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Curr Biol
2010 Feb 09;203:253-8. doi: 10.1016/j.cub.2009.12.021.
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Endocytosis is required for efficient apical constriction during Xenopus gastrulation.
Lee JY
,
Harland RM
.
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Coordinated apical constriction (AC) in epithelial sheets drives tissue invagination [1, 2] and is required for diverse morphogenetic movements such as gastrulation [3], neurulation [4, 5], and organogenesis [6]. We showed previously that actomyosin contractility drives AC in Xenopus laevis bottle cells [7]; however, it remained unclear whether it does so in concert with other processes. Here we report that endocytosis-driven membrane remodeling is required for efficient AC. We found endosomes exclusively in bottle cells in the early gastrula. Disrupting endocytosis with dominant-negative dynamin or rab5 perturbed AC, with a significant decrease in constriction rate late in the process, suggesting that endocytosis operates downstream of actomyosin contractility to remove excess membrane. Additionally, disrupting endocytosis during neurulation inhibits AC in hingepoint cells, resulting in neural tube closure defects. Thus, membrane remodeling during AC could be a general mechanism to achieve efficient invagination in embryos.
Figure 2. Dominant-Negative Dynamin and Dominant-Negative Rab5 Disrupt Bottle Cell Formation by Affecting Apical Constriction
(A) Epifluorescence images of stage 10.5 embryos, vegetal views. All embryos were injected with membrane EGFP mRNA plus dominant-negative (DN)
dynamin, wild-type (WT) dynamin, or WT + DN. Arrows point to blastopore forming in the dorsal marginal zone (DMZ). Scale bar represents 250 mm.
(B) Confocal midsagittal sections of embryos, all injected with membrane EGFP plus DN, WT, or WT + DN. Embryos were stained with anti-GFP and streptavidin.
Scale bar represents 50 mm.
(C) Quantification of blastopore depth and bottle cell morphometrics in DN dynamin-injected embryos. *p < 0.01, **p < 0.001, zp < 0.05 for GFP versus
rescue, p < 0.001 for DN versus rescue. Each bar represents the mean of five experiments. For all graphs, error bars represent 6 standard error of the mean.
(Da and Db) Confocal midsagittal sections of uninjected control embryos or embryos injected with DN rab5 mRNA and stained with anti-DM1a (tubulin) and
streptavidin. Scale bar represents 50 mm. Bottom panels (Db) show higher magnification of biotin-labeled membrane. Scale bar represents 10 mm.
(E) Quantification of blastopore depth (n = 88 control embryos; n = 53 DN rab5 embryos) and bottle cell morphometrics (n = 187 control cells; n = 113 DN rab5
cells). *p < 0.001. Each bar represents the mean of six experiments; error bars indicate 6 standard error of the mean (SEM). See also Figures S2 and S3.
Figure 3. Apical Membrane Remodeling Is Required for
Efficient Apical Constriction Downstream of Actomyosin
Contractility
(A) Apical accumulation of F-actin and myosin does not
require endocytosis. Confocal midsagittal sections of
embryos were injected with membrane EGFP alone or with
membrane EGFP plus DN dynamin and then stained with
phalloidin to visualize F-actin or anti-pMLC. Scale bar
represents 50 mm.
(B) Transmission electron micrographs of GFP and DN dynamin-
injected embryos. Animal cells do not have microvilli,
whereas bottle cells have microvilli (indicated with arrows).
The following abbreviations are used: m, mitochondria; P,
pigment granule; Y, yolk platelet. Vesicles are pseudocolored
in blue. Scale bars represent 0.5 mm.
(C) Rate of constriction is the same between wild-type and
DN dynamin bottle cells until cells become very constricted.
Top panels are stills from two time points (0 min, 15 min) of
time-lapse movies (Movies S1 and S2). Larger cells
(>250 mm2) are pseudocolored in blue and mustard; smaller
cells (<250 mm2) are in orange and purple. Line graphs indicate
the total decrease in apical surface area over 15 min.
n represents number of cells measured; *p < 0.05; error
bars indicate 6 SEM. See also Table S1.
Figure 4. Endocytosis Occurs in the Apically Constricting Dorsal-Lateral Hingepoint Cells during Neurulation, and Perturbing Endocytosis Results in Apical
Constriction and Neural Tube Closure Defects
(A) Model of apical membrane dynamics during bottle cell apical constriction.
(B) Endocytosis occurs in the neural tube. Cross-section of a stage 18 embryo is shown, dorsal side up, stained with streptavidin. Scale bar represents 50mm.
(C) Injection of DN dynamin results in a range of neural tube closure defects. Top panels show unilateral injection of membrane EGFP in whole stage
24 embryos, dorsal views; embryos are oriented anterior to the left. Bottom panels show higher magnification of the anterior neural tube, anterior up.
Asterisks indicate injected side.
(D) DN dynamin-injected hingepoint cells appear less apically constricted than GFP-injected cells. Stage 18 embryos were stained with anti-DM1a (tubulin)
and anti-GFP. Scale bar represents 50 mm. See also Figure S4 and Movie S3.
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