D: HE staining and TUNEL staining to assess the lesions of the rat spinal cord; E: Western blot to detect CD31 expression to assess angiogenesis; F–H: ELISA detects the levels of inflammatory factors; the values are expressed by the mean standard deviation. * P < 0.05 vs. TMP + oe-NC; # P < 0.05 vs. TMP + sh-NC.

Article Snippet: After being separated by 12 % SDS-PAGE, the samples were loaded onto polyvinylidene fluoride membranes (Bio-Rad, USA) which were then counteracted with primary antibodies EGFL7 (19291-1-AP, 1:500, Proteintech), CD31 (ab28364, 1:500, Abcam), and GAPDH (2118, 1:1000, CST) and co-incubated with the secondary antibody (ab6734, 1:3000, Abcam).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Heliyon

Article Title: Tetramethylpyrazine promotes angiogenesis and nerve regeneration and nerve defect repair in rats with spinal cord injury

doi: 10.1016/j.heliyon.2023.e21549

Figure Lengend Snippet: EGFL7 inhibition resists miR-497-5p silencing-induced protection against SCI. A: RT-qPCR or Western blot to detect EGFL7 expression; B: BBB score to test the motor behavior of rats; C –D: HE staining and TUNEL staining to assess the lesions of the rat spinal cord; E: Western blot to detect CD31 expression to assess angiogenesis; F–H: ELISA detects the levels of inflammatory factors; the values are expressed by the mean standard deviation. * P < 0.05 vs. TMP + miR-497-5p antagomir + sh-NC.

Article Snippet: After being separated by 12 % SDS-PAGE, the samples were loaded onto polyvinylidene fluoride membranes (Bio-Rad, USA) which were then counteracted with primary antibodies EGFL7 (19291-1-AP, 1:500, Proteintech), CD31 (ab28364, 1:500, Abcam), and GAPDH (2118, 1:1000, CST) and co-incubated with the secondary antibody (ab6734, 1:3000, Abcam).

Techniques: Inhibition, Quantitative RT-PCR, Western Blot, Expressing, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation