Dynamics of of TZ-epi-NSC transition in space and time. (A–D′) Snapshots from a time-lapse video showing the dynamics of TZ-epi-NSC transition (E-cad-mCherry, white; Neur-GFP, green). TZ cells that have not yet become epi-NSCs were manually tracked (color code refers to the distance to epi-NSCs at t = 0). (E) Cumulative plot of TZ cells switching to the epi-NSC state, based on Neur-GFP expression over time (switch was defined by a threshold of 1 SD below the mean of Neur-GFP intensity in apically constricted epi-NSCs; n = 3 brains; 34–47 cells tracked per brain). Transition appeared to be a continuous process. (F–F″) Model of mechanical coupling by Neur. Acquisition of the epi-NSC state (green; L’sc levels in blue) is a probabilistic event occurring at the single-cell level (asterisk; F). It is associated with the expression of Neur that in turn induces apical constriction (F′). Neur down-regulates Crb in a cell-autonomous manner, hence moving the boundary between low and high Crb cells, hence the enrichment of junctional MyoII (red dotted line) resulting from the Crb anisotropy: MyoII accumulation increases at the lateral junction and decreases at the medial junction (F′). This is proposed to modify tension along this interface and promote cell–cell rearrangement to direct integration of the newly fated epi-NSCs into the one-cell row of epi-NSC at the medial edge of the TZ (F″). This mechanical coupling may ensure precise tissue architecture and regular spatial progression of the differentiation front. (G–I) Immunostainings showing the epi-NSCs (Neur-GFP, green; E-cad, red; cell outlines in white) located at the interface with the Neur-negative TZ cells which were used to evaluate precision of morphogenesis (G, wild-type; H, optix-Gal4 UAS-flp sdt^GFP3: Neur-resistant cells do not express Sdt-GFP3, green; yellow star; I: rhoGEF3^KO). (J and J′) The roughness of the epi-NSC/TZ junctional boundary (red dotted line in J and J′) was measured by calculating the mean of the deviation values (d in J′) along the x axis from a reference axis (blue dotted line in J and J′) linking the first junctional vertex to the last in groups of five epi-NSCs (green in J). See also Materials and methods. (K) Plot showing the mean values of deviation, or roughness, in wild-type (n = 12), optix-Gal4 UAS-flp sdt^GFP3 (n = 10), and rhoGEF3^KO (n = 6) brains (ns, not significant; ***, P = 0.00092, one-way ANOVA, Scheffé multiple comparison tests). Upon excision of exon 3 of sdt, epi-NSCs formed a less regular junctional interface. Scale bars = 10 μm.