Multiscale analysis reveals that diet-dependent midgut plasticity emerges from alterations in both stem cell niche coupling and enterocyte size

The gut is the primary interface between an animal and food, but how it adapts to qualitative dietary variation is poorly defined. We find that the Drosophila midgut plastically resizes following changes in dietary composition. A panel of nutrients collectively promote gut growth, which sugar opposes. Diet influences absolute and relative levels of enterocyte loss and stem cell proliferation, which together determine cell numbers. Diet also influences enterocyte size. A high sugar diet inhibits translation and uncouples ISC proliferation from expression of niche-derived signals but, surprisingly, rescuing these effects genetically was not sufficient to modify diet's impact on midgut size. However, when stem cell proliferation was deficient, diet's impact on enterocyte size was enhanced, and reducing enterocyte-autonomous TOR signaling was sufficient to attenuate diet-dependent midgut resizing. These data clarify the complex relationships between nutrition, epithelial dynamics, and cell size, and reveal a new mode of plastic, diet-dependent organ resizing

Borexino : geo-neutrino measurement at Gran Sasso, Italy

Geo-neutrinos, electron anti-neutrinos produced in beta-decays of naturally occurring radioactive isotopes in the Earth, are a unique direct probe of our planet's interior. After a brief introduction of the geo-neutrinos' properties and of the main aims of their study, we discuss the features of a detector which has recently provided breakthrough achievements in the field, Borexino, a massive, calorimetric liquid scintillator detector installed at the underground Gran Sasso Laboratory. With its unprecedented radiopurity levels achieved in the core of the detection medium, it is the only experiment in operation able to study in real time solar neutrino interactions in the challenging sub-MeV energy region. Its superior technical properties allowed Borexino also to provide a clean detection of terrestrial neutrinos. Therefore, the description of the characteristics of the detected geo-neutrino signal and of the corresponding geological implications are the main core of the discussion contained in this work

Reversible regulation of stem cell niche size associated with dietary control of Notch signalling

BACKGROUND: Stem cells can respond to environmental and physiological inputs to adaptively remodel tissues. Little is known about whether stem cell niches are similarly responsive. The Drosophila ovary germline stem cell (GSC) niche is a well-studied model, which is comprised of cap cells that provide anchorage and maintenance signals for GSCs to maintain oogenesis. Previous studies have shown a strong link between diet and the regulation of oogenesis, making this a useful model system in which to investigate dietary regulation of the niche and its associated stem cells. RESULTS: We show that the Drosophila ovary GSC cap cell niche is a dynamic structure, which can contract and expand in fluctuating dietary conditions. Cap cells are lost when adult flies are shifted to nutrient poor diet and are restored after returning flies to nutrient-rich medium. Notch signalling in cap and escort cells is similarly reduced and restored by dietary shifts to nutrient poor and rich media. In old flies decreased Notch signalling is associated with decreased robustness of the niche to dietary changes. We demonstrated using a Notch temperature sensitive allele that removal and restoration of Notch signalling also leads to a reduction and re-expansion of the niche. Changes in niche size were not associated with apoptosis or cell division. We identified two distinct roles for Notch in the adult germarium. Notch can act in cap cells to prevent their loss while activation of Notch in the flanking escort cells results in expansion of the niche. CONCLUSIONS: We provide evidence that dietary changes alone are sufficient to alter Notch signalling and reversibly change niche size in the adult in wild type flies. We show Notch acts in different cells to maintain and re-expand the niche and propose a model in which cell fate transitions between cap cells and flanking somatic cells accounts for niche dynamics. These findings reveal an unexpected reversible plasticity of the GSC niche whose responses provide an integrated read out of the physiological status of the fly that is modulated by diet and age. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12861-015-0059-8) contains supplementary material, which is available to authorized users

The Drosophila ZO-1 protein Polychaetoid suppresses Deltex-regulated Notch activity to modulate germline stem cell niche formation

The developmental signalling protein Notch can be proteolytically activated following ligand-interaction at the cell surface, or can be activated independently of its ligands, following Deltex (Dx)-induced Notch endocytosis and trafficking to the lysosomal membrane. The means by which different pools of Notch are directed towards these alternative outcomes remains poorly understood. We found that theDrosophilaZO-1 protein Polychaetoid (Pyd) suppresses specifically the Dx-induced form of Notch activation bothin vivoand in cell culture assays.In vivowe confirmed the physiological relevance and direction of the Pyd/Dx interaction by showing that the expanded ovary stem cell niche phenotypes ofpydmutants require the presence of functional Dx and other components that are specific to the Dx-induced Notch activation mechanism. In S2 cells we found that Pyd can form a complex with Dx and Notch at the cell surface and reduce Dx-induced Notch endocytosis. Similar to other known activities of ZO-1 family proteins, the action of Pyd on Dx-induced endocytosis and signalling was found to be cell density dependent. Thus, together, our results suggest an alternative means by which external cues can tune Notch signalling through Pyd regulation of Dx-induced Notch trafficking.</jats:p

ISC proliferation and survival following <i>Ecc15</i> infection are compromised by inhibition of the TFs and pathways that are required for <i>upd3</i> induction.

<p>(A) Total pH3+ cell counts in unchallenged and <i>Ecc15</i> infected guts demonstrate that knockdown in ECs of <i>D-Fos</i>, <i>yki</i>, <i>sd</i>, <i>Trl</i>, and <i>sna</i> as well as upstream components of the MAPK and Dpp pathways is accompanied by a decrease in ISC mitotic activity. Statistical significance: mean values of at least 3 repeats are represented ± SEM. *p<0.05, **p<0.01, ***p<0.001 (student’s t test). (B-D) Survival curves of flies orally infected with <i>Ecc15</i> alongside RNAi-induced knockdown of indirect <i>upd3</i> regulators (B), Hippo and Dpp pathways components (C), or MAPK pathway factors (D). Curves represent averaged survival ± SE. Statistical significance: *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001 (Log-rank test).</p

Infection-induced <i>upd3</i> expression in ECs requires the TFs D-Jun and D-Fos, activated by upstream Src-MAPK pathways.

<p>(A-B) Knockdown by RNAi of multiple constituents of MAPK pathways, as well as Src kinases or the TFs D-Jun (Jra) and D-Fos (Kay) inhibits <i>upd3-lacZ</i> activity under <i>Ecc15</i> infection or UC conditions. (C) RT-qPCR measurements of total midgut <i>upd3</i> expression corroborate <i>upd3-lacZ</i> results. (D) In addition to their requirement for <i>upd3-lacZ</i> activity, activation of the MAPKs and SFKs can also induce <i>upd3-lacZ</i> expression in UC conditions. SAPKs can also induce this activity when stimulated. (E, F) Immunostaining against phosphorylated forms of ERK and Src reveals that these kinases are activated in response to infection in ECs. Scale bar is 100μm. Statistical significance: mean values of at least 3 repeats are represented ± SEM. *p<0.05, **p<0.01, ***p<0.001 (student’s t-test).</p

Combination of <i>in vivo</i>, <i>in vitro</i>, and <i>in silico</i> TF screens identifies direct and indirect regulators of <i>upd3</i> transcription.

<p>(A) Basic schematics of the RNAi (A) and yeast one-hybrid screens (A’) along with the number of positive TF hits for each. (B) Venn diagram displaying the number of positive hit TFs identified by each screen and identified by multiple approaches. (C) Summary table of important TF hits organized by whether they induced or suppressed <i>upd3</i> induction, as well as by their TF category: putative direct regulators of <i>upd3</i> that likely bind to enhancer regions of the gene, indirect regulators that lack evidence for direct binding potential but have strong phenotypes and probable cause for controlling <i>upd3</i>, and epigenetic regulators that may influence <i>upd3</i> expression by modifying genomic DNA structure. The seven genes that were positive hits for all three screens are indicated by red text. (D) Schematic representation of D-Fos and Sd binding motifs present in <i>upd3</i> enhancer regions C (Green), I (Blue), and R (Purple).</p

Infection-induced expression of <i>upd3</i> in ECs requires the Hippo and Dpp pathways.

<p>(A-C) Measurements of midgut <i>upd3-lacZ</i> activity under <i>Ecc15</i> infected and UC conditions during EC-specific knockdown or overexpression of Hippo and Dpp pathway components. Depletion of the Hippo TFs <i>sd</i> or <i>yki</i>, or overexpression of an upstream inhibitor (Msn) blocks basal and infection-induced <i>upd3-lacZ</i> expression. Likewise, knockdown of <i>trr</i>, an epigenetic enhancer of Yki/Sd activity, also inhibits infection-induced <i>upd3-lacZ</i>. Alternatively, overexpression of Yki or knockdown of its upstream inhibitors <i>wts</i> and <i>msn</i> is sufficient to induce <i>upd3-lacZ</i>. Knockdown of the Dpp pathway TF <i>Mad</i>, either of the three Dpp pathway receptors, <i>tkv</i>, <i>sax</i>, or <i>put</i>, or overexpression of the <i>Mad</i> inhibitor, Sgg all blocked <i>upd3-lacZ</i> activity. Overexpression of Dpp itself or knockdown of <i>sgg</i> induced <i>upd3-lacZ</i>. (D) RT-qPCR was used to directly measure <i>upd3</i> transcription levels, and confirm that the function of the Hippo and Dpp pathway TFs are required for <i>upd3</i> induction. Statistical significance: mean values of at least 3 repeats are represented ± SEM. *p<0.05, **p<0.01, ***p<0.001 (student’s t-test).</p

Bacterial infection, stress and the microbiota induce <i>upd3</i> through distinct enhancers.

<p>(A) RT-qPCR measured <i>upd3</i> expression is significantly induced by <i>Ecc15</i> and <i>Pe</i> infection, as well as bleomycin (bleo) treatment and DSS. (B) ISC proliferation, measured by phospho-Histone H3 (pH3) immunostaining, is triggered in response to ingestion of harmful bacteria (<i>Ecc15</i> and <i>Pe</i>) and chemical stressors (bleo and DSS). (C) RT-qPCR measurements of <i>upd3</i> transcription in the gut of germ-free (GF) flies shows reduced expression compared to their conventionally reared (CR) counterparts. (D, E) Confocal imaging shows that <i>upd3-C-GFP</i> and <i>upd3-R-GFP</i> strongly induce GFP expression in response to all presented stresses, except for DSS treatment. (F) In contrast, enhancer I responds exclusively to <i>Ecc15</i> and marginally to <i>Pe</i> infection by GFP induction. (G) Measuring GFP expression in <i>upd3-C-GFP</i> and <i>upd3-R-GFP</i> flies by RT-qPCR, normalized to the GFP expression in each line under CR conditions, reveals a reduction in basal enhancer C and R activity in GF conditions. Scale bars are 50μm. Statistical significance: mean values of at least 3 repeats are represented ± SEM. *p<0.05, **p<0.01, ***p<0.001 (student’s t-test).</p