(A) Evolution of trunk ventral cell (TVC) polarization. Panels show in vivo-rendered images of cells at the indicated embryonic stages. Leader in blue, trailer in red, non-migratory anterior tail muscles (ATMs) in white. Spheres inside cells mark the center of mass, sphere to the right indicates direction of anterior migration. Angle theta between the axis of leader/trailer and direction of migration is indicated. (B) Three hypothesized polarization modes for two-cell migration. Independent: cells polarize independently in the signal direction and move with the same speed. Faster-slower: cells polarize independently in the signal direction, but one cell moves faster than the other. Leader-trailer: one cell (leader) follows the signal direction, while the other (trailer) polarizes in the direction of the leader’s center of mass. L, leader; T, trailer. (C) Establishment of alignment between the leader/trailer axis and direction of migration. Cos(θ) is shown for indicated embryonic stages. Data were pooled from two biological replicates. Statistical analysis was performed using one-way ANOVA and Bonferroni post test. **p<0.01. (D) Left: the simulated evolution of two cells’ geometry, quantified as cosine of the angle between the line connecting the cells centroids and the signal direction, (), for the three polarization modes shown in (B). Five simulations are run for each mode, and the shaded area shows the standard error. Center: representative snapshots of two cells reaching linear arrangement or at the end of simulation using each mode. The colors of the frames correspond to the dataset on the graph. Right: scatter plot showing the time when reaches for two modes with mean and standard error and statistical analysis using Student’s t-test with Welch’s correction. (E) Top: hypothesized polarization modes for three cells. Independent: cells polarize independently in the signal direction and move with equal speeds. Faster-slower: cells polarize independently in the signal direction, in this case, the leader travels the fastest, trailer the slowest, and middle cell travels at an intermediate speed. Leader-trailer: one cell (leader) follows the signal direction, middle cell polarizes towards the leader, and trailer polarizes toward the middle cell. Bottom: simulation of the three-cell group polarization under the three hypothesized polarization modes. Left: the initial cell arrangement in silico (top) and in vivo (bottom). Note that in vivo there are always four cells prior to migration arranged in a rectangular pattern. Center: the polarization of migrating cell clusters over time is quantified by the cosine of the angle between the lines connecting the leader and the two posterior cells separately, as shown in (F). Five simulations were run for each mode, and shaded area shows the standard error. Right: representative snapshots when the three cells reach linear arrangement for the three modes examined (for the independent-same mode, linear arrangement is never reached, snapshot shows cells at the end of a simulation run). The colors of the frames correspond to the datasets on the graph. (F) Three migratory cells are linearly arranged in the direction of migration in vivo. Bar graphs show the proportion of TVCs that migrate as either three or four cells under induced MAPK signaling by Mesp>Rasca and proportion of cell groups that are linearly polarized in each subset. Data were pooled from two biological replicates. Error bars show standard error of proportion. (G) Effects of modulating cell-cell adhesion on the contacting area between the two cells and on their speed, quantified by the percentage of total surface area of the leader cell (top graph, left y-axis, blue symbols), on the ability of the cell pair to polarize in the direction of migration quantified by (top graph, right y-axis, red symbols), where is the angle between the line connecting two cells and the moving direction as shown in the top image on the left, and on the total displacement of the leader/trailer pair (bottom graph). x-axis shows the relative energy of the cell-cell junction (the adhesion parameter is rescaled here so that larger value means stronger cell-cell adhesion). Images show cell pairs with either high (top) or low (bottom) cell-cell adhesion. Arrow represents leader/trailer axis.