Fig. 3. Cell division during tube outgrowth.

a EdU pulse experiments measure frequency of cell proliferation during tubulogenesis from tube outgrowth to larva stages (as defined in Supplementary Fig.1). Number of total larvae analyzed n = 60. b–d EdU staining (magenta) in the three tube areas: stalk, middle and tip cells. Arrowheads in (c) and (d) indicate nuclei with incorporated EdU. Insert in (d) shows a transverse view (tv) of the hydropore canal from another specimen with nuclei stained with EdU. Scale bars 10 μm (b, c), 20 μm (d), 5 μm (insert). e Heatmap showing frequency of cell proliferation (white is zero, dark blue is max). f Frequency of cell proliferation in the tip cells for left and right tubes. n = 60 total number of larvae. Two-sided Student’s t-test: EL (***p-value = 0.0002), L1 (*p-value = 0.0260), L3 (*p-value = 0.0409). g, h Examples of a dividing cells from Supplementary Movie 10; magnification of a mitotic cell (g’) and the same cell undergoing cytokinesis (h’); sb = 5 μm. i Trajectory of a cell that divides, taken from Supplementary Movie 10. Arrow indicates the mitotic event, where a and b are the two daughter cells. j, k EdU staining in larvae treated with Abemaciclib to arrest cells in G1 from gastrula to late gastrula (during tube outgrowth) or from late gastrula to early larva (during tube elongation). Dotted lines indicate the tubes. Scale bar 20 μm. l EdU incorporation in presence of Abemaciclib to test inhibition of cell cycle and (m) effects on tube outgrowth and elongation. Number of larvae n = 16 (control LG); 32 (Abe G- > LG); 36 (control EL); 43 (Abe LG- > EL). LG (late gastrula); EL (early larva); L1 (larva stage 1); L2 (larva stage 2); L3 (larva stage 3). Statistical significance was assessed by a two- sided Student’s t-test (****p < 0.0001; ns not significant). In box plots the median is the middle line, box represents 25th and 75th percentiles, whiskers indicate the minimum and maximum data range. Source data are provided as a Source Data file.