Limits...
EphrinB2 affects apical constriction in Xenopus embryos and is regulated by ADAM10 and flotillin-1.

Ji YJ, Hwang YS, Mood K, Cho HJ, Lee HS, Winterbottom E, Cousin H, Daar IO - Nat Commun (2014)

Bottom Line: The Eph/ephrin signalling pathways have a critical function in cell adhesion and repulsion, and thus play key roles in various morphogenetic events during development.Here we show that a decrease in ephrinB2 protein causes neural tube closure defects during Xenopus laevis embryogenesis.This dramatic decline in ephrinB2 protein levels on the absence of flotillin-1 expression is specific, and is partly the result of an increased susceptibility to cleavage by the metalloprotease ADAM10.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell and Developmental Signaling, National Cancer Institute-Frederick, Frederick, Maryland 21702, USA.

ABSTRACT
The Eph/ephrin signalling pathways have a critical function in cell adhesion and repulsion, and thus play key roles in various morphogenetic events during development. Here we show that a decrease in ephrinB2 protein causes neural tube closure defects during Xenopus laevis embryogenesis. Such a decrease in ephrinB2 protein levels is observed on the loss of flotillin-1 scaffold protein, a newly identified ephrinB2-binding partner. This dramatic decline in ephrinB2 protein levels on the absence of flotillin-1 expression is specific, and is partly the result of an increased susceptibility to cleavage by the metalloprotease ADAM10. These findings indicate that flotillin-1 regulates ephrinB2 protein levels through ADAM10, and is required for appropriate neural tube morphogenesis in the Xenopus embryo.

Show MeSH

Related in: MedlinePlus

Loss of ADAM10 rescues F1aMO-induced neural tube defects, while over-expression of ADAM10 causes neural tube defects(a) Knockdown of ADAM10 rescues neural tube closure defects induced by F1aMO. Two dorsal cells of four cell stage embryos were injected with Alexa 488 conjugated dextran (green) and the F1aMO alone or an ADAM10 MO, or both. Presented are dorsal view images with light and fluorescent microscopy. (b) The gap width of the neural tubes in the embryos injected with the indicated MOs in (a) were measured and the average gap width and s.d. are presented in the histogram. (c) Overexpression of ADAM10 causes neural tube closure defects. Dorsal view with light and fluorescent microscopy of embryos that were injected with Alexa 488 conjugated dextran (green - tracer) alone, or with ADAM10-V5 RNA, or RNA encoding an ADAM10 mutant with compromised protease activity (ADAM10 PD-V5). (d) The width of the neural tubes in the embryos injected with the indicated reagents in (c) were measured and the average width and s.d. are presented in the histogram. (e) Western blot of lysates from the injected embryos in (c), probed with V5 antibody. (f) An ephrinB2 mutant that is resistant to ADAM10 cleavage rescues F1aMO-induced neural tube defects. Dorsal view of embryos that were injected with either control MO or F1aMO and RNA encoding flotillin-1a-Flag, or high levels of ephrinB2-HA (1.6 ng/embryo), or low levels of cleavage-resistant ephrinB2Δ197–218--HA (0.4 ng/embryo). (g) The gap widths of the neural tubes in the embryos injected with the indicated reagents in (f) were measured and the average width and s.d. are presented in the histogram. (h) Western analysis of the embryonic lysates from (f) using the indicated antibodies to show expression of flotillin-1-Flag, ephrinB2-HA, and ephrinB2Δ197–218.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4120273&req=5

Figure 10: Loss of ADAM10 rescues F1aMO-induced neural tube defects, while over-expression of ADAM10 causes neural tube defects(a) Knockdown of ADAM10 rescues neural tube closure defects induced by F1aMO. Two dorsal cells of four cell stage embryos were injected with Alexa 488 conjugated dextran (green) and the F1aMO alone or an ADAM10 MO, or both. Presented are dorsal view images with light and fluorescent microscopy. (b) The gap width of the neural tubes in the embryos injected with the indicated MOs in (a) were measured and the average gap width and s.d. are presented in the histogram. (c) Overexpression of ADAM10 causes neural tube closure defects. Dorsal view with light and fluorescent microscopy of embryos that were injected with Alexa 488 conjugated dextran (green - tracer) alone, or with ADAM10-V5 RNA, or RNA encoding an ADAM10 mutant with compromised protease activity (ADAM10 PD-V5). (d) The width of the neural tubes in the embryos injected with the indicated reagents in (c) were measured and the average width and s.d. are presented in the histogram. (e) Western blot of lysates from the injected embryos in (c), probed with V5 antibody. (f) An ephrinB2 mutant that is resistant to ADAM10 cleavage rescues F1aMO-induced neural tube defects. Dorsal view of embryos that were injected with either control MO or F1aMO and RNA encoding flotillin-1a-Flag, or high levels of ephrinB2-HA (1.6 ng/embryo), or low levels of cleavage-resistant ephrinB2Δ197–218--HA (0.4 ng/embryo). (g) The gap widths of the neural tubes in the embryos injected with the indicated reagents in (f) were measured and the average width and s.d. are presented in the histogram. (h) Western analysis of the embryonic lysates from (f) using the indicated antibodies to show expression of flotillin-1-Flag, ephrinB2-HA, and ephrinB2Δ197–218.

Mentions: One prediction from this model is that the neural tube closure defect induced by loss of flotillin-1 may be rescued by the disruption of ADAM10 function. To test this prediction, embryos were injected with either F1aMO or ADAM10 MO alone or together, and the gap width of the neural tube at stage 18 was examined. ADAM10 MO substantially rescued the neural tube closure defect induced by F1aMO, as evidenced by the morphology and the reduction in gap width between the neural folds (Fig. 10a, b). The reciprocal experiment also supports this concept. Introduction of ADAM10 RNA into two dorsal blastomeres of four cell stage embryos cause a neural tube closure defect later in development, while the control ADAM10 construct having compromised metalloprotease activity fails to cause this defect (Fig. 10c–e).


EphrinB2 affects apical constriction in Xenopus embryos and is regulated by ADAM10 and flotillin-1.

Ji YJ, Hwang YS, Mood K, Cho HJ, Lee HS, Winterbottom E, Cousin H, Daar IO - Nat Commun (2014)

Loss of ADAM10 rescues F1aMO-induced neural tube defects, while over-expression of ADAM10 causes neural tube defects(a) Knockdown of ADAM10 rescues neural tube closure defects induced by F1aMO. Two dorsal cells of four cell stage embryos were injected with Alexa 488 conjugated dextran (green) and the F1aMO alone or an ADAM10 MO, or both. Presented are dorsal view images with light and fluorescent microscopy. (b) The gap width of the neural tubes in the embryos injected with the indicated MOs in (a) were measured and the average gap width and s.d. are presented in the histogram. (c) Overexpression of ADAM10 causes neural tube closure defects. Dorsal view with light and fluorescent microscopy of embryos that were injected with Alexa 488 conjugated dextran (green - tracer) alone, or with ADAM10-V5 RNA, or RNA encoding an ADAM10 mutant with compromised protease activity (ADAM10 PD-V5). (d) The width of the neural tubes in the embryos injected with the indicated reagents in (c) were measured and the average width and s.d. are presented in the histogram. (e) Western blot of lysates from the injected embryos in (c), probed with V5 antibody. (f) An ephrinB2 mutant that is resistant to ADAM10 cleavage rescues F1aMO-induced neural tube defects. Dorsal view of embryos that were injected with either control MO or F1aMO and RNA encoding flotillin-1a-Flag, or high levels of ephrinB2-HA (1.6 ng/embryo), or low levels of cleavage-resistant ephrinB2Δ197–218--HA (0.4 ng/embryo). (g) The gap widths of the neural tubes in the embryos injected with the indicated reagents in (f) were measured and the average width and s.d. are presented in the histogram. (h) Western analysis of the embryonic lysates from (f) using the indicated antibodies to show expression of flotillin-1-Flag, ephrinB2-HA, and ephrinB2Δ197–218.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4120273&req=5

Figure 10: Loss of ADAM10 rescues F1aMO-induced neural tube defects, while over-expression of ADAM10 causes neural tube defects(a) Knockdown of ADAM10 rescues neural tube closure defects induced by F1aMO. Two dorsal cells of four cell stage embryos were injected with Alexa 488 conjugated dextran (green) and the F1aMO alone or an ADAM10 MO, or both. Presented are dorsal view images with light and fluorescent microscopy. (b) The gap width of the neural tubes in the embryos injected with the indicated MOs in (a) were measured and the average gap width and s.d. are presented in the histogram. (c) Overexpression of ADAM10 causes neural tube closure defects. Dorsal view with light and fluorescent microscopy of embryos that were injected with Alexa 488 conjugated dextran (green - tracer) alone, or with ADAM10-V5 RNA, or RNA encoding an ADAM10 mutant with compromised protease activity (ADAM10 PD-V5). (d) The width of the neural tubes in the embryos injected with the indicated reagents in (c) were measured and the average width and s.d. are presented in the histogram. (e) Western blot of lysates from the injected embryos in (c), probed with V5 antibody. (f) An ephrinB2 mutant that is resistant to ADAM10 cleavage rescues F1aMO-induced neural tube defects. Dorsal view of embryos that were injected with either control MO or F1aMO and RNA encoding flotillin-1a-Flag, or high levels of ephrinB2-HA (1.6 ng/embryo), or low levels of cleavage-resistant ephrinB2Δ197–218--HA (0.4 ng/embryo). (g) The gap widths of the neural tubes in the embryos injected with the indicated reagents in (f) were measured and the average width and s.d. are presented in the histogram. (h) Western analysis of the embryonic lysates from (f) using the indicated antibodies to show expression of flotillin-1-Flag, ephrinB2-HA, and ephrinB2Δ197–218.
Mentions: One prediction from this model is that the neural tube closure defect induced by loss of flotillin-1 may be rescued by the disruption of ADAM10 function. To test this prediction, embryos were injected with either F1aMO or ADAM10 MO alone or together, and the gap width of the neural tube at stage 18 was examined. ADAM10 MO substantially rescued the neural tube closure defect induced by F1aMO, as evidenced by the morphology and the reduction in gap width between the neural folds (Fig. 10a, b). The reciprocal experiment also supports this concept. Introduction of ADAM10 RNA into two dorsal blastomeres of four cell stage embryos cause a neural tube closure defect later in development, while the control ADAM10 construct having compromised metalloprotease activity fails to cause this defect (Fig. 10c–e).

Bottom Line: The Eph/ephrin signalling pathways have a critical function in cell adhesion and repulsion, and thus play key roles in various morphogenetic events during development.Here we show that a decrease in ephrinB2 protein causes neural tube closure defects during Xenopus laevis embryogenesis.This dramatic decline in ephrinB2 protein levels on the absence of flotillin-1 expression is specific, and is partly the result of an increased susceptibility to cleavage by the metalloprotease ADAM10.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell and Developmental Signaling, National Cancer Institute-Frederick, Frederick, Maryland 21702, USA.

ABSTRACT
The Eph/ephrin signalling pathways have a critical function in cell adhesion and repulsion, and thus play key roles in various morphogenetic events during development. Here we show that a decrease in ephrinB2 protein causes neural tube closure defects during Xenopus laevis embryogenesis. Such a decrease in ephrinB2 protein levels is observed on the loss of flotillin-1 scaffold protein, a newly identified ephrinB2-binding partner. This dramatic decline in ephrinB2 protein levels on the absence of flotillin-1 expression is specific, and is partly the result of an increased susceptibility to cleavage by the metalloprotease ADAM10. These findings indicate that flotillin-1 regulates ephrinB2 protein levels through ADAM10, and is required for appropriate neural tube morphogenesis in the Xenopus embryo.

Show MeSH
Related in: MedlinePlus