Fig. 5: MD cells in non-cyclorrhaphan flies divide out-of-plane to reduce head expansion.

a, Presence of the CF (yellow arrows) or out-of-plane division (magenta arrows) as shown with nuclear staining (DRAQ5) in the anterior half of D. melanogaster (n = 13), M. abdita (n = 30), H. illucens (n = 57), C. fuscipes (n = 24), C. riparius (n = 24) and C. albipunctata (n = 13). b, Schematic representation of MD1 and MD2 in the C. riparius embryo head domain. c,d, Sum projection of MD2 showing MD2o and MD2i at tissue scale (c, purple box in b) and cellular views during telophase (d). Asterisks indicate the first dividing cells. Cell outlines, LifeAct-mCherry; cytokinetic rings, Sqh–eGFP; arrows, cytokinesis with cytokinetic rings perpendicular (in-plane) or parallel (out-of-plane) to the apical surface. n = 6 embryos. e,f, Apical cell surface area (e, n = 18 and 24 cells from 3 embryos) or subdomain surface area (f, paired two-sided t-test on maxima; *P = 0.0145; n = 5) comparing MD2o and MD2i as a function of time, normalized to surface area at the blastoderm stage. Bold lines indicate means and the shaded region indicates standard errors. Note in e, the summed areas of two daughter cells were plotted for in-plane divisions after completion of division, whereas only the apical daughter cell was plotted for out-of-plane divisions. Scale bars, 25 µm (C. fuscipes, C. riparius and C. albipunctata) and 50 µm (D. melanogaster, M. abdita and H. illucens) (a,b); 10 µm (c); and 5 µm (d).