Figure 8. Loss of radial patterning of cell behaviours in salivary gland placodes lacking Fkh.

(A–B’) Examples of fkh mutant and wild-type placodes illustrating the lack of pit formation. A control placode at early stage 11 shows clear constriction and pit formation in the dorsal-posterior corner (yellow dotted line in cross-sections in B, B’), whereas even at late stage 11 a fkh mutant placode (beyond the time frame of our quantitative analysis) does not show a pit in the dorsal-posterior corner, instead a shallow central depression forms with some constricted central apices (yellow dotted line in cross-sections in A), (A’). Myosin II (sqhGFP) is in green and DE-Cadherin in magenta, white dotted lines in main panels outline the placode boundary. (C, D) Stills of segmented and tracked time lapse movies for control (C) and fkh[6] mutant (D) placodes, apical cell outlines are shown and colour-coded by apical cell area. Note the lack of contraction at the tissue and cell level in the fkh[6] mutant. (E–F’’) Analysis of cumulative apical strains in fkh mutant placodes (from five movies) in comparison to the analysis of wild-type embryos (as shown in Figure 2). Over the first 36 min of tube budding centred around the first appearance of tissue-bending in the wild-type and an equivalent time point in the fkh mutants, the fkh mutant placodes show only a slight expansion at the tissue level in the cells far from the predicted pit position (F) due to cell shape changes (F’) with very little intercalation contributing to the change (E’’, F’’). 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 8—figure supplement 2]), is indicated by shaded boxes at the top of each panel: wt ‘rad’ vs fkh ‘rad’ (dark grey) and wt ‘circ’ vs fkh ‘circ’ (light grey).

Figure 8—figure supplement 1. Loss of radial patterning of cell behaviours in salivary gland placodes lacking Fkh.

(A–C) Fkh expression (magenta in A, B) initiates around the future point of invagination in the dorsal-posterior corner at late stage 10 (A’) and then spreads radially across the secretory part of the placode in early stage 11 (B’). (C) Schematic of radially expanding expression pattern of Fkh. (D–E’’) Comparison of Crumbs (red) and myosin II (sqhGFP; green) localisation in control (D–D’’) and fkh mutant (E–E’’) placodes. In the control, Crumbs is strongest near the invagination point and levels drop with radial distance from the pit, whereas in the fkh[6] mutant beyond late stage 11 Crumbs is stronger in the central shallow depression, with less towards the periphery. Areas of remaining circumferential myosin II polarisation are visible in the fkh[6] mutant (E’’). (F–G) Example fkh[6] mutant placode showing remaining myosin II (green) and Bazooka (magenta) polarisation, (G) shows a schematic representation of the area magnified in (F’–F’’’). Dotted lines in panels indicate the placode boundary. Scale bars in (A, B, D, E and F) are 5 µm.

Figure 8—figure supplement 2. Loss of radial patterning of cell behaviours in salivary gland placodes lacking Fkh.

(A–B’’) Spatial summary of average apical strain rates in fkh[6] mutants covering 15 min prior to 18 min post-commencement of tissue bending, showing the radial (A–A’’) as well as circumferential (B–B’’) contributions, expressed as proportion (pp) change per minute. Expansion is magenta, contraction is green. (C) Schematic of time windows covered by the time-lapse movies analysed for the apical strain rate calculations in fkh[6] mutant embryos. (D–E’) Instantaneous apical strain rates in fkh[6] mutants corresponding to the cumulative plots in Figure 8E–F’’. Error bars depict the mean of within-embryo variances of five 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.