Extended Data Fig. 7: Aggregative sheets are comparable in morphology to control sheets.

(a) Mean sheet area at 1X (grey) and 2X (turquoise) salinities over a 2-hour aggregation experiment. (b) Cell density measured in n = 12 splash pools (SpA to SpL, Exped-C) compared to the range used in laboratory aggregation experiments (grey area). Diamonds are average estimates, and bars indicate uncertainty (ranging from minimal to maximal estimates). (c) Mean sheet area at 1X (grey) and 2X (turquoise) salinities across different cell densities. (d-g) 3D reconstructions of confocal z-stacks of aggregative (d-e) and control sheets (f-g), with a membrane dye labelling the cell body and flagella (FM 4-64FX, magenta) and F-actin dye labelling the collar (Phalloidin 488, white). (d’-g’). Mid-z cross-sections (dashed lines) in d-g. Note that cells within aggregative sheets are connected by collar-collar contacts (white arrowheads) and have aligned apico-basal axes as in control sheets. (h) Quantification of cell number per colony in d-g. (h’) Quantification of sheet area in d-g. (i) Quantification of percentage of aligned cells within colonies in d-g. (i’) Quantification of mean collar-collar angles between cells per sheet in d-g. (j) Quantification of circularity of sheets in d-g. Black circles: means. Error bars: standard deviations. Diamonds: mean values of each independent replicate. p-values in a and h-j by the Mann-Whitney U test (n.s., non-significant). All experiments shown (except in b) were performed in N = 3 biological replicates, two technical replicates each (n = 41 aggregative sheets; n = 46 control sheets).