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First published online February 18, 2004
doi: 10.1242/10.1242/dev.01005

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Active cell migration drives the unilateral movements of the anterior visceral endoderm

Shankar Srinivas^1,3,,, Tristan Rodriguez^1,*,, Melanie Clements^1,, James C. Smith^2,3 and Rosa S. P. Beddington^1,

¹ Division of Mammalian Development, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
² Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
³ Wellcome Trust/Cancer Research UK Institute and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK

View larger version (114K): [in a new window]   Fig. 1. Representative frames (A-C) from a movie of a cultured 5.5 dpc embryo (see Movie 1 at http://dev.biologists.org/supplemental/). (D) Drawing depicting the orientation of the embryo. The embryo was imaged every 15 minutes with phase-contrast and fluorescence optics. Time from the start of imaging is indicated in hours at the bottom right of each frame. EGFP fluorescence (green) marks the anterior visceral endoderm (AVE). The embryo develops normally and the AVE moves unilaterally to the extra-embryonic ectoderm in approximately 4 hours. It does not move beyond the epiblast for the remainder of the culture period. A, prospective anterior; P, prospective posterior. Scale bar in A: 50 µm for A-D.

View larger version (93K): [in a new window]   Fig. 2. Frontal view (A-K) of a developing 5.5 dpc embryo (see Movies 2 and 3). (L) Drawing depicting the orientation of the embryo, with anterior facing the reader. The embryo was imaged every 12 minutes with phase-contrast and fluorescence optics. In this figure the phase-contrast images have been made darker so that the fluorescent AVE cells can be seen more clearly. Time from the start of imaging is indicated in hours and minutes at the bottom right of each frame. AVE cells move proximally until they reach the junction of the epiblast with the extra-embryonic ectoderm and then start spreading laterally. Cells cover this distance in about 5 hours and project filopodia in the direction of motion (see Fig. 3). Note that fluorescent cells can be seen interspersed with non-fluorescent cells at time points later than 2 hours. Scale bar in A: 50 µm for A-L.

View larger version (43K): [in a new window]   Fig. 5. Pre-streak stage mouse embryos imaged with epifluorescence and confocal microscopy. Hex-GFP expressed in the AVE is green, nuclei are blue (stained with DAPI) and cell borders are red (stained for actin with TRITC-Phalloidin). Each panel shows a different embryo, with anterior always to the left. (A) An epifluorescence and phase-contrast image of an embryo showing the AVE clearly discernible as a thickening of the visceral endoderm. (B) A 3D-volume rendering of a confocal image stack of an embryo at an equivalent stage, showing the plane at which the confocal sections in panels C and D were acquired. (C) A confocal section through the distal tip of an embryo, illustrating the columnar nature of the single layer of cells at the distal tip. The cells are clearly polarised, their nuclei closer to the epiblast. GFP-expressing and non-expressing cells can be seen intermingled in the AVE. (D) An embryo in which the AVE has started migrating proximally. AVE cells migrate in contact with the epiblast at all times, and are never seen on top of other visceral endoderm cells. Scale bar in D: 40 µm for A; 10 µm for C; 15 µm for D.

View larger version (57K): [in a new window]   Fig. 3. Details of migrating AVE cells, showing filopodial processes. In all the panels, proximal is to the top and distal to the bottom. (A,B) Cells from one embryo, separated by an interval of 10 minutes. (C,D) Cells from a different embryo, separated by an interval of 12 minutes. Filopodia (marked with arrows) form primarily in the proximal direction (the direction of motion of the cells). (E,F) Panels separated by an interval of 10 minutes showing an AVE cell dividing (arrowhead). The orientation of the division of AVE cells is not consistently aligned to the direction of motion of the AVE, and divisions are not observed frequently enough to drive the movement of the AVE. Scale bar in A: 10 µm for A-F. (G) A polar plot of the direction and length of 23 filopodia observed in seven embryos. The lengths of the filopodia are expressed as a fraction of the radius of the cell. Filopodia form predominantly in the proximal direction of the embryo and are often greater than one cell radius in length.

View larger version (74K): [in a new window]   Fig. 4. Movement of individual cells of the AVE. (A,B) Tracks of eight cells overlaid on the last frame of the movie so as to show the paths they took to reach their final position (Fig. 2, Movies 2 and 3). Cells are labelled c1 to c8. Cells were tracked after 1 hour and 48 minutes had elapsed because they could not be reliably distinguished before this. Cell movements are rather direct during distal to proximal migration, but become highly convoluted once they start spreading laterally. Cells 4 and 7, and 5 and 6 are sister pairs, and hence share a common track prior to their division. (C) A quantitative measurement of cell behaviour was obtained by calculating, for each cell and at each time interval of 12 minutes, the ratio of its proximal displacement to its lateral displacement. The mean values for all eight cells were calculated for each time point, and are plotted against time in culture. Initially, the ratio tends to be substantially greater than unity, indicating that displacement is predominantly proximal. At approximately 5 hours (red arrow), when cells reach the boundary of epiblast and extra-embryonic ectoderm, the ratio decreases to less than one, indicating that motion is predominantly lateral. (D) Inspection of panels A and B suggests that cells move to the left on reaching the boundary between epiblast and extra-embryonic ectoderm. This is an artefact introduced by the fact that the embryo `rolled' slightly to the left during culture (see Movie 2, at 7 hours of culture). The effects of rolling can be abrogated by calculating the separation between cells, and D plots the separation between two representative pairs of cells, as well as the average distance moved by the four cells during each 12-minute time interval. Cells 1 and 3 move apart during culture, whereas 2 and 4 come closer together, indicating that cells do not behave in a coordinated manner on reaching the extra-embryonic ectoderm. Though the distance covered by cells in each time interval varies widely, it does not show any trend over the course of culture, indicating that the cells do not slow down or speed up. Scale bar: 50 µm.

View larger version (150K): [in a new window]   Fig. 6. Whole-mount in situ hybridisation of wild-type 5.5 dpc embryos showing expression of Hex (A) and Cerl (B). Note that not all AVE cells express these two markers of the anterior visceral endoderm. Scale bar: 50 µm.

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