Hemogenic Endothelium within the Zebrafish Caudal Hematopoietic Tissue illustrates the Common Ties of the Vascular and Hematopoietic Systems

Hemogenic endothelium involves the specification of a hematopoietic stem cell (HSC) from an existing endothelial cell. It, along with common developmental origins, co-regulation, and shared niches are examples of the close ties the hematopoietic and endothelial lineages share in development. As a significant portion of total HSCs are generated via a hemogenic endothelium intermediate, modulation of this pathway is expected to impact both hematopoietic and endothelial development. Currently, our understanding of how endothelial cells transition to the HSC lineage is still limited. We found that non-aortic endothelial cells, specifically venous endothelial cells, are capable of undergoing the transition to an HSC lineage, suggesting that the hemogenic capacity is a more general characteristic of endothelial cells then previously appreciated. When we analyzed these cells further, we found that they were positive for venous specific markers and well as hematopoietic transcription factors. Further, we identified a potential homolog of Platelet Endothelial Cell Adhesion Molecule 1 (PECAM1) in zebrafish and characterized its expression during development. We found that this molecule is expressed in a manner consistent with PECAM1 and is involved in flow-dependent process. Finally, we find that loss of this PECAM1-like molecule is capable of modulating hematopoiesis, suggesting that the vascular and hematopoietic share common machinery responsible for the observed response to blood flow.Doctor of Philosoph

Mutant-specific gene expression profiling identifies SRY-related HMG box 11b (SOX11b) as a novel regulator of vascular development in zebrafish

Previous studies have identified two zebrafish mutants, cloche and groom of cloche, which lack the majority of the endothelial lineage at early developmental stages. However, at later stages, these avascular mutant embryos generate rudimentary vessels, indicating that they retain the ability to generate endothelial cells despite this initial lack of endothelial progenitors. To further investigate molecular mechanisms that allow the emergence of the endothelial lineage in these avascular mutant embryos, we analyzed the gene expression profile using microarray analysis on isolated endothelial cells. We find that the expression of the genes characteristic of the mesodermal lineages are substantially elevated in the kdrl+ cells isolated from avascular mutant embryos. Subsequent validation and analyses of the microarray data identifies Sox11b, a zebrafish ortholog of SRY-related HMG box 11 (SOX11), which have not previously implicated in vascular development. We further define the function sox11b during vascular development, and find that Sox11b function is essential for developmental angiogenesis in zebrafish embryos, specifically regulating sprouting angiogenesis. Taken together, our analyses illustrate a complex regulation of endothelial specification and differentiation during vertebrate development

Vascular endothelial growth factor signaling regulates the segregation of artery and vein via ERK activity during vascular development

Segregation of two axial vessels, the dorsal aorta and caudal vein, is one of the earliest patterning events occur during development of vasculature. Despite the importance of this process and recent advances in our understanding on vascular patterning during development, molecular mechanisms that coordinate the segregation of axial vessels remain largely elusive. In this report, we find that Vascular Endothelial Growth Factor-A (Vegf-A) signaling regulates the segregation of dorsal aorta and axial vein during development. Inhibition of Vegf-A pathway components including ligand Vegf-A and its cognate receptor Kdrl, caused failure in segregation of axial vessels in zebrafish embryos. Similarly, chemical inhibition of Mitogen-activated protein kinase kinase (Map2k1)/Extracellular-signal-regulated kinases (Erk) and Phosphatidylinositol 3-kinases (PI3K), which are downstream effectors of Vegf-A signaling pathway, led to the fusion of two axial vessels. Moreover, we find that restoring Erk activity by over-expression of constitutively active MEK in embryos with a reduced level of Vegf-A signaling can rescue the defects in axial vessel segregation. Taken together, our data show that segregation of axial vessels requires the function of Vegf-A signaling, and Erk may function as the major downstream effector in this process

Inter-Cellular Exchange of Cellular Components via VE-Cadherin-Dependent Trans-Endocytosis

<div><p>Cell-cell communications typically involve receptor-mediated signaling initiated by soluble or cell-bound ligands. Here, we report a unique mode of endocytosis: proteins originating from cell-cell junctions and cytosolic cellular components from the neighboring cell are internalized, leading to direct exchange of cellular components between two adjacent endothelial cells. VE-cadherins form transcellular bridges between two endothelial cells that are the basis of adherence junctions. At such adherens junction sites, we observed the movement of the entire VE-cadherin molecule from one endothelial cell into the other with junctional and cytoplasmic components. This phenomenon, here termed trans-endocytosis, requires the establishment of a VE-cadherin homodimer <i>in trans</i> with internalization proceeding in a Rac1-, and actomyosin-dependent manner. Importantly, the trans-endocytosis is not dependent on any known endocytic pathway including clathrin-dependent endocytosis, macropinocytosis or phagocytosis. This novel form of cell-cell communications, leading to a direct exchange of cellular components, was observed in 2D and 3D-cultured endothelial cells as well as in the developing zebrafish vasculature.</p></div

VEC trans-endocytosis is Rac1 dependent.

<p>(<b>A</b>) Co-culture of HUVECs expressing VEC-EGFP and HUVECs expressing iRFP with 100 µM of NSC23766. NSC23766, a specific inhibitor for Rac1 activation, inhibited trans-endocytosis of VEC-EGFP, though cell-cell junctions remained intact. Arrow shows intact cell-cell junction. Cells were first pre-treated for an hour with 100 µM of NSC23766 before co-culture, then were mixed and incubated for 4 hours with 100 µM of NSC23766. Scale bar  = 10 µm. (<b>B</b>) Quantitative analysis of the number of trans-endocytosed structures with Rac1 or Cdc42 inhibitors. ML141, a specific inhibitor of Cdc42, and NSC23766 were used at concentrations around their IC₅₀ values. The IC₅₀ values of ML141 and NSC23766 are 2.6 µM and 50 µM, respectively. NSC23766 inhibited trans-endocytosis of VEC in a dose-dependent manner. The number of trans-endocytosed structures was counted for over 10-13 different fields of view per time point; n = 24-35 (ML141) and n = 19-38 (NSC23766). *, p<0.01 vs DMSO. Data were expressed as mean ± SD. (<b>C</b>) Co-culture of HUVECs expressing PA-Rac1-CA and HUVECs expressing VEC-EGFP. PA-Rac1-CA was accumulated at cell-cell junctions and co-localized with VEC-EGFP after its activation at 0 time point. Arrowheads show co-localization of PA-Rac1-CA with VEC-EGFP at cell-cell junctions. Scale bar  = 10 µm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090736#pone.0090736.s010" target="_blank">Movie S4</a>. (<b>D</b>) Co-culture of HUVECs expressing PA-Rac1-DN and HUVECs expressing VEC-EGFP. PA-Rac1-DN was not accumulated at cell-cell junctions, nor co-localized with VEC-EGFP after its activation at 0 time point. Scale bar  = 10 µm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090736#pone.0090736.s010" target="_blank">Movie S4</a>.</p

VEC trans-endocytosis mediates transport of junctional proteins.

<p>(<b>A</b>) Co-culture of HUVECs expressing VEC-Y658E-EGFP and HUVECs expressing VEC-Y658F-TagRFPT. Arrow heads show VEC-Y658F-TagRFPT molecules were trans-endocytosed by VEC-Y658E-EGFP expressing cells. (<b>B</b>) Co-culture of HUVECs expressing p120-EGFP and HUVECs expressing VEC-TagRFPT. Arrow shows p120-EGFP molecules were trans-endocytosed by VEC-TagRFPT expressing cells. (<b>C</b>) Co-culture of HUVECs expressing β-catenin-EGFP and HUVECs expressing VEC-TagRFPT. Arrows show β-catenin -EGFP molecules were trans-endocytosed by VEC-TagRFPT expressing cells. (<b>D</b>) Co-culture of HUVECs expressing VEC-EGFP and HUVECs expressing VEC/α-catenin-FLAG. Arrowheads show VEC/α-catenin-FLAG molecules were trans-endocytosed by VEC-EGFP expressing cells. (<b>E</b>) Co-culture of HUVECs expressing EGFP and HUVECs expressing VEC-TagRFPT. Arrow shows EGFP molecules were trans-endocytosed and co-localized with VEC-TagRFPT in the recipient cell. (<b>F</b>) Co-culture of HUVECs labeled with iRFP and VEC-EGFP expressing HUVECs transfected with Cy3-labeled scramble siRNA. Arrowhead shows siRNA molecules were trans-endocytosed and co-localized with VEC-EGFP in the recipient cell. (<b>A–F</b>) Lower images are higher magnification of the indicated area in upper images. Scale bars  = 20 µm, upper images; 5 µm, lower images.</p

VEC molecules are internalized by adjacent cells via trans-endocytosis.

<p>(<b>A</b>) Co-culture of primary microvascular endothelial cells from mice lungs. The primary microvascular endothelial cells were isolated separately from VEC-EGFP knock-in mice and Rosa26-mTmG mice expressing mTomato fluorescent protein in all cells and fixed after 24 hours of the co-culture. The trans-endocytosed VEC-EGFP molecules in mTomato fluorescent protein expressing cells were stained by anti-GFP antibody without (upper images) and with permeabilization (lower images). (<b>B</b>) Higher magnification of images of the indicated area in A. Arrows in upper images show VEC-EGFP molecule could not be stained by anti-GFP antibody in non-permeabilized cells. Arrowheads in lower images show VEC-EGFP molecule stained by anti-GFP antibody in permeabilized cells. (<b>C</b>) Co-culture of HUVECs expressing VEC-EGFP and HUVECs expressing VEC-TagRFPT. Trans-endocytosis of VEC occurred in HUVECs (scale bar  = 20 µm). (<b>D</b>) Higher magnification of the indicated area in C. Arrow shows VEC-EGFP molecules were trans-endocytosed by VEC-TagRFPT expressing cells. Asterisks show the extended filopodia or adherens junctions from the neighboring cell. Arrow heads show VEC-TagRFPT molecules were trans-endocytosed by VEC-EGFP expressing cells. Scale bar  = 5 µm.</p

VEC trans-endocytosis occurs in sprouting HUVECs and endothelial cells in zebrafish embryos.

<p>(<b>A</b>) A single z-plane, x-z and y-z cross-sectional images of the tube-like structure of sprouting HUVECs. HUVECs expressing VEC-EGFP and HUVECs expressing VEC-TagRFPT were co-cultured in three-dimension fibrin gels. The Z-stack images were taken at the connection between HUVECs expressing VEC-EGFP and HUVECs expressing VEC-TagRFPT. Images were collected at 0.3 µm intervals with the 488 nm and 561 nm lasers to create a stack in the Z axis with a 60x objective. Scale bar  = 20 µm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090736#pone.0090736.s011" target="_blank">Movie S5</a>. (<b>B</b>) Higher magnification of a 3-dimensional projection image of the indicated area in A. Arrows show VEC-EGFP molecules were trans-endocytosed by HUVECs expressing VEC-TagRFPT. Scale bar  = 10 µm. (<b>C</b>) A three dimensional projection image, x-z and y-z cross-sectional images of the connection between the dorsal longitudinal anastomotic vessel (DLAV) and an intersegmental vessel (ISV) of a zebrafish embryo. Zebrafish VEC-EGFP (zVEC-EGFP) plasmids were injected into Tg(flkl:myr-mCherry) zebrafish using Tol2 system for transient mosaic expression of zVEC-EGFP. Images were collected at 1 µm intervals using the 488 nm and 561 nm lasers to create a stack in the Z axis with a 60x objective. Scale bar  = 5 µm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090736#pone.0090736.s012" target="_blank">Movie S6</a>. (<b>D</b>) Sequential 3-dimensional projection image of the zebrafish vessel in C. Arrows show a zVEC-EGFP positive structure budding to inside of the endothelial cell. Scale bars  = 5 µm.</p

VEC trans-endocytosis is not dependent on known endocytic pathway.

<p>(<b>A</b>) Co-culture of HUVECs expressing VEC-EGFP and HUVECs expressing VEC-TagRFPT, then stained with anti-Rab5 antibody. The trans-endocytosed VEC molecules by the adjacent cell co-localized with Rab5 in the recipient cells to a low extent. Lower images are higher magnification of the indicated area in upper images. Scale bars  = 20 µm, upper images; 5 µm, lower images. (<b>B</b>) Quantitative analysis of the number of trans-endocytosed VEC molecules co-localized with Rab5 in the recipient cells. About 15–20% of trans-endocytosed VEC molecules co-localized with Rab5 in the recipient cells. The percentage of Rab5 co-localization with VEC-TagRFPT was counted over 6-9 different fields of view for each time point; n = 6 (3 h), n = 8 (5 h) and n = 10 (7 h). Data were expressed as mean ± SD. (<b>C</b>) Co-culture of COS7 cells expressing VEC-EGFP and COS7 cells expressing VEC-TagRFPT with or without various inhibitors. Arrows show the trans-endocytosed VEC-EGFP molecules by VEC-TagRFPT expressing cells. Arrowheads show trans-endocytosed VEC-TagRFPT molecules by VEC-EGFP expressing cells. The trans-endocytosis occurred even with several inhibitors for clathrin-dependent endocytosis, macropinocytosis or phagocytosis. Dynasore (20 µM), an inhibitor for clathrin/dynamin dependent endocytosis, 5-(N-ethyl-N-isopropyl) amiloride (25 µM), the macropinocytosis inhibitor, Y27632 (10 µM), ROCK inhibitor, and bafilomycin A1 (200 nM), a specific inhibitor of the vacuolar type H(+)-ATPase for phagocytosis were used. Scale bars  = 20 µm. (<b>D</b>) Quantification of the number of trans-endocytosis positive cells with indicated inhibitors. The percentage of trans-endocytosis positive cells was counted over 10 different fields of view for each inhibitor; n = 88 (DMSO), n = 65 (dynasore), n = 57 (amiloride), n = 63 (Y27632) and n = 57 (bafilomycin A1). Data were expressed as mean ± SD.</p