InteractiveFly: GeneBrief

ahuizotl: Biological Overview | References


Gene name - ahuizotl

Synonyms -

Cytological map position - 43F6-43F6

Function - signaling

Keywords - calcium dependent protein that ensures the elimination of less fit cells, fitness-based cell culling is naturally used to maintain tissue health, delay aging, and extend lifespan, wings

Symbol - azot

FlyBase ID: FBgn0033238

Genetic map position - chr2R:7,949,839-7,950,465

Classification - EF-Hand calcium-binding domain

Cellular location - cytoplasmic



NCBI link: EntrezGene

azot orthologs: Biolitmine
Recent literature
Coelho, D. S. and Moreno, E. (2020). Neuronal Selection Based on Relative Fitness Comparison Detects and Eliminates Amyloid-beta-Induced Hyperactive Neurons in Drosophila. iScience 23(9): 101468. PubMed ID: 32866827
Summary:
During adult life, damaged but viable neurons can accumulate in the organism, creating increasingly heterogeneous and dysfunctional neural circuits. One intriguing example is the aberrant increased activity of cerebral networks detected in vulnerable brain regions during preclinical stages of Alzheimer's disease. The pathophysiological contribution of these early functional alterations to the progression of Alzheimer's disease is uncertain. A unique cell selection mechanism based on relative fitness comparison between neurons is able to target and remove aberrantly active neurons generated by heterologous human amyloid-β in Drosophila. Sustained neuronal activity is sufficient to compromise neuronal fitness and upregulate the expression of the low fitness indicators Flower(LoseB) and Azot in the fly. Conversely, forced silencing of neurons restores brain fitness and reduces amyloid-β-induced cell death. The manipulation of this cell selection process, which was already proved to be conserved in humans, might be a promising new avenue to treat Alzheimer's.
Merino, M. M. (2023). Azot expression in the Drosophila gut modulates organismal lifespan. Commun Integr Biol 16(1): 2156735. PubMed ID: 36606245
Summary:
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. This study found a potential connection between gut-specific Azot expression and lifespan which may uncover a more widespread organ-specific mechanism modulating organismal survival.
Merino, M. M. (2023). Azot expression in the Drosophila gut modulates organismal lifespan. Commun Integr Biol 16(1): 2156735. PubMed ID: 36606245
Summary:
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. This study found a potential connection between gut-specific Azot expression and lifespan which may uncover a more widespread organ-specific mechanism modulating organismal survival.
BIOLOGICAL OVERVIEW

Viable yet damaged cells can accumulate during development and aging. Although eliminating those cells may benefit organ function, identification of this less fit cell population remains challenging. Previously, a molecular mechanism, based on 'fitness fingerprints' displayed on cell membranes, was identifed that allows direct fitness comparison among cells in Drosophila. This study reports the physiological consequences of efficient cell selection for the whole organism. The study found that fitness-based cell culling is naturally used to maintain tissue health, delay aging, and extend lifespan in Drosophila. A gene, ahuizotl (azot), was identified that ensures the elimination of less fit cells. Lack of azot increases morphological malformations and susceptibility to random mutations and accelerates tissue degeneration. On the contrary, improving the efficiency of cell selection is beneficial for tissue health and extends lifespan (Merino, 2015).

Individual cells can suffer insults that affect their normal functioning, a situation often aggravated by exposure to external damaging agents. A fraction of damaged cells will critically lose their ability to live, but a different subset of cells may be more difficult to identify and eliminate: viable but suboptimal cells that, if unnoticed, may adversely affect the whole organism (Merino, 2015).

What is the evidence that viable but damaged cells accumulate within tissues? The somatic mutation theory of aging proposes that over time cells suffer insults that affect their fitness, for example, diminishing their proliferation and growth rates, or forming deficient structures and connections. This creates increasingly heterogeneous and dysfunctional cell populations disturbing tissue and organ function. Once organ function falls below a critical threshold, the individual dies. The theory is supported by the experimental finding that clonal mosaicism occurs at unexpectedly high frequency in human tissues as a function of time, not only in adults due to aging, but also in human embryos (Merino, 2015).

Does the high prevalence of mosaicism in our tissues mean that it is impossible to recognize and eliminate cells with subtle mutations and that suboptimal cells are bound to accumulate within organs? Or, on the contrary, can animal bodies identify and get rid of unfit viable cells (Merino, 2015)?

One indirect mode through which suboptimal cells could be eliminated is proposed by the 'trophic theory,' which suggested that Darwinian-like competition among cells for limiting amounts of survival-promoting factors will lead to removal of less fit cells. However, it is apparent from recent work that trophic theories are not sufficient to explain fitness-based cell selection, because there are direct mechanisms that allow cells to exchange 'cell-fitness' information at the local multicellular level (Merino, 2015).

In Drosophila, cells can compare their fitness using different isoforms of the transmembrane protein Flower. The 'fitness fingerprints' are therefore defined as combinations of Flower isoforms present at the cell membrane that reveal optimal or reduced fitness (Merino, 2013; Rhiner, 2010). The isoforms that indicate reduced fitness have been called FlowerLose isoforms, because they are expressed in cells marked to be eliminated by apoptosis called 'Loser cells' (Rhiner, 2010). However, the presence of FlowerLose isoforms at the cell membrane of a particular cell does not imply that the cell will be culled, because at least two other parameters are taken into account: (1) the levels of FlowerLose isoforms in neighboring cells: if neighboring cells have similar levels of Lose isoforms, no cell will be killed (Merino, 2013; Rhiner, 2010); (2) the levels of a secreted protein called Sparc, the homolog of the Sparc/Osteonectin protein family, which counteracts the action of the Lose isoforms (Portela, 2010; Merino, 2015 and references therein).

Remarkably, the levels of Flower isoforms and Sparc can be altered by various insults in several cell types, including: (1) the appearance of slowly proliferating cells due to partial loss of ribosomal proteins, a phenomenon known as cell competition; (2) the interaction between cells with slightly higher levels of d-Myc and normal cells, a process termed supercompetition; (3) mutations in signal transduction pathways like Dpp signaling ; or (4) viable neurons forming part of incomplete ommatidia. Intriguingly, the role of Flower isoforms is cell type specific, because certain isoforms acting as Lose marks in epithelial cells (Rhiner, 2010) are part of the fitness fingerprint of healthy neurons (Merino, 2013). Therefore, an exciting picture starts to appear, in which varying levels of Sparc and different isoforms of Flower are produced by many cell types, acting as direct molecular determinants of cell fitness. This study aimed to clarify how cells integrate fitness information in order to identify and eliminate suboptimal cells. Subsequently, the physiological consequences were analyzed of efficient cell selection for the whole organism (Merino, 2015).

In order to discover the molecular mechanisms underlying cell selection in Drosophila, this study analyzed genes transcriptionally induced using an assay where WT cells (tub>Gal4) are outcompeted by dMyc-overexpressing supercompetitors (tub>dmyc) due to the increased fitness of these dMyc-overexpressing cells (Rhiner, 2010). The expression of CG11165 was strongly induced 24 hr after the peak of flower and sparc expression. In situ hybridization revealed that CG11165 mRNA was specifically detected in Loser cells that were going to be eliminated from wing imaginal discs due to cell competition. The gene, which was named ahuizotl (azot) after a multihanded Aztec creature selectively targeting fishing boats to protect lakes, consists of one exon. azot's single exon encodes for a four EF-hand-containing cytoplasmic protein of the canonical family that is conserved, but uncharacterized, in multicellular animals (Merino, 2015).

To monitor Azot expression, a translational reporter was designed resulting in the expression of Azot::dsRed under the control of the endogenous azot promoter in transgenic flies. Azot expression was not detectable in most wing imaginal discs under physiological conditions in the absence of competition. Mosaic tissue was generated of two clonal populations, which are known to trigger competitive interactions resulting in elimination of otherwise viable cells. Cells with lower fitness were created by confronting WT cells with dMyc-overexpressing cells, by downregulating Dpp signaling, by overexpressing FlowerLose isoforms (Rhiner, 2010), in cells with reduced Wg signaling, by suppressing Jak-Stat signaling or by generating Minute clones. Azot expression was not detectable in nonmosaic tissue of identical genotype, nor in control clones overexpressing UASlacZ. On the contrary, Azot was specifically activated in all tested scenarios of cell competition, specifically in the cells undergoing negative selection. Azot expression was not repressed by the caspase inhibitor protein P35 (Merino, 2015).

Because Flower proteins are conserved in mammals (Petrova, 2012), tests were made to see if they are also able to regulate azot. Mouse Flower isoform 3 (mFlower3) has been shown to act as a 'classical' Lose isoform, driving cell elimination when expressed in scattered groups of cells (Petrova, 2012), a situation where azot was induced in Loser cells but is not inducing cell selection when expressed ubiquitously a scenario where azot was not expressed. This shows that the mouse FlowerLose isoforms function in Drosophila similarly to their fly homologs (Merino, 2015).

Interestingly, azot is not a general apoptosis-activated gene because its expression is not induced upon eiger, hid, or bax activation, which trigger cell death. Azot was also not expressed during elimination of cells with defects in apicobasal polarity or undergoing epithelial exclusion-mediated apoptosis (dCsk) (Merino, 2015).

azot expression was analyzed during the elimination of peripheral photoreceptors in the pupal retina, a process mediated by Flower-encoded fitness fingerprints (Merino, 2013). Thirty-six to 38hr after pupal formation (APF), when FlowerLose-B expression begins in peripheral neurons (Merino, 2013), no Azot expression was detected in the peripheral edge. At later time points (40 and 44hr APF), Azot expression is visible and restricted to the peripheral edge where photoreceptor neurons are eliminated. This expression was confirmed with another reporter line, azot{KO; gfp}, where gfp was directly inserted at the azot locus using genomic engineering techniques (Merino, 2015).

From these results, it is concluded that Azot expression is activated in several contexts where suboptimal and viable cells are normally recognized and eliminated (Merino, 2015).

To understand Azot function in cell elimination, azot knockout (KO) flies were generated by deleting the entire azot gene. Next, Azot function was analyzed using dmyc-induced competition. In the absence of Azot function, loser cells were no longer eliminated, showing a dramatic 100-fold increase in the number of surviving clones. Loser cells occupied more than 20% of the tissue 72hr after clone induction (ACI). Moreover, using azot{KO; gfp} homozygous flies (that express GFP under the azot promoter but lack Azot protein), it was found that loser cells survived and showed accumulation of GFP. From these results, it is concluded that azot is expressed by loser cells and is essential for their elimination.

In addition, clone removal was delayed in an azot heterozygous background (50-fold increase, 15%), compared to control flies with normal levels of Azot. Cell elimination capacity was fully restored by crossing two copies of Azot::dsRed into the azot-/- background demonstrating the functionality of the fusion protein. Silencing azot with two different RNAis was similarly able to halt selection during dmyc-induced competition. Next, in order to determine the role of Azot's EF hands, a mutated isoform of Azot (Pm4Q12) was generated and overexpressed, that carryed, in each EF hand, a point mutation known to abolish Ca2+ binding. Although overexpression of wild-type azot in negatively selected cells did not rescue the elimination, overexpression of the mutant AzotPm4Q12 reduced cell selection, functioning as a dominant-negative mutant. This shows that Ca2+ binding is important for Azot function. Finally, staining for apoptotic cells corroborated that the lack of Azot prevents cell elimination, because cell death was reduced 8-fold in mosaic epithelia containing loser cells (Merino, 2015).

The role of azot in elimination of peripheral photoreceptor neurons in the pupal retina was examined using homozygous azot KO flies. Pupal retinas undergoing photoreceptor culling (44hr APF) of azot+/+ and azot-/- flies were stained for the cell death marker and the proapoptotic factor. Consistent with the expression pattern of Azot, the number of Hid and TUNEL-positive cells was dramatically decreased in azot-/- retinas compared to azot+/+ retinas (Merino, 2015).

Those results show that Azot is required to induce cell death and Hid expression during neuronal culling. Therefore, tests were performed to see that was also the case in the wing epithelia during dmyc-induced competition. Hid was found to be expressed in loser cells and the expression was found to be strongly reduced in the absence of Azot function (Merino, 2015).

Finally, forced overexpression of FlowerLose isoforms from Drosophila were unable to mediate WT cell elimination when Azot function was impaired by mutation or silenced by RNAi (Merino, 2015).

These results suggested that azot function is dose sensitive, because heterozygous azot mutant flies display delayed elimination of loser cells when compared with azot WT flies. Therefore advantage was taken of the functional reporter Azot::dsRed to test whether cell elimination could be enhanced by increasing the number of genomic copies of azot. Tissues with three functional copies of azot were more efficient eliminating loser cells during dmyc-induced competition and most of the clones were culled 48hr ACI. From these results, it is concluded that azot expression is required for the elimination of Loser cells and unwanted neurons (Merino, 2015).

Next, it was asked what could be the consequences of decreased cell selection at the tissue and organismal level. To this end, advantage was taken of the viability of homozygous azot KO flies. An increase of several developmental aberrations was observed. Focus was placed on the wings, where cell competition is best studied and, because aberrations, including melanotic areas, blisters, and wing margin nicks, were quantified. Wing defects of azot mutant flies could be rescued by introducing two copies of azot::dsRed, showing that the phenotypes are specifically caused by loss of Azot function (Merino, 2015).

Next, it was reasoned that mild tissue stress should increase the need for fitness-based cell selection after damage. First, in order to generate multicellular tissues scattered with suboptimal cells, larvae were exposed to UV light and Azot expression was monitored in wing discs of UV-irradiated WT larvae that were stained for cleaved caspase-3, 24hr after treatment. Under such conditions, Azot was found to be expressed in cleaved caspase-3-positive cells. All Azot-positive cells showed caspase activation and 17% of cleaved caspase-positive cells expressed Azot. This suggested that Azot-expressing cells are culled from the tissue. To confirm this, later time points (3 days after irradiation) were examined; the increase in Azot-positive cells was no longer detectable. The elimination of azot-expressing cells after UV irradiation required azot function, because cells revealed by reporter azot{KO; gfp}, that express GFP instead of Azot, persisted in wing imaginal discs from azot-null larvae. Tests were performeed to see if lack of azot leads to a faster accumulation of tissue defects during organ development upon external damage. azot-/- pupae 0 stage were irradiated, and the number of morphological defects in adult wings was compared to those in nonirradiated azot KO flies. It was found that aberrations increased more than 2-fold when compared to nonirradiated azot-/- flies (Merino, 2015).

In order to functionally discriminate whether azot belongs to genes regulating apoptosis in general or is dedicated to fitness-based cell selection, whether azot silencing prevents Eiger/TNF-induced cell death was exanubed. Inhibiting apoptosis (UASp35) or eiger (UASRNAieiger) rescued eye ablation, whereas azot silencing and overexpression of AzotPm4Q12 did not. Furthermore, azot silencing did not impair apoptosis during genitalia rotation or cell death of epithelial precursors in the retina. These results highlight the consequences of nonfunctional cell-quality control within developing tissues (Merino, 2015).

The next part of the analysis demonstrated that the azot promoter computes relative FlowerLose and Sparc Levels. Epistasis analyses were performed to understand at which level azot is transcriptionally regulated. For this purpose, the assay where WT cells are outcompeted by dMyc-overexpressing supercompetitors was used. It was previously observed that azot induction is triggered upstream of caspase-3 activation and accumulates in outcompeted cells unable to die. Then, upstream events of cell selection were genetically modified. Silencing fweLose transcripts by RNAi or overexpressing Sparc both blocked the induction of Azot::dsRed in WT loser cells. In contrast, when outcompeted WT cells were additionally 'weakened' by Sparc downregulation using RNAi, Azot is detected in almost all loser cells compared to its more limited induction in the presence of endogenous Sparc. Inhibiting JNK signaling with UASpuc did not suppress Azot expression (Merino, 2015).

The activation of Azot upon irradiation was examined. Strikingly, it was found that all Azot expression after irradiation was eliminated when Flower Lose was silenced and also when relative differences of Flower Lose where diminished by overexpressing high levels of Lose isoforms ubiquitously. On the contrary, Azot was not suppressed after irradiation by expressing the prosurvival factor Bcl-2 or a p53 dominant negative. These results show that Azot expression during competition and upon irradiation requires differences in Flower Lose relative levels (Merino, 2015).

Finally, the regulation of Azot expression in neurons was analyzed. Silencing fwe transcripts by RNAi blocked the induction of Azot::dsRed in peripheral photoreceptors. Because Wingless signaling induces FlowerLose-B expression in peripheral photoreceptors (Merino, 2013), tests were performed to see if overexpression of Daxin, a negative regulator of the pathway, affected Azot levels. Axin overespression completely inhibited Azot expression. Similarly, overexpression of the cell competition inhibitor Sparc also fully blocked Azot endogenous expression in the retina. Finally, ectopic overexpression of FlowerLose-B in scattered cells of the retina was sufficient to trigger ectopic Azot activation. These results show that photoreceptor cells also can monitor the levels of Sparc and the relative levels of FlowerLose-B before triggering Azot expression (Merino, 2015).

These results suggest that the azot promoter integrates fitness information from neighboring cells, acting as a relative 'cell-fitness checkpoint.'

To test if fitness-based cell selection is a mechanism active not only during development, but also during adult stages, WT adult flies were exposed to UV light and monitor Azot and Flower expression were monitored in adult tissues. UV irradiation of adult flies triggered cytoplasmic Azot expression in several adult tissues including the gut and the adult brain. Likewise, UV irradiation of adult flies triggered Flower Lose expression in the gut and in the brain. Irradiation-induced Azot expression was unaffected by Bcl-2 but was eliminated when Flower Lose was silenced or when relative differences of Flower Lose where diminished in the gut. This suggests that the process of cell selection is active throughout the life history of the animal. Further confirming this conclusion, Azot function was essential for survival after irradiation, because more than 99% of azot mutant adults died 6 days after irradiation, whereas only 62.4% of WT flies died after the same treatment. The percentage of survival correlated with the dose of azot because adults with three functional copies of azot had higher median survival and maximum lifespan than WT flies, or null mutant flies rescued with two functional azot transgenes (Merino, 2015).

The next part of the study addressed the role of cell selection during aging. Lack of cell selection could affect the whole organism by two nonexclusive mechanisms. First, the failure to detect precancerous cells, which could lead to cancer formation and death of the individual. Second, the time-dependent accumulation of unfit but viable cells could lead to accelerated tissue and organ decay. We therefore tested both hypotheses (Merino, 2015).

It has been previously shown that cells with reduced levels for cell polarity genes like scrib or dlg are eliminated but can give rise to tumors when surviving. Therefore this study checked if azot functions as a tumor suppressing mechanism in those cells. Elimination of dlg and scrib mutant cells was not affected by RNAi against azot or when Azot function was impaired by mutation, in agreement with the absence of azot induction in these mutant cells. However, azot RNAi or the same azot mutant background efficiently rescued the elimination of clones with reduced Wg signaling (Merino, 2015).

Moreover, the high number of suboptimal cells produced by UV treatment did not lead to tumoral growth in azot-null background. Thus, tumor suppression mechanisms are not impaired in azot mutant backgrounds, and tumors are not more likely to arise in azot-null mutants (Merino, 2015).

Also tests were performed to see whether the absence of azot accelerates tissue fitness decay in adult tissues. Focused was placed on the adult brain, where neurodegenerative vacuoles develop over time and can be used as a marker of aging. The number was compared of vacuoles appearing in the brain of flies lacking azot (azot-/-), WT flies (azot+/+), flies with one extra genomic copy of the gene (azot+/+; azot+), and mutant flies rescued with two genomic copies of azot (azot-/-;azot+/+). For all the genotypes analyzed, a progressive increase was observed in the number and size of vacuoles in the brain over time. Interestingly, azot-/- brains showed higher number of vacuoles compared to control flies (azot+/+ and azot-/-;azot+/+) and a higher rate of vacuole accumulation developing over time. In the case of flies with three genomic copies of the gene (azot+/+; azot+), vacuole number tended to be the lowest (Merino, 2015).

The cumulative expression of azot was analyzed during aging of the adult brain. Positive cells were detected as revealed by reporter azot{KO; gfp}, in homozygosis, that express GFP instead of Azot. A time-dependent accumulation of azot-positive cells was observed (Merino, 2015).

From this, it is concluded that azot is required to prevent tissue degeneration in the adult brain and lack of azot showed signs of accelerated aging. This suggested that azot could affect the longevity of adult flies. Flies lacking azot (azot-/-) had a shortened lifespan with a median survival of 7.8 days, which represented a 52% decrease when compared to WT flies (azot+/+), and a maximum lifespan of 18 days, 25% less than WT flies (azot+/+). This effect on lifespan was azot dependent because it was completely rescued by introducing two functional copies of azot. On the contrary, flies with three functional copies of the gene (azot+/+; azot+) showed an increase in median survival and maximum lifespan of 54% and 17%, respectively (Merino, 2015).

In conclusion, azot is necessary and sufficient to slow down aging, and active selection of viable cells is critical for a long lifespan in multicellular animals (Merino, 2015).

The next part of the study demonstrates that death of unfit cells is sufficient and required for multicellular fitness maintenance. The results cited above show the genetic mechanism through which cell selection mediates elimination of suboptimal but viable cells. However, using flip-out clones and MARCM, this study found that Azot overexpression was not sufficient to induce cell death in wing imaginal discs. Because Hid is downstream of Azot, it was wondered whether expressing Hid under the control of the azot regulatory regions could substitute for Azot function (Merino, 2015).

In order to test this hypothesis, the whole endogenous azot protein-coding sequence was replaced by the cDNA of the proapoptotic gene hid (azot{KO; hid}) flies. In a second strategy, the whole endogenous azot protein-coding sequence was replaced by the cDNA of transcription factor Gal4, so that the azot promoter can activate any UAS driven transgene (azot{KO; Gal4} flies. The number of morphological aberrations was compared in the adult wings of six genotypes: first, homozygous azot{KO; Gal4} flies that lacked Azot; second, azot{KO; hid} homozygous flies that express Hid with the azot pattern in complete absence of Azot; third, azot+/+ WT flies as a control; and finally three genotypes where the azot{KO; Gal4} flies were crossed with UAShid, UASsickle, another proapoptotic gene, or UASp35, an apoptosis inhibitor. In the case of UASsickle flies, a second azot mutation was introduced to eliminate azot function. Interestingly, the number of morphological aberrations was brought back to WT levels in all the situations where the azot promoter was driving proapoptotic genes (azot{KO; hid}, azot{KO; Gal4} × UAShid, azot{KO; Gal4} × UASsickle with or without irradiation. On the contrary, expressing p35 with the azot promoter was sufficient to produce morphological aberrations despite the presence of one functional copy of azot. Likewise, p35-expressing flies (azot{KO; Gal4}/azot+; UASp35) did not survive UV treatments, whereas a percentage of the flies expressing hid (26%) or sickle (28%) in azot-positive cells were able to survive (Merino, 2015).

From this, it is concluded that specifically killing those cells selected by the azot promoter is sufficient and required to prevent morphological malformations and provide resistance to UV irradiation (Merino, 2015).

The next part of the study demonstrated that death of unfit cells extends lifespan It was asked whether the shortened longevity observed in azot-/- flies could be also rescued by killing azot-expressing cells with hid in the absence of Azot protein. It was found that azot{KO; hid} homozygous flies had dramatically improved lifespan with a median survival of 27 days at 29°C, which represented a 125% increase when compared to azot-/- flies, and a maximum lifespan of 34 days, 41% more than mutant flies (Merino, 2015).

Similar results were obtained at 25°C. It was found that flies lacking azot (azot-/-) had a shortened lifespan with a median survival of 25days, which represented a 24% decrease when compared to WT flies (azot+/+), and a maximum lifespan of 40 days, 31% less than WT flies (azot+/+). On the contrary, flies with three functional copies of the gene (azot+/+; azot+) or flies where azot is replaced by hid (azot{KO; hid} homozygous flies) showed an increase in median survival of 54% and 63% and maximum lifespan of 12% and 24%, respectively (Merino, 2015).

Finally, the effects of dietary restriction on longevity of those flies was tested. It was found that dietary restriction could extend both the median survival and the maximum lifespan of all genotypes. Interestingly, dietary restricted flies with three copies of the gene azot showed a further increase in maximum lifespan of 35%. This shows that dietary restriction and elimination of unfit cells can be combined to maximize lifespan (Merino, 2015).

In conclusion, eliminating unfit cells is sufficient to increase longevity, showing that cell selection is critical for a long lifespan in Drosophila (Merino, 2015).

This study has shown that active elimination of unfit cells is required to maintain tissue health during development and adulthood. The gene (azot), whose expression is confined to suboptimal or misspecified but morphologically normal and viable cells. When tissues become scattered with suboptimal cells, lack of azot increases morphological malformations and susceptibility to random mutations and accelerates age-dependent tissue degeneration. On the contrary, experimental stimulation of azot function is beneficial for tissue health and extends lifespan. Therefore, elimination of less fit cells fulfils the criteria for a hallmark of aging (Merino, 2015).

Although cancer and aging can both be considered consequences of cellular damage, no evidence was found for fitness-based cell selection having a role as a tumor suppressor in Drosophila. The results rather support that accumulation of unfit cells affect organ integrity and that, once organ function falls below a critical threshold, the individual dies (Merino, 2015).

Azot expression in a wide range of 'less fit' cells, such as WT cells challenged by the presence of 'supercompetitors,' slow proliferating cells confronted with normal proliferating cells, cells with mutations in several signaling pathways (i.e., Wingless, JAK/STAT, Dpp), or photoreceptor neurons forming incomplete ommatidia. In order to be expressed specifically in 'less fit' cells, the transcriptional regulation of azot integrates fitness information from at least three levels: (1) the cell's own levels of FlowerLose isoforms, (2) the levels of Sparc, and (3) the levels of Lose isoforms in neighboring cells. Therefore, Azot ON/OFF regulation acts as a cell-fitness checkpoint deciding which viable cells are eliminated. It is proposed that by implementing a cell-fitness checkpoint, multicellular communities became more robust and less sensitive to several mutations that create viable but potentially harmful cells. Moreover, azot is not involved in other types of apoptosis, suggesting a dedicated function, and - given the evolutionary conservation of Azot - pointing to the existence of central cell selection pathways in multicellular animals (Merino, 2015).


REFERENCES

Search PubMed for articles about Drosophila Azot

Merino, M. M., Rhiner, C., Portela, M. and Moreno, E. (2013). "Fitness fingerprints" mediate physiological culling of unwanted neurons in Drosophila. Curr Biol 23: 1300-1309. PubMed ID: 23810538

Merino, M.M., Rhiner, C., Lopez-Gay, J.M., Buechel, D., Hauert, B. and Moreno, E. (2015). Elimination of unfit cells maintains tissue health and prolongs lifespan. Cell 160: 461-476. PubMed ID: 25601460

Petrova, E., Lopez-Gay, J. M., Rhiner, C. and Moreno, E. (2012). Flower-deficient mice have reduced susceptibility to skin papilloma formation. Dis Model Mech 5: 553-561. PubMed ID: 22362363

Portela, M., Casas-Tinto, S., Rhiner, C., Lopez-Gay, J. M., Dominguez, O., Soldini, D. and Moreno, E. (2010). Drosophila SPARC is a self-protective signal expressed by loser cells during cell competition. Dev Cell 19: 562-573. PubMed ID: 20951347

Rhiner, C., Lopez-Gay, J. M., Soldini, D., Casas-Tinto, S., Martin, F. A., Lombardia, L. and Moreno, E. (2010). Flower forms an extracellular code that reveals the fitness of a cell to its neighbors in Drosophila. Dev Cell 18: 985-998. PubMed ID: 20627080


Biological Overview

date revised: 22 June 2023

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