dach1 overexpression  (OriGene)

Journal: Genes & Development

Article Title: DACH1 stimulates shear stress-guided endothelial cell migration and coronary artery growth through the CXCL12–CXCR4 signaling axis

doi: 10.1101/gad.301549.117

Figure Lengend Snippet: Dach1 mutants have small coronary arteries. ( A ) Whole-mount confocal images of hearts from E17.5 or P0 immunolabeled with VE-cadherin (endothelial cells; red) and SM-MHC (smooth muscle; blue). Dach1 mutant coronary arteries (CA) were smaller in diameter and exhibited abnormal looping structures (arrowheads). ( B , C ) There were no apparent structural defects in Dach1 mutant capillaries, as quantified by vessel coverage ( B ) and junction density ( C ). n = 9 wild type; n = 14 knockout. ( D ) Quantification of the diameters of the left (LCA) and right (RCA) main coronary arteries (primary branches). n = 9 wild type; n = 28 heterozygous; n = 14 knockout. ( E ) Smooth muscle coverage of the coronary artery was unaffected at E17.5 by Dach1 deficiency. n = 8 wild type; n = 10 knockout. ( F ) Linear regression lines of artery diameters when moving from primary (1°) to secondary (2°) to tertiary (3°) branches showed a shallower slope, indicating a potential defect in hierarchical patterning in knockout hearts at E17.5. Dots represent individual arteries. LCA: n = 9 wild type, n = 11 knockout; RCA: n = 9 wild type, n = 4 knockout. ( G ) Quantification of persistent arterial loops (indicated by arrowheads in A ) in E17.5 hearts. ( H ) Representative images of tissue sections from Dach1 knockout and wild-type littermate hearts show that Dach1 -deficient hearts did not display any gross structural defects. Error bars indicate standard deviation. (ns) Nonsignificant; (*) P < 0.05; (**) P < 0.01; (****) P < 0.0001. Bars: A , 200 µm; H , 500 µm.

Article Snippet: For Dach1 overexpression experiments, pCMV6 mouse Dachshund1 GFP-tagged (transcript variant 1, OriGene Technologies, MG216327) was purchased, and the Dach1 -coding region was subcloned into a lentiviral-ready pLJM-eGFP plasmid (Addgene, plasmid 19319).

Techniques: Immunolabeling, Mutagenesis, Knock-Out, Standard Deviation

Journal: Genes & Development

Article Title: DACH1 stimulates shear stress-guided endothelial cell migration and coronary artery growth through the CXCL12–CXCR4 signaling axis

doi: 10.1101/gad.301549.117

Figure Lengend Snippet: DACH1 is expressed in coronary endothelial cells. ( A ) Schematic of arterial remodeling where small plexus vessels create a remodeling zone (RZ) that transforms into a mature coronary artery (CA). Blue arrows indicate relative direction and magnitude of blood flow. ( B , C , D ) Whole-mount confocal images localizing DACH1 during arteriogenesis of the right coronary artery at developmental stages E14.5 ( B ), E15.5 ( C ), and E16.5 ( D ). Boxed regions ( middle ) highlight remodeling zones undergoing arteriogenesis in the low-magnification views ( left ). DACH1 was localized to coronary endothelial cells but decreased in arteries as they matured, while VE-cadherin levels were unchanged. ( B ′, C ′, D ′) Quantification of fluorescent intensity. n = 6 hearts per time point. ( E ) Tissue section from an adult heart showing the absence of DACH1 in endothelial cells of large coronary arteries but maintenance of expression in capillaries (arrowheads). ( F ) Representative images of proliferating endothelial cells (arrowheads) in different regions of the coronary vasculature. Colocalization analysis was used to exclude 5-ethynyl-2′-deoxyuridine-positive (EdU + ) staining in nonendothelial cells (see the Materials and Methods). ( G ) Quantification of endothelial proliferation rates shown in F . n = 4 wild-type hearts. Statistical comparisons are between the subepicardial and the indicated regions. ( H ) Endothelial polarity in developing vessels was determined by the orientation of the nuclei (ERG) and Golgi (GOLPH4) relative to the direction of local blood flow from the aorta. Arrows indicate either polarization against flow (green), static/unpolarized (yellow), or polarization with flow (red). ( I ) Quantification of polarity as indicated in H . n = 4 wild-type arteries. (Ao) Aorta; (EC) endothelial cell. Error bars indicate standard deviation. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001. Bars: B , C , 200 µm; D , 300 µm; E , 100 µm; F , 50 µm; H , 25 µm.

Article Snippet: For Dach1 overexpression experiments, pCMV6 mouse Dachshund1 GFP-tagged (transcript variant 1, OriGene Technologies, MG216327) was purchased, and the Dach1 -coding region was subcloned into a lentiviral-ready pLJM-eGFP plasmid (Addgene, plasmid 19319).

Techniques: Expressing, Staining, Standard Deviation

Journal: Genes & Development

Article Title: DACH1 stimulates shear stress-guided endothelial cell migration and coronary artery growth through the CXCL12–CXCR4 signaling axis

doi: 10.1101/gad.301549.117

Figure Lengend Snippet: Endothelial-specific deletion of Dach1 disrupts arterial remodeling and endothelial cell polarization. ( A ) DACH1 was completely eliminated in the endothelial cells of Dach1 eCKO mice. ( B ) Dach1 eCKO coronary arteries recapitulated the smaller arterial diameter phenotype present in those of global Dach1 knockouts. ( C ) Quantification of the diameters of the left (LCA) and right (RCA) main coronary arteries at E17.5. n = 32 control; n = 18 Dach1 eCKO . ( D ) Smooth muscle coverage of the coronary artery was unaffected by endothelial Dach1 deficiency at E17.5. n = 11 control; n = 7 Dach1 eCKO . ( E ) Classification of JAG1 + arterial remodeling zones (early, late, and mature) in whole-mount confocal images at E15.5 during coronary arteriogenesis. Genotypes of hearts are indicated. ( F ) Classifying remodeling zones in wild-type and Dach1 mutant hearts revealed a delay in remodeling that was resolved by E17.5 but resulted in smaller arteries. A remodeling delay was also present in the Dach1 eCKO s at E14.5. The number of hearts classified is shown. ( G ) Representative images of endothelial polarity quantification in mutant coronary arteries. Arrows indicate against flow (green), unpolarized/static (yellow), or with flow (red). ( H ) Quantification of polarity as indicated in G showed a reduction in polarity against flow in Dach1 eCKO arteries. n = 7 control arteries; n = 9 Dach1 eCKO arteries. Statistical comparisons are between the same categories in control and Dach1 eCKO . ( I ) Migration in human coronary artery endothelial cells (HCAECs) was decreased when Dach1 was depleted using lentiviral-delivered Dach1 -specific shRNAs. (Ao) Aorta; (CA) coronary artery; (EC) endothelial cell; (RZ) remodeling zone. Error bars indicate standard deviation. (ns) Nonsignificant; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001. Bars: A , B , E , 200 µm; G , 25 µm.

Article Snippet: For Dach1 overexpression experiments, pCMV6 mouse Dachshund1 GFP-tagged (transcript variant 1, OriGene Technologies, MG216327) was purchased, and the Dach1 -coding region was subcloned into a lentiviral-ready pLJM-eGFP plasmid (Addgene, plasmid 19319).

Techniques: Mutagenesis, Migration, Standard Deviation

Journal: Genes & Development

Article Title: DACH1 stimulates shear stress-guided endothelial cell migration and coronary artery growth through the CXCL12–CXCR4 signaling axis

doi: 10.1101/gad.301549.117

Figure Lengend Snippet: Dach1 overexpression stimulates shear stress-guided endothelial cell behaviors. ( A ) List of GO terms enriched in Dach1 -overexpressing cells, indicating a prominent role in cell motility (highlighted in red). Cell–cell signaling-related terms are in bold italic type. ( B – F ) HCAECs were infected with either Lenti-GFP or Lenti-Dach1-GFP , cultured under uniform laminar shear stress (35 dyn/cm 2 ), and subjected to time-lapse imaging. ( B ) Plotting migration tracks (40 cells per condition) showed that Lenti-Dach1-GFP cells migrated against flow throughout the culture period. ( C ) Migration along the Y -axis and total migration. n = 120 cells from three experiments. ( D ) Still images from time-lapse movies showing that Dach1-GFP cells aligned earlier than controls. ( E ) Rose plots depicting cell alignment angle at different time points following the onset of flow. n = ∼350 cells from one representative experiment of three total experiments. ( F ) Measurements of cell length versus width showing that Dach1 expression enhanced elongation in response to flow. ( G – J ) HCAECs were infected with either Lenti-GFP or Lenti-Dach1-GFP , cultured under uniform laminar shear stress for 72 h, and immunostained to label Golgi and nuclei. ( G ) Schematic showing how cell polarity was quantified using localization of nuclei (blue) and Golgi (red). ( H ) Example of categorization as described in G . Arrows indicate cells oriented against flow (green), static/unpolarized cells (yellow), or cells oriented with flow (red). ( I ) Lenti-Dach1-GFP cells exhibited a significantly increased percentage of cells in the “against flow” group relative to control cells. ( J ) An increased percentage of highly polarized cells was also evident. Error bars indicate standard deviation. (ns) Nonsignificant; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001. Bars: D , 100 µm; H , 50 µm.

Article Snippet: For Dach1 overexpression experiments, pCMV6 mouse Dachshund1 GFP-tagged (transcript variant 1, OriGene Technologies, MG216327) was purchased, and the Dach1 -coding region was subcloned into a lentiviral-ready pLJM-eGFP plasmid (Addgene, plasmid 19319).

Techniques: Over Expression, Infection, Cell Culture, Imaging, Migration, Expressing, Standard Deviation

Journal: Genes & Development

Article Title: DACH1 stimulates shear stress-guided endothelial cell migration and coronary artery growth through the CXCL12–CXCR4 signaling axis

doi: 10.1101/gad.301549.117

Figure Lengend Snippet: The CXCL12–CXCR4 signaling axis is downstream from DACH1. ( A ) RNA sequencing data from whole E17.5 hearts revealed that Cxcl12 was down-regulated in Dach1 knockout animals. n = 3 wild-type hearts; n = 3 knockout hearts. ( B ) RNA sequencing from HCAECs overexpressing Dach1 showed that Cxcl12 was up-regulated relative to controls. n = 3 biological replicates per condition. ( C ) The Cxcl12 reporter gene ( DsRed ) was decreased in Dach1 knockout hearts. Artery widths are indicated by yellow lines. ( D ) Quantification of endogenous DsRed fluorescence. n = 3 wild type; n = 8 heterozygous; n = 5 knockout. ( E ) In situ hybridization for Cxcl12 in Dach1 eCKO hearts showed decreased expression in arterial endothelial cells (arrowheads). n = 9 control; n = 5 Dach1 eCKO . ( F ) CXCR4 inhibition with AMD3100 reduced the migration against flow induced by Dach1 overexpression. n = 120 cells total from three independent experiments. ( G ) AMD3100 reduced the percentage of cells polarized against flow in all conditions. Statistical comparisons comparing the same category of cells between control and drug-treated HCAECs within the same lentiviral treatment. Error bars indicate standard deviation. (ns) Nonsignificant; (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001. Bars, 200 µm.

Article Snippet: For Dach1 overexpression experiments, pCMV6 mouse Dachshund1 GFP-tagged (transcript variant 1, OriGene Technologies, MG216327) was purchased, and the Dach1 -coding region was subcloned into a lentiviral-ready pLJM-eGFP plasmid (Addgene, plasmid 19319).

Techniques: RNA Sequencing Assay, Knock-Out, Fluorescence, In Situ Hybridization, Expressing, Inhibition, Migration, Over Expression, Standard Deviation

Journal: Genes & Development

Article Title: DACH1 stimulates shear stress-guided endothelial cell migration and coronary artery growth through the CXCL12–CXCR4 signaling axis

doi: 10.1101/gad.301549.117

Figure Lengend Snippet: DACH1 is specifically decreased by arterial-type flow. ( A ) Schematic of a parallel plate flow chamber that models arterial-type flow (uniform laminar). ( B ) DACH1 immunofluorescence showed that uniform laminar flow decreased expression compared with static controls. DAPI is in blue. ( C ) Nuclear DACH1 was significantly reduced as shear stress increased. n = 8 or more fields of view (FOVs) per condition from three experiments. ( D ) Schematic of impinging flow chamber that models flow in the early remodeling plexus and vascular branch points (nonuniform gradient). ( E ) DACH1 immunofluorescence was not obviously changed in regions experiencing gradient shear stress. ( F ) Quantification showing that nuclear DACH1 was only mildly reduced in areas of gradient shear stress. n = 14 FOVs per position from seven experiments. ( G ) DACH1 levels plotted as a function of shear stress (SS) in parallel and impinging flow. Error bars indicate standard deviation. (*) P < 0.05; (****) P < 0.0001. Bars, 150 µm.

Article Snippet: For Dach1 overexpression experiments, pCMV6 mouse Dachshund1 GFP-tagged (transcript variant 1, OriGene Technologies, MG216327) was purchased, and the Dach1 -coding region was subcloned into a lentiviral-ready pLJM-eGFP plasmid (Addgene, plasmid 19319).

Techniques: Immunofluorescence, Expressing, Standard Deviation

Journal: Genes & Development

Article Title: DACH1 stimulates shear stress-guided endothelial cell migration and coronary artery growth through the CXCL12–CXCR4 signaling axis

doi: 10.1101/gad.301549.117

Figure Lengend Snippet: DACH1 is high at branch points in vitro and in mouse and human arteries. ( A ) Schematic of the Ibidi Y-shaped chamber slide. Branch points create regions that experience uniform (boxes) and nonuniform (dashed-line boxes) laminar shear stresses. ( B ) Images corresponding to the regions indicated in the schematic. Sites experiencing nonuniform or low shear stress increased nuclear DACH1. ( C ) Quantification of nuclear DACH1 levels at individual sites along the chamber from two independent experiments. P -values were compared with regions 1 and 2. ( D , E ) Immunofluorescence of retinal arteries (RA; dotted lines) of the postnatal eye ( D ) and quantification of fluorescence levels ( E ) showed that DACH1 was significantly higher in endothelial cells specifically at branch points (orange arrowheads in D ; yellow column in E ). n = 10 branches from two animals. ( F ) Immunofluorescence of P0 coronary arteries (CA; dotted lines) showing that DACH1 was up-regulated at branch points (orange arrowheads). ( G , H ) DACH1 was increased at branch points in human coronary arteries. ( G ) En face preparations imaged as shown in the schematic, with the boxed regions in the right panels. ( H ) Quantification of DACH1 fluorescence. n = 8 straight FOVs from four human coronary arteries; n = 16 branch FOVs from four human coronary arteries. ( I ) Working model for the interaction between blood flow and DACH1 expression during arterial remodeling. (EC) Endothelial cell. Error bars indicate standard deviation. (ns) Nonsignificant; (*) P < 0.05; (**) P < 0.01; (****) P < 0.0001. Bars: B , 200 µm; D , 10 µm; F , G , 100 µm.

Article Snippet: For Dach1 overexpression experiments, pCMV6 mouse Dachshund1 GFP-tagged (transcript variant 1, OriGene Technologies, MG216327) was purchased, and the Dach1 -coding region was subcloned into a lentiviral-ready pLJM-eGFP plasmid (Addgene, plasmid 19319).

Techniques: In Vitro, Immunofluorescence, Fluorescence, Expressing, Standard Deviation