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Files in this Data Supplement:
Fig. S1. Lateral view of the moving cell mass. (A) View of the moving cell mass of the posterior half of a hemisegment of segment 2 and an anterior part of segment 3. Anterior is to the left. The red dashed line indicates the area that is shown in a lateral section in B. (B) Lateral section through the moving cell mass. Top is to the right. The histoblasts move as a single cell layer. Among other cells, muscle precursors and oenocytes are positioned in a layer underneath.
Fig. S2. Distance maps. The average distance to each cell’s six closest neighbours was plotted at 30 (A) and 40 (B) hours APF (pupa #1). The histoblasts are spaced more evenly at the end of their movement. In the posterior part of the segment, cells are closer together than in the anterior part. The average distance between cells is 7 µm. Anterior is to the left.
Fig. S3. Cell migration of histoblasts in segment 3. The trajectories of cells at the very tip of one hemisegment of segment 3 are shown. Similar to segment 2 (see Fig.1F), the cells move dorsally and then turn anteriorly. Anterior is to the left. Scale bar: 50 µm.
Fig. S4. Division angles. In situ plots of all divisions of the tracked cells of pupa #2 (A) and pupa #4 (B). The banded LUT shows the angles on a 1° to 180° scale indicating the direction of the deviation of the division from the DV axis (90°). Comparing both pupae, there seem to be no regions where cells divide similarly.
Fig. S5. Analysis of ‘wild-type’ clones marked with DsRed. The marked clones of cells have an elongated shape in the DV axis and do not split. Clones of cells are marked with nuclear DsRed. All nuclei are marked with Histone::GFP. Besides the clones in the epithelium, individual cells underneath the epithelium also express DsRed. Anterior is to the left. The yellow dashed line indicates the dorsal midline.
Fig. S6. Model of the changes in the arrangement of cells in the moving cell mass. The arrows indicate the direction of movement and their colour indicates cell velocity. The purple lines indicate adhesive forces between cells that might be graded in the AP direction (Lawrence et al., 1999). The pink lines indicate compartment boundaries. The depicted LECs express higher levels of Sqh::GFP and are only consecutively removed, which might canalise the movement of the histoblasts in the dorsal direction. Cells in posterior areas of the moving cell mass move dorsally for a longer time. The most anterior cells start their anterior movement first. Differential adhesion at the compartment boundary (Morata and Lawrence, 1975) might enable cells to move more freely, leading to more extensive rearrangements near the boundary.
Movie 1. Spreading of histoblasts. Please also use the arrow keys to play the movies slowly as details might only be visible by scrolling through the movies frame by frame. This movie shows the entire migration of the histoblasts (18 to 47 hours APF) using a Histone::GFP marker (segments 2 and 3). At the beginning of the movie, the anterior and posterior dorsal histoblast nests fuse. Then the histoblasts move as a single epithelial layer towards the midline and replace the LECs. One row of LECs separates the histoblasts of neighbouring segments laterally from each other and is only subsequently removed. To be able to film the entire process, the pupa was rolled once during filming to allow a view of the dorsal midline although filming started laterally. Maximum projection of a z-stack of 23 frames covering 57.5 µm. Anterior is to the left. Image area is 555 µm2. See Fig. 1A,B.
Movie 2. Tracking histoblasts of segment 2 and distinguishing the A and P compartments. Histone::GFP marks the nuclei of all cells. Cells that were tracked using SIMI Biocell are marked with a white dot. Additionally, en.Gal4 drives DsRed expression in cells of the P compartment. Twenty-five to 41 hours APF. Maximum projection of a z-stack of 15 frames (45 µm). Anterior is to the left. Dorsal view. Image area is 328 µm2. See Fig. 1C,D.
Movie 3. 3D representations of the development of segment 2. Movies of the 3D representations shown in Fig. 2A. (A) Left panel and (B) right panel.
Movie 4. The velocity of histoblasts. Movie showing the velocities of all cells calculated at consecutive time intervals plotted at the cell's position using a banded LUT (see Fig. 2B). The velocities of the cells change slightly over time. Histoblasts that are more posterior in the segment tend to move faster. Cells are coloured grey if the second of the two coordinates used to calculate velocity is missing. Anterior is to the left.
Movie 5. Imaging the development of segment 2 using a membrane marker. The small histoblasts replace the large larval cells. One can see how the cell-cell junctions change their lengths constantly and also how junctions disappear and form (see also Movies 6, 7). Towards the end of the movie, the segmental fold is forming. Bristle cells show a cadherin-rich structure. DE-cadherin::GFP. Maximum projection of a z-stack of four frames (10 µm). Anterior is to the left. Dorsal view. Image area is 244 µm2. 16.8 hours of development are shown.
Movie 6. Dynamics of cell-cell junctions. Detail from Movie 5. The membrane of one cell is coloured white. Red dots highlight the junctions. The marked cell rearranges relative to the cell positioned dorsal to it. At the end of the movie, it is positioned anterior to it. DE-Cadherin::GFP. Anterior is left, dorsal is up.
Movie 7. Cell rearrangement by remodelling of cell-cell junctions. Detail from Movie 5. The featured cell, the membranes of which are highlighted in white, temporarily changes its position relative to its neighbour (asterisk) and a junction is built anew (red line). Please note the dynamics of the process. DE-Cadherin::GFP. Three minutes per frame. Anterior is left, dorsal is up.
Movie 8. Cells push each other locally. Detail from Movie 2. Two histoblasts are marked using red and blue arrows. The ‘red’ nucleus moves above the ‘blue’ one and divides (M). In general, divisions occur on the apical side of the epithelium. In this example, the initial positions of cells are restored in the end.
Movie 9. Some histoblasts change their neighbours during development. Detail from Movie 5. A cell (red asterisk) not far away from the A/P boundary is marked. It divides (M) and its more medially positioned daughter is tracked using red asterisks. Two immediate neighbours are marked with a white and a yellow asterisk, respectively. Later, these cells lose contact. Anterior is left, dorsal is up.
Movie 10. The trajectories of the movements of the LECs. The paths the LECs moved in 30-minute intervals are plotted, similarly to Movie 11 (pupa #1). The movement of the histoblasts is shown in grey for reference. Anterior is to the left. See also Fig. 6C.
Movie 11. The trajectories of the movements of the histoblasts. The paths of all cells within a 30-minute interval are plotted by connecting the two coordinates of each cell at the beginning and the end of this interval with a line. The colour of the line represents the velocity of the cells. In addition to the global movement towards the midline, local differences in velocity and direction can be seen. This movie shows pupa #4. Anterior is to the left. See also Fig. 6A.
Movie 12. Cell death of histoblasts. Detail of Movie 2. The white arrow shows a cell that is going to die. Before the cell disappears from the epithelium, the nucleus fragments (red arrow).
Movie 13. Disappearing cells constrict apically. Detail of Movie 5. The membrane marker (DE-cadherin::GFP) shows how a disappearing histoblast constricts apically (arrow).
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