Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
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.
Balklava,
Genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic.
2007, Pubmed
Balklava,
Genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic.
2007,
Pubmed
Burnside,
Microtubules and microfilaments in newt neuralation.
1971,
Pubmed
Chavrier,
Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments.
1990,
Pubmed
Copp,
Neurulation in the cranial region--normal and abnormal.
2005,
Pubmed
Damke,
Induction of mutant dynamin specifically blocks endocytic coated vesicle formation.
1994,
Pubmed
Gilchrist,
Defining a large set of full-length clones from a Xenopus tropicalis EST project.
2004,
Pubmed
,
Xenbase
Hagemann,
Rab5-mediated endocytosis of activin is not required for gene activation or long-range signalling in Xenopus.
2009,
Pubmed
,
Xenbase
Haigo,
Shroom induces apical constriction and is required for hingepoint formation during neural tube closure.
2003,
Pubmed
,
Xenbase
Jullien,
Morphogen gradient interpretation by a regulated trafficking step during ligand-receptor transduction.
2005,
Pubmed
,
Xenbase
Keller,
An experimental analysis of the role of bottle cells and the deep marginal zone in gastrulation of Xenopus laevis.
1981,
Pubmed
,
Xenbase
Keller,
How we are shaped: the biomechanics of gastrulation.
2003,
Pubmed
,
Xenbase
Kerman,
Ribbon modulates apical membrane during tube elongation through Crumbs and Moesin.
2008,
Pubmed
Kida,
Daam1 regulates the endocytosis of EphB during the convergent extension of the zebrafish notochord.
2007,
Pubmed
Kim,
Ryk cooperates with Frizzled 7 to promote Wnt11-mediated endocytosis and is essential for Xenopus laevis convergent extension movements.
2008,
Pubmed
,
Xenbase
Kimberly,
Bottle cells are required for the initiation of primary invagination in the sea urchin embryo.
1998,
Pubmed
Krieg,
The cellular anatomy of embryos of the nematode Caenorhabditis elegans. Analysis and reconstruction of serial section electron micrographs.
1978,
Pubmed
Kurth,
Bottle cell formation in relation to mesodermal patterning in the Xenopus embryo.
2000,
Pubmed
,
Xenbase
Lee,
Actomyosin contractility and microtubules drive apical constriction in Xenopus bottle cells.
2007,
Pubmed
,
Xenbase
Lee,
Mechanisms of cell positioning during C. elegans gastrulation.
2003,
Pubmed
LEWIS,
Mechanics of invagination.
1947,
Pubmed
Li,
Structure-function relationship of the small GTPase rab5.
1993,
Pubmed
Lu,
Endocytic control of epithelial polarity and proliferation in Drosophila.
2005,
Pubmed
Matteoni,
Translocation and clustering of endosomes and lysosomes depends on microtubules.
1987,
Pubmed
Minsuk,
Surface mesoderm in Xenopus: a revision of the stage 10 fate map.
1997,
Pubmed
,
Xenbase
Mu,
EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine "fingers" and contains a calmodulin-binding IQ motif.
1995,
Pubmed
Myat,
Organ shape in the Drosophila salivary gland is controlled by regulated, sequential internalization of the primordia.
2000,
Pubmed
Odell,
The mechanical basis of morphogenesis. I. Epithelial folding and invagination.
1981,
Pubmed
Ogata,
TGF-beta signaling-mediated morphogenesis: modulation of cell adhesion via cadherin endocytosis.
2007,
Pubmed
,
Xenbase
Pelissier,
Trafficking through Rab11 endosomes is required for cellularization during Drosophila embryogenesis.
2003,
Pubmed
Schoenwolf,
Quantitative analyses of changes in cell shapes during bending of the avian neural plate.
1984,
Pubmed
Shaye,
Modulation of intracellular trafficking regulates cell intercalation in the Drosophila trachea.
2008,
Pubmed
Shook,
Pattern and morphogenesis of presumptive superficial mesoderm in two closely related species, Xenopus laevis and Xenopus tropicalis.
2004,
Pubmed
,
Xenbase
Shook,
Epithelial type, ingression, blastopore architecture and the evolution of chordate mesoderm morphogenesis.
2008,
Pubmed
Tsarouhas,
Sequential pulses of apical epithelial secretion and endocytosis drive airway maturation in Drosophila.
2007,
Pubmed
Ulrich,
Wnt11 functions in gastrulation by controlling cell cohesion through Rab5c and E-cadherin.
2005,
Pubmed
Voiculescu,
The amniote primitive streak is defined by epithelial cell intercalation before gastrulation.
2007,
Pubmed
,
Xenbase
Wallingford,
Neural tube closure requires Dishevelled-dependent convergent extension of the midline.
2002,
Pubmed
,
Xenbase
