Neur marks emerging epithelial NSCs in the TZ. (A) Diagram of the third instar larval brain displaying the OL NE. P, posterior; A, anterior; D, dorsal; V, ventral. (B) The proneural wave sweeps the NE from medial to lateral, inducing NE–NSC transition in a region called the TZ defined by the graded expression of L’sc (red nuclei). The medial TZ cell appears in green. M, medial; L, lateral. (C–E″) Surface (C–D′) and cross section (E–E″; A, apical; B, basal) views showing that Neur-GFP (green) becomes expressed in the medial-most TZ cells (L’sc, red in D–E″). These cells showed increased E-cad levels (red in C). Lower levels of Neur-GFP were observed in NSCs (C′, D′, and E″). (F–G′) Most Neur-positive TZ cells (Neur-GFP, green) coexpressed Wor (red, F), while the NSC marker Dpn was up-regulated only in NSCs past the TZ (red, G). Arrows point to medial TZ cells. (H and H′) TZ cells (L’sc, H′) expressed CycB-RFP (red, H) and E2F1-GFP (green, H), indicating that they are in G2 phase. (I–J′) Medial TZ cells (Neur-GFP, green) appeared to undergo the first round of asymmetric cell division (pH3, phospho-histone H3 in white; DAPI, blue). This division was oriented along the apical-basal axis, and Mira localized asymmetrically (I–I″; Mira, red). The arrow in I′ indicates the basal crescent of Mira. A large apical cell (J) and a small basal cell (J′), likely corresponding to the first-born GMC, appeared to be produced by this asymmetric division. (K–M″) Cross-section views showing that the medial-most TZ cells marked by Neur-GFP (green) and L’sc (red in K) were apically constricted and had a characteristic teardrop shape while retaining apical AJs (K; E-cad, white). E-cad remained detectable as these cells divided (M–M″; E-cad, red; DAPI, blue). These apically constricted epithelial cells undergoing asymmetric cell division were named epi-NSCs (L). Scale bars = 10 µm.