The DM brain NBs generate a large number of progeny during larval development. (a) Lineage labeling of a NB by MARCM. Left: schematic representation of a NB lineage in transgenic flies carrying a repressor transgene GAL80 distal to an FRT site in heterozygous (±) conditions. Ubiquitous expression of GAL80 under tubulin promoter control (pink) prevents GAL4-driven expression of the mCD8::GFP marker gene (green). Heat shock-induced FLP recombinase (FLP) at a given time point mediates the FRT site-specific mitotic recombination. Segregation of recombinant chromosomes at mitosis may result in the loss of the GAL80 repressor transgene in the NB daughter, which allows stable expression of the marker in this cell and its progeny. After several rounds of division such a positively labeled clone contains the NB, one or more GMCs and numerous post-mitotic neurons (N). Right: following random heat-shock induced NB recombination in newly hatched larvae, the size and composition of isolated NB lineages were examined at different time points during larval development. (b) NB clones were examined in all parts of the brain and ventral ganglia with the exception of optic lobes. The latter are easily recognizable in a single brain hemisphere by their lateral position and the high density of cells that express the progenitor marker Miranda (magenta, lower panels). On confocal images of brain hemispheres at low magnification (lower panels), GFP-labeled NB clones are easily identifiable by the presence of a large Miranda-positive NB and an associated cluster of clonal progeny. Unusually large clones could be identified in the dorsomedial part of the brain hemispheres (arrowheads). Anterior is to the top and lateral is to the left for each view. OPC and IPC, outer and inner proliferating centers, respectively. Scale bars: 50 μm. (c) The size of NB lineages was determined by counting cells in isolated clones plotted on the diagram according to their position in the nervous system (x axis). Each dot represents a clone with the mean ± standard deviation indicated by dots and error bars next to each group. DM, dorsomedial NB lineage; MB, mushroom body NB lineage; n, number of clones examined in each area. (d) Growth rate of different lineages examined at different time points after clone induction. Dots and bars represent the average size and standard deviation determined from the indicated number of clones.