Clonal expansion of IPs analyzed by MARCM. (a) Schematic representation of the different types of MARCM clones that can be recovered following FLP-mediated recombination in a NB (red arrow) and segregation of homozygous GAL80 chromosomes into one of its two daughter cells (green). A multicellular clone lacking the NB (right panel) reveals the ability of the IP daughter cell to undergo several rounds of division. Not shown are FLP-mediated recombination events in the GMC or in the IP that give rise to multicellular clones only in the latter case. Recombination in the GMC gives a single labeled cell. (b) Top: schematic organization of multicellular GFP-labeled clones (green) after time-controlled recombination (heat-shocked FLP, black arrows) in two developing NB lineages. Bottom: unlike NB clones (upper lineage), IP clones were identified as GFP-labeled cell clusters lacking the large Miranda-positive NB and pushed away from this founder cell by proliferation (non-NB clone, lower lineage). The size and composition of clonal progenies were examined 48 hours after two independent heat-induced recombination events. (c) Size distribution of multicellular non-NB clones generated by recombination at 24 h (light grey bars) or 48 h (dark grey bars) ALH and assayed 48 hours later. The similar histogram profile reveals the comparable mitotic potential of progenitors present in the DM lineage at 24 or 48 h ALH. (d, d') Representative confocal image of NB clones induced at 48 h ALH and examined at 96 h in a dissected brain stained for the markers indicated (dorsal view, lateral to the left, anterior to the top). DM NBs are identifiable in the most medial row of large cells (arrowheads) by their association to a large cluster of Miranda-positive progenitors (various DM lineages are outlined by dots in (d); the GFP channel was omitted for clarity). The GFP-labeled progeny of a single DM NB follows the orientation of the Miranda-positive cell cluster. A typical non-DM NB clone is found on the lateral site of the brain (asterisk). This single large NB is associated with a few Miranda-positive GMCs. (e) Representative IP clone of four cells among the presumptive progeny of the nearest DM NB (arrowheads); same scale and conditions as in (d). A magnification of the area boxed in (e) is shown in (e', e"), with one channel omitted for clarity. The cells in the clone have undetectable level of Miranda (red) and all express the neuronal marker ELAV (blue). Scale bars: 15 μm.