Analysis of cell length and polarity changes during leader-to-follower transition. (A) Plot of the mean nucleus-centrosome distance (mean length±s.d. of the nuclear-centrosomal vectors) as a function of cell distance from the leading edge showing elongation from front to rear of the pLLP. (B) Nuclear-centrosomal vectors are colour coded according to their length, illustrating this progressive elongation. (C) Polar angles of cell polarity vectors colour coded and shown in the side view of the rosette-centred template pLLP (top) and corresponding colour coding of nuclei positions in top view (bottom). This measure of how upright cells stand shows the concentric organisation of the rosettes. (D) Polarity vectors colour coded according to their azimuth angles on a top view of the rosette-centred template pLLP (top) and plot of nuclei positions displayed in blue for rear-oriented cells and in red for front-oriented cells (bottom). This analysis of vector orientation in the migration plane demonstrates an evolution from rear-oriented to radially organised cells via a transition zone of mixed polarities. (E) Time-lapse images showing dynamically an EMTB-3GFP-expressing cell undergoing the transition from rear oriented to front oriented. The shifting cell nucleus is indicated by asterisks; chevrons indicate the brightest point of EMTB labelling that corresponds to the microtubule-organising centre. Scale bar: 10 µm.

Live imaging of Cdh2 reveals adherens junction assembly during organogenesis. (A) cdh2:Cdh2-GFP BAC recapitulates endogenous Cdh2 expression in the pLLP, showing enriched apical localisation of Cdh2-GFP. The maximal intensity projection of the pLLP is coupled with orthogonal z views. (B) Ratiometric images of cdh2:Cdh2-TagRFP and the membrane marker cldnb:lyn-GFP on confocal z plane show a relative increase of Cdh2 in apical rosette centres (top) that is not seen on middle z planes (bottom). (C) Box plots showing the distribution of the mean ratios in the rosette centre middle z plane and apical z plane (n=13, Wilcoxon paired test, ^**P<0.01). (D) Concatenated time-lapse images of a Cdh2-GFP pLLP depicting (1) the appearance of apical dots just behind the leading cells and (2) the progressive clustering during rosette formation. The more mature rosette in (3) is shown on the left 3 hours later (top) together with an enlarged view of a single confocal plane (bottom) highlighting the appearance of tiny apical poles. (E) Images of Cdh2-GFP-labelled pLLP before (top) and after (bottom) segmentation of Cdh2-GFP clusters. Histogram (blue) and distribution probability (green) of segmented apical Cdh2-GFP clusters (n=27 pLLP) displayed together with the histogram of the percentage of front-oriented cells (red) in the front part of the pLLP. The transition zone defined in Fig. 2D, where front-oriented cells appear (grey) corresponds to the domain where the first apical clusters are detected. (F) Time-lapse images of Cdh2-GFP, centrin-tdTomato clones of cells in the pLLP. The indicated cell (asterisk) undergoes a transition from rear oriented to front oriented. An enlarged time-lapse view of this cell (bottom, outlined; scale bar: 5 µm) shows the appearance a Cdh2 cluster (green chevron) before the cell shifts. Red chevrons indicate the cell centrosome. Scale bars: 20 µm, unless otherwise stated.

Tandem fluorescent protein timers (tFTs) reveal changes in cadherin stabilisation at subcellular and tissue scales. (A) Images from a time-lapse movie showing the progressive front to rear loss of the apical Cdh2-GFP foci after colcemid treatment (right panels). A deposited neuromast maintaining Cdh2-GFP adherens junctions after colcemid treatment is shown on the left. (B) Box plots of the time required for the loss of the 1st, 2nd and 3rd rosettes after treatment with colcemid (n=12 pLLP, Friedman rank sum test, ^**P<0.001). (C) BAC cdh2:Cdh2-tFTs embryo illustrating the Cdh2 age differences between tissues. Inset, magnification of the squared area containing the pLLP. Maximal intensity projections. (D) Ratiometric images of cdh2:Cdh2-tFTs show a relative increase of the red/green ratio from the front to the back pLLP rosettes and deposited neuromasts. (E) Box plots of the distribution of the lifetime ratios (red/green) of apical clusters and subjacent membranes in the corresponding area demonstrating the increased lifetime of Cdh2 in the clusters compared with membranes (n=175, paired t-test, ^***P<0.001). (F) Mean and s.e.m. of the age difference between clusters and membranes (red/green ratios of clusters minus membrane) normalised on the pLLP leading most cluster (rosette1) showing a gradual stabilisation of Cdh2 in the clusters compared with membrane as rosettes mature into neuromasts (n=35 pLLP, Friedman rank sum test, ^***P<0.001). NM, neuromast. Scale bars: 200 µm in C; 20 µm in A,D.

Generation of live adherens junction reporter lines (A) Immunohistochemistry of Cdh1 in the pLLP. (B) Immunohistochemistry of Cdh2 in a 30hpf embryo and in the pLLP (lower panel). (C) Double antibody staining for Cdh2 and ZO1 in the pLLP. The twice-enlarged view of a rosette highlights the broader localisation of Cdh2 around ZO1. (D) Whole embryo overview of the cadherin2:Cadherin2-GFP BAC that recapitulates endogenous Cdh2 expression. Brackets highlight the position of the pLLP. (E) Rescue of the parachute mutant (cdh2tm101b-/-, PAC-/-) by cadherin2:Cadherin2-GFP transgenics. Left panels show a clutch of embryos from an incross of cadherin2:Cadherin2-GFP;cdh2tm101b-/-. Non-transgenic embryos (*) show characteristic mutant phenotype illustrated in the top right panels (curled tail and brain defects, white arrow) whereas transgenic siblings develop normally (bottom right) and survive. Scale bars embryo=200µm, pLLP=20µm. (F) Images from a time-lapse movie of a cldnb:lyn-GFP;cdh2tm101b-/- showing the migration of the pLLP at 30hpf and the formation and deposition of neuromasts in the absence of Cdh2. Scale bar=50µm.(G) 48hpf cldnb:lyn-GFP;cdh2tm101b-/- embryos depicting the posterior lateral line neuromast pattern after the pLLP has reached the tail.

Reversible loss of rosette constrictions upon microtubule depolymerisation (A) Time-lapse images of a control and a pLLP treated with colcemid and aphidicolin, which prevents cell entry into S phase, illustrating rosette disassembly even in absence of mitosis. Arrows indicate rosette constrictions. (B) (i) Concatenated time-lapse images at 15-minute intervals of a cldnb:lyn-GFP pLLP treated with colcemid showing loss of rosettes (red arrowheads). (ii) Image of the same pLLP just before washout and inactivation of the drug (WO). (iii) Concatenated time-lapse images of the pLLP after washout, appearing rosettes are highlighted with red arrows. (iv) Image of the pLLP after washout showing normal tissue organisation. (C) Immunohistochemistry for the tight junction protein ZO1 in a control and a 2 hour-colcemid treated pLLP showing the loss of its apical concentration in a front to back manner after microtubule depolymerisation. Arrows point at apical ZO1 concentrations. Scale bars=20µm.