Fig. 8. Brain NBs develop from neighbouring neuroectodermal cells (in mitotic domain B). (A-C) Anti -Tubulin/anti Dpn double staining of a late stage 8 flat preparation; anterior (a) is towards the top and dorsal (d) towards the left. (A) Anti--Tubulin (brown) highlights the outline of ectodermal cells and indicates cells in mitosis (e.g. some posterior cells of domain 5) (Foe, 1989). Position of mitotic domains 1, 5, 9 and B is indicated. Dpn protein (blue) is expressed in ectodermal domains; rectangle encloses most of the ocular Dpn expression that is part of mitotic domain B and gives rise to first brain NBs (compare with Fig. 2A and Fig. 5). At that stage, domain B cells are normally distinguished from domain 1 and 5 cells (most of which have already undergone mitosis) by their larger size. (B,C) Higher magnification of the region framed in A at two different focal planes: the apical level of the peripheral ectoderm (B) and about 20 µm deeper at the level of NBs (C). Red dots in B indicate transient apical cellular processes of three adjacent cells in the centre of domain B. In C, individual Dpn-positive NBs deriving from ocular Dpn domains are assigned. The adjacent apical cellular processes in B belong to the neighbouring NBs marked by red inscription in C. (D-G) 4D microscopic analysis indicates that the population of stage 8 protocerebral NBs derives from adjacent neuroectodermal progenitor cells of mitotic domain B. The origin of identified late stage 8 NBs (D,E) is traced back to the neuroectoderm at stage 6 (st6; F,G) using z-stack timelapse recordings (see Materials and Methods). (D,F) In vivo Nomarski pictures (lateral view; anterior is towards the left and dorsal is towards the top) at the level of NBs (D, focal plane at about 25 µm depth) and peripheral ectoderm (F; focal plane at about 8 µm depth). (E,G) Corresponding 3D computer models. Nuclei of identified NBs (as assigned in E) and of their corresponding neuroectodermal precursors (F,G) are indicated by the same colour code. Nuclei of other ectodermal cells are dark blue (E,G). The borders of mitotic domains 1, 2, 5, 9 and B are outlined in G. a, d, m: anterior, dorsal, medial.