Figure 3

The J16 mutation leads to defects in neuroblast cell fate. (A, B) The size asymmetry of neuroblast divisions is disrupted in J16 clones. DNA staining (blue); Miranda (red); CD8::GFP (green, A', B'); Bar: 10 μm. Wild type clones (A) contain a single large neuroblast and a number of smaller progeny. Only the neuroblast and occasionally GMCs express Miranda. J16 clones (B) contain multiple cells expressing Miranda, all of which are of a similar size, and many of which appear to be arrested in metaphase. (C, D) Cells in J16 clones are intermediate in size. Neuroblasts in wild type clones visualised with anti-GFP staining (C) are approximately 12 μm in diameter, while neurons are approximately 4 μm across. In contrast, cells in J16 clones (D) are 7–9 μm in diameter. (E, F) The J16 chromosome carries an allele of polo, but this does not cause the cell size phenotype. DNA staining (blue); Miranda (red); CD8::GFP (green, F). J16/Df(3L)Exel9636 hemizygote brains (E) show the polo metaphase arrest phenotype. J16 clones generated in larvae carrying a polo genomic rescue construct (F) are not arrested in metaphase (arrow indicates cell in anaphase; note that Miranda does not correctly form a crescent in this cell) but still show the cell size phenotype. (G, H) Loss of neuroblast marker expression in J16 embryos. Flat preparations of stage 8–9 wild type (G) and homozygous J16 (H) embryos stained with anti-Worniu (black) and anti-Engrailed (brown). Worniu staining in wild type embryos reveals a stereotypical array of neuroblasts. In J16 embryos there is a loss of Worniu expression at a low frequency (arrowheads) suggesting a loss of neuroblast cell fate. (I, J) Loss of neuroblast progeny in J16 embryos. Flat preparations of stage 16 wild type (I) and J16 embryos (J) stained with anti-Eve. Eve is a marker for the progeny of four embryonic neuroblasts (see text). In J16 embryos, Eve staining is absent at a low frequency in the EL neurons (arrowheads), RP2 neurons (arrow) and CQ neurons (not shown).