Endocytosis Is Required for Efficient Apical Constriction During Xenopus

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provided by Elsevier - Publisher Connector Current Biology 20, 253–258, February 9, 2010 ª2010 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2009.12.021 Report Endocytosis Is Required for Efficient Apical Constriction during Xenopus Gastrulation

Jen-Yi Lee1,* and Richard M. Harland1,* To determine where endosomes traffic in bottle cells, we 1Department of Molecular and Cell Biology, labeled stage 9 embryos with biotin, quenched the reaction, University of California, Berkeley, Berkeley, CA 94720, USA and fixed the embryos at 30 min intervals (‘‘chase’’). As cells underwent AC, the number of endosomes also increased, strengthening the correlation between endocytosis and AC. Summary Endosomes trafficked away from the apical membrane, and some vesicles colocalized with the basolateral membrane Coordinated apical constriction (AC) in epithelial sheets (Figure 1D). drives tissue invagination [1, 2] and is required for diverse Previously, we showed that intact but not dynamic microtu- morphogenetic movements such as gastrulation [3], neuru- bules were required for AC [7]. If intact microtubules function as lation [4, 5], and organogenesis [6]. We showed previously tracks for endosomes, then nocodazole treatment should that actomyosin contractility drives AC in Xenopus laevis prevent endocytosis, whereas taxol treatment should not. bottle cells [7]; however, it remained unclear whether it Indeed, in nocodazole-treated embryos, endosomes remained does so in concert with other processes. Here we report at the apical membrane (see Figure S1 available online). In that endocytosis-driven membrane remodeling is required contrast, taxol-treated embryos exhibited internalized vesicles for efficient AC. We found endosomes exclusively in bottle indistinguishable from controls (Figure S1). Our data confirm cells in the early gastrula. Disrupting endocytosis with that intact microtubules are required for endocytosis and dominant-negative dynamin or rab5 perturbed AC, with a show that endosomes move toward the basolateral membrane significant decrease in constriction rate late in the process, in bottle cells. suggesting that endocytosis operates downstream of acto- myosin contractility to remove excess membrane. Addition- Perturbing Endocytosis Disrupts Apical Constriction ally, disrupting endocytosis during neurulation inhibits AC Because intact microtubules are required for both AC [7] and in hingepoint cells, resulting in neural tube closure defects. endocytosis (Figure S1), we postulated that endocytosis could Thus, membrane remodeling during AC could be a general be required for AC. To test this, we perturbed the initial step of mechanism to achieve efficient invagination in embryos. endocytosis with dominant-negative (DN) dynamin, which weakly binds GTP, preventing endosome scission [15, 16]. Results and Discussion Targeting DN dynamin to the DMZ perturbed blastopore formation (Figure 2A) but did not affect cell morphology when Endosomes Are Present Only in Bottle Cells expressed in non-DMZ cells (Figure S2A). Despite the signifi- and Traffic along Microtubules cant effect on AC (see below) and delay in blastopore forma- In Xenopus laevis embryos, cells in the dorsal marginal zone tion, DN dynamin-injected embryos (referred to as DN dynamin (DMZ) undergo apical constriction (AC) to form bottle cells, embryos) developed with no obvious defects (Figure S2F). initiating blastopore invagination [8, 9]. Previously, we showed This is consistent with the finding that development occurs that taxol, which stabilizes microtubules, does not affect bottle normally even after surgical removal of bottle cells [9]. cell formation, whereas nocodazole, a microtubule depolyme- To confirm that DN dynamin disrupts endocytosis, we rizing agent, disrupts AC; therefore, microtubules must be counted endosomes in GFP and DN dynamin embryos. DN intact but need not be dynamic for AC [7]. Because microtu- dynamin embryos had significantly fewer endosomes than bules are necessary for vesicular trafficking [10], we hypothe- control embryos (Figure 2B; Figure S2B), confirming that sized that microtubules may facilitate AC by functioning as endocytosis was inhibited. tracks for endosomes. To determine the effect of inhibiting endocytosis on AC, we To determine whether endocytosis occurs in bottle cells, measured blastopore depth and bottle cell morphometric we employed a cell-impermeable activated biotin that was parameters, including apical width, apicobasal length, and previously used to surface label Xenopus embryos [11, 12]; apical index (length-over-width ratio, AI) [7]. DN dynamin blas- subsequently, internalized membrane could be visualized topores were 36% shallower than GFP blastopores (Figures with fluorescent streptavidin. We observed biotin-labeled 2B and 2C). Additionally, DN dynamin bottle cells were signif- vesicles exclusively in bottle cells (Figure 1A), starting in stage icantly less constricted than control embryos, whereas apico- 10 gastrulae (data not shown); therefore, the appearance of basal length was indistinguishable between the two groups vesicles accompanies the onset of constriction. (Figure 2C). The wider apices in DN dynamin bottle cells ac- To confirm that biotinylated vesicles represented endo- counted for the significant decrease in AI compared to control somes, we used endosome markers EEA1 [13] and rab5- (Figure 2C). These effects were rescuable with wild-type dyna- EGFP [14]; labeled vesicles colocalized with both markers, min, illustrating specificity (Figures 2A–2C). Interestingly, confirming that they are endosomes (Figures 1B and 1C). We these measurements recapitulate what we previously ob- therefore exploited the specificity and low background of served in nocodazole-treated embryos [7], suggesting an biotinylation to assay endocytosis. overlapping function between endocytosis and microtubules during AC. These results suggest that dynamin-mediated endocytosis is required for efficient AC in bottle cells. *Correspondence: [email protected] (J.-Y.L.), [email protected] To test whether DN dynamin affects bottle cell AC by per- (R.M.H.) turbing cell fate [16], we examined chordin, xbra, and sox17 Current Biology Vol 20 No 3 254

these deleterious side effects, we chose to use DN dynamin in our experiments as an effective, specific reagent to block dy- namin function. To further elaborate the role of endocytosis during AC, we in- hibited rab5, a small GTPase that is required for early endo- some sorting, by injection of DN rab5, which binds GTP weakly [18]. Internalized biotin was nearly undetectable in DN rab5 embryos, confirming inhibition of endocytosis (Figure 2D; Figure S2C). DN rab5 embryos had 33% shallower blastopores and less constricted bottle cells relative to uninjected embryos, with no differences in apicobasal length (Figure 2E). As with DN dynamin, cell fates were unaltered by DN rab5 (Figure S2E), consistent with the finding that DN rab5 does not affect activin signaling [19]. Compared to controls, DN rab5 tailbud-stage embryos were short and kinked (Figure S2G), indicative of defective convergent extension [20]. Interestingly, overexpres- sion of constitutively active rab5 had no effect on blastopore formation or bottle-cell morphometrics (Figures S3D and S3E), suggesting that upregulating endocytosis at the level of rab5 cannot increase the rate or extent of AC. Together, the DN dynamin and DN rab5 results strongly argue that endocy- tosis is required for AC in bottle cells.

Endocytosis Is Required for Apical Constriction Downstream of Actomyosin Contractility Although endocytosis is required for efficient AC, it did not Figure 1. Biotinylated Vesicles Are Present Only in Bottle Cells in Xenopus completely abolish contraction. Thus, we hypothesized that Early Gastrula-Stage Embryos actomyosin contractility folds the apical membrane into micro- (A) Vegetal view (left, epifluorescence), midsagittal section (center, villi and that endocytosis is then required to remove the excess confocal), and higher magnification of midsagittal section (right) of stage membrane. 10 embryos labeled with NHS-LC-sulfo biotin and anti-DM1a (tubulin). This hypothesis makes three predictions. First, actin and Arrow indicates bottle cells. Scale bar represents 25 mm. myosin should be apically localized in both GFP and DN dyna- (B) Confocal midsagittal sections of embryos stained with anti-EEA1 and min bottle cells. Indeed, both GFP and DN dynamin bottle cells biotin. (C) Confocal midsagittal sections of embryos injected with rab5 enhanced exhibited apical localization of F-actin and activated myosin green fluorescent protein (EGFP) mRNA, then fixed and stained with anti- (Figure 3A), suggesting that endocytosis acts downstream of GFP and biotin. Arrows in (B) and (C) indicate colocalization of biotin with the establishment of the apical actomyosin machinery. The early endosome markers. correct apical localization of F-actin and myosin in Xenopus (D) Confocal midsagittal sections of embryos following pulse-chase labeling bottle cells also indicates that endocytosis is not required for with biotin. Embryos were fixed at the indicated time points and stained with some aspects of polarity, as it is in Caenorhabditis elegans anti-DM1a and streptavidin. Arrows indicate endosomes that appear to be on or near the basolateral membrane. In all midsagittal sections, images are [21] and Drosophila [22]. oriented vegetal to the lower left; scale bars for (B)–(D) represent 50 mm. See The second prediction is that if endocytosis is required also Figure S1. downstream of actomyosin contractility and membrane folding, then microvilli should be present at the apical surface expression as readouts of organizer, mesoderm, and endo- of both GFP and DN dynamin bottle cells. Microvilli have been derm fate, respectively. Expression was indistinguishable observed in many apically constricting cells, including bottle between controls and DN dynamin embryos (Figure S2E). cells [23], where they contribute to reduction in apical surface Thus, DN dynamin does not affect AC indirectly through area. Transmission electron microscopy revealed that, altering cell fate. whereas animal cells exhibited unfolded apical membrane, To independently assess the role of dynamin during AC, we both GFP and DN dynamin bottle cells contained numerous injected translation-blocking antisense morpholino oligonu- microvilli (Figure 3B). Apart from the difference in apical width, cleotides (MO) targeting dnm-like and dnm-2, the two most there were no apparent disparities between GFP and DN dyna- abundant transcripts during gastrulation [17]. Whereas single min bottle cells, suggesting that membrane folding is not MO injections at 160 ng did not affect endocytosis or AC, significantly disrupted in DN dynamin bottle cells. double injections of 80 ng of each MO significantly perturbed The final prediction is that both GFP and DN dynamin bottle both processes (Figures S3A–S3C). cells should initially constrict at similar rates but, as membrane Because only the double morphants exhibited endocytosis accumulates, the DN dynamin bottle cells should slow down. and AC defects, we suggest that both isoforms are involved To test this prediction, we monitored the apical surface area in bottle cell AC; moreover, the effect on endocytosis and at the blastopores of embryos injected with membrane- AC is specific and not due to off-target effects. However, enhanced GFP (EGFP) alone or GFP plus DN dynamin (Movies both single and double morphants exhibited a range of devel- S1 and S2; Figure 3C). Measurement of apical surface areas opmental phenotypes such as failure to close the blastopore, at 3 min intervals over the course of 15 min revealed that defective convergent extension, and embryonic lethality, both GFP and DN dynamin bottle cells with apices greater presumably caused by MO-related toxicity (data not shown). than 250 mm2 exhibited nearly identical rates of AC, but, as Because DN dynamin effectively inhibited endocytosis without expected, the constriction rate decreased as cells became Endocytosis Is Required for Apical Constriction 255

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 strep- tavidin. 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. smaller (Figure 3C; Table S1). In cells with apical surfaces Endocytosis Occurs in the Apically Constricting Neural smaller than 250 mm2, constriction rates were initially indistin- Tube guishable between GFP and DN dynamin bottle cells. How- We propose that actomyosin contractility initiates AC, folding ever, as AC proceeded, GFP bottle cells continued to constrict apical plasma membrane into microvilli (Figure 4A). As the at a constant rate, whereas DN dynamin bottle cells stalled, apical surface area decreases, excess membrane must be constricting significantly more slowly than GFP cells (Fig- removed by endocytosis to accommodate further shrinkage. ure 3C; Table S1). Therefore, as we predicted, DN dynamin When endocytosis is inhibited, either by microtubule depoly- cells initially constrict at the same rate as their GFP counter- merization or by blocking core members of the endocytic parts but then stall as they get smaller, presumably as a result pathway, AC initiates properly but slows because of a buildup of excess membrane that impedes constriction. Our results of excess membrane. suggest that AC involves a dual mechanism: actomyosin To determine whether this mechanism is specific to bottle contraction accompanied by membrane removal to drive cells or whether it is applicable elsewhere, we examined the efficient invagination. role of endocytosis during Xenopus neural tube (NT) closure, Current Biology Vol 20 No 3 256

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 dyna- min-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 pseudocol- ored 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 indi- cate 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.

closure (58 of 58 embryos), whereas DN dynamin embryos exhibited a range of phenotypes, varying from wild-type (15 of 50 embryos) to kinked (21 of 50 embryos) to open anterior NT or mild exence- phaly (28 of 50 embryos)(Figure 4C). The kinked phenotype probably results from the uninjected side crossing over the midline to compensate for less efficient folding on the DN dynamin-injected side. In cross-section, DN dynamin hingepoint cells appeared less constricted and less elon- gated compared to GFP-injected cells (Figure 4D) and to their uninjected counterparts (Figure S4). Together, our results show that inhibiting endocy- tosis with DN dynamin causes NT closure defects and suggest that inefficient AC in the dorsolateral hingepoint cells may be the underlying cause of these defects.

Endocytosis and Morphogenesis Endocytosis is important for many cellular processes during development such as mainte- nance of epithelial polarity [22], signaling during convergent extension [20, 26–28], and cellulariza- tion [29]. In particular, endocytosis at the apical membrane is crucial for Drosophila tracheal morphogenesis to modulate membrane tension [30], to clear proteins from the lumen [31], and where cells undergo AC to bend the neural plate [24]. In mice, to remodel adherens junctions during intercalation [32]. defective AC results in NT closure defects such as exence- Whereas previous reports showed that endocytosis is impor- phaly and spina bifida [25]. To confirm that endocytosis occurs tant for organ shape, our results point to an additional in the Xenopus NT [12], we biotinylated neurula-stage embryos mechanism where endocytosis is required during AC to affect and observed endocytic vesicles exclusively in the NT (Fig- cell shape. ure 4B). Next, we perturbed endocytosis by targeting DN dyna- In the context of AC, endocytosis may be a general mecha- min mRNA to the anterior NT. Unilaterally injected embryos nism to accelerate constriction during bending and invagina- were selected so that the uninjected side could serve as an tion. Interestingly, the two C. elegans endoderm precursors internal control. Neural plates expressing GFP alone exhibited that ingress during gastrulation do so via AC [33], but they NT morphology and closure rates indistinguishable from the do not undergo obvious shape changes or contain microvilli uninjected side, whereas DN dynamin neural folds were irreg- [34], suggesting that membrane folding and endocytosis may ularly shaped and formed more slowly than the uninjected side only be necessary in cases of AC associated with cell-sheet (Movie S3). At stage 24, all GFP embryos had completed NT invagination. Furthermore, efficient AC may be more important Endocytosis Is Required for Apical Constriction 257

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 represents50mm. (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. for embryos that rely on ingression en masse to drive morpho- type and DN dynamin were coinjected. For neural tube experiments, genesis, such as in the chick primitive streak [35] and in the 100 pg membrane EGFP and 200 pg DN dynamin1 mRNA were injected gastrulating axolotl [36]. That inefficient AC is not more delete- into one animal-dorsal cell at the eight-cell stage. rious for Xenopus embryos is consistent with the view that several engines cooperate to drive gastrulation, and removal Staining of just one may have modest outcomes [37]. Nonetheless, Embryos were labeled with 2.5 mg/ml sulfo-NHS-LC-biotin (Pierce) [11]. Biotin was detected with 1:500 Alexa Fluor 555 streptavidin (Molecular our results implicate endocytosis-driven membrane remodel- Probes). Embryos were stained [7] with primary antibodies against mouse ing as a mechanism essential for efficient AC and subsequent DM1a (1:500; Sigma), rabbit EEA1 (1:200; Abcam), rabbit phospho20 myosin invagination of cell sheets. light chain (1:500; Abcam), and chicken GFP (1:500; Abcam). Goat Alexa Fluor secondary antibodies (Molecular Probes) were used at 1:500. Standard antibody specificity controls were performed. Experimental Procedures

Embryo Culture and Manipulation Imaging and Morphometrics Embryo culture and inhibitor experiments were performed as described Embryos were imaged and quantified as described [7]. Statistical signifi- [7, 38]. Capped mRNA was injected dorsovegetally in two cells at the cance was determined by two-tailed t tests. For time-lapse movies, images four-cell stage. Amounts injected were 250 pg GFP (membrane-tethered were captured every 30 s with the 633 objective on a Leica DM RE confocal EGFP) [39], 250–500 pg DN dynamin1-K44E, 1 ng DN rab5-S34N, and microscope with Leica TCS software. Apical surface areas were measured 500 pg rab5-EGFP [16]. For rescue experiments, equal amounts of wild- with NIH ImageJ. Current Biology Vol 20 No 3 258

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