Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) Cas9/sgRNA/Oligo-targeting site near the SOX7 stop codon (underlined in red). The SgRNA sequence is highlighted in red (this contains the stop codon), while the protospacer-adjacent motif (PAM) sequence is shown in blue. The oligo donor containing the V5 tag (green box), is flanked by 60 bp homology arms. The red arrows represent PCR sequencing primers for genotyping (SF, V5R and SR). (B) PCR genotyping of CRISPR Sox7 -V5 injected live-born pups. Left: PCR genotyping with primer pair SF and V5R showed the desired band at the correct size in postnatal (P) 3, P4, P6, P7 and P10. Right: PCR genotyping with primer pair SF and SR produced slightly larger products in P4, P6 and P10 compared to P3 and P7, indicating successful 42 bp V5 tag integration. At least 3 bands were discovered in P3 and P7, but not in P4, P6 and P10 when separated by 3.5% gel electrophoresis (not shown). Red arrows indicate the expected PCR product sizes. (C) Sequencing of PCR product using SF primer (for both SF/V5R and SF/SR) confirmed the correct fusion of V5 tagged to the last codon in both Sox7 alleles of the Sox7 -V5 founders.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Sequencing, CRISPR, Injection, Produced, Nucleic Acid Electrophoresis

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) At E10.5, SOX7 (white) is expressed in intersomitic vessels (ISVs), the dorsal aorta (DA) (delineated by arterial marker, SOX17), cardinal vein (CV), and the surrounding migrating endothelial cells (red arrowheads) (B) SOX7 was not detected in PROX1+ migrating lymphatic endothelial cells (LECs) (asterisks). (C) At E16.5, SOX7 is downregulated in the SOX17+ main arterial branch (boxed area with dotted line), but continues to be expressed in the less mature arteries near the midline (box area with solid line). Arteries are delineated by arterial marker SOX17; major veins and vascular plexus are delineated by endomucin (EMCN). (D) SOX7/ β -galactosidase ( β -gal) (green) also stains the SOX17 (white) positive major arteries in the skin plexus of E14.5 Sox7 +/- lac-Z reporter mice. Dors., dorsal; Med., medial. Scale bars = 100 μm.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Marker

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) Representative images of E10.5 Sox7 iECKO mutants and sibling controls, injected with tamoxifen at E9.5. The endothelium is delineated by endomucin (EMCN) (blue) and endothelial cells by ETS-related gene (ERG) (red). (Top right panel) The percentage of SOX7 positive endothelial cells over the total number of endothelial cells quantified from 10 sequential transverse sections in 2 control and 3 Sox7 iECKO mutants. (Bottom right panel) Graph indicating the levels of Sox7 transcript in sibling controls and Sox7 iECKO mutants. Mean ± SEM; scored sibling control, n=9; Sox7 iECKO mutants, n=7; Mann-Whitney U- test. P <0.0005 (***). (B) Bright-field images of Sox7 iECKO mutants and sibling controls at E14.5, after pulsing with tamoxifen at E9.5 and E10.5. Scored sibling controls, n=8; Sox7 iECKO mutants, n=5. (C) Brightfield images and whole-mount immunostaining of Sox7 iECKO mutant and sibling control skin at E13.5, after Cre induction at E9.5 and E10.5. Dermal lymphatic structures are marked by Neuropilin 2 (NRP2) (membranous white), lymphatic endothelial cells by Prospero-related homeobox 1 (PROX1) (red), and blood vessels by EMCN (green). Dash line represents the midline of the embryo. Scored sibling controls, n=7; Sox7 iECKO mutants, n=7 (D-H) Quantification of (D) lymphatic sprout migration distance from the midline (E) lymphatic branch points/area (F) lymphatic vessel width (μm) (G) PROX1+ nuclei/area and (H) PROX1+ nuclei/lymphatic vessels across the whole skin. Total area = 4200 x 1500 μm on both sides of the midline, in sibling controls (n=3) and Sox7 iECKO mutants (n=5-6). (F,H) Average of width and PROX1+ nuclei was obtained from 7 random representative leading lymphatic vessels, of fixed length at 200 μm, from both sides of the midline in each skin. (I,J) Quantification of (I) disconnected lymphatic endothelial cell (LEC) clusters (<100 μm) and vessel branches (>100μm)/area and (J) PROX1+ nuclei in each LEC cluster. Total area quantified = 4200 x 2200 μm on both side of midline in sibling controls (n=3) and Sox7 iECKO mutants (n=5). PROX1+ nuclei were quantified from n=29 and n=122 LEC clusters, respectively. (D-J) Skins were from the cervico-thoracic regions of E13.5 embryos, defloxed at E9.5, E10.5. Mean ± SEM, Mann-Whitney U –test. P <0.05 (*). LV, lymphatic vessel. Scale bars = 100 μm (immunofluorescence images A,C), 1 mm (bright-field images B,C).

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Injection, MANN-WHITNEY, Immunostaining, Mutagenesis, Migration, Immunofluorescence

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) Whole-mount immunostaining of Sox7 iECKO mutant and sibling control embryonic skin at E14.5, following injection with tamoxifen at E9.5 and E10.5. Dermal lymphatic structures are marked by NRP2 (membranous white), lymphatic endothelial cells by PROX1 (red), blood vessels by EMCN (green) and arterioles by SOX7 (white nuclei). Dashed line represents the midline of the embryo. (B) Whole-mount immunostaining of Sox7 iECKO mutant and sibling control for EMCN (grey), showing dermal blood vessels at E13.5, after injection with tamoxifen at E9.5 and E10.5. Endothelial cells are marked by ERG (red), proliferative cells are stained by phospho-histone 3, H3 (green). (Right panel) Quantitation of EMCN+ (blood) vessel front density (μm 3 ), number of H3+ proliferative and ERG+ endothelial cells in this region. Scored sibling control, n=5; Sox7 iECKO mutants, n=4; Mean ± SEM; Mann-Whitney U -test. P <0.05 (*); ns = not significant. (C) Whole-mount immunostaining of Sox7 iECKO ; mT/mG mutant and Cre+; mT/mG control embryonic skin at E14.5, after injection with tamoxifen at E12.5. Blood vessels are stained with EMCN (red), cells after Cre excision are shown in green, and lymphatic endothelial cells are marked by PROX1 (white). (D-F) Graphs showing quantitation of the Cre activity in (D) blood vessels, (E) lymphatic vessels and (F) EMCN+ (blood) vessel density (μm 3 ), from n=4 Cre+; mT/mG controls and n=3 Sox7 iECKO ; mT/mG mutants. Mean ± SEM; Mann-Whitney U -test; ns = not significant. (G) Whole-mount immunostaining of Sox7 iECKO mutant and sibling control embryonic skin at E14.5, after injection with tamoxifen at E11.5 and E12.5. Dermal lymphatic structures are marked by the NRP2 (membranous white), lymphatic endothelial cells by PROX1 (red), and blood vessels are stained by EMCN (green). Dashed line represents the midline of the embryo. (H) Immunostaining of Sox7 iECKO mutant and sibling control coronal sections, at E11.5, after injection with tamoxifen at E9.5, E10.5. Lymphatic progenitor cells in the cardinal veins (CVs) are PROX1+ (white). Yellow arrowheads highlight the presence of LEC progenitors in the region near the dorsal aorta (DA) in the Sox7 iECKO mutant, which is typically absent in the sibling control. Scale bars = 100 μm.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Immunostaining, Mutagenesis, Injection, Staining, Quantitation Assay, MANN-WHITNEY, Activity Assay

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A-B) Nebulosa plots from single-nuclei RNA-Seq on E14.5 embryonic skins, showing the expression of Sox7 and Vegfc. Both Sox7 and Vegfc are found expressed in BECs (red arrowheads), but not LECs (empty red arrowheads). (C) Vegfc (red) fluorescent RNA in situ hybridisation on mouse cross-sections at E11.5. The endothelium is delineated by PECAM (white) and LECs by PROX1 (green nuclei). BEC-specific endogenous levels of Vegfc (red arrowheads) are higher than LECs (asterisks). (D) qPCR on FACs-sorted PECAM+CD45- endothelial cells of Sox7 iECKO mutants and sibling controls at E14.5, injected with tamoxifen at E11.5 and E12.5. Expression is normalised to endothelial marker Pecam and Cdh5 . Scored sibling controls, n=9; Sox7 iECKO mutants, n=8. (E-F) qPCR on human arterial endothelial cells (HUAECs) transfected with SiSOX7 or SiCTRL for (E) 30 h and (F) 17 h. (G) qPCR on human venous endothelial cells (HUVECS) transfected with SiSOX7 or SiCTRL for 17 h. (E-G) Expression is relative to HPRT and GAPDH. Data from one siRNA experiment performed in triplicates. (D–G) Mean ± SEM; t -test. P <0.05 (*); P <0.005 (**); P <0.0005 (***). BEC, blood endothelial cell; LEC, lymphatic endothelial cell; DA, dorsal aorta; CV, cardinal vein; Dors., dorsal; Med., medial. Scale bars = 100 μm.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: RNA Sequencing Assay, Expressing, In Situ, Hybridization, Injection, Marker, Transfection

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A-D) Nebulosa plots from single-nuclei RNA-Seq on E14.5 embryonic skins, showing markers for BECs ( Emcn ), LECs ( Prox1 ), SMCs ( Acta2 ) and neuronal cells ( Npas3 ). (E) Microarray analysis of dermal BEC and LEC populations sorted from different embryonic stages reveals that Sox7 and Vegfc mRNA are preferentially expressed by BECs. BECs, blood endothelial cells; LECs, lymphatic endothelial cells; and SMC, smooth muscle cells.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: RNA Sequencing Assay, Microarray

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A-D) SOX7 transcriptionally activates Notch effector, HEY1, to repress Vegfc . (A) qPCR on FAC-sorted PECAM+CD45- endothelial cells of Sox7 iECKO mutants and sibling controls at E14.5, injected with tamoxifen at E11.5 and E12.5. Expression is normalised to the endothelial marker Pecam and Cdh5 . Scored sibling controls, n=9; Sox7 iECKO mutants, n=8. (B-C) qPCR on (B) human arterial endothelial cells (HUAECs) and (C) human venous endothelial cells (HUVECs) transfected with SiSOX7 or SiCTRL for 17 h. In addition to HEY1, DLL4 levels were also downregulated in the human cell line experiments. Expression is relative to HPRT and GAPDH. Data from one siRNA experiment performed in triplicates. Mean ± SEM; t -test. P <0.05 (*); P <0.005 (**); P <0.0005 (***). (D) HEY1 represses human VEGFC promoter activity. HeLa cells were co-transfected with human or mouse VEGFC-luc and either EV (empty vector) or HEY1 expression constructs as indicated. VEGFC luciferase activity was measured and normalised to Renillla luciferase activity, which was then made relative to the promoter-less vector, pGL3-basic, which was set to 1. Biological replicates, n=3 independent repeats of the same experiment. Mean ± SEM; t -test. P <0.05 (*). (E-G) SOX7 physically interacts with transcription repressor, HEY1. (E) Amplified Luminescent Proximity Homogenous Assay (ALPHAScreen) shows the heatmap of SOX7 pairwise protein-protein interaction tested, where red indicates strong interaction and light blue indicates an absence of interaction. (F) Single molecule spectroscopy reveals that SOX7 is able to directly interact with HEY1. SOX7 and the target transcription factors were tagged with GFP or Cherry. GFP-SOX7 and HEY1-Cherry show co-incidence at 0.66, suggesting a 1:2 interaction. Peaks centered on 0 (green) or 1 (red) correspond to the GFP or Cherry-tagged proteins only. (G) Co-immnunoprecipitation analysis of SOX7 and HEY1. HEK293 cells were transfected with indicated plasmids and harvested 24 h after transfection, immunoprecipitated by anti-GFP or Ig control before immunoblot analysis to determine the presence of bait/prey (top) and the input (bottom). N=2.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Injection, Expressing, Marker, Transfection, Activity Assay, Plasmid Preparation, Construct, Luciferase, Amplification, Amplified Luminescent Proximity Homogenous Assay, Spectroscopy, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) SOX7 represses human VEGFC promoter activity. HeLa cells were co-transfected with human or mouse VEGFC-luc, EV (empty vector), or SOX7 expression constructs as indicated. VEGFC luciferase activity was measured and normalised to Renillla luciferase activity, which was then made relative to the promoter-less vector, pGL3-basic, which was set to 1. Biological replicates, n=3. Mean ± SEM; t -test. P <0.05 (*). (B) Schematic representation of the human VEGFC locus 255 kb upstream from the transcription start site (TSS) (denoted as VEGFC-255 region) from the UCSC Genome browser. The H3K27Ac is denoted in light blue, DNAseI hypersensitive hotspots are indicated by black/grey boxes, where the darkness is proportional to the maximum signal strength observed in any cell line. The chromatin state in HUVECs is shown in purple (indicates insulator), green (poised enhancer) and pink (a Polycomb-repressed region). The HUVECs CCCTC-binding factor (CTCF) binding location is shown in orange. Multiple species conservation is shown in blue peaks and alignments with black stripes. (C) The human luciferase VEGFC-255 BCR transgene. B (black), binding region; C (blue), conserved region and R (pink), repressive region. (D) Schematic representation of the mouse Vegfc locus 152kb upstream from the TSS (denoted as Vegfc- 152). The chromatin state in mouse at E11 from ENCODE is denoted in pink (indicates a heterochromatin region enriched for H3K9me3 repressive marker). The ATAC-Seq (in black) indicates accessible DNA regions in E11 mouse hearts. Multiple species conservation is shown in blue peaks and alignments with black stripes. (E) The mouse luciferase Vegfc-152 RBC transgene. R (pink), repressive region; B (black), binding region and C (blue), conserved region. (F) SOX7 represses VEGFC promoter activity through VEGFC-255 and Vegfc-152 . HeLa cells were co-transfected with human or mouse VEGFC-luc, VEGFC-255 BC-luc, VEGFC-255 BCR-luc, Vegfc-152 BC-luc, Vegfc-152 RBC-luc, EV (empty vector), or SOX7 expression constructs as indicated. VEGFC luciferase activity was measured and normalised to Renillla luciferase activity, which was then made relative to the promoter-less vector, pGL3-basic, which was set to 1. Biological replicates, n=3. Mean ± SEM; t -test. P <0.05 (*); P <0.005 (**); ns = not significant.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Construct, Luciferase, Binding Assay, Marker

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) MSigDB pathway analysis on the top 2k peaks from human SOX7 HUVEC ChIP-Seq identified enrichment for genes within the VEGF/VEGFR gene set. These include VEGFR1 , VEGFR2 , NRP1 , NRP2 , PDGFC , VEGFA and VEGFC . (B-F) Binding profiles and motif analysis of mouse SOX7-V5 ChIP-Seq (B) Image from Integrative Genome Viewer (IGV) illustrating ChIP-Seq tracks at E9.5 (pink) and E10.5 (light blue) mapped to the mouse reference genome GRCm38/mm10 (top track, dark blue). The height of a graph peak indicates the number of “positive bindings” on a given chromosomal location. Total number of binding events is indicated on the left under each track name. Top DNA-binding motifs in SOX7 - V5 ChIP-Seq by MEME-ChIP in (C) E9.5 and (D) E10.5 embryos. The different height of the letters in the position weight matrix reveals information content of each position (in bits), correlated by the degree of certainty of the nucleotide at a given position. (E) Spaced motif analysis (SPAMO) found a SOX7 primary motif amongst the topmost enriched motif next to a MEF2A/MEF2C binding motif found in E9.5 SOX7 - V5 ChIP-Seq, with 2093 total occurrences. SOX7 is best gapped at 134 bp from the MEF2A/MEF2C binding motif (24/2093). (F) ChIP-PCR on both bound fraction (left) and corresponding assumed unbound controls (right), as predicted by the peak location from SOX7-V5 ChIP-Seq. (G) A total of 712 genes were found overlapping between E9.5 and E10.5 SOX7-V5 mouse ChIP-Seq. (H-J) KEGG pathway analysis (H) and gene ontological analysis by DAVID bioinformatics database, showing the top biological processes (I) and molecular functions (J) associated to the 712 overlapping genes from the SOX7-V5 mouse ChIP-Seq.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) At 2 days post Cre induction, E11.5 Sox7 iECKO mutants showed ectopic expression of PROX1+ (red) in medial ventral regions of the cardinal veins (CVs), close to dorsal aorta (DA) (yellow arrowheads). (B) Rose diagram indicates the % of PROX1+ cells over total number of EMCN+ endothelial cells, within each indicated region. The medial region closest to the DA is indicated by 0/360°; most lateral region, 180°; dorsal, 90° and ventral, 270°. Scored sibling controls, n=5; Sox7 iECKO mutants, n=4. Mean ± SEM; Mann-Whitney U -test. P <0.05 (*). (C) Graph indicates the % of total PROX1+ cells over the total number of EMCN+ endothelial cells within each embryo analysed. A total of 2907 endothelial cells quantified, from n=5 sibling controls and 1780 endothelial cells from n=4 Sox7 iECKO mutants. Mean ± SEM; Mann-Whitney U -test. P =0.0635. (D) Whole-mount immunostaining of control and Sox7 iECKO embryonic skin at E12.5 after Cre induction at E9.5, E10.5. Dermal lymphatic structures are stained with NRP2 (membranous white), lymphatic endothelial cells, PROX1 (red), and blood vessels, both EMCN (green) and SOX7 (white nuclei). Arrowheads indicate EMCN low/- PROX1 + NRP2 +/low individual LECs on blood vessels (type I), and arrow shows EMCN + PROX1 + NRP2 low/- LEC within the vessel wall (type II). (E) Graph shows the total events of PROX1+ single LECs or LEC clusters cells associated with EMCN+ blood vessel plexus within each embryo. PROX1+ cells were quantified from n=4 controls and n=5 Sox7 iECKO mutants. Mean ± SEM; Mann-Whitney U -test. P<0.05 (*). Dors., dorsal; Med., medial. Scale bars = 100 μm.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Expressing, MANN-WHITNEY, Immunostaining, Staining

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A-B) Whole-mount immunostaining of Sox7 iECKO mutant or control embryonic skin, showing the different types of LECs emerging from the blood capillary plexus. Skins were harvested from embryos at E12.5, after Cre-induction at E9.5 and E10.5. Dermal lymphatic structures are marked by NRP2 (membranous white), lymphatic endothelial cells by PROX1 (red), and blood vessels are stained by EMCN (green) and SOX7 (white nuclei). Arrowheads indicate EMCN low/- PROX1 + NRP2 +/low individual LECs on blood vessels (type I), arrows show the single EMCN + PROX1 + NRP2 low/- LEC within the blood vessel wall (type II) and empty arrowheads mark the EMCN +/- PROX1 + NRP2 +/low LEC clusters (2-4 cells) exiting from the blood vessel network (type III). (C-E) Graph shows the quantitation of different types of PROX1+ LEC progenitors, comparing between control and Sox7 iECKO mutant skins. PROX1+ LEC progenitors were quantified from n=4 controls and n=5 Sox7 iECKO mutants. Mean ± SEM; Mann-Whitney U -test. P <0.05 (*); ns = not significant. Scale bars = 100 μm.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Immunostaining, Mutagenesis, Staining, Quantitation Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) Table from HOMER motif database showing the top 10 most enriched transcription motifs associated to SOX7 binding sites (B, G) Graphs showing intersections between top 2k SOX7 ChIP-Seq peak locations and HUVEC or mouse histone markers, respectively from the ENCODE consortium. Only peaks with at least 50% overlapping region with the histone markers are considered. Intersection was performed using the EpiExplorer online resource tool. (C, H, J) Venn diagrams showing intersections of different active histone markers and (D, I, K) repressive histone markers, both displayed at least 50% overlapped, with the top 2k of SOX7 ChIP-Seq binding regions. Diagram was generated using online resource stool, Venny 2.1.0. (E, L) Graphs showing the overall proportion of top 2k SOX7 ChIP-Seq peaks, with at least an active or a repressive histone mark. (F) Graphs showing intersections between top 2k SOX7 HUVEC ChIP-Seq peak locations and chromatin states in HUVECs from the ENCODE consortium. Only peaks with at least 50% overlapping region with each corresponding chromatin state are considered. Intersection was performed using the EpiExplorer online resource tool.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Binding Assay, ChIP-sequencing, Generated

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A and B) LEC progenitor organisation is impaired in both early stage and organ-specific lymphangiogenesis. ( A ) In physiological conditions, LEC progenitors emerge in the dorsal lateral part of the CVs and migrate towards the dorso-lateral aspects of the embryo. In the absence of functional SOX7, blood vascular endothelial cells from the arterial compartment increase the local expression of endothelial VEGFC, perturbing its tissue distribution. This induces and/or expands the number of LEC progenitors in ventro-medial aspects of the CVs. ( B ) Dermal lymphatics are dysmorphic in the absence of SOX7 function in blood vascular endothelial cells. An increase in VEGFC in the dermal blood endothelial cells causes hyperproliferation of local LEC progenitors. This is shown by an increase in the emergence of local LEC progenitors from the endomucin-positive blood capillary plexus at E12.5 (red asterisks). Changes in expressions of other SOX7-dependent lymphangiocrines also contribute to the migration defects in the dermal lymphatics. ( C ) Model showing the molecular mechanisms of SOX7-dependent repression of VEGFC transcription identified in this study: 1) SOX7 binds to distal regulatory elements (silencing regions) to suppress VEGFC transcription. 2) SOX7 represses VEGFC promoter activity. 3) SOX7 acts upstream of Notch effector and repressor, HEY1. SOX7-induced expression of HEY1 causes VEGFC downregulation and 4) Protein-protein interaction between SOX7 and HEY1 forms a complex to repress VEGFC transcription. LEC, lymphatic endothelial cells; AEC, arterial endothelial cells; VEC, venous endothelial cells; Lat., lateral; Med, medial.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Functional Assay, Expressing, Migration, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Laccaic Acid A Is a Direct, DNA-competitive Inhibitor of DNA Methyltransferase 1 *

doi: 10.1074/jbc.M113.480517

Figure Lengend Snippet: qPCR validation of LCA microarray analysis and changes in gene expression caused by treatment with control compound, anthraquinone-2-carboxylic acid

Article Snippet: Microarray Analysis Total RNA was isolated from 2 × 10 5 cells and used for microarray analysis (University of Iowa DNA Core Facility) in hybridization to Human Gene ST1.0 Array GeneChips (Affymetrix).

Techniques: Biomarker Discovery, Microarray, Gene Expression, Control

Journal: Microbiology

Article Title: Global transcriptional analysis of the stringent response in Enterococcus faecalis

doi: 10.1099/mic.0.060236-0

Figure Lengend Snippet: qRT-PCR primers used to validate the microarray results

Article Snippet: The slides were hybridized to a mixture containing equal amounts of test and reference cDNA for 16 h at 42 °C in a MicroArray User Interface (MAUI) hybridization chamber (BioMicro Systems).

Techniques: Microarray

Journal: Microbiology

Article Title: Global transcriptional analysis of the stringent response in Enterococcus faecalis

doi: 10.1099/mic.0.060236-0

Figure Lengend Snippet: qRT-PCR validation of microarray data

Article Snippet: The slides were hybridized to a mixture containing equal amounts of test and reference cDNA for 16 h at 42 °C in a MicroArray User Interface (MAUI) hybridization chamber (BioMicro Systems).

Techniques: Biomarker Discovery, Microarray