Cell rearrangements, cell divisions and cell death in a migrating epithelial sheet in the abdomen of Drosophila

Abstract

During morphogenesis, cell movements, cell divisions and cell death work together to form complex patterns and to shape organs. These events are the outcome of decisions made by many individual cells, but how these decisions are controlled and coordinated is elusive. The adult abdominal epidermis of Drosophila is formed during metamorphosis by divisions and extensive cell migrations of the diploid histoblasts, which replace the polyploid larval cells. Using in vivo 4D microscopy, we have studied the behaviour of the histoblasts and analysed in detail how they reach their final position and to what extent they rearrange during their spreading. Tracking individual cells, we show that the cells migrate in two phases that differ in speed, direction and amount of cellular rearrangement. Cells of the anterior (A) and posterior (P) compartments differ in their behaviour. Cells near the A/P border are more likely to change their neighbours during migration. The mitoses do not show any preferential orientation. After mitosis, the sisters become preferentially aligned with the direction of movement. Thus, in the abdomen, it is the extensive cell migrations that appear to contribute most to morphogenesis. This contrasts with other developing epithelia, such as the wing imaginal disc and the embryonic germband in Drosophila, where oriented mitoses and local cell rearrangements appear to direct morphogenesis. Furthermore, our results suggest that an active force created by the histoblasts contributes to the formation of the adult epidermis. Finally, we show that histoblasts occasionally undergo apoptosis.

Fig. 1.

4D microscopy of histoblast migration during abdominal epidermis formation in Drosophila. (A,B) Development of dorsal histoblast nests. Dorsolateral view of segments 2 and 3. Nuclei of larval cells (large) and histoblasts (small) are visualised by Histone::GFP (green). Insets illustrate the location of the region shown. (A) At 18 hours after puparium formation (APF), the anterior and posterior dorsal histoblast nests fuse shortly before histoblast migration begins (arrow). (B) At 47 hours APF, the histoblasts have met at the dorsal midline and the whole abdomen is covered with adult cells. The segmental fold develops (arrows). See also Movie 1 in the supplementary material. (C,D) Tracking cells in a pupa in which the P compartment cells are marked with en.Gal4 driving DsRed (pupa #4, segment 2). White dots indicate cells that were tracked using SIMI Biocell. See also Movie 2 in the supplementary material. (E,F) Representative paths of cells. (E) Cells move in a more or less straight line towards the dorsal midline. Some small detours are visible (pupa #4). (F) Cells of segment 2 (white) and 3 (blue) turn anteriorly when they approach the midline. Cells of segment 1 (red) do not turn. The most anterior cell of the opposite hemisegment (yellow) is positioned next to the most anterior cell of this hemisegment, illustrating the matching of cells of the two hemisegments at the midline (pupa #1). To achieve this registration, the white cell, which is positioned more posteriorly during its dorsal migration, moves further in an anterior direction than the yellow cell. In all images, anterior is to the left. Yellow dashed lines indicate the dorsal midline. Scale bars: 50 μm.

Fig. 2.

The velocity and duration of the dorsal migration of cells determine their position. (A) Topology of the cell mass. The left and right panels show the same 3D representations of pupa #4 differently colour-coded to illustrate the change in cell positioning from 25 to 41 hours APF (n=273 and 443 cells, respectively). Spheres indicate anterior (A) compartment cells, and ovals indicate posterior (P) compartment cells. At the left, the cells are colour-coded arbitrarily in stripes with respect to the DV axis at the beginning of the recording; at the right, colour-coding is with respect to the AP axis at the end of the recording. The more posteriorly that cells are positioned the more dorsally they will move. Furthermore, cells do not change their positions along the AP axis. Cells in the P compartment appear to rearrange more extensively than those in A. Cells close to the A/P boundary move the furthest. See also Movies 3A,B in the supplementary material. (B) The velocity of each cell is plotted at its position at three consecutive time points (pupa #1). The number of hours after the start of the recording is indicated in the top right corner. The velocities of the cells change over time. Histoblasts which are more posterior in the segment tend to move faster. Cells are coloured grey if their velocity cannot be calculated because one of the coordinate-pair is missing. See also Movie 4 in the supplementary material. (C) The trajectories of all cells are plotted by connecting the coordinates of a cell in 30-minute intervals with a line (pupa #1). The colour of the line represents the velocity of the cells. More anteriorly positioned cells turn anteriorly earlier. The cell that turns first is marked with an arrow. More posteriorly positioned cells tend to move faster and turn anteriorly later. In all images, anterior is to the left.

Fig. 3.

The anterior migration of histoblasts. (A) The average velocity of all cells in 30-minute time intervals (pupa #1). Error bars show s.d. The red arrow indicates the time point when the first, most anteriorly positioned cells start to move anteriorly (see also arrow in Fig. 2C). About this time, the histoblasts slow down. The box shows the moving cell mass at this time point; one row of larval epithelial cells (LECs) still separates the histoblasts of neighbouring segments (white arrows). (B) The whole of segment 2 laterally of the midline. DE-cadherin::GFP marks the cell membranes. The upper panel shows the cell mass, which is moving in a DV direction, shortly before the histoblasts meet at the midline. The lower panel shows the same region 12 hours later, well after the cells have started to move anteriorly. Cells are much more organised now and tend to be uniformly shaped and elongated in the DV axis. The segmental groove can be seen (arrow). See also Movie 5 in the supplementary material. In all images, anterior is to the left. Scale bars: 25 μm.

Fig. 4.

Changes in neighbourhood relations between histoblasts. (A) (Top) Neighbourhood map. To calculate the neighbourhood map, the distances to the six closest neighbours at 30 hours APF were calculated for each cell. Then, the distances to the same six cells were calculated at 40 hours APF. For each of the six neighbours, the difference between these two values was calculated and then the average was plotted at the position of the cell at 30 hours APF (pupa #1). This value is a measure of changes in neighbourhood relations. Cells that were present at 30 hours APF but disappeared before 40 hours APF are coloured in grey. (Bottom) The averages shown in the neighbourhood map are plotted against the x-coordinates of the cells (error bars indicate s.d.). Black bars indicate the same regions in neighbourhood map and graph. The bars differ in size because at the bottom the coordinates are spread out along the x-axis. Most changes occur close to the A/P boundary (arrow) and in the centre of the cell mass, where cells are densest (asterisk). Some of the changes in the centre might be due to the spreading out of the cells, which is more extensive here because cells are very close together at the beginning (see Fig. S2 in the supplementary material). (B) Sister cells sometimes lose contact. Different sister cell pairs are colour-coded: blue, light blue and white remain neighbours, whereas red and yellow lose contact. (C) The division of a neighbouring cell (red line) separates sister cells (yellow).

Fig. 5.

Cell division orientation and change in position of sister cells relative to each other. (A) Determination of division orientation and the position of sister cells relative to each other (ρ). One frame after division, the angle relative to the DV axis was calculated (division angle ∂). Shortly before the first division of the sisters (or the end of the movie), the angle defining the position of the two sister cells relative to each other along the DV axis was calculated (ρ). In between division and the end of their cell cycle, the sister cells might rearrange relative to each other. (B) Bar chart showing the orientation of cell divisions (∂) and the position of sister cells (ρ) relative to the DV axis. Angles are shown on a 0° to 90° scale. The average angles are ∂=43±26° and ρ36±26° (n=747 sister pairs). The groups differ significantly (see Table S1 in the supplementary material). After rearrangement, more sister pairs are oriented in the direction of movement (0°) than after mitosis. (C) Diagram illustrating possible outcomes of the rearrangement of sister cells after cell division. (1) Sisters do not change their relative position after a division along the DV axis (direction of movement). (2) Sisters rearrange themselves (red arrow) orthogonal to the DV axis after a division along the DV axis. (3) Sisters do not change their relative position after a division orthogonal to the DV axis. (4) Sisters rearrange themselves along the DV axis after a division orthogonal to the DV axis. Blue and red circles indicate the groups analysed in D. (D) Sister pairs that arose from divisions along the DV axis and retained this arrangement (blue) and sister pairs that arose from divisions orthogonal to the DV axis but rearranged along the DV axis (red). Most sisters whose mother divided in the direction of movement retained this arrangement, whereas ∼50% of the sister cells whose mother divided orthogonally to the direction of movement rearrange. Most of these rearrangements occur within the first 24 minutes after division (n=747 sister pairs). (E) Analysis of the relationship of division orientation (∂) and the position of sister cells relative to each other (ρ). The division angles (in groups of 15°) are plotted against the corresponding angles representing the position of sisters relative to each other, shown as box plots (n=747 sister pairs). The medians indicate that ∂ correlates with ρ, which suggests that sisters are more likely to be arranged along the DV axis when the division of their mother had already biased their positioning. In all images, anterior is to the left and dorsal up. Error bars show s.d.

Fig. 6.

Movement of histoblasts and LECs. (A) Six consecutive frames of Movie 11 (see Movie 11 in the supplementary material), which show the trajectories of all cells colour-coded according to their velocity (pupa #4). Cells move straight towards the midline (green arrow). Approaching the midline, they are hampered by the slowly retreating LECs, whereupon they slow down and undergo a whirling movement (arrowheads). Once the LECs have retreated, the cells suddenly move quickly anteriorly (blue arrow) and, in more posterior areas, towards the midline (red arrow). (B) The epithelial sheet is often folded (asterisk) near the last row of histoblasts touching the LECs (arrow). A yellow dashed line indicates the dorsal midline. (C) Trajectories of the dying and disappearing LECs illustrated by the same method as used in Fig. 2C (pupa #1). The approaching histoblasts are outlined with green squares. The LECs retreat more or less straight towards the midline. In the posterior part of the segment, they move slightly faster. At the midline, they tend to move anteriorly. The black dashed line indicates the dorsal midline. See also Movie 10 in the supplementary material. (D) Left hemisegment of segment 2 of a pupa expressing Sqh::GFP. White arrows indicate histoblasts. The LECs close to the segment borders (red arrows) express a higher level of Sqh::GFP than other LECs. In all images, anterior is to the left. Scale bar: 50 μm.