Fig. 1.

Single cell labelling and imaging of delaminating otic NBs. (A) Schematics of otic NB delamination in the neurogenic domain (red) at 20 hpf and SAG shape (green) at 54 hpf. (B) Labelling of the otic epithelial membranes at 24 hpf (left) and the SAG at 54 hpf (right), the latter being located behind the otic vesicle (white dashed oval). Both images are lateral views. An NB (pseudocoloured green) delaminating from the neurogenic domain displays a distinct shape compared with non-delaminating otic cells (pseudocoloured purple). The boxed area represents the region from where images in C and D are taken. Different SAG lobes are outlined with a green line. (C) Single-labelled delaminating NB undergoing apical thinning (white arrowhead). The NB exits neuroepithelia leaving membrane traces inside neuroepithelia (white arrows). The images shown in C are from Movie 1, and are also depicted in Fig. S1C (top) with Pard3-GFP fluorescence. (D) NB from the neurogenic domain extending dynamic filopodia and blebs (white arrowheads) (representative of n=7). See also Movie 2. (E) Delaminating NB undergoing apical thinning and Pard3 relocation. Pard3 moves from apical to basal domains concomitantly with membrane zippering (white arrowheads) and accumulation of Pard3 in basal domains (yellow arrowhead). Right-hand panel represents a scheme of this phenomenon. (F) Collective delamination of NBs. NBs extend filopodia in the collective front while delaminating (white arrowheads) and Pard3 relocates (white arrows) without losing its punctate pattern (representative of n=7). (G) After delamination, a group of contacting NBs separate (blue and orange dots) concomitantly with Pard3 rearrangements (representative of n=3). (H) Arl13 staining in magenta. Non-neurogenic (upper dashed square) and neurogenic (lower dashed square) anterior domains are indicated, representing the region of insets in H′ and H″, respectively, from a different embryo. The Arl13b reporter is observed inside the neuroepithelia in the neurogenic domain, but is absent in the non-neurogenic domain. A spatial section across time (white transverse line corresponding to kymographs) shows the presence of Arl13 reporter in the neurogenic region only (representative of n=10). a, apical; aLL, anterior Lateral Line ganglion; b, basal. Anterior is to the left, posterior to the right in all images. Scale bars: 20 µm (B,H); 10 µm (C-G,H′,H″).