UASt regulatory sequences drive expression of a Pi3K92E cDNA fragment, with a mutation (D954A) introduced the putative ATP binding site. The open reading frame is tagged at the N-terminal end with Tag:MYC.
There is an increase in the frequency of clone splitting in the Pi3K92ED954A.Scer\UAS.T:Hsap\MYC-expressing 'loser' cells when cell competition is induced in the pupal notum through generation of Pi3K92ED954A.Scer\UAS.T:Hsap\MYC-expressing clones in wild type tissues compared with wild type clones in wild type tissue. The 'loser' clones show reduced compactness over time. The maximum speed of relaxation of junctions after laser nanoablation is reduced in loser-loser and loser-winner junctions compared with winner-winner junctions.
In contrast to wild-type, transgenic flies expressing Scer\GAL4ppl.PP>Pi3K92ED954A.Scer\UAS.T:Hsap\MYC and reared on medium supplemented with Apis mellifera royal jelly do not show increased body size. However, similarly to wild-type flies in response to royal jelly, they display shortened developmental time compared with flies reared on control medium.
Expression of Pi3K92ED954A.Scer\UAS.T:Hsap\MYC under the control of Scer\GAL4amn-c651 results in a reduction in the size of the prothoracic gland in larvae compared with controls at the equivalent time point of 5 days after egg deposition. These animals show only a moderate developmental delay and pupate, eclosing as larger than normal flies. The defects in developmental rate and adult size are suppressed by feeding 20-hydroxyecdysone to larvae.
Hemocytes expressing Pi3K92ED954A.Scer\UAS.T:Hsap\MYC (under the control of Scer\GAL4crq.PO) exhibit normal distribution at all stages of development, and appear morphologically indistinguishable from their wild-type counterparts. Pi3K92ED954A.Scer\UAS.T:Hsap\MYC-expressing hemocytes migrate along the ventral midline in a pattern identical to that seen in wild-type embryos, demonstrating velocity, directionality, and polarity equal to wild-type cells.
Laser-ablated Pi3K92ED954A.Scer\UAS.T:Hsap\MYC-expressing embryos (under the control of Scer\GAL4crq.PO) fail to chemotax toward the wound site and the wound remains largely undetected by the hemocytes up to 1 hour after laser ablation. The same result is obtained when beads are implanted in the embryo. There is no significant different in hemocyte number between Pi3K92ED954A.Scer\UAS.T:Hsap\MYC-expressing embryos and wild-type.
When Pi3K92ED954A.Scer\UAS.T:Hsap\MYC is driven by Scer\GAL4P0206, animals exhibit increased growth: adults have 17% greater weight on average. This increase was attributable to an increase in cell number in the wing and eye. The timing of embryonic and larval development in these mutants is comparable to controls. The duration of pupal development is advanced by less than four hours. A 1 to 3 hour delay is seen in the time Pi3K92ED954A.Scer\UAS.T:Hsap\MYC (without driver) mutants enter pupal development.
Cells in sector C of the wing expressing Pi3K92ED954A.Scer\UAS.T:Hsap\MYC under the control of Scer\GAL4dpp.blk1 are 12.6% smaller than control cells. The average area of the mutant sector is reduced by 18.7% compared to controls and the average number of cells in the mutant sector is reduced by 13.3%. In addition to the autonomous reduction in the area of the sector expressing Pi3K92ED954A.Scer\UAS.T:Hsap\MYC under the control of Scer\GAL4dpp.blk1 (sector C), a non-autonomous reduction of all other wing sectors is seen. In the B sector of mosaic wings the reduction in area is 20%, while the global reduction in area of the wing is 7%. Expression of Pi3K92ED954A.Scer\UAS.T:Hsap\MYC under the control of Scer\GAL4ap-md544 results in a clear decrease in size and number of chaetae compared to controls. In the acrostical region there is an increase in density of microchaetae compared to wild type, which is due to both reduced cell size and fewer cells between the microchaetae than normal (4.16 in mutant tissue compared to 5.34 in wild type).
When expression is driven by Scer\GAL4dpp.blk1 the area between wing veins LIII and LIV is significantly reduced due to a decrease in both cell number and cell size. When expression is driven by Scer\GAL4Bx-MS1096 the whole wing is significantly reduced in size.
When expression is driven in imaginal disc cells by Scer\GAL4Act5C.PP the size of the cells is slightly decreased (as measured by FSC (forward scatter)). Overexpression of Pi3K92ED954A.Scer\UAS.T:Hsap\MYC is sufficient to reduce the rate of increase of cell number. When expression is driven by Scer\GAL4en-e16E, the size of the posterior compartment of the wing disc is significantly reduced. There is no comparable effect on the anterior compartment.
Third instar larval eye-antennal imaginal disc clones co-expressing dlg1GD4689 and Ras85DV12.Scer\UAS under the control of Scer\GAL4Act5C.CoinFLP-FRT, in combination with Dicer-2 for efficient RNAi, form invasive tumors and induce autophagosomes (assessed by a Atg8a fluorescence reporter) in a cell non-autonomous manner in neighboring disc tissue, which are suppressed and not suppressed, respectively, by the additional clonal co-expression of Pi3K92ED954A.Scer\UAS.T:Hsap\MYC.