Regulation of Hemocytes in Drosophila Requires dappled Cytochrome b5

A major category of mutant hematopoietic phenotypes in Drosophila is melanotic tumors or nodules, which consist of abnormal and overproliferated blood cells, similar to granulomas. Our analyses of the melanotic mutant dappled have revealed a novel type of gene involved in blood cell regulation. The dappled gene is an essential gene that encodes cytochrome b5, a conserved hemoprotein that participates in electron transfer in multiple biochemical reactions and pathways. Viable mutations of dappled cause melanotic nodules and hemocyte misregulation during both hematopoietic waves of development. The sexes are similarly affected, but hemocyte number is different in females and males of both mutants and wild type. Additionally, initial tests show that curcumin enhances the dappled melanotic phenotype and establish screening of endogenous and xenobiotic compounds as a route for analysis of cytochrome b5 function. Overall, dappled provides a tractable genetic model for cytochrome b5, which has been difficult to study in higher organisms

Toll mediated infection response is altered by gravity and spaceflight in Drosophila.

Space travel presents unlimited opportunities for exploration and discovery, but requires better understanding of the biological consequences of long-term exposure to spaceflight. Immune function in particular is relevant for space travel. Human immune responses are weakened in space, with increased vulnerability to opportunistic infections and immune-related conditions. In addition, microorganisms can become more virulent in space, causing further challenges to health. To understand these issues better and to contribute to design of effective countermeasures, we used the Drosophila model of innate immunity to study immune responses in both hypergravity and spaceflight. Focusing on infections mediated through the conserved Toll and Imd signaling pathways, we found that hypergravity improves resistance to Toll-mediated fungal infections except in a known gravitaxis mutant of the yuri gagarin gene. These results led to the first spaceflight project on Drosophila immunity, in which flies that developed to adulthood in microgravity were assessed for immune responses by transcription profiling on return to Earth. Spaceflight alone altered transcription, producing activation of the heat shock stress system. Space flies subsequently infected by fungus failed to activate the Toll pathway. In contrast, bacterial infection produced normal activation of the Imd pathway. We speculate on possible linkage between functional Toll signaling and the heat shock chaperone system. Our major findings are that hypergravity and spaceflight have opposing effects, and that spaceflight produces stress-related transcriptional responses and results in a specific inability to mount a Toll-mediated infection response

Antifungal AMPs.

<p><b>A.. </b><i>Metchnikowin</i> and <b>B.. </b><i>Drosomycin</i> transcript levels were assessed by qPCR in space and Earth flies infected with fungus (F) or bacteria (B), or uninfected (U), and standardized by comparison to the level of ribosomal protein gene <i>rp49</i>. Error bars = SEM for 3 experiments.</p

Microarray-based analysis of response to <i>B. bassiana</i>.

<p>The total number of genes upregulated or downregulated in Earth flies only (Earth) or space flies only (Space) or in both (overlap) are indicated by Venn diagrams. Pathway analysis of each of these groups is shown on the right side of the figure. The number of genes in each functional category is depicted in bar graphs (primary y-axis), and the <i>P</i> values corresponding to statistical over-representation of each category are presented as a line graph (secondary y-axis). Note that certain genes annotated into more than one of these categories.</p

Effects of hyper g on post-infection survival and energy stores.

<p><b>A.</b> Survival after infection with <i>B. bassiana</i> is increased by exposure to hyper g (4 g) in wild type (wt) and the rescued <i>yuri</i> strain, <i>yuri</i>^c263; UAS-<i>yuri</i> (UAS), but not in the gravitaxis mutant <i>yuri</i>, <i>yuri</i>^c263 (<i>yuri</i>). +infected, −uninfected. Error bars = SEM for 3 experiments. <b>B.</b> Additional strains tested also survive infection longer at hyper g: <i>imd</i>, using <i>imd¹</i>, and for <i>Thor</i>, which encodes the Drosophila translational regulator 4E-BP, using <i>Thor²</i>, the null allele, and its control, the revertant strain Thor^1rev1. <i>P</i> values for log rank. <b>C.</b> Post-infection energy stores of trigycerides, protein and carbohydrates are not significantly different at hyper g. Error bars = SEM for 3 experiments.</p

Analysis of transcriptional modulations produced by spaceflight.

<p>Transcriptional profiles of uninfected space and Earth flies were compared and differentially expressed genes were grouped by hierarchical clustering. Pathway analysis was utilized to identify statistically enriched biological themes. The number of genes in each category is depicted in bar graphs (primary y-axis), and the <i>P</i> values corresponding to statistical over-representation of each category are presented as a line graph (secondary y-axis).</p

Transcriptional response of space flies without an infection.

<p>Transcriptional response of space flies without an infection.</p

Microarray-based analysis of response to <i>E. coli</i>.

<p>The total number of genes upregulated or downregulated in Earth flies only (Earth) or space flies only (space) or in both (overlap) are indicated by Venn diagrams. Pathway analysis is shown on the right side of the figure. The number of genes in each functional category is depicted in bar graphs (primary y-axis), and the <i>P</i> values corresponding to statistical over-representation of each category are presented as a line graph (secondary y-axis).</p

Transcriptional profiling of genes associated with the Toll pathway.

<p><b>A.</b>Relative expression levels of selected Toll associated genes as detected by microarray are shown in uninfected (U, circles) Earth (blue) and space (tan) flies, and following fungal (F, triangles) or bacterial (B, squares) infection of space and Earth flies. Transcriptional regulation <b>A.</b> not shared or <b>B.</b> shared by space and earth flies infected with fungus.</p