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EphB4 forward signalling regulates lymphatic valve development.

Zhang G, Brady J, Liang WC, Wu Y, Henkemeyer M, Yan M - Nat Commun (2015)

Bottom Line: Here we show that EphB4-dependent forward signalling regulates lymphatic valve development, a process previously thought to be regulated by ephrinB2-dependent reverse signalling.We develop antibodies that selectively target EphB4 and ephrinB2.Furthermore, a chemical genetic approach is used to unequivocally show that the kinase activity of EphB4 is essential for lymphatic valve development.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Division of Research, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

ABSTRACT
Bidirectional signalling is regarded as a notable hallmark of the Eph-ephrin signalling system: Eph-dependent forward signalling in Eph-expressing cells and ephrin-dependent reverse signalling in Ephrin-expressing cells. The notion of ephrin-dependent reverse signalling derives from genetic experiments utilizing mice carrying mutations in the intracellular region of ephrinBs. Here we show that EphB4-dependent forward signalling regulates lymphatic valve development, a process previously thought to be regulated by ephrinB2-dependent reverse signalling. We develop antibodies that selectively target EphB4 and ephrinB2. We find that mice bearing genetically altered cytoplasmic region of ephrinB2 have significantly altered EphB4-dependent forward signalling. Selective inhibition of EphB4 using a functional blocking antibody results in defective lymphatic valve development. Furthermore, a chemical genetic approach is used to unequivocally show that the kinase activity of EphB4 is essential for lymphatic valve development.

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Lymphatic valves are present in ephrinB2lacZ/lacZ mice.(a) Visualization of mesenteric lymphatic vessels and valves by immunostaining for Prox-1 in E18 embryos. Scale bar, 500 μm. Right panel, quantification of lymphatic valves, two-tailed, unpaired student's t-test, n=3 per genotype (error bars, s.d.). (b) Elevated EphB4 phosphorylaiton in ephrinB2lacZ/lacZ embryos. Total tissue lysates from E12.5 embryos were subjected to phospho-EphB4 and total EphB4 immunoblotting analysis. Ratios of pEphB4:total EphB4 are graphed. **P<0.01 (two-tailed, unpaired student's t-test), n=6 for ephrinB2+/+, n=4 for ephrinB2lacZ/lacZ, error bars, s.d. NS, not significant.
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f5: Lymphatic valves are present in ephrinB2lacZ/lacZ mice.(a) Visualization of mesenteric lymphatic vessels and valves by immunostaining for Prox-1 in E18 embryos. Scale bar, 500 μm. Right panel, quantification of lymphatic valves, two-tailed, unpaired student's t-test, n=3 per genotype (error bars, s.d.). (b) Elevated EphB4 phosphorylaiton in ephrinB2lacZ/lacZ embryos. Total tissue lysates from E12.5 embryos were subjected to phospho-EphB4 and total EphB4 immunoblotting analysis. Ratios of pEphB4:total EphB4 are graphed. **P<0.01 (two-tailed, unpaired student's t-test), n=6 for ephrinB2+/+, n=4 for ephrinB2lacZ/lacZ, error bars, s.d. NS, not significant.

Mentions: The ephrinB2lacZ allele, in which the entire cytoplasmic domain of ephrinB2 is replaced by β-gal, has also been used to interrogate the functional importance of ephrinB2-dependent reverse signalling2028. As ephrinB2lacZ/lacZ mice suffer perinatal lethality, we examined the lymphatic valves in E18 embryos. To our surprise, the lymphatic valves were abundantly present (Fig. 5a), which is in sharp contrast to ephrinB26YFΔV/6YFΔV mice that completely lack the lymphatic valves. In addition, ephrinB2lacZ/lacZ mice do not phenocopy ephrinB26YFΔV/6YFΔV mice with respect to midline development29. As both ephrinB26YFΔV/6YFΔV and ephrinB2lacZ/lacZ mice lack the critical signalling elements in the cytoplasmic region of ephrinB2, their distinct phenotypes argue against the view that compromised ephrinB2-dependent reverse signalling is the underlying cause of the observed developmental defects.


EphB4 forward signalling regulates lymphatic valve development.

Zhang G, Brady J, Liang WC, Wu Y, Henkemeyer M, Yan M - Nat Commun (2015)

Lymphatic valves are present in ephrinB2lacZ/lacZ mice.(a) Visualization of mesenteric lymphatic vessels and valves by immunostaining for Prox-1 in E18 embryos. Scale bar, 500 μm. Right panel, quantification of lymphatic valves, two-tailed, unpaired student's t-test, n=3 per genotype (error bars, s.d.). (b) Elevated EphB4 phosphorylaiton in ephrinB2lacZ/lacZ embryos. Total tissue lysates from E12.5 embryos were subjected to phospho-EphB4 and total EphB4 immunoblotting analysis. Ratios of pEphB4:total EphB4 are graphed. **P<0.01 (two-tailed, unpaired student's t-test), n=6 for ephrinB2+/+, n=4 for ephrinB2lacZ/lacZ, error bars, s.d. NS, not significant.
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f5: Lymphatic valves are present in ephrinB2lacZ/lacZ mice.(a) Visualization of mesenteric lymphatic vessels and valves by immunostaining for Prox-1 in E18 embryos. Scale bar, 500 μm. Right panel, quantification of lymphatic valves, two-tailed, unpaired student's t-test, n=3 per genotype (error bars, s.d.). (b) Elevated EphB4 phosphorylaiton in ephrinB2lacZ/lacZ embryos. Total tissue lysates from E12.5 embryos were subjected to phospho-EphB4 and total EphB4 immunoblotting analysis. Ratios of pEphB4:total EphB4 are graphed. **P<0.01 (two-tailed, unpaired student's t-test), n=6 for ephrinB2+/+, n=4 for ephrinB2lacZ/lacZ, error bars, s.d. NS, not significant.
Mentions: The ephrinB2lacZ allele, in which the entire cytoplasmic domain of ephrinB2 is replaced by β-gal, has also been used to interrogate the functional importance of ephrinB2-dependent reverse signalling2028. As ephrinB2lacZ/lacZ mice suffer perinatal lethality, we examined the lymphatic valves in E18 embryos. To our surprise, the lymphatic valves were abundantly present (Fig. 5a), which is in sharp contrast to ephrinB26YFΔV/6YFΔV mice that completely lack the lymphatic valves. In addition, ephrinB2lacZ/lacZ mice do not phenocopy ephrinB26YFΔV/6YFΔV mice with respect to midline development29. As both ephrinB26YFΔV/6YFΔV and ephrinB2lacZ/lacZ mice lack the critical signalling elements in the cytoplasmic region of ephrinB2, their distinct phenotypes argue against the view that compromised ephrinB2-dependent reverse signalling is the underlying cause of the observed developmental defects.

Bottom Line: Here we show that EphB4-dependent forward signalling regulates lymphatic valve development, a process previously thought to be regulated by ephrinB2-dependent reverse signalling.We develop antibodies that selectively target EphB4 and ephrinB2.Furthermore, a chemical genetic approach is used to unequivocally show that the kinase activity of EphB4 is essential for lymphatic valve development.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Division of Research, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

ABSTRACT
Bidirectional signalling is regarded as a notable hallmark of the Eph-ephrin signalling system: Eph-dependent forward signalling in Eph-expressing cells and ephrin-dependent reverse signalling in Ephrin-expressing cells. The notion of ephrin-dependent reverse signalling derives from genetic experiments utilizing mice carrying mutations in the intracellular region of ephrinBs. Here we show that EphB4-dependent forward signalling regulates lymphatic valve development, a process previously thought to be regulated by ephrinB2-dependent reverse signalling. We develop antibodies that selectively target EphB4 and ephrinB2. We find that mice bearing genetically altered cytoplasmic region of ephrinB2 have significantly altered EphB4-dependent forward signalling. Selective inhibition of EphB4 using a functional blocking antibody results in defective lymphatic valve development. Furthermore, a chemical genetic approach is used to unequivocally show that the kinase activity of EphB4 is essential for lymphatic valve development.

Show MeSH
Related in: MedlinePlus