Figure 3. Strain rate analysis of early tubulogenesis using a 3D proxy.

(A–B) Epithelial cells of the placode are about 2–5 µm in apical diameter but extend about 15 µm into the embryo along their apical-basal axis. To assess behaviour of the tissue and cells at depth, we analysed a mid-basal level, about 8 µm basal to the apical surface. Scale bars are 5 µm. (C) Illustration of the method used: we segmented and tracked cells at a depth of ~8 µm, and repeated the strain rate analysis at this mid-basal level (blue) to compare with apical (pink). (D–D’) Comparison of apical (pink, as shown in Figure 2) and basal (blue) cumulative strains, for cell shape changes (D, E) and cell intercalation (D’, E’), near the pit (D,D’) and far from the pit (E,E’). Note how cell shape changes apical versus basal near the pit suggest progressive cell wedging as apices contract isotropically more rapidly than basally (D), and how the rate of cell intercalation across the tissue is highly coordinated between apical and basal, especially in the cells far from the pit (D’,E’). Statistical significance based on a mixed-effects model and a p<0.05 threshold (calculated for instantaneous strain rates [see Figure 2—figure supplement 1 and Figure 3—figure supplement 2]), is indicated by shaded boxes at the top of each panel: apical ‘rad’ vs basal ‘rad’ (dark grey) and apical ‘circ’ vs basal ‘circ’ (light grey).

Figure 3—figure supplement 1. Strain rate analysis of early tubulogenesis using a 3D proxy.

(A) Schematic of time windows covered by the time-lapse movies analysed for the basal strain rate calculations. (B–C’’) Spatial summary of the basal strain rates (calculated from −15 to + 18 min), showing circumferential contribution (B–B’’, circ) and radial contribution, (C–C’’, ‘rad’). Constriction (green) still dominates the pit region at the tissue level (B, C), due to cell constriction (B’, C’), with strong expansion (magenta) towards the pit radially, predominantly due to cell intercalation (C’’). (D–D’) Comparison of the average basal and apical cumulative strain of all pooled placodal cells. Overall, basally (blue curves) the tissue elongates towards the pit whilst contracting circumferentially, in a more isotropic fashion than apically (pink curves). Basally, cell shape change is weakly contractile radially, like apical, while it is near zero circumferentially, unlike apical (D’), with intercalation extending the tissue towards the pit and contracting it circumferentially almost identically in apical and basal (D’’). (E, F). Comparison of apical (pink, as shown in Figure 2) and basal (blue) cumulative strain, for tissue shape change (E, F). Statistical significance based on a mixed-effects model and a p<0.05 threshold (calculated for instantaneous strain rates [Figure 2—figure supplement 1 and Figure 3—figure supplement 2]), is indicated by shaded boxes at the top of each panel: apical ‘rad’ vs basal ‘rad’ (dark grey) and apical ‘circ’ vs basal ‘circ’ (light grey).

Figure 3—figure supplement 2. Strain rate analysis of early tubulogenesis using a 3D proxy.

(A–B’) Instantaneous mid-basal strain rates corresponding to the cumulative strain plots in Figure 3D–E’ and Figure 3—figure supplement 1E,F. Error bars depict the mean of within-embryo variance of six movies. Significance using a mixed-effects model of p<0.05 is indicated by shaded boxes at the top of each panel: tissue vs cell shape (green) and tissue vs intercalation (orange). See Materials and methods for details of statistics.