Azot expression in the Drosophila gut modulates organismal lifespan

ABSTRACT Cell Competition emerged in Drosophila as an unexpected phenomenon, when confronted clones of fit vs unfit cells genetically induced. During the last decade, it has been shown that this mechanism is physiologically active in Drosophila and higher organisms. In Drosophila, Flower (Fwe) eliminates unfit cells during development, regeneration and disease states. Furthermore, studies suggest that Fwe signaling is required to eliminate accumulated unfit cells during adulthood extending Drosophila lifespan. Indeed, ahuizotl (azot) mutants accumulate unfit cells during adulthood and after physical insults in the brain and other epithelial tissues, showing a decrease in organismal lifespan. On the contrary, flies carrying three functional copies of the gene, unfit cell culling seems to be more efficient and show an increase in lifespan. During aging, Azot is required for the elimination of unfit cells, however, the specific organs modulating organismal lifespan by Azot remain unknown. Here we found a potential connection between gut-specific Azot expression and lifespan which may uncover a more widespread organ-specific mechanism modulating organismal survival.

Flower (Fwe) proteins have been described as regulators of competitive cell interactions in Drosophila and higher organisms [4][5][6][19][20][21][22]. The fwe gene in Drosophila encodes for three different Fwe isoforms; Fwe-Ubi, Fwe-LoseA and Fwe-LoseB [6,22,23]. Initially, Fwe role in Cell Competition was found by using competitive settings which were genetically induced in the developing Drosophila wing [21]. Further experiments showed then a primary physiological role for Flower in the developing retina, mediating the elimination of unwanted postmitotic neurons [6]. Fwe-dependent competition eliminates unfit cells by using tissue-specific fingerprints, in which different Fwe isoforms are required for unfit cell recognition and elimination [6,21].
Fwe proteins are multiple pass transmembrane proteins in which the C-terminus is extracellular [6,21,22]. Moreover, the Fwe proteins are also deployed in the tissue during wing development, coordinating growth and death phenomena [22,24]. The Decapentaplegic (Dpp) morphogen regulates organ growth in the Drosophila wing [25][26][27][28][29][30][31]. Dpp forms gradients in the target tissues and the ranges of these gradients keep proportional during organ growth [25,26] (i.e. they scale). In order to preserve this proportionality, there is a machinery which tunes the "size" of the Dpp gradient while the tissue grows keeping gradient and tissue proportional [25,26]. The scaling machinery consists of two proteins, Dally and Pentagone (Pent) and scaling homeostasis is ensured by molecular associations of these scaling factors with Fwe. In such a way that Dally/Pent scaling activity is tuned down by Fwe, while Fwe associated with the scaling partners inhibits its killing function [22,24,26,32,33]. Consistently, deleting Fwe extracellular sequences required for the association with the scaling partners is sufficient to trigger Fwe killing role [22,24].
In Drosophila, Fwe eliminates unfit cells during development, regeneration and disease states during neurodegeneration [4][5][6]34,35]. Furthermore, studies suggest that Fwe signaling is required to eliminate unfit cells during adulthood extending Drosophila lifespan [5,34]. Drosophila mutants (i.e. ahuizotl) abrogating Fwe signal transduction pathway show developmental defects and compromised survival [5,34]. Ahuizotl, an EF-hand calcium binding protein has been characterized as a marker of unfit cells in Competition assays as well as in physiological scenarios [4,5,34]. Indeed, ahuizotl mutant flies accumulate unfit cells during adulthood, after physical insults and neurodegenerative setups in the brain and other epithelial tissues, showing a decrease in organismal lifespan [4,5,34]. On the contrary, flies carrying three functional copies of the gene, unfit cell culling seems to be more efficient and these flies show an increase in lifespan [5,34]. We named the gene ahuizotl after a mythological creature guardian of the lakes, said to protect the species from fishermen. ahuizotl (azot) name comes from the Aztec language Nahuatl and it means "spiny aquatic thing" [5,36,37] (Figure 1a).
During aging, Azot is required for the elimination of unfit cells, as previously described by using azot mutant flies [4,5]. However, specific organs modulating organismal lifespan by Azot remain unknown [4,5]. Is organ-specific expression of Azot sufficient to regulate organismal lifespan? To explore this question and eliminate genetic background effects, we used mifepristone (formerly known as RU-486) as inducing agent to activate azot transcription, taking advantage of the conditional RU486-dependent Gal4 system [38][39][40][41] (GeneSwitch (GS)). First, in order to overexpress Azot at the organismal level, we used UASazot driven by the ubiquitous inducible driver act-GS-Gal4.  Table 1,2). These data are consistent with previous studies at the organismal level, where by increasing the number of functional copies of azot, flies eliminate more efficiently unfit cells and show an increase in lifespan compared to control ones [4,5].
To test possible lifespan phenotypes overexpressing Azot in organ-specific manner, we focus on the Drosophila gut. In recent years, the link between intestinal homeostasis and organismal lifespan has been described by a great number of studies in the field, supporting that the intestine is a key organ regulating organismal lifespan [42][43][44][45][46][47]. We therefore targeted the gut enteroblast/enterocyte (EB/EC) cells by using the RU486-inducible 5966-GS-Gal4 driver [44,48] Tables 1, 2).

Lifespan analyses
Drosophila lines and crosses were kept on standard cornmeal fly food vials. Cohorts of 100 females flies (1-3 days old) per genotype were collected and kept at 29°C under light-dark cycle. Deaths were scored at regular intervals (three times per week) and surviving flies were transferred to new vials. GS system was used to minimize background effects when comparing different Drosophila genotypes [38][39][40]. For UAS induction (RU486-induced), the stock solution of RU486 (Mifepristone, Sigma, prepared in 80% ethanol) was diluted in Mili-Q water to a final concentration of 100 μM. Then, 300 μL of the diluted solution was added to the surface of the fly food and allowed to dry at room temperature for 48 h. Similarly control flies (RU486-uninduced) received vehicle control [34,56].

Statistical analyses
To carry out statistical analyses and quantify lifespan values we used the application OASIS 2. The log-rank test was used to analyze differences between survival curves and determine P values [57].

Acknowledgments
We thank Eduardo Moreno and Heinrich Jasper for sharing reagents and Drosophila lines. M. Merino was supported by the Swiss National Science Foundation (SNSF) (SystemsX.ch, Transition Postdoc Fellowship) and Novartis Foundation Fellowships.
We apologize to all colleagues whose work has not been unintentionally cited.

Disclosure statement
No potential conflict of interest was reported by the author(s).