Notch1 functions as a negative regulator of lymphatic endothelial cell differentiation in the venous endothelium

Abstract

In development, lymphatic endothelial cells originate within veins and differentiate via a process requiring Prox1. Notch signaling regulates cell-fate decisions, and expression studies suggested that Jag1/Notch1 signaling functions in veins during lymphatic endothelial specification. Using an inducible lymphatic endothelial Prox1CreERT2 driver, Notch signaling was suppressed by deleting Notch1 or expressing dominant-negative Mastermind-like in Prox1+ endothelial cells. Either loss of Notch1 or reduced Notch signaling increased Prox1+ lymphatic endothelial progenitor cell numbers in the veins, leading to incomplete separation of venous and lymphatic vessels. Notch loss of function resulted in excessive Prox1+ lymphatic cells emerging from the cardinal vein and significant lymphatic overgrowth. Moreover, loss of one allele of Notch1 in Prox1 heterozygous mice rescued embryonic lethality due to Prox1 haploinsufficiency and significantly increased Prox1+ lymphatic endothelial progenitor cell numbers. Expression of a constitutively active Notch1 protein in Prox1+ cells suppressed endothelial Prox1 from E9.75 to E13.5, resulting in misspecified lymphatic endothelial cells based upon reduced expression of podoplanin, LYVE1 and VEGFR3. Notch activation resulted in the appearance of blood endothelial cells in peripheral lymphatic vessels. Activation of Notch signaling in the venous endothelium at E10.5 did not arterialize the cardinal vein, suggesting that Notch can no longer promote arterialization in the cardinal vein during this developmental stage. We report a novel role for Notch1 in limiting the number of lymphatic endothelial cells that differentiate from the veins to assure proper lymphatic specification.

DLL4 promotes continuous adult intestinal lacteal regeneration and dietary fat transport

“…Notch signaling regulates many aspects of BEC biology, including arteriovenous differentiation and angiogenesis (36,37). The role of Notch during sprouting lymphangiogenesis remains controversial: Notch signaling blockade was shown to both inhibit and promote lymphatic growth, suggesting a highly context-dependent role of this pathway (38)(39)(40)(41). In BECs, the Notch ligand DLL4 is highly expressed in tip cells, where it regulates sprouting through the induction of Notch signaling in stalk cells (42)(43)(44)(45)(46); however, the expression pattern of DLL4 in the small intestine vasculature has not to our knowledge been studied.…”

Development of the lymphatic system: new questions and paradigms

“…The jagged 1/Notch1 pathway was shown to act as negative regulator of lymphangiogenesis in mice by repressing the Coup-TFII/Prox1 signaling axis (Murtomaki et al, 2013), thereby inducing maintenance of a venous cell identity. In addition, in vitro studies demonstrate that Notch overactivation represses the expression of lymphatic markers via downstream effectors of Notch signaling (Kang et al, 2010).…”

Notch signaling functions in lymphatic valve formation

“…HdLECs isolated from human neonatal foreskins (Columbia IRBAAAB-1700) were maintained in EGM-2MV BulletKit (Lonza) with VEGFC (10 ng/ml, RnD) (Murtomaki et al, 2013). HdLECs were infected with adenovirus expressing constitutively active Notch1 (N1IC) or Notch4 (N4int3), or Hey1, Hey2, lacZ or GFP (Shawber et al, 2007;Tung et al, 2009).…”

“…Notch regulates lymphatic sprouting events in physiological and pathological settings (Niessen et al, 2011;Zheng et al, 2011). We have shown that Notch functions in embryonic venous endothelium to restrict the number of venous ECs that adopt the lymphatic fate (Murtomaki et al, 2013).…”