A conserved gene regulatory network controls tissue homeostasis in flies and mammals.

<p>(<b>A</b>) In both flies and mammals, the gut epithelium produces immune effectors, including reactive oxygen species (ROS) and antimicrobial peptides (AMPs). Epithelial cells and immune cells secrete cytokines that stimulate tissue regeneration. The Hippo pathway is a conserved regulator of intestinal stem cell (ISC) activity. In <i>Drosophila</i>, activation of the JAK-STAT pathway by the cytokine Unpaired 3 (Upd3) triggers the release of epidermal growth factors (EGFs) by the stem cell niche, which then induces stem cell proliferation. JAK-STAT activation also directly stimulates ISC proliferation and differentiation. The Wingless (Wnt/Wg) pathway is a major regulator of ISC proliferation in mammals and also promotes tissue regeneration through cMyc in the infected <i>Drosophila</i> midgut. The dashed arrows indicate presumed activities but as yet are undefined. (<b>B</b>) Gut microbes and viruses coordinately stimulate a PDGF-VEGF Receptor and Extracellular Signal-Regulated Kinase dependent (pvf2/PVR/ERK) antiviral response through the Immune Deficiency (Imd) pathway and Cyclin-dependent-kinase 9 (Cdk9) induction, respectively, in the midgut of <i>Drosophila</i>. The ERK-stimulated antiviral activities/effectors have not been determined. In addition, exogenous insulin initiates ERK mediated antiviral activity.</p

Relative size of commensal bacteria populations in 36 inbred fly lines.

<p>Relative commensal bacterial level was determined using quantitative PCR and was calculated relative to levels of a single copy <i>Drosophila</i> gene (Drosophila gene Ct—Bacterial gene Ct). The y-axis shows the residuals of these measurements from a model accounting for block effect (mean ± 1 S.D.). Units are on a log2 scale. Higher values correspond to a higher ratio of bacterial DNA to fly DNA. In all plots, the lines are ordered according the rank order of the <i>L</i>. <i>plantarum</i> residuals.</p

The repression machinery controlling <i>ZAM</i> and <i>Idefix</i> acts post-transcriptionally, before translation.

<p>A: Transcripts from the pGFP-ZU transgene were examined in northern blot experiments. A typical result is shown in A. GFP transcripts revealed by a riboprobe complementary to GFP mRNAs are detected in the U line and not in the S line. Actin is used as a loading control. B: Northern blots and quantification based on three northern blot experiments performed on flies containing pGFP-IdU and pGFP-IdUAS transgenes. Their structures are presented above the graph. No GFP transcripts synthesized from the pGFP-IdU transgene are detected by the GFP riboprobe in an S background, whereas their amount is high in a U background. An even higher amount of GFP transcripts is observed in an S or U background when the <i>Idefix</i> fragment is inserted in the opposite orientation (pGFP-IdUAS transgenes). C and D- RNase protection assays reveal the presence of small RNAs (20 to 30 nt long) that are homologous to <i>ZAM</i> and <i>Idefix.</i> These RNAs are detected in S lines and, at a much lower level, in the U line. Small RNAs homologous to the antisense strand of the 5′UTR of <i>ZAM</i> are presented in C. 20 to 30 nt long antisense strand RNAs (−) homologous to the 5′UTR or the <i>gag</i> gene of <i>Idefix</i> are detected. Sense strands (+) are absent or present in very small amounts. A typical experiment is presented in D. Signs (+) and (−) indicate respectively sense-strand and anti-sense strand RNAs of ZAM or Idefix revealed by the riboprobes.</p

<i>Drosophila</i> Genotype Influences Commensal Bacterial Levels

<div><p>Host genotype can influence the composition of the commensal bacterial community in some organisms. Composition, however, is only one parameter describing a microbial community. Here, we test whether a second parameter—abundance of bacteria—is a heritable trait by quantifying the presence of four commensal bacterial strains within 36 gnotobiotic inbred lines of <i>Drosophila melanogaster</i>. We find that <i>D</i>. <i>melanogaster</i> genotype exerts a significant effect on microbial levels within the fly. When introduced as monocultures into axenic flies, three of the four bacterial strains were reliably detected within the fly. The amounts of these different strains are strongly correlated, suggesting that the host regulates commensal bacteria through general, not bacteria-specific, means. While the correlation does not appear to be driven by simple variation in overall gut dimensions, a genetic association study suggests that variation in commensal bacterial load may largely be attributed to physical aspects of host cell growth and development.</p></div

GO category enrichment of genes strongly associated with bacterial levels in the fly as calculated by Panther.

<p>GO category enrichment of genes strongly associated with bacterial levels in the fly as calculated by Panther.</p

<i> ZAM</i> and <i>Idefix</i> are regulated by a PIWI-dependent pathway in the reproductive apparatus.

<p><i>In situ</i> hybridization experiments reveal <i>ZAM</i> and <i>Idefix</i> expression in female gonads from third instar larvae. <i>ZAM</i> and <i>Idefix</i> transcripts are not detected in S flies with a wild-type <i>piwi</i> gene (left). As shown by the black staining, <i>ZAM</i> and <i>Idefix</i> mRNAs are detected in U flies with a wild-type <i>piwi</i> gene (middle). In S lines homozygous for the <i>piwi</i>³ allele, <i>ZAM</i> or <i>Idefix</i> transcripts are no longer repressed, and their transcription is visualised in gonads (right). Probes used in these experiments are indicated on the left.</p

Significant genetic variation and heritability in abundance of commensal gut bacteria across DGRP lines of <i>Drosophila</i>.

<p>Significant genetic variation and heritability in abundance of commensal gut bacteria across DGRP lines of <i>Drosophila</i>.</p

Transgenes with a GFP reporter gene fused to a <i>ZAM</i> sequence act as sensors of the repression.

<p>The genomic structures of the transgenes pGFP-Zenv and pGFP-Idgag used in this study are presented at the tops of both panels: The grey boxes correspond to the UASt promoter, the dotted boxes to the GFP gene, and the white box to the <i>env</i> fragment of <i>ZAM</i> or the <i>gag</i> fragment of <i>Idefix</i>. Triangles indicate the FRT sites. Focal plane of the follicles dissected from a line in which the pGFP-Zenv transgene is driven by the ubiquitous Actin-Gal4 driver. Expression of the pGFP-Zenv transgene in an S genetic background before (A) or after (B) <i>flp</i>-recombinase action, or in a U genetic background before the <i>flp</i> treatment (C). GFP expression in the ovarioles of a transgenic line bearing the pGFP-Idgag transgene driven by the ubiquitous Actin-Gal4 driver. Expression of the pGFP-Idgag transgene in an S genetic background before (D) or after (E) <i>flp</i>-recombinase action, or in a U genetic background before the <i>flp</i> treatment (F). No GFP is detected in ovaries of the S lines. Its expression is recovered after the flp treatment or when the COM locus is mutated, as in the U genetic background.</p

The silencing mechanism targeting ZAM and Idefix is active in somatic tissues throughout fly development.

<p>A) In an S/S genetic background, the pGFP-IdU sensor transgene driven by 24B-Gal4 is not expressed in embryos, larvae, or adults (top, middle and bottom panels, respectively). Only a very faint level of fluorescence, corresponding to the background expression of GFP, is detected. B) In a U/U genetic background, the GFP-IdU transgene silencing is released and GFP fluorescence is clearly observed in the three stages analyzed. The fluorescence pattern recapitulates the expression of the HOW gene in muscle and tendon cells, as expected for the 24B-Gal4 driver <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001526#pone.0001526-Brand1" target="_blank">[22]</a>. C) In an S/S genetic background, the pGFP-IdUAS sensor transgene carrying the 5′UTR of <i>Idefix</i> in the opposite orientation is not subjected to the silencing exerted on the <i>Idefix</i> sequences. pGFP-IdUAS is correctly expressed and GFP is detected in embryos, larvae, and adult flies.</p

Transgenes bearing <i>ZAM</i> or <i>Idefix</i> sequences placed in an antisense orientation are not targeted by the repression.

<p>Expression of sensor transgenes carrying <i>ZAM</i> or <i>Idefix</i> fragments inserted in a sense and an antisense orientation. The genomic structure of the so-called pGFP-Zenv, pGFP-ZenvAS, pGFP-IdU, pGFP-IdUAS transgenes are depicted on the left. The orientation of the fragment is indicated by an arrow. The repression mechanism is able to discriminate between sense and antisense targeted sequences. In an S/S genetic background, only transgenes with ZAM and Idefix in an antisense orientation are correctly expressed. Clear fluorescence due to GFP expression is detected in the ovarian follicles of pGFP-ZenvAS and pGFP-IdUAS transgenes, as illustrated on the right.</p