Combination of hypomorphic mutations of the Drosophila homologues of aryl hydrocarbon receptor and nucleosome assembly protein family genes disrupts morphogenesis, memory and detoxification

Abstract

Aryl hydrocarbon receptor is essential for biological responses to endogenous and exogenous toxins in mammals. Its Drosophila homolog spineless plays an important role in fly morphogenesis. We have previously shown that during morphogenesis spineless genetically interacts with CG5017 gene, which encodes a nucleosome assembly factor and may affect cognitive function of the fly. We now demonstrate synergistic interactions of spineless and CG5017 in pathways controlling oxidative stress response and long-term memory formation in Drosophila melanogaster. Oxidative stress was induced by low doses of X-ray irradiation of flies carrying hypomorphic mutation of spineless, mutation of CG5017, and their combination. To determine the sensitivity of these mutants to pharmacological modifiers of the irradiation effect, we irradiated flies growing on standard medium supplemented by radiosensitizer furazidin and radioprotector serotonin. The effects of irradiation were investigated by analyzing leg and antenna morphological structures and by using real-time PCR to measure mRNA expression levels for spineless, Cyp6g1 and Gst-theta genes. We also examined long-term memory in these mutants using conditioned courtship suppression paradigm. Our results show that the interaction of spineless and CG5017 is important for regulation of morphogenesis, long-term memory formation, and detoxification during oxidative stress. Since spineless and CG5017 are evolutionary conserved, these results must be considered when evaluating the risk of combining similar mutations in other organisms, including humans.

Figure 1. Wild type and mutant leg and antenna phenotypes.

Micrographs show the normal morphology of the tarsal structures of the wild type Canton S, ss^a40ahm and milkah-1 flies. Antennal structures of the milkah-1 mutant flies do not show any difference from the wild type. The distal segment of the antenna – the arista - of the ss^a40ahm flies presents a certain thickening of the proximal end. The arista of the ss^a40ahmmilkah-1 hybrid flies suffered homeotic transformation into an unsegmented tarsus. The tarsal structures of the ss^a40ahmmilkah-1 hybrids show altered segment fusion.

Figure 2. Wild type and mutant antenna and leg in flies in normal conditions and following irradiation.

Simultaneous combination of mutations at both ss and CG5017 loci increases sensitivity to even low doses of X-ray radiation (1 and 10 R), which is manifested as an increase in the mutant phenotype (photographs within the red frame).

Figure 3. Effect of X-rays and pharmacological agents on Cyp6g1, GST-theta (CG1681) and ss mRNA expression.

The relative level of expression of mRNA was measured using real-time PCR. A, B, C – level of mRNA expressed in Canton S, ss^a40a and ss^aSc flies respectively. As a control, we used RNA from flies, which have not been exposed to radiation (cont.). F, 5-HT – level of mRNA expressed in flies, grown on medium with added furazidin (F) and 5-hydroxytryptamine (5-HT), respectively; R – radiation dose in Roentgens; 1-10R, level of expressed mRNA of the genes under study in flies irradiated with a dose of 1-10 R. F1-10R, 5-HT1-10R – level of expressed mRNA of the genes under study in flies grown on medium with added F or 5-HT and radiated with 1-10 R, respectively. The bars show the level of mRNA expression. The error bars represent the standard error of the mean of triplicate experiments. * - P<0.05, compared to control group, - P<0.05, compared to F or 5-HT group.

Figure 4. Dynamics of acquisition and retention of conditioned courtship suppression in mutant males.

Males from wild type Canton S (A-B), milkah-1 (C–D), ss^a40a (E–F) and ss^a40amilkah-1 (G–H) lines were tested. (A, C, E, G) – of tested males. (B, D, F, H) – of tested males. On the X-axis: time following training, in days; on the Y axis: – courtship index, – learning index, standard units. Open columns – of naive males, hatched columns – () of trained males. Each point represents 20 males. ** - or significantly lower than for wild type (two-sided randomization test, ) in similar condition; - in a test immediately following training or in a deferred test, significantly lower than the CI of naive males (two-sided randomization test, ); - in the delayed test significantly lower than in test immediately following training (two-sided randomization test,.