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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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ephrinB2 and flotillin-1 morphants show neural tube closure defects(a) Neural tube closure defects in ephrinB2 or flotillin-1 morphants. Dorsal view of stage 18 control, ephrinB2, flotillin-1a or flotillin-1b MO-injected embryos, and those co-injected with morpholino-resistant RNA (B211MT-HA for B2MO, F1aΔUTR-Flag for F1aMO and F1bΔUTR-Flag for F1bMO) Left is anterior, and right is posterior. (b) Quantification of neural tube closure defects in ephrinB2 or flotillin-1 morphants. Embryos showing a single dorsal line were counted as having closed neural tubes. At least three independent experiments were performed and error bars indicate standard deviation (s.d.) (c) ephrinB2, flotillin-1a or flotillin-1b MOs were injected into embryos, and neural tube closure was examined at neurula (st. 18/19) and swimming tadpole stages. Note the anterior neural tube defects. (d) Percentage of neural tube closure in ephrinB2 MO, flotillin-1a MO or flotillin-1b MO-injected embryos. Note that the percentage of neural tube closure is somewhat elevated at later stages in B2MO, F1aMO and F1bMO-injected embryos, indicating that some of the embryos displayed a profound delay in neural tube closure, while others retain an open neural tube. Error bars indicate s. d. (e) ephrinB1 MO or flotillin-2 MO injected embryos show normal neural tube closure.
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Figure 7: ephrinB2 and flotillin-1 morphants show neural tube closure defects(a) Neural tube closure defects in ephrinB2 or flotillin-1 morphants. Dorsal view of stage 18 control, ephrinB2, flotillin-1a or flotillin-1b MO-injected embryos, and those co-injected with morpholino-resistant RNA (B211MT-HA for B2MO, F1aΔUTR-Flag for F1aMO and F1bΔUTR-Flag for F1bMO) Left is anterior, and right is posterior. (b) Quantification of neural tube closure defects in ephrinB2 or flotillin-1 morphants. Embryos showing a single dorsal line were counted as having closed neural tubes. At least three independent experiments were performed and error bars indicate standard deviation (s.d.) (c) ephrinB2, flotillin-1a or flotillin-1b MOs were injected into embryos, and neural tube closure was examined at neurula (st. 18/19) and swimming tadpole stages. Note the anterior neural tube defects. (d) Percentage of neural tube closure in ephrinB2 MO, flotillin-1a MO or flotillin-1b MO-injected embryos. Note that the percentage of neural tube closure is somewhat elevated at later stages in B2MO, F1aMO and F1bMO-injected embryos, indicating that some of the embryos displayed a profound delay in neural tube closure, while others retain an open neural tube. Error bars indicate s. d. (e) ephrinB1 MO or flotillin-2 MO injected embryos show normal neural tube closure.

Mentions: To assess whether there is a functional effect mediated by the interaction between flotillin-1 and ephrinB2, we examined the effect of knocking down ephrinB2 in Xenopus embryos, which express ephrinB2 at early stages of neural tube formation. When embryos were injected with the ephrinB2 MO (B2MO), only 10.2 % displayed a closed neural tube by stage 18/19 (Fig. 7a, b), while 96.5 % of the CoMO injected embryos had closed neural tubes (Fig. 7a, b). At later stages, profound delays or failure of neural tube closure were observed (Fig. 7c, d). This effect is specific to the B2MO, since MO-mediated knockdown of ephrinB1 did not disrupt neural tube closure (Fig. 7e). Further evidence of the specificity of the B2MO and the requirement for ephrinB2 in neural tube closure is provided by the rescue of neural tube defects (59.6 % closed neural tubes) by re-expressing ephrinB2 using B211MT RNA, which is resistant to the B2MO (Fig. 7a, b).


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)

ephrinB2 and flotillin-1 morphants show neural tube closure defects(a) Neural tube closure defects in ephrinB2 or flotillin-1 morphants. Dorsal view of stage 18 control, ephrinB2, flotillin-1a or flotillin-1b MO-injected embryos, and those co-injected with morpholino-resistant RNA (B211MT-HA for B2MO, F1aΔUTR-Flag for F1aMO and F1bΔUTR-Flag for F1bMO) Left is anterior, and right is posterior. (b) Quantification of neural tube closure defects in ephrinB2 or flotillin-1 morphants. Embryos showing a single dorsal line were counted as having closed neural tubes. At least three independent experiments were performed and error bars indicate standard deviation (s.d.) (c) ephrinB2, flotillin-1a or flotillin-1b MOs were injected into embryos, and neural tube closure was examined at neurula (st. 18/19) and swimming tadpole stages. Note the anterior neural tube defects. (d) Percentage of neural tube closure in ephrinB2 MO, flotillin-1a MO or flotillin-1b MO-injected embryos. Note that the percentage of neural tube closure is somewhat elevated at later stages in B2MO, F1aMO and F1bMO-injected embryos, indicating that some of the embryos displayed a profound delay in neural tube closure, while others retain an open neural tube. Error bars indicate s. d. (e) ephrinB1 MO or flotillin-2 MO injected embryos show normal neural tube closure.
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Figure 7: ephrinB2 and flotillin-1 morphants show neural tube closure defects(a) Neural tube closure defects in ephrinB2 or flotillin-1 morphants. Dorsal view of stage 18 control, ephrinB2, flotillin-1a or flotillin-1b MO-injected embryos, and those co-injected with morpholino-resistant RNA (B211MT-HA for B2MO, F1aΔUTR-Flag for F1aMO and F1bΔUTR-Flag for F1bMO) Left is anterior, and right is posterior. (b) Quantification of neural tube closure defects in ephrinB2 or flotillin-1 morphants. Embryos showing a single dorsal line were counted as having closed neural tubes. At least three independent experiments were performed and error bars indicate standard deviation (s.d.) (c) ephrinB2, flotillin-1a or flotillin-1b MOs were injected into embryos, and neural tube closure was examined at neurula (st. 18/19) and swimming tadpole stages. Note the anterior neural tube defects. (d) Percentage of neural tube closure in ephrinB2 MO, flotillin-1a MO or flotillin-1b MO-injected embryos. Note that the percentage of neural tube closure is somewhat elevated at later stages in B2MO, F1aMO and F1bMO-injected embryos, indicating that some of the embryos displayed a profound delay in neural tube closure, while others retain an open neural tube. Error bars indicate s. d. (e) ephrinB1 MO or flotillin-2 MO injected embryos show normal neural tube closure.
Mentions: To assess whether there is a functional effect mediated by the interaction between flotillin-1 and ephrinB2, we examined the effect of knocking down ephrinB2 in Xenopus embryos, which express ephrinB2 at early stages of neural tube formation. When embryos were injected with the ephrinB2 MO (B2MO), only 10.2 % displayed a closed neural tube by stage 18/19 (Fig. 7a, b), while 96.5 % of the CoMO injected embryos had closed neural tubes (Fig. 7a, b). At later stages, profound delays or failure of neural tube closure were observed (Fig. 7c, d). This effect is specific to the B2MO, since MO-mediated knockdown of ephrinB1 did not disrupt neural tube closure (Fig. 7e). Further evidence of the specificity of the B2MO and the requirement for ephrinB2 in neural tube closure is provided by the rescue of neural tube defects (59.6 % closed neural tubes) by re-expressing ephrinB2 using B211MT RNA, which is resistant to the B2MO (Fig. 7a, b).

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