QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00211

This Article Summary Full Text Full Text (PDF) Movies Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, J.-Y. Articles by Goldstein, B. Search for Related Content PubMed PubMed Citation Articles by Lee, J.-Y. Articles by Goldstein, B.

Mechanisms of cell positioning during C. elegans gastrulation

Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA

View larger version (36K): [in a new window]   Fig. 1. C. elegans gastrulation. (A) Early embryonic lineage of C. elegans development. Daughters are annotated `a' for anterior, `p' for posterior, `l' for left, and `r' for right. Anterior is towards the left. The endoderm precursors, Ea and Ep, are labeled in blue. The neighboring cells that close the gastrulation cleft are in green (ABplpa, ABplpp, MSap, MSpa, MSpp and P4). The scale on the left refers to time, in minutes, since fertilization. (B) Confocal images of lateral view of gastrulating embryos labeled with the membrane marker SynaptoRed to better visualize cell boundaries. Ea and Ep ingress towards the center of the embryo, and are eventually surrounded by MSap and P4. Asterisks indicate Ea and Ep and neighboring cells are labeled with arrows in B-D. (C,D) Panels show DIC time-lapse views of gastrulation. (C) Lateral view of gastrulation, similar to B. Images are at 10 minute intervals. (D) Ventral view of gastrulation. From left to right, time intervals are 0, 12 and 32 minutes. As Ea and Ep `sink' into the embryo, six cells close up the ventral cleft. Note that while ABplpa and ABplpp start moving toward the cleft, they divide and the posterior daughters of these cells finish the movement. In this and all figures, embryos are oriented anterior towards the left, and posterior towards the right. Scale bars: 10 µm. Movies of time-lapse images are available online at http://dev.biologists.org/supplemental/.

View larger version (15K): [in a new window]   Fig. 7. ML-7 and ML-9, myosin light chain kinase inhibitors, are more potent inhibitors of gastrulation than H-7, a PKA inhibitor. Embryos were assayed as gastrulation-inhibited if the ventral surfaces of Ea and Ep were not fully covered by P4 and MSxx by 1 hour after exposure to inhibitor. Ki (inhibition coefficient, expressed in µM) of the drugs against MLCK are as follows: ML-7, 0.3; ML-9, 3.8; H-7, 97 (Mabuchi and Takano-Ohmuro, 1990). We found that the doses required to completely inhibit gastrulation were as follows: ML-7, 250 µM; ML-9, 750 µM; H-7, 4 mM. We note that as much as 300 µM was required to inhibit MLCK in Xenopus growth cones (Ruchhoeft and Harris, 1997), and high concentrations may be required to out-compete the reservoir of endogenous ATP, as both ML-7 and ML-9 are ATP analogs (Saitoh et al., 1987). Each data point represents between four and 13 embryos.

View larger version (72K): [in a new window]   Fig. 8. Microsphere-marking of cell surfaces reveals that the ventral surfaces of Ea and Ep contract during gastrulation. (A) Kymograph derived from movements of microspheres (white) during gastrulation from one film of a wild-type P1 isolate. In the kymograph, the image in each frame of the time-lapse recording is used to generate horizontal lines of image data that are pasted together in descending order; hence, time is represented on the y-axis. Each pixel in a horizontal line of image data was created by selecting the brightest pixel in a 20-pixel high region. As a result, horizontal movement of microspheres can be seen as horizontal movement of white dots as the kymograph proceeds from top to bottom. First frame and last frame used for the kymograph are above and below the kymograph, respectively. Ea and Ep are labeled with asterisks, the Ea/Ep boundary is marked by yellow arrowhead in still frames and by the yellow line in the kymograph, and the white arrow indicates the direction of MSxx movement. The microspheres on Ep can be seen converging towards each other during gastrulation movements. (B) Summary of microsphere movements traced from all ten simultaneous DIC/GFP films. Each arrow indicates the total displacement and angle of movement by each microsphere. `X' indicates microspheres that did not exhibit any displacement relative to the displacement of the cell. (C) Average of the vectors (see Materials and Methods). Insets show the directions of microsphere movement from each quadrant, with the average direction in gray. The average velocity for each set of vectors is shown below the box.

View larger version (118K): [in a new window]   Fig. 2. Gastrulation in intact embryos, devitellinized embryos and P1 isolates. Asterisks indicate Ea and Ep, arrows indicate MSxx and P4. Time-lapse images were time-standardized between all four sets, with 0 minutes indicating the start of gastrulation movements. (A-D) Time-lapse images of gastrulation in an intact embryo (same embryo as Fig. 1C), (E-H) in a devitellinized embryo, and (I-P) in P1 isolates. Variation of starting orientation between intact and devitellinized embryos is due to devitellinization (A versus E). The two sets of P1 isolate images represent two different division patterns, either in a dumb-bell orientation (I-L) or in a linear orientation (M-P). Linear orientation occurs in less than 10% of all P1 isolates (data not shown). Other division patterns were variations between these two extremes. Scale bars: 10 µm. Movies of time-lapse images available online at http://dev.biologists.org/supplemental/.

View larger version (29K): [in a new window]   Fig. 3. Timing and extent of gastrulation movements. (A) Timing of the beginning of gastrulation movements in intact embryos (light gray circles), devitellinized embryos (medium gray circles) and P1 isolates (black circles) with respect to the length of the Ea and Ep cell cycle. The timing of gastrulation was normalized between embryos (see Materials and Methods). Black ticks along Ea/Ep lineage represent a fifth of the total cell cycle. The early lineage is shown for reference. (B,C) Quantification of P4 and MSxx movement relative to Ea and Ep in wild-type (WT) intact, devitellinized, P1 isolates and mom-2 P1 isolates. The data are shown by quadrant graphs, with gray unbroken lines representing individual cases and black broken lines representing the mean for all embryos. A diagram representing the measurement method is shown above the graphs. (B) Quantification of P4 movement relative to Ea and Ep from the time P4 is born until MSxx is born (12 minutes). (C) Quantification of MSxx and P4 movement relative to Ea and Ep after MSxx is born (30 minutes).

View larger version (38K): [in a new window]   Fig. 4. MSxx and P4 do not chemotax towards each other during gastrulation. Asterisks indicate Ea and Ep, an `X' indicates the removed cells, and the remaining neighbor is labeled with an arrow. (A-C) P4 moves with respect to Ea and Ep in the absence of MS. (D-F) MSxx moves with respect to Ea and Ep in the absence of P4. (G) Angle quantification of six sets of each manipulation, as in Fig. 3. The angle measurements for P4 represent only the first 12 minutes of movement, as this number was significant from mom-2 P4 (see Results, Table 1). Scale bars: 10 µm.

View larger version (73K): [in a new window]   Fig. 5. Microfilaments are required for gastrulation. Asterisks indicate Ea and Ep. Confocal imaging of live embryos documents the qualitative, not quantitative, effects of pharmacological agents on tubulin and actin distribution. (A-C) Effects of drugs on tubulin in live embryos expressing Tubulin::GFP strain, treated with (A) 1% DMSO (control), (B) 50 µM taxol and (C) 10 µM nocodazole. (D-F) Effects of drugs on actin in live embryos expressing Actin::YFP, treated with (D) 1% DMSO, (E) 5 µg/ml cytochalasin D and (F) 10 µM latrunculin B. 1% DMSO treatment was the control as this was the final concentration of DMSO used for the drug studies. Gastrulation progressed normally in the presence of 1% DMSO. To the right of each image are the numbers of embryos assayed for gastrulation. The embryos imaged were different from those used for the assay. Scale bar: 10 µm.

View larger version (105K): [in a new window]   Fig. 6. No lamellipodia or filopodia form in P1 isolates. Ea and Ep are labeled with asterisks, and arrows indicate presumptive leading edges of MSxx and P4. (A) Scanning electron micrograph of a P1 isolate. (B) F-actin localization. (C) Phosphotyrosine localization. An enrichment of phosphotyrosine was seen at the Ep/P4 boundary, but is unlikely to be functionally significant as it was not polarized in the direction of movement and it was not present at the Ea/MSxx boundary. Additionally, it was recently shown that phosphotyrosine accumulates at the EMS/P2 boundary due to a signaling pathway that is not implicated in gastrulation (Bei et al., 2002). Scale bars: 10 µm.

View larger version (28K): [in a new window]   Fig. 9. Ea and Ep polarity directs the movement of their neighbors. On the left side of each panel is a schematic drawing of part of a P1 isolate and the experiment performed. Cells in the isolates were separated at the site indicated by the broken line, rotated along an axis (unbroken gray line in A), and recombined in their normal orientations. The arrow below the cells indicates the direction of cell movement by the reference cell. On the right of each panel is the corresponding side view, in which the P1 isolates are oriented as if looking down from one end, either P4 or MSxx (as indicated). Yellow arrows indicate the direction of movement of P4 cells and the green arrows indicate the direction of movement of MSxx cells. Separation and recombination of (A) Ea and Ep, with P4 as the reference cell; (B) MSxx and Ea, with P4 as the reference cell; or (C) P4 and Ep, with MSxx as the reference cell.

View larger version (20K): [in a new window]   Fig. 10. Model of cell contraction during C. elegans gastrulation. (Top) Ea and Ep with myosin enriched at the ventral side. Hatching represents myosin, while gray arc underneath the cells represents the eggshell. Myosin-based contraction causes the ventral side of Ea and Ep to constrict, bringing neighbors closer to each other, which pushes Ea and Ep into the center of the embryo (bottom).