Mechanotransduction activates canonical Wnt/ß-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves.

Lymphatic vasculature regulates fluid homeostasis by returning interstitial fluid to blood circulation. Lymphatic endothelial cells (LECs) are the building blocks of the entire lymphatic vasculature. LECs originate as a homogeneous population of cells predominantly from the embryonic veins and undergo stepwise morphogenesis to become the lymphatic capillaries, collecting vessels or valves. The molecular mechanisms underlying the morphogenesis of the lymphatic vasculature remain to be fully understood. Here we show that canonical Wnt/ß-catenin signaling is necessary for lymphatic vascular morphogenesis. Lymphatic vascular-specific ablation of ß-catenin in mice prevents the formation of lymphatic and lymphovenous valves. Additionally, lymphatic vessel patterning is defective in these mice, with abnormal recruitment of mural cells. We found that oscillatory shear stress (OSS), which promotes lymphatic vessel maturation, triggers Wnt/ß-catenin signaling in LECs. In turn, Wnt/ß-catenin signaling controls the expression of several molecules, including the lymphedema-associated transcription factor FOXC2. Importantly, FOXC2 completely rescues the lymphatic vessel patterning defects in mice lacking ß-catenin. Thus, our work reveals that mechanical stimulation is a critical regulator of lymphatic vascular development via activation of Wnt/ß-catenin signaling and, in turn, FOXC2.

RASA1-driven cellular export of collagen IV is required for the development of lymphovenous and venous valves in mice.

RASA1, a negative regulator of Ras-MAPK signaling, is essential for the development and maintenance of lymphatic vessel valves. However, whether RASA1 is required for the development and maintenance of lymphovenous valves (LVV) and venous valves (VV) is unknown. In this study, we show that induced disruption of Rasa1 in mouse embryos did not affect initial specification of LVV or central VV, but did affect their continued development. Similarly, a switch to expression of a catalytically inactive form of RASA1 resulted in impaired LVV and VV development. Blocked development of LVV was associated with accumulation of the basement membrane protein, collagen IV, in LVV-forming endothelial cells (EC), and could be partially or completely rescued by MAPK inhibitors and drugs that promote collagen IV folding. Disruption of Rasa1 in adult mice resulted in venous hypertension and impaired VV function that was associated with loss of EC from VV leaflets. In conclusion, RASA1 functions as a negative regulator of Ras signaling in EC that is necessary for EC export of collagen IV, thus permitting the development of LVV and the development and maintenance of VV.

YAP and TAZ maintain PROX1 expression in the developing lymphatic and lymphovenous valves in response to VEGF-C signaling.

Lymphatic vasculature is an integral part of digestive, immune and circulatory systems. The homeobox transcription factor PROX1 is necessary for the development of lymphatic vessels, lymphatic valves (LVs) and lymphovenous valves (LVVs). We and others previously reported a feedback loop between PROX1 and vascular endothelial growth factor-C (VEGF-C) signaling. PROX1 promotes the expression of the VEGF-C receptor VEGFR3 in lymphatic endothelial cells (LECs). In turn, VEGF-C signaling maintains PROX1 expression in LECs. However, the mechanisms of PROX1/VEGF-C feedback loop remain poorly understood. Whether VEGF-C signaling is necessary for LV and LVV development is also unknown. Here, we report for the first time that VEGF-C signaling is necessary for valve morphogenesis. We have also discovered that the transcriptional co-activators YAP and TAZ are required to maintain PROX1 expression in LVs and LVVs in response to VEGF-C signaling. Deletion of Yap and Taz in the lymphatic vasculature of mouse embryos did not affect the formation of LVs or LVVs, but resulted in the degeneration of these structures. Our results have identified VEGF-C, YAP and TAZ as a crucial molecular pathway in valve development.

GATA2 controls lymphatic endothelial cell junctional integrity and lymphovenous valve morphogenesis through miR-126.

Mutations in the transcription factor GATA2 cause lymphedema. GATA2 is necessary for the development of lymphatic valves and lymphovenous valves, and for the patterning of lymphatic vessels. Here, we report that GATA2 is not necessary for valvular endothelial cell (VEC) differentiation. Instead, GATA2 is required for VEC maintenance and morphogenesis. GATA2 is also necessary for the expression of the cell junction molecules VE-cadherin and claudin 5 in lymphatic vessels. We identified miR-126 as a target of GATA2, and miR-126-/- embryos recapitulate the phenotypes of mice lacking GATA2. Primary human lymphatic endothelial cells (HLECs) lacking GATA2 (HLECΔGATA2) have altered expression of claudin 5 and VE-cadherin, and blocking miR-126 activity in HLECs phenocopies these changes in expression. Importantly, overexpression of miR-126 in HLECΔGATA2 significantly rescues the cell junction defects. Thus, our work defines a new mechanism of GATA2 activity and uncovers miR-126 as a novel regulator of mammalian lymphatic vascular development.

Correlative Fluorescence and Scanning Electron Microscopy to Study Lymphovenous Valve Development.

Lymph collected from throughout the body is exclusively returned to blood circulation via two pairs of bilaterally located lymphovenous valves. Lymphovenous valves share numerous similarities with lymphatic and venous valves and are defective in multiple mouse models of lymphedema or lymphatic dysfunction. Here we describe a protocol that combines the strengths of fluorescence microscopy and scanning electron microscopy to precisely locate and analyze the topography of developing lymphovenous valves at high resolution.

Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/ß-Catenin Signaling.

Wnt/ß-catenin signaling is necessary for lymphatic vascular development. Oscillatory shear stress (OSS) enhances Wnt/ß-catenin signaling in cultured lymphatic endothelial cells (LECs) to induce expression of the lymphedema-associated transcription factors GATA2 and FOXC2. However, the mechanisms by which OSS regulates Wnt/ß-catenin signaling and GATA2 and FOXC2 expression are unknown. We show that OSS activates autocrine Wnt/ß-catenin signaling in LECs in vitro. Tissue-specific deletion of Wntless, which is required for the secretion of Wnt ligands, reveals that LECs and vascular smooth muscle cells are complementary sources of Wnt ligands that regulate lymphatic vascular development in vivo. Further, the LEC master transcription factor PROX1 forms a complex with ß-catenin and the TCF/LEF transcription factor TCF7L1 to enhance Wnt/ß-catenin signaling and promote FOXC2 and GATA2 expression in LECs. Thus, our work defines Wnt sources, reveals that PROX1 directs cell fate by acting as a Wnt signaling component, and dissects the mechanisms of PROX1 and Wnt synergy.