Dynamic analyses of Drosophila gastrulation provide insights into collective cell migration

Abstract

The concerted movement of cells from different germ layers contributes to morphogenesis during early embryonic development. Using an optimized imaging approach and quantitative methods, we analyzed the trajectories of hundreds of ectodermal cells and internalized mesodermal cells within Drosophila embryos over 2 hours during gastrulation. We found a high level of cellular organization, with mesoderm cell movements correlating with some but not all ectoderm movements. During migration, the mesoderm population underwent two ordered waves of cell division and synchronous cell intercalation, and cells at the leading edge stably maintained position. Fibroblast growth factor (FGF) signaling guides mesodermal cell migration; however, we found some directed dorsal migration in an FGF receptor mutant, which suggests that additional signals are involved. Thus, decomposing complex cellular movements can provide detailed insights into collective cell migration.

Fig. 1

Two-photon microscopy and analysis of histone2A (H2A)–GFP expressing embryos captures key events in gastrulation. (A and B) Cross-sections of wild-type (A) and htl mutant (B) embryos stained with antibody to Twist. (C and D) Confocal 1PEF (C) fails to image internalized mesoderm cells, whereas 2PEF (D) captures the positions of the internalized cells. (E) A 50-μm-deep and 10-μm-thick lateral slice through an H2A-GFP embryo demonstrates the signal-to-noise ratio (anterior, left). (F) Segmentation of mesoderm nuclei (orange spheres) by the use of Imaris software (Bitplane AG, Zurich, Switzerland). Each sphere was defined by the fluorescent intensity of H2A-GFP. Furrow formation, furrow collapse as a result of an EMT, and spreading of the mesoderm to form a monolayer are illustrated from top to bottom, respectively. (G to J) Tracking cell positions in three dimensions over time. Shown are dorsal (G) and posterior (H) views of mesoderm tracks (blue and yellow indicate early and late time points, respectively) and dorsal (I) and posterior (J) views of mesoderm (orange) and ectoderm (gray) net displacement vectors. Scale bars, 20 μm.

Fig. 2

Decomposition and correlative analysis of cell movements with the use of cylindrical coordinates. (A and B) The use of cylindrical coordinates allows the positioning of cells according to the body plan of the embryo at stage 6. (C to E) Cell trajectories (blue lines) reveal that each axis corresponds to a morphogenetic movement. (C) r is the radial position over time (for example, furrow collapse and intercalation; 0 indicates the center of the embryo). (D) θ is the angular movement (for example, mesoderm spreading and ectoderm convergence; 0 indicates the position of the ventral midline). (E) L corresponds to the movement of cells along the length of the embryo (for example, germ-band elongation). In (C) to (E), time (t) = 0 is set as the point when AP movement begins. (F to H) Correlation of the velocity (v) of each mesoderm cell with its six nearest ectodermal neighbors along the (F) radial, (G) angular, and (H) AP axes, with correlation values of 0.21 ± 0.43, 0.08 ± 0.18, and 0.90 ± 0.06, respectively (n = 3 embryos). (I) Dorsal view of mesoderm cell displacement before (orange) and after (blue) subtraction of local ectoderm cell movements.

Fig. 3

Quantitative analysis of morphogenetic events reveals a high level of organization in wild-type embryos. (A) A color code marks the angular position of cells in the furrow at stage 7 and shows the spatial organization as cells move over time. rad, radians. Each line represents the trajectory of one cell. (B) Position and timing of each cell division (colored circle). The color code represents the radial position in the furrow at stage 7. DNA morphology during cell division in H2A-GFP embryos is shown (top left). (C) Analysis of intercalation events within the mesoderm over time shown as a percentage of mesoderm cells intercalating (n = 3 embryos). (D) The position of mesoderm cells before and after intercalation events.

Fig. 4

Furrow collapse and spreading of mesoderm cells are disrupted in htl mutants. (A) Position of mesoderm cells (circles) at stage 7 and stage 10 in wild-type and htl embryos shown with a radial color code. (B to G) Angular movement of cells over time analyzed in wild-type [(B) to (D)] and htl mutant [(E) to (G)] embryos within the entire [(B) and (E)], upper [(C) and (F)], and lower [(D) and (G)] furrow (black line indicates the average mesoderm displacement with respect to the midline). (H and I) Spreading profile of wild-type (H) and htl (I) embryos. The color code represents the distance from the ectoderm at the end of spreading (red indicates far from ectoderm and green indicates close to ectoderm). The gray line represents a spreading coefficient of A = 2, where θ_end = A(θ_start)+ B [B, constant (13)]. Cells that do not spread within the collective are represented within gray regions of the graph (13). In general, cells located close to the ectoderm fall along the gray line. (J) The radial position (r) of two particular groups of mesoderm cells from the upper furrow of htl mutants is depicted over time. One group exhibits normal spreading behavior (light blue), and the other group exhibits aberrant spreading (dark blue). (K) The furrow collapse in htl mutants is disrupted, resulting in cells falling randomly to one side of the embryo. Upper-furrow cells that reach the ectoderm (light blue) undergo normal spreading, whereas cells that remain far from the ectoderm spread abnormally (dark blue). Red cells are Red indicates lower-furrow cells.