Segregated Foxc2, NFATc1 and Connexin expression at normal developing venous valves, and Connexin-specific differences in the valve phenotypes of Cx37, Cx43, and Cx47 knockout mice

Abstract

Venous valves (VVs) are critical for unidirectional blood flow from superficial and deep veins towards the heart. Congenital valve aplasia or agenesis may, in some cases, be a direct cause of vascular disease, motivating an understanding of the molecular mechanisms underlying the development and maintenance of VVs. Three gap junction proteins (Connexins), Cx37, Cx43, and Cx47, are specifically expressed at VVs in a highly polarized fashion. VVs are absent from adult mice lacking Cx37; however it is not known if Cx37 is required for the initial formation of valves. In addition, the requirement of Cx43 and Cx47 for VV development has not been studied. Here, we provide a detailed description of Cx37, Cx43, and Cx47 expression during mouse vein development and show by gene knockout that each Cx is necessary for normal valve development. The valve phenotypes in the knockout lines exhibit Cx-specific differences, however, including whether peripheral or central VVs are affected by gene inactivation. In addition, we show that a Cx47 null mutation impairs peripheral VV development but does not affect lymphatic valve formation, a finding of significance for understanding how some CX47 mutations cause inherited lymphedema in humans. Finally, we demonstrate a striking segregation of Foxc2 and NFATc1 transcription factor expression between the downstream and upstream faces, respectively, of developing VV leaflets and show that this segregation is closely associated with the highly polarized expression of Cx37, Cx43, and Cx47. The partition of Foxc2 and NFATc1 expression at VV leaflets makes it unlikely that these factors directly cooperate during the leaflet elongation stage of VV development.

Defective lymphatic valve development and chylothorax in mice with a lymphatic-specific deletion of Connexin43

“…In this study, we noted that Cx47 levels were greatly reduced in thoracic duct valves when Cx43 was eliminated from LECs, suggesting that the expression, assembly, or targeting of Cx47 may depend on Cx43. Furthermore, a Cx47 null mutation alone does not cause LV defects or lymphedema in mice, but a deficiency in both Cx47 and Cx43 does lead to mild lymphedema in some embryos, consistent with a dominant negative model (Munger et al, 2016). Thus, our study not only demonstrates that LEC Cx43 is required for normal development of LVs, it also raises the possibility that a dominant negative Cx47 mutation associated with inherited lymphedema, if it inhibits Cx43, might disrupt lymphatic function by negatively affecting LV development and function.…”

“…A similar role for Cx43 may occur during venous valve development (Munger et al, 2016). In cardiac neural crest cells and embryonic cortical neurons, Cx43 is required for directed cell migration, raising the possibility that Cx43 could play an analogous role during valve formation (Kameritsch et al, 2011; Matsuuchi and Naus, 2012; Kotini and Mayor, 2015).…”

“…However, in contrast to Cx37, which in cultured LECs is jointly regulated by oscillating shear stress and the forkhead transcription factor Foxc2, Cx43 expression is repressed by oscillating shear stress and is not dependent on Foxc2, indicating that the two family members are regulated by different signaling pathways (Sabine et al, 2012). During embryogenesis, there is widespread early expression of Cxs in the lymphatic as well as venous endothelium (Kanady et al, 2011; Munger et al, 2016). As development proceeds, Cx37 and Cx43 become highly enriched at valves but are differentially expressed on the two sides of the valve leaflet, with Cx43 present on the upstream face of the valve leaflet and Cx37 on the downstream face (Kanady et al, 2011; Munger et al, 2016).…”

“…During embryogenesis, there is widespread early expression of Cxs in the lymphatic as well as venous endothelium (Kanady et al, 2011; Munger et al, 2016). As development proceeds, Cx37 and Cx43 become highly enriched at valves but are differentially expressed on the two sides of the valve leaflet, with Cx43 present on the upstream face of the valve leaflet and Cx37 on the downstream face (Kanady et al, 2011; Munger et al, 2016). …”

“…Brachial veins and superficial caudal epigastric veins were examined for venous valves using a dissecting microscope, as previously described (Munger et al, 2016). …”