Abstract
Poly(ADP-ribose) polymerase (PARP) may play important roles in nuclear events such as cell cycle, cell proliferation, and maintenance of chromosomal stability. However, the exact biological role played by PARP or how PARP is involved in these cellular functions is still unclear. To elucidate the biological functions of PARP in vivo, we have constructed transgenic flies that overexpress Drosophila PARP in the developing eye primordia. These flies showed mild roughening of the normally smooth ommatidial lattice and tissue polarity disruption caused by improper rotation and chirality of the ommatidia. To clarify how this phenotypical change was induced, here we analyzed transgenic flies overexpressing PARP in the developing eye, embryo, and adult in detail. PARP mRNA level and the phenotype were enhanced in flies carrying more copies of the transgene. Developing eyes from third instar larvae were analyzed by using the neural cell marker to examine the involvement of PARP in cell fate. Morphological disorder of non-neuronal accessory cells was observed in PARP transgenic flies. Interestingly, overexpression of PARP did not interfere with the cell cycle or apoptosis, but it did disrupt the organization of cytoskeletal F-actin, resulting in aberrant cell and tissue morphology. Furthermore, heat-induced PARP expression disrupted organization of cytoskeletal F-actin in embryos and tissue polarity in adult flies. Because these phenotypes closely resembled mutants or transgenic flies of the tissue polarity genes, genetic interaction of PARP with known tissue polarity genes was examined. Transgenic flies expressing either PARP or RhoA GTPase in the eye were crossed, and co-expression of PARP suppressed the effect of RhoA GTPase. Our results indicate that PARP may play a role in cytoskeletal or cytoplasmic events in developmental processes of Drosophila.
Highlights
** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Oncology, Institute of Basic Medical Science, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japan
Our results indicate that overexpression of Poly(ADP-ribose) polymerase (PARP) interferes with the organization of cytoskeletal filamentous actin (F-actin) and disrupts tissue polarity
PARP Overexpression Induced Mild Rough-eye Phenotype with Incorrect Chirality—For targeted expression of D.PARP in the developing eye of Drosophila, D.PARP cDNA was placed under the control of GMR such that PARP was expressed only in and posterior to the morphogenetic furrow (MF) [23, 24]
Summary
Fly Stocks and Genetics—Flies were cultured at 25 °C. Oregon-R and w1118 were used as wild-type unless otherwise stated. To analyze the effect of PARP overexpression in the adult fly, a 1-h heat pulse was given at 37 °C every 12 h from the early third instar to the end of the pupal stage. Northern and Western Blotting—Total RNA was isolated from eyeantennal discs of wild-type and GMR-PARP third instar larvae as described [28]. For Western blotting, protein was extracted from wild type and GMR-PARP eye antenal discs from third instar larvae, separated on SDS-PAGE using a 7.5% gel, and electrotransferred onto an Immobilon-P membrane (Millipore, Bedford, MA). Detection of Programmed Cell Death—TdT-mediated dUTP nick-end labeling (TUNEL) of third instar larval eye discs was carried out using an In Situ Cell Death Detection Kit (Roche Diagnostics) according to the manufacturer’s protocol
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