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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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Neuroepithelial cells of flotillin-1 and ephrinB2 morphants fail to apically constrict in the presumptive neural tube(a) Apical constriction defects of ephrinB2 and flotillin-1 morphants. B2MO or F1bMO were injected into one side of the embryos along with GFP RNA as a tracer. At the neurula stage, embryos were stained with anti-tubulin (green) antibody to outline the cells and with anti-GFP (red) antibody to label the injected side. Cell shapes are outlined below. Blue; uninjected cells, red; MO injected cells. Scale bar represents 50 µm. (b) The average ratios of the apical surfaces to the perimeters among five different cells on each side of the midline were calculated for five different embryos. Blue bars; uninjected side, red bars; MO injected side. (c) Decreased actin intensity in ephrinB2 or flotillin-1 morphants. Each embryo was injected with the indicated MO and GFP RNA with or without the appropriate rescue RNA as indicated. GFP (green) shows the injected side, and actin is stained red (phalloidin staining). In the third column, the neuroepithelum is outlined in green on the MO-injected side and red on the uninjected side. Red boxed regions from embryos in the second column are presented as enlarged images in the fourth and fifth columns. Horizontal scale bar represents 300 µm and vertical scale bar 50 µm. (d) Total actin intensity is decreased in ephrinB2 or flotillin-1 morphants. Percent total actin intensities were calculated for the uninjected or injected side of the embryo using the methodology described in the Methods section. A two-tailed t-test was used to generate the P value. These results represent three independent experiments. Error bars represent s.d.
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Figure 8: Neuroepithelial cells of flotillin-1 and ephrinB2 morphants fail to apically constrict in the presumptive neural tube(a) Apical constriction defects of ephrinB2 and flotillin-1 morphants. B2MO or F1bMO were injected into one side of the embryos along with GFP RNA as a tracer. At the neurula stage, embryos were stained with anti-tubulin (green) antibody to outline the cells and with anti-GFP (red) antibody to label the injected side. Cell shapes are outlined below. Blue; uninjected cells, red; MO injected cells. Scale bar represents 50 µm. (b) The average ratios of the apical surfaces to the perimeters among five different cells on each side of the midline were calculated for five different embryos. Blue bars; uninjected side, red bars; MO injected side. (c) Decreased actin intensity in ephrinB2 or flotillin-1 morphants. Each embryo was injected with the indicated MO and GFP RNA with or without the appropriate rescue RNA as indicated. GFP (green) shows the injected side, and actin is stained red (phalloidin staining). In the third column, the neuroepithelum is outlined in green on the MO-injected side and red on the uninjected side. Red boxed regions from embryos in the second column are presented as enlarged images in the fourth and fifth columns. Horizontal scale bar represents 300 µm and vertical scale bar 50 µm. (d) Total actin intensity is decreased in ephrinB2 or flotillin-1 morphants. Percent total actin intensities were calculated for the uninjected or injected side of the embryo using the methodology described in the Methods section. A two-tailed t-test was used to generate the P value. These results represent three independent experiments. Error bars represent s.d.

Mentions: CoMO-injected embryos and B2MO- or F1MO-injected embryos appear to form normal neural plates until stage 14. Strikingly, after this stage we observed that ephrinB2 and flotillin-1 morphants develop broad neural grooves and lack elevated neural folds (Fig. 7a–d), indicating a possible problem in the process of neural plate bending, which is driven by apical constriction29. When one side of the embryo is injected with B2MO or F1bMO, it displays apical constriction defects of the neuroepithelium, while the uninjected side retains normal morphology and wedge shaped cells (Fig. 8a). Cell lengths are significantly decreased and cell widths are increased on the B2MO- or F1bMO-injected side when compared to the uninjected side. The average ratio of the length of the apical surface to the cell perimeter was calculated using five different cells adjacent to the midline. The ratio increased for cells harboring the B2MO or the F1bMO compared to uninjected cells (Fig. 8b), indicating that loss of ephrinB2 or flotillin-1b reduces apical constriction in the neuroepithelium.


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)

Neuroepithelial cells of flotillin-1 and ephrinB2 morphants fail to apically constrict in the presumptive neural tube(a) Apical constriction defects of ephrinB2 and flotillin-1 morphants. B2MO or F1bMO were injected into one side of the embryos along with GFP RNA as a tracer. At the neurula stage, embryos were stained with anti-tubulin (green) antibody to outline the cells and with anti-GFP (red) antibody to label the injected side. Cell shapes are outlined below. Blue; uninjected cells, red; MO injected cells. Scale bar represents 50 µm. (b) The average ratios of the apical surfaces to the perimeters among five different cells on each side of the midline were calculated for five different embryos. Blue bars; uninjected side, red bars; MO injected side. (c) Decreased actin intensity in ephrinB2 or flotillin-1 morphants. Each embryo was injected with the indicated MO and GFP RNA with or without the appropriate rescue RNA as indicated. GFP (green) shows the injected side, and actin is stained red (phalloidin staining). In the third column, the neuroepithelum is outlined in green on the MO-injected side and red on the uninjected side. Red boxed regions from embryos in the second column are presented as enlarged images in the fourth and fifth columns. Horizontal scale bar represents 300 µm and vertical scale bar 50 µm. (d) Total actin intensity is decreased in ephrinB2 or flotillin-1 morphants. Percent total actin intensities were calculated for the uninjected or injected side of the embryo using the methodology described in the Methods section. A two-tailed t-test was used to generate the P value. These results represent three independent experiments. Error bars represent s.d.
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Figure 8: Neuroepithelial cells of flotillin-1 and ephrinB2 morphants fail to apically constrict in the presumptive neural tube(a) Apical constriction defects of ephrinB2 and flotillin-1 morphants. B2MO or F1bMO were injected into one side of the embryos along with GFP RNA as a tracer. At the neurula stage, embryos were stained with anti-tubulin (green) antibody to outline the cells and with anti-GFP (red) antibody to label the injected side. Cell shapes are outlined below. Blue; uninjected cells, red; MO injected cells. Scale bar represents 50 µm. (b) The average ratios of the apical surfaces to the perimeters among five different cells on each side of the midline were calculated for five different embryos. Blue bars; uninjected side, red bars; MO injected side. (c) Decreased actin intensity in ephrinB2 or flotillin-1 morphants. Each embryo was injected with the indicated MO and GFP RNA with or without the appropriate rescue RNA as indicated. GFP (green) shows the injected side, and actin is stained red (phalloidin staining). In the third column, the neuroepithelum is outlined in green on the MO-injected side and red on the uninjected side. Red boxed regions from embryos in the second column are presented as enlarged images in the fourth and fifth columns. Horizontal scale bar represents 300 µm and vertical scale bar 50 µm. (d) Total actin intensity is decreased in ephrinB2 or flotillin-1 morphants. Percent total actin intensities were calculated for the uninjected or injected side of the embryo using the methodology described in the Methods section. A two-tailed t-test was used to generate the P value. These results represent three independent experiments. Error bars represent s.d.
Mentions: CoMO-injected embryos and B2MO- or F1MO-injected embryos appear to form normal neural plates until stage 14. Strikingly, after this stage we observed that ephrinB2 and flotillin-1 morphants develop broad neural grooves and lack elevated neural folds (Fig. 7a–d), indicating a possible problem in the process of neural plate bending, which is driven by apical constriction29. When one side of the embryo is injected with B2MO or F1bMO, it displays apical constriction defects of the neuroepithelium, while the uninjected side retains normal morphology and wedge shaped cells (Fig. 8a). Cell lengths are significantly decreased and cell widths are increased on the B2MO- or F1bMO-injected side when compared to the uninjected side. The average ratio of the length of the apical surface to the cell perimeter was calculated using five different cells adjacent to the midline. The ratio increased for cells harboring the B2MO or the F1bMO compared to uninjected cells (Fig. 8b), indicating that loss of ephrinB2 or flotillin-1b reduces apical constriction in the neuroepithelium.

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