Atg1Δ3D somatic clones in the eye do not lead to loss of photoreceptor cells, as compared to controls.
Homozygous mushroom body gamma neuron clones show normal axon pruning.
Atg1Δ3D mutant somatic clones generated in the fat body undergo normal endocytosis; endocytic tracer is seen throughout the cytosol of both control and mutant cells.
Atg1Δ3D is able to completely suppress the formation of autophagosomes in the muscles of both untreated and chloroquine-treated larvae starved on low-nutrient food for 6 hours.
Ovarian follicle cell clones of Atg1Δ3D exhibit impaired autophagy induction.
Chimeric ovaries composed from an Atg1Δ3D hemizygous germline and wild-type follicle cells are defective in autophagy, as starvation does not induced lysotracker staining in the mutant germline cells, but in the enveloping wild-type follicle cells. Atg1Δ3D germline chimeras develop functional ovaries, and their egg-laying behavior and hatching rates are indistinguishable from sibling control chimeras, albeit the offspring develop with a delay of 2 days. When the Atg1Δ3D germline chimeras are crossed with Atg1Δ3D heterozygous males, the resulting Atg1Δ3D homozygous mutant animals die in late larval stages, similar to Atg1Δ3D homozygous mutants derived from heterozygous mothers. There are no defects in egg chamber development or egg morphology.
Heat-shock induced mitotic recombination results in Atg1Δ3D homozygous mutant follicle cell clones in 69% of the egg chambers with most of them being mosaic. Flies containing Atg1Δ3D mutant follicle cell clones lay very few eggs that resemble those generated by irradiation, lacking dorsal appendages and embryonic cuticle, and only 5% of the eggs hatched. Approximately 89% of the eggs laid by females containing Atg1Δ3D mutant follicle cell clones exhibit dorsal appendage defects; consequently, only 11% of the chimeric eggs hatched. Defective dorsal appendage formation is only observed in 15% of the eggs containing control clones, and 58% of the control eggs hatched. Given that 15% of the control eggs show egg defects, the frequency of the egg phenotype that is solely due to the Atg1Δ3D deletion (74%) is in accordance with the frequency of follicle cell clones observed in Atg1Δ3D chimeras (69%), suggesting that almost every egg chamber containing Atg1Δ3D mutant follicle cell clones results in defective eggs.
Females carrying homozygous germline clones have a significant increase in the number of stage 14 egg chambers that have persisting TUNEL-negative nurse cell nuclei compared to wild-type egg chambers at the same stage, in which nurse cell nuclei are rarely detected (and those that are detected are all TUNEL positive). The persisting nurse cell nuclei show condensed nuclear staining.
Primary hemocytes dissected from homozygous third instar larvae fail to spread, remaining round in shape.
36% of Atg1Δ3D germ line clone stage 14 egg chambers show persisting nurse cell nuclei. 2% of egg chambers display a dumpless phenotype where nurse cell cytoplasm has not been transferred to the oocyte.
Atg1Δ3D/+ results in a significant decrease in life span as compared to control flies.
Cell death in the germarium of Atg1Δ3D germline clones is reduced to 8.4% (compared to 26% of the control) and to 3.5% for mid-stage egg chambers (compared to 9.7% of the control).
Mutants are viable until pupal stages.
The abundance of Lysotracker-positive foci (a marker for autophagosomes) is strongly reduced in Atg1Δ3D guts from larvae exposed to paraquat-containing food or from starved larvae, compared to wild type larvae under the same conditions.
Fewer apoptotic (TUNEL positive) cells are seen in region two cysts in nutrient-deprived Atg1Δ3D mutant germline clones than are seen in wild type. Degenerating stage eight egg chambers in starved Atg1Δ3D clones show low or no TUNEL-positive staining compared with controls, although nuclear DNA condensation is still observed in these egg chambers.
No mitotic defects are evident in the larval brain and imaginal discs of Atg1Δ3D mutant animals.
In normally fed animals, the size of homozygous Atg1Δ3D clones in the wing disc is not significantly different from their twin spot controls, but in animals grown on medium containing 2μm rapamycin, homozygous Atg1Δ3D clones have a 59% larger average clone size compared to the wild-type twin spot.
Cell size in homozygous clones in the fat body is normal in normally fed animals, but is increased compared to controls under starvation conditions.
Starvation-induced autophagy fails to occur in the larval fat body. Larvae show reduced viability and do not develop beyond pupation. During their period of viability, larvae are hypersensitive to starvation.