Abstract
Cell death is a fundamental progress that regulates cell number, tissue homeostasis and organ size in development. The c-Jun N-terminal kinase (JNK) pathway has been evolutionarily conserved from fly to human, and plays essential roles in regulating cell death. To characterize additional genes that regulate JNK signaling, we performed a genetic screen in Drosophila and identified dGLYAT, a novel gene whose function was previously unknown, as a modulator of JNK-mediated cell death. We found that loss of dGLYAT suppressed JNK activation and cell death triggered by over-expression of Egr or Hep, or depletion of puc or lgl in development, suggesting dGLYAT regulates both ectopic and physiological functions of JNK pathway. Furthermore, we showed that loss of dGLYAT inhibits JNK-mediated ROS production, suggesting dGLYAT regulates multiple functions of JNK signaling in vivo.
Highlights
MethodsAll stocks were raised on a standard cornmeal and agar medium at 25 °C unless otherwise indicated
We identified dGLYAT as a crucial modulator of JNK pathway in vivo by using Drosophila as a model organism
We showed that loss of dGLYAT blocks ectopic Egr- or Hep-induced JNK activation and cell death, and depletion-of-puc or lgl-triggered physiological JNK activation and cell death in development
Summary
All stocks were raised on a standard cornmeal and agar medium at 25 °C unless otherwise indicated. For experiments involving tub-Gal80ts, eggs were allowed to develop at 25 °C for 2–3 days, transferred to 29 °C for 2 days to inactivate Gal[80]. Ptc-Gal[442], GMR-Gal[447], UAS-EgrRegg[119], UAS-GFP and UAS-HepCA48, UAS-Dp5333, tub-Gal80ts and pucE6933 have been used previously; UAS-puc-IR (V3018) was obtained from Vienna Drosophila RNAi Center; UAS-bsk-IR (NIG5680R-2) was obtained from National Institute of Genetics (NIG-FLY); PBac{PB}CG34010c02982 was obtained from Harvard (the Exelixis Collection); UAS-dGLYAT-IR was obtained from Tsinghua Fly Center. Eye and wing discs were dissected from 3rd instar larvae in 1 × PBS (phosphate-buffered saline) and incubated in 1 × 10−5 M acridine orange (AO) for 5 minutes at room temperature.
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