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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.

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Loss of flotillin-1 leads to a reduction in ephrinB2 expression(a) Injection scheme. CoMO with ephrinB2 RNA is injected into one cell of two-cell stage embryos, and F1MO along with ephrinB2 RNA and GFP RNA were injected into the other cell. (b) ephrinB2 expression is reduced in the absence of Flotillin-1a. F1aMO, ephrinB2-HA RNA and GFP RNA co-injected cells are green. CoMO and ephrinB2-HA RNA co-injected cells are not green. EphrinB2-HA expression is visualized in red. Cells containing F1aMO, but displaying reduced ephrinB-HA expression are outlined in white dots. Scale bar represents 20 µm. (c) F1bMO decreases ephrinB2 expression. F1bMO, ephrinB2-Flag RNA and GFP RNA co-injected cells fluoresce green. CoMO and ephrinB2-Flag RNA co-injected cells are not green. EphrinB2-HA expression is visualized in red. Arrowheads indicate GFP-expressing cells that harbor the F1bMO. Scale bar represents 20 µm. (d) Flotillin-1 knockdown specifically causes ephrinB2 loss. Embryos were injected with HA-tagged ephrinB1, -B2 or -B3 RNAs along with either CoMO or F1aMO. Western analysis was performed with anti-HA antibody and anti-Erk2 as a loading control. (e) EphrinB2 loss due to the F1aMO is rescued by re-expression of flotillin-1a. Western analysis of embryos injected with ephrinB2-HA RNA and F1aMO, with or without F1aΔUTR-Flag RNA. (f) Decrease in ephrinB2 due to F1bMO is rescued by F1bΔUTR-Flag expression. Western analysis of embryos injected with ephrinB2-HA RNA and F1bMO, with or without F1bΔUTR-Flag RNA.. (g) Western analysis of endogenous ephrinB2 protein in neural folds that have been injected with the indicated MOs, and blotted using the indicated antibodies. Erk2 is used as a loading control. (h) Western analysis of endogenous ephrinB1 protein in neural folds that have been injected with the indicated MOs, and blotted using the indicated antibodies. Erk2 is used as a loading control.
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Figure 2: Loss of flotillin-1 leads to a reduction in ephrinB2 expression(a) Injection scheme. CoMO with ephrinB2 RNA is injected into one cell of two-cell stage embryos, and F1MO along with ephrinB2 RNA and GFP RNA were injected into the other cell. (b) ephrinB2 expression is reduced in the absence of Flotillin-1a. F1aMO, ephrinB2-HA RNA and GFP RNA co-injected cells are green. CoMO and ephrinB2-HA RNA co-injected cells are not green. EphrinB2-HA expression is visualized in red. Cells containing F1aMO, but displaying reduced ephrinB-HA expression are outlined in white dots. Scale bar represents 20 µm. (c) F1bMO decreases ephrinB2 expression. F1bMO, ephrinB2-Flag RNA and GFP RNA co-injected cells fluoresce green. CoMO and ephrinB2-Flag RNA co-injected cells are not green. EphrinB2-HA expression is visualized in red. Arrowheads indicate GFP-expressing cells that harbor the F1bMO. Scale bar represents 20 µm. (d) Flotillin-1 knockdown specifically causes ephrinB2 loss. Embryos were injected with HA-tagged ephrinB1, -B2 or -B3 RNAs along with either CoMO or F1aMO. Western analysis was performed with anti-HA antibody and anti-Erk2 as a loading control. (e) EphrinB2 loss due to the F1aMO is rescued by re-expression of flotillin-1a. Western analysis of embryos injected with ephrinB2-HA RNA and F1aMO, with or without F1aΔUTR-Flag RNA. (f) Decrease in ephrinB2 due to F1bMO is rescued by F1bΔUTR-Flag expression. Western analysis of embryos injected with ephrinB2-HA RNA and F1bMO, with or without F1bΔUTR-Flag RNA.. (g) Western analysis of endogenous ephrinB2 protein in neural folds that have been injected with the indicated MOs, and blotted using the indicated antibodies. Erk2 is used as a loading control. (h) Western analysis of endogenous ephrinB1 protein in neural folds that have been injected with the indicated MOs, and blotted using the indicated antibodies. Erk2 is used as a loading control.

Mentions: Since ephrinB2 is normally localized to the basolateral region of plasma membranes, we examined whether flotillin-1 influences ephrinB2 localization or expression (Fig. 2a–c; Supplementary movie 1). EphrinB2 RNA was injected into both sides of an embryo, and the F1aMO or F1bMO along with GFP RNA was introduced into only one side. Surprisingly, ephrinB2 protein levels were greatly reduced in the plasma membrane of F1aMO and F1bMO bearing cells, as evidenced by the dramatically reduced ephrinB2 fluorescence (red) found in the F1MO bearing cells (green) when compared to CoMO bearing cells (non-green) (Fig. 2b, c). Western analysis also showed that ephrinB2-HA protein expression was greatly diminished when flotillin-1a was knocked down, while exogenous expression of ephrinB1 and ephrinB3 was comparable in the presence of F1aMO or CoMO (Fig. 2d). Moreover, the introduction of MO-resistant flotillin-1a or -1b RNA along with the respective MO rescued ephrinB2 protein levels (Fig. 2e, f), indicating that flotillin-1 specifically inhibits the loss of ephrinB2 protein expression.


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 flotillin-1 leads to a reduction in ephrinB2 expression(a) Injection scheme. CoMO with ephrinB2 RNA is injected into one cell of two-cell stage embryos, and F1MO along with ephrinB2 RNA and GFP RNA were injected into the other cell. (b) ephrinB2 expression is reduced in the absence of Flotillin-1a. F1aMO, ephrinB2-HA RNA and GFP RNA co-injected cells are green. CoMO and ephrinB2-HA RNA co-injected cells are not green. EphrinB2-HA expression is visualized in red. Cells containing F1aMO, but displaying reduced ephrinB-HA expression are outlined in white dots. Scale bar represents 20 µm. (c) F1bMO decreases ephrinB2 expression. F1bMO, ephrinB2-Flag RNA and GFP RNA co-injected cells fluoresce green. CoMO and ephrinB2-Flag RNA co-injected cells are not green. EphrinB2-HA expression is visualized in red. Arrowheads indicate GFP-expressing cells that harbor the F1bMO. Scale bar represents 20 µm. (d) Flotillin-1 knockdown specifically causes ephrinB2 loss. Embryos were injected with HA-tagged ephrinB1, -B2 or -B3 RNAs along with either CoMO or F1aMO. Western analysis was performed with anti-HA antibody and anti-Erk2 as a loading control. (e) EphrinB2 loss due to the F1aMO is rescued by re-expression of flotillin-1a. Western analysis of embryos injected with ephrinB2-HA RNA and F1aMO, with or without F1aΔUTR-Flag RNA. (f) Decrease in ephrinB2 due to F1bMO is rescued by F1bΔUTR-Flag expression. Western analysis of embryos injected with ephrinB2-HA RNA and F1bMO, with or without F1bΔUTR-Flag RNA.. (g) Western analysis of endogenous ephrinB2 protein in neural folds that have been injected with the indicated MOs, and blotted using the indicated antibodies. Erk2 is used as a loading control. (h) Western analysis of endogenous ephrinB1 protein in neural folds that have been injected with the indicated MOs, and blotted using the indicated antibodies. Erk2 is used as a loading control.
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Figure 2: Loss of flotillin-1 leads to a reduction in ephrinB2 expression(a) Injection scheme. CoMO with ephrinB2 RNA is injected into one cell of two-cell stage embryos, and F1MO along with ephrinB2 RNA and GFP RNA were injected into the other cell. (b) ephrinB2 expression is reduced in the absence of Flotillin-1a. F1aMO, ephrinB2-HA RNA and GFP RNA co-injected cells are green. CoMO and ephrinB2-HA RNA co-injected cells are not green. EphrinB2-HA expression is visualized in red. Cells containing F1aMO, but displaying reduced ephrinB-HA expression are outlined in white dots. Scale bar represents 20 µm. (c) F1bMO decreases ephrinB2 expression. F1bMO, ephrinB2-Flag RNA and GFP RNA co-injected cells fluoresce green. CoMO and ephrinB2-Flag RNA co-injected cells are not green. EphrinB2-HA expression is visualized in red. Arrowheads indicate GFP-expressing cells that harbor the F1bMO. Scale bar represents 20 µm. (d) Flotillin-1 knockdown specifically causes ephrinB2 loss. Embryos were injected with HA-tagged ephrinB1, -B2 or -B3 RNAs along with either CoMO or F1aMO. Western analysis was performed with anti-HA antibody and anti-Erk2 as a loading control. (e) EphrinB2 loss due to the F1aMO is rescued by re-expression of flotillin-1a. Western analysis of embryos injected with ephrinB2-HA RNA and F1aMO, with or without F1aΔUTR-Flag RNA. (f) Decrease in ephrinB2 due to F1bMO is rescued by F1bΔUTR-Flag expression. Western analysis of embryos injected with ephrinB2-HA RNA and F1bMO, with or without F1bΔUTR-Flag RNA.. (g) Western analysis of endogenous ephrinB2 protein in neural folds that have been injected with the indicated MOs, and blotted using the indicated antibodies. Erk2 is used as a loading control. (h) Western analysis of endogenous ephrinB1 protein in neural folds that have been injected with the indicated MOs, and blotted using the indicated antibodies. Erk2 is used as a loading control.
Mentions: Since ephrinB2 is normally localized to the basolateral region of plasma membranes, we examined whether flotillin-1 influences ephrinB2 localization or expression (Fig. 2a–c; Supplementary movie 1). EphrinB2 RNA was injected into both sides of an embryo, and the F1aMO or F1bMO along with GFP RNA was introduced into only one side. Surprisingly, ephrinB2 protein levels were greatly reduced in the plasma membrane of F1aMO and F1bMO bearing cells, as evidenced by the dramatically reduced ephrinB2 fluorescence (red) found in the F1MO bearing cells (green) when compared to CoMO bearing cells (non-green) (Fig. 2b, c). Western analysis also showed that ephrinB2-HA protein expression was greatly diminished when flotillin-1a was knocked down, while exogenous expression of ephrinB1 and ephrinB3 was comparable in the presence of F1aMO or CoMO (Fig. 2d). Moreover, the introduction of MO-resistant flotillin-1a or -1b RNA along with the respective MO rescued ephrinB2 protein levels (Fig. 2e, f), indicating that flotillin-1 specifically inhibits the loss of ephrinB2 protein expression.

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