Figure 3

Molecular characterization of NB-like and GMC-like progenitors in the progeny of DM NBs. Confocal images of MARCM-labeled NB clones in the dorsal part of larval brains stained for the markers indicated on the top of the columns. Representative views of (a-f) non-DM lineages are used as a reference for (g-i") the DM lineages. Clones were labeled with CD8::GFP (membrane marker, green in all panels) and CNN::GFP (centrosomes visualized as bright green spots in e, f, i-i"). Proliferative cells are detected by anti-Cyclin (red in e, f, i-i') and anti-PH3 during mitosis (blue in all panels). In a non-DM NB clone, mitosis is restricted to two cell types: the NB and a single GMC in close proximity (a-f, asterisks and arrowheads, respectively). NBs show a unique pattern of polarized expression of Prospero and Miranda at the cell cortex during mitosis (a, c) and stable expression of Cyclin E throughout the cell cycle (e, mitosis; f, interphase). In contrast, the GMC is uniquely defined when engaged in mitosis (PH3 positive) by nuclear localization of Prospero (b, inset), weak uniform cortical localization of Miranda (d, inset) and lack of Cyclin E (f, inset). (g-i) In DM clones many progenitors other than the NB are identified as PH3-positive nuclei. These cells show patterns of marker expression usually found in mitotic NBs (IP; arrows) or mitotic GMCs (arrowheads). Lower panels show close up views of the areas boxed in (g-i). The two types of mitotic progenitors can be detected simultaneously in a single DM lineage (images) and are found at a comparable ratio when quantified in multiple clones using the three independent markers (histograms). IP, small NB-associated intermediate progenitor with NB-like marker expression. Scale bars: 10 μm (a-f) or 15 μm (g-i).