Developmental Expression of \u3cem\u3eDrop-Dead\u3c/em\u3e is Required for Early Adult Survival and Normal Body Mass in \u3cem\u3eDrosophila melanogaster\u3c/em\u3e

In Drosophila melanogaster, mutations in the gene drop-dead (drd) result in early adult lethality, with flies dying within 2 weeks of eclosion. Additional phenotypes include neurodegeneration, tracheal defects, starvation, reduced body mass, and female sterility. The cause of early lethality and the function of the drd protein remain unknown. In the current study, the temporal profiles of drd expression required for adult survival and body mass regulation were investigated. Knockdown of drd expression by UAS-RNAi transgenes and rescue of drd expression on a drd mutant background by a UAS-drd transgene were controlled with the Heat Shock Protein 70 (Hsp70)-Gal4 driver. Flies were heat-shocked at different stages of their life cycle, and the survival and body mass of the resulting adult flies were assayed. Surprisingly, the adult lethal phenotype did not depend upon drd expression in the adult. Rather, expression of drd during the second half of metamorphosis was both necessary and sufficient to prevent rapid adult mortality. In contrast, the attainment of normal adult body mass required a different temporal pattern of drd expression. In this case, manipulation of drd expression solely during larval development or metamorphosis had no effect on body mass, while knockdown or rescue of drd expression during all of pre-adult (embryonic, larval, and pupal) development did significantly alter body mass. Together, these results indicate that the adult-lethal gene drd is required only during development. Furthermore, the mutant phenotypes of body mass and lifespan are separable phenotypes arising from an absence of drd expression at different developmental stages

Neurodegeneration in \u3cem\u3eDrop-Dead\u3c/em\u3e Mutant \u3cem\u3eDrosophila melanogaster\u3c/em\u3e Is Associated with the Respiratory System but Not with Hypoxia

Mutations in the gene drop-dead (drd) cause diverse phenotypes in adult Drosophila melanogaster including early lethality, neurodegeneration, tracheal defects, gut dysfunction, reduced body mass, and female sterility. Despite the identification of the drd gene itself, the causes of early lethality and neurodegeneration in the mutant flies remain unknown. To determine the pattern of drd expression associated with the neurodegenerative phenotype, knockdown of drd with various Gal4 drivers was performed. Early adult lethality and neurodegeneration were observed upon knockdown of drd in the tracheal system with two independent insertions of the breathless-Gal4 driver and upon knockdown in the tracheal system and elsewhere with the DJ717-Gal4 driver. Surprisingly, rescue of drd expression exclusively in the tracheae in otherwise mutant flies rescued the neurodegenerative phenotype but not adult lethality. Gut dysfunction, as measured by defecation rate, was not rescued in these flies, and gut function appeared normal upon tracheal-specific knockdown of drd. Finally, the hypothesis that tracheal dysfunction in drd mutants results in hypoxia was tested. Hypoxia-sensitive reporter transgenes (LDH-Gal4 and LDH-LacZ) were placed on a drd mutant background, but enhanced expression of these reporters was not observed. In addition, manipulation of drd expression in the tracheae did not affect expression of the hypoxia-induced genes LDH, tango, and similar. Overall, these results indicate that there are at least two causes of adult lethality in drd mutants, that gut dysfunction and neurodegeneration are independent phenotypes, and that neurodegeneration is associated with tracheal expression of drd but not with hypoxia

Pleiotropic and Novel Phenotypes in The \u3cem\u3eDrosophila\u3c/em\u3e Gut Caused by Mutation of \u3cem\u3eDrop-Dead\u3c/em\u3e

Normal gut function is vital for animal survival, and deviations from such function can contribute to malnutrition, inflammation, increased susceptibility to pathogens, diabetes, neurodegenerative diseases, and cancer. In the fruit fly Drosophila melanogaster, mutation of the gene drop-dead (drd) results in defective gut function, as measured by enlargement of the crop and reduced food movement through the gut, and drd mutation also causes the unrelated phenotypes of neurodegeneration, early adult lethality and female sterility. In the current work, adult drd mutant flies are also shown to lack the peritrophic matrix (PM), an extracellular barrier that lines the lumen of the midgut and is found in many insects including flies, mosquitos and termites. The use of a drd-gal4 construct to drive a GFP reporter in late pupae and adults revealed drd expression in the anterior cardia, which is the site of PM synthesis in Drosophila. Moreover, the ability of drd knockdown or rescue with several gal4 drivers to recapitulate or rescue the gut phenotypes (lack of a PM, reduced defecation, and reduced adult survival 10–40 days post-eclosion) was correlated to the level of expression of each driver in the anterior cardia. Surprisingly, however, knocking down drd expression only in adult flies, which has previously been shown not to affect survival, eliminated the PM without reducing defecation rate. These results demonstrate that drd mutant flies have a novel phenotype, the absence of a PM, which is functionally separable from the previously described gut dysfunction observed in these flies. As the first mutant Drosophila strain reported to lack a PM, drd mutants will be a useful tool for studying the synthesis of this structure

Temporal And Spatial Requirements Of Drop-Dead Expression For Adult Survival In Drosophila Melanogaster

Mutations in the gene drop-dead (drd) cause diverse phenotypes in adult Drosophila melanogaster including early lethality, neurodegeneration, fragile tracheae, gut dysfunction, reduced body mass, and female sterility. The cause of early lethality and the function of the drd protein remain unknown. To elucidate a molecular function of DRD, the temporal and spatial requirements of the drd gene for survival were determined. drd expression was manipulated with the Gal4-UAS system, using UAS-RNAi transgenes and a UAS-drd transgene on a drd mutant background to knock down and rescue expression of drd, respectively. To investigate the temporal requirements of drd expression for adult survival, the UAS-RNAi and UAS-drd rescue line were controlled with the Heat Shock Protein 70-Gal4 driver. Flies were heat-shocked at different stages of their lifecycle, and the survival of the resulting adult flies was assayed. Expression of drd during the second half of metamorphosis was necessary and sufficient to prevent rapid adult mortality. To determine the spatial requirements of drd expression for adult survival, drd expression was knocked down with various tissue-specific Gal4 drivers and the progeny were screened for early lethality. Neurodegeneration and early lethality and gut dysfunction and early lethality were observed upon knockdown of drd in the tracheae and anterior cardia, respectively. The tracheal phenotype in drd mutants is hypothesized to cause hypoxia-induced neurodegeneration. Hypoxia-sensitive reporter transgenes were placed on a drd mutant background, but expression of these reporters was not observed, suggesting these flies are not hypoxic. Rescue of drd expression in the tracheae rescued neurodegeneration but not adult lethality. However, rescue of drd in the tracheae and anterior cardia rescued both causes of early lethality, neurodegeneration and starvation, indicating that drd expression in these two tissues is sufficient for survival. From these data, it is hypothesized that drd is required for extracellular matrix (ECM) integrity. Both critical tissues produce an ECM; tracheae produce a cuticle to line the lumen and the anterior cardia synthesizes the peritrophic matrix, which lines the midgut. Additionally, cuticle processing occurs in metamorphosis, the critical period for drd expression. Future direction will focus on establishing a role for DRD in ECM synthesis

Neurodegeneration in <i>Drop-Dead</i> Mutant <i>Drosophila melanogaster</i> Is Associated with the Respiratory System but Not with Hypoxia

<div><p>Mutations in the gene <i>drop-dead</i> (<i>drd</i>) cause diverse phenotypes in adult <i>Drosophila melanogaster</i> including early lethality, neurodegeneration, tracheal defects, gut dysfunction, reduced body mass, and female sterility. Despite the identification of the <i>drd</i> gene itself, the causes of early lethality and neurodegeneration in the mutant flies remain unknown. To determine the pattern of <i>drd</i> expression associated with the neurodegenerative phenotype, knockdown of <i>drd</i> with various Gal4 drivers was performed. Early adult lethality and neurodegeneration were observed upon knockdown of <i>drd</i> in the tracheal system with two independent insertions of the <i>breathless-Gal4</i> driver and upon knockdown in the tracheal system and elsewhere with the <i>DJ717-Gal4</i> driver. Surprisingly, rescue of <i>drd</i> expression exclusively in the tracheae in otherwise mutant flies rescued the neurodegenerative phenotype but not adult lethality. Gut dysfunction, as measured by defecation rate, was not rescued in these flies, and gut function appeared normal upon tracheal-specific knockdown of <i>drd</i>. Finally, the hypothesis that tracheal dysfunction in <i>drd</i> mutants results in hypoxia was tested. Hypoxia-sensitive reporter transgenes (<i>LDH-Gal4</i> and <i>LDH-LacZ</i>) were placed on a <i>drd</i> mutant background, but enhanced expression of these reporters was not observed. In addition, manipulation of <i>drd</i> expression in the tracheae did not affect expression of the hypoxia-induced genes <i>LDH</i>, <i>tango</i>, and <i>similar</i>. Overall, these results indicate that there are at least two causes of adult lethality in <i>drd</i> mutants, that gut dysfunction and neurodegeneration are independent phenotypes, and that neurodegeneration is associated with tracheal expression of <i>drd</i> but not with hypoxia.</p> </div

Expression of <b><i>drd</i></b><b> in the tracheae does not rescue survival, but does rescue neurodegeneration.</b>

<p>Tracheal specific expression of <i>drd</i> by crossing <i>btl-Gal4(II)/CyO</i> males with <i>drd^lwf/FM7a;UAS-drd</i> females did not rescue the early lethality phenotype (a) or the neurodegeneration phenotype (b: Control on top, degeneration was observed in 7 of 7 brains; Rescue on bottom, degeneration was observed in 7 of 7 brains). Two copies of the <i>btl-Gal4(II)</i> driver where utilized and the above experiments repeated. <i>drd^lwf;UAS-drd btl-Gal4(II)/btl-Gal4(II)</i> flies still exhibited the early lethality phenotype. Single copy Gal4 rescue and sibling controls are the same data as in Fig. 2a on this graph (c). However, these flies no longer display neurodegeneration (d). n = 0 of 3 degenerating brains. Neurodegeneration was assayed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068032#pone-0068032-g002" target="_blank">Figure 2</a>. For survival curves, n = 50–56 flies/genotype.</p

Knockdown of <b><i>drd</i></b><b> in the tracheae causes neurodegeneration.</b>

<p>Brain sections of 4 day old flies were stained with haematoxylin and eosin. Neurodegeneration was observed in <i>btl-Gal4(II)/51184 UAS-Dcr-2</i> (b) and <i>btl-Gal4(II)/UAS-Dcr-2 37404</i> (d), but not in the sibling controls (a and c, respectively). Arrows indicate holes.</p

Hypoxia-induced genes are not upregulated in <b><i>drd</i></b><b> mutants or affected by tracheal expression of </b><b><i>drd</i></b><b>.</b>

<p><i>LDH</i>, <i>sima</i>, and <i>tango</i> are all upregulated after 5 hours of anoxic treatment in Canton S flies (a) while the genes are not consistently upregulated in <i>drd^lwf</i> males (b). Control and anoxic treated data are the same in Fig. 5a and 5b. Male <i>51184 UAS-Dcr-2/btl-Gal4(II)</i> (c), <i>UAS-Dcr-2 37404/btl-Gal4(II)</i> (d), and <i>w drd^lwf;UAS-drd btl-Gal4(II)/btl-Gal4(II)</i> flies (e) were assayed to determine the expression levels of hypoxia-induced genes. Asterices indicate significant difference by 1-way ANOVA and Bonferroni’s post-test between experimental and sibling controls in a,c,d,e and between control or anoxic Canton S and <i>drd^lwf</i> in b. ns p>0.05; *p<0.05; **p<0.01. n = 3. Error bars represent SEM.</p

Knockdown of <b><i>drd</i></b><b> in the tracheae does not cause starvation.</b>

<p>Triglyceride (a,b) and glycogen (c,d) levels of male progeny from crossing <i>btl-Gal4/CyO</i> females with either <i>w;{GD15915}v51184 UAS-Dcr-2</i> (a,c) or <i>w;UAS-Dcr-2 {GD3367}v37404</i> (b,d) males. No significant effect of genotype was seen by 2-way ANOVA. n = 10 flies/point. Pairs of male flies of the same genotype from the cross above were also utilized for defecation assays during the second day post-eclosion. Defecation assays were also performed on <i>drd^lwf;UAS-drd btl-Gal4(II)/btl-Gal4(II)</i> (e). Asterices indicate significant difference by 1-way ANOVA and Bonferroni’s post-test. ns p>0.05; *p<0.05; ***p<0.001. n = 6 pairs of flies/condition. Error bars represent SEM.</p

<b><i>drd</i></b><b> expression is required in the tracheae but not the brain for survival.</b>

<p>There was no effect on lifespan when <i>drd</i> expression was knocked down by either <i>w;{GD15915}v51184 UAS-Dcr-2 </i>or <i>w;UAS-Dcr-2 {GD3367}v37404</i> with the <i>elav-Gal4</i> (a), <i>repo-Gal4</i> (b), <i>17A-Gal4</i> (c), or <i>elav-Gal4; 17A-Gal4</i> (d) drivers. Early lethality was observed when <i>drd</i> expression was knocked down with the <i>btl-Gal4(II)</i> (e), <i>btl-Gal4(III)</i> (f), and <i>DJ717-Gal4</i> (g) drivers. n = 50–81 flies/genotype.</p