Multiple mouse models of primary lymphedema exhibit distinct defects in lymphovenous valve development
R. Sathish Srinivasan
Lymph is returned to the blood circulation exclusively via four lymphovenous valves (LVVs). Despite their vital importance, the architecture and development of LVVs is poorly understood. We analyzed the formation of LVVs at the molecular and ultrastructural levels during mouse embryogenesis and identified three critical steps. First, LVV-forming endothelial cells (LVV-ECs) differentiate from PROX1+ progenitors and delaminate from the luminal side of the veins. Second, LVV-ECs aggregate, align perpendicular to the direction of lymph flow and establish lympho-venous connections. Finally, LVVs mature with the recruitment of mural cells. LVV morphogenesis is disrupted in four different mouse models of primary lymphedema and the severity of LVV defects correlate with that of lymphedema. In summary, we have provided the first and the most comprehensive analysis of LVV development. Furthermore, our work suggests that aberrant LVVs contribute to lymphedema.We had previously described several key anatomical landmarks of lymphovenous valves (LVVs) in mouse embryos (Srinivasan and Oliver 2011). These landmarks are schematically shown in Supplementary Figure 1. Arteries and lymphatic valves are excluded from this figure for simplicity. A total of four LVVs are present in mice, with an “LVV-complex” containing two LVVs on either side of the body immediately lateral to the thymic lobules (orange structures). One of these locations is enlarged on the left to show the structures. The internal jugular vein, external jugular vein and subclavian vein merge together into the superior vena cava that drains deoxygenated blood into the right atrium of the heart. Venous valves (VV, depicted in green) guard the entry of veins into the junction. The lymph sac is split into two vessels by an artery just before entering the venous junction via LVVs (magenta). One LVV is located between the subclavian and external jugular veins. The other LVV is located between the external jugular vein and internal jugular vein. Using this information, we characterized the architecture of LVVs at the ultrastructural level.We employed ProxTom lymphatic vessel reporter mice in correlative fluorescence and scanning electron microscopy (SEM) experiments. In these transgenic mice, the Prox1 promoter drives expression of the tdTomato (Tom) transgene, which encodes red fluorescent protein (Truman et al. 2012). We prepared cross sections encompassing the entire junction of veins, including the LVVs, from P0 ProxTom newborn mice (Supplementary Figure 1, between the dotted lines). We performed immunohistochemistry on these sections for the lymphatic endothelial cell (LEC) marker LYVE1 (lymphatic vessel endothelial hyaluronan receptor 1) and for the pan-endothelial cell marker CD31. Subsequently, we visualized the immunostained sections by confocal imaging in the anterior-to-posterior orientation (Ant) to observe the lymph sacs and the upstream side of LVVs. The samples were also analyzed in the posterior-to-anterior orientation (Pos) to observe the venous junction and the downstream side of LVVs and VVs en face.In the Ant orientation, we observed weak Tom and LYVE1 expression in the LECs of the lymph sacs (Supplementary Figure 1 Ant, dotted lines). In contrast, we observed strong Tom expression in VV (Supplementary Figure 1 Ant, arrowhead) and LVVs (Supplementary Figure 1 Ant, white arrows). LYVE1 expression is absent in VVs, but is strongly expressed in LVVs (Supplementary Figure 1 Ant, white arrows). We also observed strong Tom and LYVE1 expressions in the lymphatic valve seen at the junction of a lymphatic vessel draining into the lymph sac (Supplementary Figure 1 Ant, red arrow). The scattered LYVE1+ Tom− cells are macrophages.In the Pos orientation, the site of overlap between Tom+ cells and LYVE1+ cells indicates the LVV, the entry point of the lymph sac into the vein (Supplementary Figure 1 Pos, white arrows). VVs and the lymphatic valve are also seen (Supplementary Figure 1 Pos, arrowheads and red arrow respectively).After identifying the various anatomical structures by fluorescence microscopy as described above, the samples were carefully removed from the slides and processed for SEM. In the Ant orientation, the lymph sacs appear positioned like a bird’s nest at the branch point of the major veins (Figure 1A, pseudo colored in yellow). An artery is seen splitting the lymph sac into two lymphatic vessels. The upstream side of the LVVs is seen as narrow crevices in the middle of these vessels (Figure 1A, white and yellow arrows, the higher magnification picture of a LVV is pseudo colored in magenta). Cells at the entrance of LVVs appear elongated perpendicular to the direction of lymph flow (Figure 1A LVV, arrows). In comparison to LVVs, the VV at the entrance of the external jugular vein has a much wider opening (Figure 1A, pseudo colored in green). A higher magnification picture of this VV is also presented (Figure 1A VV). Cells upstream to the VV in the external jugular vein are rectangular in shape and appear to have aligned parallel to blood flow (Figure 1A VV, arrowheads and Supplementary Figure 2A). This is expected for endothelial cells exposed to laminar blood flow. Arterial endothelial cells are also aligned in the direction of blood flow (Supplementary Figure 2B). In contrast, cells on the upstream side of the VV have a round morphology (Figure 1A VV, arrows). Cells within the superior vena cava, downstream of VVs, also display a round morphology (Supplementary Figure 2A).We were able to identify the downstream side of a lymphatic valve that is located at the entrance of a lymphatic vessel into lymph sacs (Figure 1A, red arrow). At higher magnification (Figure 1A LV, pseudo colored in blue), lymphatic valve cells are aligned perpendicular to the direction of fluid flow.SEM in the Pos orientation revealed the downstream side of VV (Figure 1B, green) and LVVs (Figure 1B, magenta). VVs have longer leaflets compared to LVVs. At higher magnification, cells in the downstream side of VVs and LVVs appear elongated and perpendicular to the direction of fluid flow (Figure 1B, white and yellow arrowheads respectively). At the opening of the LVVs, we observe cloudy aggregates reminiscent of fibrin clots (Figure 1B, red arrowhead).Unlike venous and lymphatic valves, each of which is composed of a homogeneous population of cells, LVVs are formed from a mixed population of endothelial cells: LECs from lymph sacs and the PROX1+LYVE1− LVV-forming endothelial cells (LVV-ECs) from veins (Srinivasan and Oliver 2011). We performed transmission electron microscopy (TEM) using E18.5 embryos, which are developmentally very close to P0 pups, to visualize the interaction between LECs and LVV-ECs. In these sections LVV-ECs with distinct nuclei are seen on the outer side of the valve (Figure 1C, pseudo colored in magenta). However, very few nuclei are seen in the LECs (Figure 1C, pseudo colored in yellow). LECs appear to be profoundly stretched within LVVs. An extracellular matrix compartment is seen separating the LECs and LVV-ECs (Figure 1C).In summary, LVVs share some similarities with VVs and lymphatic valves. Cells on the downstream side of these valves are elongated and arranged perpendicular to the direction of fluid flow. But, some important previously unanticipated differences exist. Most conspicuously, VVs have longer leaflets and larger opening compared to LVVs. And, cells at the upstream entrance of VVs appear cuboidal whereas the cells at the entrance of LVVs appear elongated.We thank Dr. Guillermo Oliver and St. Jude Children’s research hospital for generous support during the early stages of this work. We thank Drs. Rodger McEver and Lorin Olson for critical reading of the manuscript and for insightful comments, Dr. Ying Yang for vibratome sectioning and staining protocol, Mr. Michael McDaniel for help with confocal microscopy, Dr. Jane Song for data analysis using ImageJ and GraphPad Prism, Dr. Pierre Chambon for CreERT2 cDNA, Ms. Lisa Whitworth (Microscopy Laboratory, Oklahoma State University, Stillwater) for SEM and Pickersgill & Andersen, Life Science Editors, for editorial assistance. RSS is supported by institutional funds of OMRF, Oklahoma Center for Adult Stem Cell Research (OCASCR, 4340), American Heart Association (15BGIA25710032) and NIH/NIGMS COBRE (P20 GM103441 PI: Dr. McEver).Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. 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