PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature

Abstract

The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of embryonic blood vascular morphogenesis. However, the molecular mechanisms required for ephrinB2 transduced cellular signaling in vivo have not been characterized. To address this question, we generated two sets of knock-in mice: ephrinB2 ⌬V mice expressed ephrinB2 lacking the C-terminal PDZ interaction site, and ephrinB2 5F mice expressed ephrinB2 in which the five conserved tyrosine residues were replaced by phenylalanine to disrupt phosphotyrosine-dependent signaling events. Our analysis revealed that the homozygous mutant mice survived the requirement of ephrinB2 in embryonic blood vascular remodeling. However, ephrinB2 ⌬V/⌬V mice exhibited major lymphatic defects, including a failure to remodel their primary lymphatic capillary plexus into a hierarchical vessel network, hyperplasia, and lack of luminal valve formation. Unexpectedly, ephrinB2 5F/5F mice displayed only a mild lymphatic phenotype. Our studies define ephrinB2 as an essential regulator of lymphatic development and indicate that interactions with PDZ domain effectors are required to mediate its functions.[Keywords: PDZ; ephrin; Eph; lymphatic valve; collecting lymphatic vessel; smooth muscle] Supplemental material is available at http://www.genesdev.org.

Role for ephrinB2 in postnatal lung alveolar development and elastic matrix integrity

“…ephrinB2 ⌬V/⌬V animals in the Bl/6 background display severe postnatal lymphatic abnormalities including chylothorax (Makinen et al, 2005). However, because ephrinB2 ⌬V/⌬V mutants typically died before 2 weeks of age, a lethality not always seen in other mouse lines with chylothorax (Huang et al, 2000;Ayadi et al, 2001), we reasoned that perhaps these animals had a second problem which contributed to early lethality.…”

“…EphrinB2, a transmembrane protein endowed with its own signaling potential, is expressed on arterial and lymphatic endothelial cells (ECs) as well as perivascular cells, and its receptor, EphB4, is largely confined to venous and lymphatic endothelium (Wang et al, 1998;Adams et al, 1999;Gerety et al, 1999;Makinen et al, 2005) Experiments using gene targeted mice have shown that these proteins are crucial for embryonic sprouting angiogenesis, and together with other family members could contribute greatly to specialization and maturation of vascular beds (Heroult et al, 2006).…”

“…We are studying hypomorphic alleles of ephrinB2, in which mutant cDNAs encoding modified ephrinB2 protein are knocked into the ephrinB2 locus (Makinen et al, 2005;Bouzioukh et al, 2007). The ephrinB2 ⌬V allele (Makinen et al, 2005) specifies ephrinB2 lacking the carboxyterminal valine, thus disturbing a binding site for PDZ domain containing proteins.…”

“…The ephrinB2 ⌬V allele (Makinen et al, 2005) specifies ephrinB2 lacking the carboxyterminal valine, thus disturbing a binding site for PDZ domain containing proteins. Mice homozygous for this allele display major lymphatic defects, including severe skin lymphatic malformations, while the skin blood vascular plexus is only mildly affected.…”

Inhibition of β1 integrin and IL-3Rβ common subunit interaction hinders tumour angiogenesis

“…Besides affecting EPC expansion, IL-3 also induces their arterial differentiation (Zeoli et al, 2008). Indeed, IL-3-cultured EPCs acquired arterial commitment (EphrinB1, EphrinB2 and Notch4 expression), but failed to undergo venous (negative EphB4 expression) or lymphatic (negative vascular endothelial growth factor receptor-3 (VEGFR3) and lymphatic vessel endothelial hyaluronan receptor-1 expression) specification ( Figure 3d) (Gro¨ger et al, 2004;Ma¨kinen et al, 2005). Consequently, the effects of soluble Fc proteins on the expression of specific arterial markers were evaluated.…”