αE-catenin regulates cell-cell adhesion and membrane blebbing during zebrafish epiboly

Abstract

αE-catenin is an actin-binding protein associated with the E-cadherin-based adherens junction that regulates cell-cell adhesion. Recent studies identified additional E-cadherin-independent roles of αE-catenin in regulating plasma membrane dynamics and cell migration. However, little is known about the roles of αE-catenin in these different cellular processes in vivo during early vertebrate development. Here, we examined the functions of αE-catenin in cell-cell adhesion, cell migration and plasma membrane dynamics during morphogenetic processes that drive epiboly in early Danio rerio (zebrafish) development. We show that depletion of αE-catenin caused a defect in radial intercalation that was associated with decreased cell-cell adhesion, in a similar manner to E-cadherin depletion. Depletion of αE-catenin also caused deep cells to have protracted plasma membrane blebbing, and a defect in plasma membrane recruitment of ERM proteins that are involved in controlling membrane-to-cortex attachment and membrane blebbing. Significantly, depletion of both E-cadherin and αE-catenin suppressed plasma membrane blebbing. We suggest that during radial intercalation the activities of E-cadherin and αE-catenin in the maintenance of membrane-to-cortex attachment are balanced, resulting in stabilization of cell-cell adhesion and suppression of membrane blebbing, thereby enabling proper radial intercalation.

Fig. 1.

ctnna morphants display morphological defects. (A-C) DIC images of live zebrafish embryos at the end of gastrulation. Arrow indicates yolk bulge. (D) Western blot of αE-catenin and tubulin in whole-embryo extracts at the same stages as those shown in A-C. (E,F) Lateral view images of live embryos at the mid-segmentation stage. (G,H) Images of live embryos, dorsal view. Horizontal arrow indicates somite, vertical arrow indicates notochord. (I) Percentage of embryos exhibiting epiboly delay at the end of gastrulation, as shown in B,C. Three independent experiments with n>70: ^*P<10^–6 versus control, ^#P<10^–5 versus rescue. ##, not significantly different from control by Student’s t-test. (J) Fraction of embryos dead or delayed by mid-segmentation, as shown in E,F. Three independent experiments, n>70 embryos. ^*P<10^–6, # control or rescue vs morphant P<10^–5. ^**, not significant. Error bars indicate s.d.

Fig. 2.

ctnna morphants are delayed in epiboly. (A-C) Immunofluorescence images of fixed zebrafish embryos stained for β-catenin. AP, animal pole; VP, vegetal pole. Upper arrow indicates deep cell margin, lower arrow indicates EVL margin. (D-F) Confocal fluorescence images of Ctnna antibody staining of the most external epiblast layer at the animal pole of embryos at 50% epiboly. Scale bar: 10 μm. (G) Rescue embryos stained for the HA tag; same view as D-F. Scale bar: 10 μm. (H) Percentage of epiboly of the deep cell (DC) and EVL margins. Three independent experiments, n=25 embryos. ^*P<0.006, ^#P<0.001. Error bars indicate s.d.

Fig. 3.

αE-catenin depletion causes defects in EVL cell morphology. (A-E) Confocal images of cells at EVL margin at 80% epiboly stained for F-actin with rhodamine phalloidin. Scale bar: 10 μm. (F) Quantification of length-width ratio (LWR) of cells at the EVL margin. Three independent experiments, n=40 cells from eight embryos. ^*P<0.002; ^#P<0.003. Error bars indicate s.d.

Fig. 4.

αE-catenin and E-cadherin depletion causes defects in radial intercalation (RI). (A-C) Confocal images from time-lapse movies at the most external epiblast layer of the animal pole at 50% epiboly. Membranes are labeled with mbGFP. Positive numbers indicate cells appearing in the focal plane (undergoing RI). Negative numbers indicate cells migrating back into the in lower epiblast layer (undergoing revRI). Scale bars: 8 μm. (D) Quantification of RI rate and revRI. n=9 embryos. ^#P<0.0005. Asterisk indicates not significant. Error bars indicate s.e.m.

Fig. 5.

αE-catenin depletion triggers protracted bleb-like protrusions. (A-C) Selected confocal images from time-lapse movies at the most external epiblast layer of the animal pole at 50% epiboly. Membranes are labeled with mbGFP. Arrows indicate membrane blebs. Scale bars: 8 μm. (D) Percentage of cells exhibiting bleb-like protrusions over 5 minutes. n=12 embryos. ^#P<10^–5 control versus rescue, ^*P<10^–5 control versus morphant. Error bars indicate s.e.m. (E) Average number of blebs per cell over 5 minutes. n=12 embryos. ^#P<10^–8 control versus rescue, ^*P<10^–8 control versus morphant. Error bars indicate s.e.m.

Fig. 6.

Membrane blebbing triggered by αE-catenin depletion is cell autonomous and myosin II-dependent. (A,B) Selected confocal images from time-lapse movies of transplanted cells expressing mbGFP in the most external epiblast external layer of the host embryos at 50% epiboly. Arrows indicate the blebs. (A) Wild-type (WT) cell in ctnna morphant host embryo. (B) ctnna morphant cell in WT host embryo. Scale bars: 5 μm. (C,D) DIC images from time-lapse movies of untreated (DMSO) embryos or embryos treated with blebbistatin (50 μM) imaged at the most external epiblast layer at the animal pole at 50% epiboly. Scale bars: 8 μm. (E) Percentage of cells exhibiting bleb-like protrusions in untreated embryos or embryos treated with blebbistatin. n=12 embryos. ^*P<10^–8, ^#P<10^–5. Note that no cell exhibited membrane blebbing in embryos treated with blebbistatin. Error bars indicate s.e.m. (F) Average number of blebs per cell untreated embryos or embryos treated with blebbistatin. n=12 embryos. ^*P<10^–9. Note that no cell exhibited membrane blebbing in embryos treated with blebbistatin. Error bars indicate s.e.m.

Fig. 7.

cdh1 and ctnna morphant DCs displayed increased phospho-MLC at cell contacts. (A-C) Confocal images of the epiblast most external layer at the animal pole of embryos at 50% epiboly stained for phospho-MLC. Scale bar: 10 μm. (D) Fluorescence intensity (FI) at cell contacts of embryos stained for pMLC. Three independent experiments, n=300 cell contacts (15 embryos) per experiment. ^*P<10^–3 versus the control (normalized to 1). Error bars indicate s.d.

Fig. 8.

Blebbing triggered by αE-catenin depletion is E-cadherin dependent. (A) Selected confocal images from time-lapse movies of transplanted ctnna morphant cells expressing mbGFP in the most external epiblast external layer of the host cdh1 morphant embryos at 50% epiboly. Scale bar: 5 μm. (B) Confocal images from time-lapse movies at the most external epiblast layer of the animal pole of live embryos at 50% epiboly. Membranes are labeled with mbGFP. Arrows indicate membrane blebs. Embryos were co-injected with cdh1 and ctnna mo. Scale bar: 8 μm. (C) Percentage of cells exhibiting bleb-like protrusions. n=12 embryos. ^*P<10^–5. Error bars indicate s.e.m. (D) Average number of blebs per cell. n=12 embryos. ^*P<10^–8. Error bars indicate s.e.m.

Fig. 9.

αE-catenin and E-cadherin depletion influence ERM protein localization and membrane blebbing in ezr morphants. (A) Percentage of cells with bleb-like membrane protrusions. n=12 embryos. ^*P<10^–6 ezr or ctnna mo low dose versus ctnna/ezr mo low dose, ^#P<10^–6 ezr mo high dose versus cdh1/ezr mo high dose. (B) Average number of blebs per cell. n=12 embryos. ^*P<10^–6 ezr or ctnna mo low dose versus ctnna/ezr mo low dose, ^#P<10^–5 ezr mo high dose versus cdh1/ezr mo high dose. Error bars indicate s.e.m. (C-E) Confocal images of the most external epiblast layer at the animal pole of embryos at 50% epiboly stained for phospho-ERM proteins. (C) Uninjected control. (D) ctnna morphant. (E) cdh1 morphant. Scale bar: ∼10 μm.