mad2G6595, Sas-4s2214 double homozygotes exhibit a developmental delay, until arresting and dying at the larval\pupal transition, as compared to controls. Their third instar larval, but not second instar larval or embryonic, brains display a severe decrease in overall volume, a severe increase in the proportion of apoptotic cells (assessed by Hid-GFP, cleaved Casp-3 and Dcp-1), a severe increase in the overall mitotic index (including of the central brain neuroblast population), and a significant increase in the proportion of DNA damaged cells, as compared to controls. Third instar larval brains also display severe tissue organization defects, which mostly affect the optic lobes, as compared to controls: loss or strong reduction of the medulla layer, which exhibits very few neuroblasts and decreased numbers of disorganized neuroepithelial cells; apparent loss of the lamina layer; decreased size of the medullar neuropil, associated with decreased size and disorganization of the axons from medullar neurons; decreased size and complexity of the outer optic anlagen (but not at the second instar larval stage); decreases in number and in organization of central brain neuroblasts, which are able to divide symmetrically, but occasionally exhibit an increased cell size and differentiation defects (i.e. expressing both Dpn and Pros proteins); but not any obvious defects in the mushroom body. Homozygosity for mad2G6595 strongly enhances the frequency of ploidy defects, but not the delayed entry into anaphase, observed in Sas-4s2214-homozygous third instar larval central brain neuroblasts.
The increased number of brain neuroblast (NB) cells characteristic for aurA8839 mutant third instar larvae is slightly elevated further by combination with homozygous mad2G6595, whereas the increased mitotic index, the highly condensed chromosome appearance in mitotic cells and the tumorigenic potential of the double mutant tissue in an allograft assay are not significantly affected compared to aurA8839 single mutant. The delay in the timing of mitotic cell cycle events is ameliorated in aurA8839;mad2G6595 brain NBs compared to aurA8839 cells but not wild-type level, no lagging chromatids (indicative of incorrect spindle attachment) are observed during anaphase and telophase.
aurA8839;Sas-4s2214;mad2G6595 triple mutants show high rate of ploidy defects in third instar larval brains, the number of neuroblasts or the mitotic cells is highly increased relative to wild-type but significantly different from aurA8839;Sas-4s2214 double mutants.
The increased brain neuroblast (NB) number observed in Sas-4s2214 mutant third instar larvae is fully suppressed by combination with mad2G6595 (the NB number in the double mutants is even lower than in wild-type controls and the brains are also smaller), while the NB mitotic spindle defects are enhanced and numerous aneuploid or polyploid cells are frequently observed. The double mutant brain tissue also cannot (unlike that from Sas-4s2214 single mutants) induce tumor formation in an allograft assay.
The presence of a TEV protease cleavable form of vtd (by expressing vtd271TEV.tub.T:Zzzz\TEV.CS,T:Hsap\MYC and Zzzz\NIaScer\UAS.T:SV5\V5,T:SV40\nls2 in a vtdex3 mutant background) suppresses the chromosome segregation defects seen in embryos expressing aldScer\UAS.cAa under the control of Scer\GAL4mat.αTub67C.T:Hsim\VP16 in a mad2G6595 mutant background. This suppression does not occur in the absence of Zzzz\NIaScer\UAS.T:SV5\V5,T:SV40\nls2.
BubR1A1204K.PR-mad2G6595 double mutants are not delayed in prometaphase (as in BubR1A1204K.PR single mutants) but instead show a rapid mitotic transit time and early onset of CycB degradation (as in mad2G6595 single mutants). Double mutants are uniformly larval/pupal lethal and double mutant brains exhibit a lower mitotic density with neuroblast aneuploidy and abnormal anaphases more frequent than in both single mutants.
BubR1KEN.T:Disc\RFP-mRFP mad2G6595 double mutants are viable and fertile, with aneuploidy levels no higher than in the single mutants. Anaphase figures also appear normal in double mutants. The time from nuclear envelope breakdown to anaphase is markedly reduced (by 40%) compared to BubR1KEN.T:Disc\RFP-mRFP single mutant or wild-type cells and 30% reduced compared to mad2G6595 single mutants. This acceleration is accompanied by an earlier onset of CycB breakdown. In contrast, the gap from CycB breakdown to anaphase is relatively constant between the double mutant and controls.
Homozygous mad2G6595 suppresses the developmental rate defects seen in larvae expressing SAKUbi.T:Avic\GFP, producing instead a slight increase in the rate of development as is seen in mad2G6595 mutants alone. The mitotic index in mad2G6595 SAKUbi.T:Avic\GFP brains is lower than in either wild type or SAKUbi.T:Avic\GFP expressing neuroblasts. The number of multipolar and defective spindles is dramatically increased compared to flies expressing SAKUbi.T:Avic\GFP and polyploidy, aneuploidy and lagging chromosomes are all seen during anaphase. The number of centrosomes per cell is increased compared to SAKUbi.T:Avic\GFP.
mad2G6595 cnnHK21 double mutants exhibit a severely delayed spindle assembly pathway, resulting in a higher mitotic index and a broad peak anaphase onset time of 8-16 minutes. Larval brains of mad2G6595 cnnHK21 double mutants have a very high incidence of polyploidy and mad2G6595 cnnHK21 individuals die as early pupae.