Combining Foxc2 and Connexin37 deletions in mice leads to severe defects in lymphatic vascular growth and remodeling.

Connexins (Cxs), proteins that are vital for intercellular communication in vertebrates, have recently been shown to play a critical role in lymphatic development. However, our knowledge is currently limited regarding the functional relationships of Cxs with other proteins and signaling pathways. Cell culture studies have shown that Cx37 is necessary for coordinated activation of the transcription factor NFATc1, which cooperates with Foxc2 (another transcription factor) during lymphatic endothelial development. These data suggest that Cxs, Foxc2, and NFATc1 are part of a common developmental pathway. Here, we present a detailed characterization of Foxc2(+/-)Cx37(-/-) mice, demonstrating that lymphatic network architecture and valve formation rely on the concurrent embryonic expression and function of Foxc2 and Cx37. Foxc2(+/-)Cx37(-/-) mice have lymphedema in utero, exhibit craniofacial abnormalities, show severe dilation of intestinal lymphatics, display abnormal lacteal development, lack lymphatic valves, and typically die perinatally (outcomes not seen in Foxc2(+/-) or Cx37(-/-) mice separately). We provide a rigorous, quantitative documentation of lymphatic vascular network changes that highlight the specific structural alterations that occur in Foxc2(+/-)Cx37(-/-) mice. These data provide further evidence suggesting that Foxc2 and Cx37 are elements in a common molecular pathway directing lymphangiogenesis.

Absence of venous valves in mice lacking Connexin37.

Venous valves play a crucial role in blood circulation, promoting the one-way movement of blood from superficial and deep veins towards the heart. By preventing retrograde flow, venous valves spare capillaries and venules from being subjected to damaging elevations in pressure, especially during skeletal muscle contraction. Pathologically, valvular incompetence or absence of valves are common features of venous disorders such as chronic venous insufficiency and varicose veins. The underlying causes of these conditions are not well understood, but congenital venous valve aplasia or agenesis may play a role in some cases. Despite progress in the study of cardiac and lymphatic valve morphogenesis, the molecular mechanisms controlling the development and maintenance of venous valves remain poorly understood. Here, we show that in valved veins of the mouse, three gap junction proteins (Connexins, Cxs), Cx37, Cx43, and Cx47, are expressed exclusively in the valves in a highly polarized fashion, with Cx43 on the upstream side of the valve leaflet and Cx37 on the downstream side. Surprisingly, Cx43 expression is strongly induced in the non-valve venous endothelium in superficial veins following wounding of the overlying skin. Moreover, we show that in Cx37-deficient mice, venous valves are entirely absent. Thus, Cx37, a protein involved in cell-cell communication, is one of only a few proteins identified so far as critical for the development or maintenance of venous valves. Because Cxs are necessary for the development of valves in lymphatic vessels as well, our results support the notion of common molecular pathways controlling valve development in veins and lymphatic vessels.

Connexin37 and Connexin43 deficiencies in mice disrupt lymphatic valve development and result in lymphatic disorders including lymphedema and chylothorax.

Intraluminal valves are required for the proper function of lymphatic collecting vessels and large lymphatic trunks like the thoracic duct. Despite recent progress in the study of lymphvasculogenesis and lymphangiogenesis, the molecular mechanisms controlling the morphogenesis of lymphatic valves remain poorly understood. Here, we report that gap junction proteins, or connexins (Cxs), are required for lymphatic valvulogenesis. Cx37 and Cx43 are expressed early in mouse lymphatic development in the jugular lymph sacs, and later in development these Cxs become enriched and differentially expressed by lymphatic endothelial cells on the upstream and downstream sides of the valves. Specific deficiencies of Cx37 and Cx43 alone or in combination result in defective valve formation in lymphatic collecting vessels, lymphedema, and chylothorax. We also show that Cx37 regulates jugular lymph sac size and that both Cx37 and Cx43 are required for normal thoracic duct development, including valve formation. Another Cx family member, Cx47, whose human analog is mutated in some families with lymphedema, is also highly enriched in a subset of endothelial cells in lymphatic valves. Mechanistically, we present data from Foxc2-/- embryos suggesting that Cx37 may be a target of regulation by Foxc2, a transcription factor that is mutated in human lymphedema-distichiasis syndrome. These results show that at least three Cxs are expressed in the developing lymphatic vasculature and, when defective, are associated with clinically manifest lymphatic disorders in mice and man.