The autophagy-related gene Atg101 in Drosophila regulates both neuron and midgut homeostasis

Atg101 is an autophagy-related gene identified in worms, flies, mice, and mammals, which encodes a protein that functions in autophagosome formation by associating with the ULK1-Atg13-Fip200 complex. In the last few years, the critical role of Atg101 in autophagy has been well-established through biochemical studies and the determination of its protein structure. However, Atg101's physiological role, both during development and in adulthood, remains less understood. Here, we describe the generation and characterization of an Atg101 loss-of-function mutant in Drosophila and report on the roles of Atg101 in maintaining tissue homeostasis in both adult brains and midguts. We observed that homozygous or hemizygous Atg101 mutants were semi-lethal, with only some of them surviving into adulthood. Both developmental and starvation-induced autophagy processes were defective in the Atg101 mutant animals, and Atg101 mutant adult flies had a significantly shorter lifespan and displayed a mobility defect. Moreover, we observed the accumulation of ubiquitin-positive aggregates in Atg101 mutant brains, indicating a neuronal defect. Interestingly, Atg101 mutant adult midguts were shorter and thicker and exhibited abnormal morphology with enlarged enterocytes. Detailed analysis also revealed that the differentiation from intestinal stem cells to enterocytes was impaired in these midguts. Cell type–specific rescue experiments disclosed that Atg101 had a function in enterocytes and limited their growth. In summary, the results of our study indicate that Drosophila Atg101 is essential for tissue homeostasis in both adult brains and midguts. We propose that Atg101 may have a role in age-related processes.


cro ARTICLE
Drosophila has increasingly become an attractive model system for studying autophagy, especially the physiological functions of autophagy in tissue homeostasis and neurogenesis (24 -26). In this study, in which we generated an Atg101 lossof-function mutant using the CRISPR/Cas9 approach in Drosophila, we report on the roles of Atg101 in regulating neuron and midgut homeostasis.

Generation and characterization of Atg101 loss-of-function mutant alleles
Drosophila Atg101 is located in an intron of the S6KL gene, which encodes an S6 kinase-like protein (Fig. 1A). To explore the physiological function of Atg101, we generated an Atg101

Atg101 regulates both neuron and midgut homeostasis
loss-of-function mutant allele, Atg101 6h , using the recently developed CRISPR/Cas9 system (Fig. 1A). Atg101 6h contains a 13-nucleotide deletion in the coding region, which causes a frameshift mutation (Fig. 1A). To examine whether this Atg101 mutation affects the expression of its host gene, S6KL, we extracted total RNA from both WT and Atg101 6h mutant animals and performed quantitative PCR analysis using a pair of exon-specific primers for S6KL. Our quantitative PCR results showed that the mature S6KL level was ϳ1.2 times of WT level in Atg101 6h mutants (Fig. S1A). Previous studies have reported that overexpression and mutation of S6KL leads to a decreased and increased bouton number at the larval neuromuscular junctions (NMJ), respectively (27,28). To further determine whether loss of Atg101 affects S6KL function, we performed immunostaining analysis and examined larval NMJ development in Atg101 6h mutants. For this analysis, an anti-CSP antibody was used to label presynaptic components at larval NMJs. Our results revealed that there were no significant changes in the number of boutons for the muscle 4 NMJ in Atg101 6h mutant larvae as compared with the control (Fig. S1, B and C). This indicated that Atg101 6h mutants exhibited no detectable defects during larval NMJ development and further confirmed that loss of Atg101 does not affect S6KL function.
Homozygous or hemizygous Atg101 6h mutants were found to be semi-lethal (Table S1). To determine whether the lethality we observed in Atg101 6h mutants was caused by the specific loss of Atg101 function, we made a UAS-Atg101 rescue transgene. Overexpression of UAS-Atg101 with a ubiquitously expressed Daughterless-Gal4 (Da-Gal4) in Atg101 6h mutants restored the viability of the mutant animals, indicating that the lethality is a likely consequence of loss of Atg101 (Table S1).
To analyze the lethality phenotype in more detail, we first examined the viability of WT and Atg101 6h mutants at different developmental stages under growth control conditions. Reduced viability was observed in both embryonic and post-embryonic stages in Atg101 6h mutants compared with the control, although the reduction of viability during the larval and pupal stages was subtle (Fig. S1D). Loss of Atg101 resulted in lethality before or during eclosion, similar to previously reported Atg17 mutants (Fig. S1, E and F) (29). Atg101 6h mutant adult flies also displayed an abnormal wing posture, which has been described in Atg17 mutants (Fig. S1, G and F) (29). This wing posture defect was rescued by overexpressing Atg101 with Da-Gal4 (Fig. S11). Some of the newly enclosed Atg101 6h mutant flies fell down easily into the food at the bottom of the vial (data not shown). In addition, we found that Atg101 6h mutant animals developed relatively slower compared with the control and the time needed from the newly hatched first instar larvae to become pupae or adults was increased by 12-24 h on average (Fig. S1, J and K).
We also determined the expression level of Atg101 at different developmental stages by performing RT-PCR analysis on RNA prepared from embryos, larvae, pupae, and adults. Atg101 mRNA was expressed at all developmental stages, with the highest level in 0 -12-h embryos (Fig. S11). In addition, the expression level of Atg101 was comparable with the levels of Atg1 and Atg8a but higher than the levels of Atg3, Atg4a, and Atg7 in 0 -12-h embryos (Fig. S1M).

Atg101 mutants exhibit defects in starvation-induced and developmental autophagy
Atg101 is an important component of the ULK1/Atg1 kinase complex in higher eukaryotes and plays essential roles in the initiation of autophagy by interacting with Atg13 (13)(14)(15). To demonstrate the role of Atg101 during the process of autophagy in vivo, we analyzed starvation-induced and developmentally triggered autophagy in the Atg101 6h mutant and WT control larval fat bodies. The dual-tagged GFP-RFP-Atg8a reporter is commonly used to follow autophagic flux and monitor autophagic activities (30). In this system, GFP fluorescence is normally quenched in the acidic environment of the autolysosome, but RFP fluorescence is pH-independent. Thus, autophagosomes can be labeled by both GFP and RFP signals and appear as yellow. Autolysosomes are positive only for RFP and appear as red. We took this approach and examined the effects of Atg101 on autophagic activities. Loss of Atg101 prevented the accumulation of both yellow and red punctate structures in fat body cells of both starved third instar larvae and late wandering third instar larvae, consistent with a previous report in which knockdown of Atg101 by RNAi blocked both starvation-induced and developmental autophagy ( Fig. 1, B-I) (17). It has been shown that Atg8a-positive structures are larger in size upon Atg101 knockdown. However, we found that the size of Atg8a punctate structures was smaller in Atg101 mutant fat body cells than in the control (Fig. 1, B-I). It is possible that some remaining Atg101 activities might affect the size of Atg8a-positive structures.
Previous studies have shown that inhibition of autophagy leads to a delay in larval midgut cell death during Drosophila metamorphosis. Consistent with this finding, morphological analysis of Atg101 6h mutant midguts revealed a delay in midgut cell death, as the gastric caeca still persisted at 4 h RPF (after puparium formation) in Atg101 6h mutant animals (Fig. S2, A and B). Together, these data confirmed that animals lacking Atg101 function are impaired in their abilities to induce autophagy during normal development and in response to starvation.

Atg101 mutant flies show decreased lifespans and impaired mobility
Mutations in several Drosophila autophagy-related genes, including Atg7, Atg8, and Atg17, lead to reduced lifespans in adult flies (29, 31-33). We therefore examined the effect on Drosophila lifespan when Atg101 was deleted. 50 -70% of the Atg101 6h mutants were able to develop to adulthood, and these flies were used for lifespan measurement. Compared with the control, Atg101 6h mutant flies exhibited a reduced adult lifespan, and most of mutant animals died at 3 weeks of age (Fig. 2,  A and B). In addition, we noticed that Atg101 6h mutant animals also showed a movement disorder, and most of them were found at the bottom of vials (data not shown). To confirm this finding, we performed a negative geotaxis assay with WT and mutant flies. When tapped to the bottom of a vial, WT flies responded by climbing to the top (Fig. 2C). However, most Atg101 6h mutant flies failed to do so (Fig. 2C). At 2 days of age, 70 -80% of the Atg101 6h mutant flies had impaired climbing Atg101 regulates both neuron and midgut homeostasis ability in a climbing assay (Fig. 2D). Their performance was much worse at day 10, with 100% of the flies impaired (Fig.  2D). These mobility defects were restored in the rescued flies ( Fig. 2, C and D). Collectively, these data indicate that loss of Atg101 leads to a reduced lifespan and impaired mobility in adult flies.

Atg101 mutants show neurodegeneration defects
The age-related decline of mobility could be a reflection of neurodegeneration in the adult brain. Previous studies have shown that an accumulation of ubiquitinated proteins is associated with progressive neurodegeneration in Atg5 and Atg7 mutant mice as well as Atg7, Atg8, and Atg17 mutant flies (29, [31][32][33][34][35]. To determine whether the mobility defects in Atg101 6h mutant were attributable to neurodegeneration, we used an antibody against ubiquitin to examine Atg101 6h mutants. Ubiquitinated proteins accumulated in 1-week-old Atg101 6h mutant fly head extracts as compared with the WT control (Fig. 3A). To confirm the disruption of autophagy in the Atg101 mutant fly head, we also examined the protein level of Ref(2)p in both WT and mutant fly heads. Consistently, Atg101 6h mutants had an obvious increase in the Ref (2) level as compared with the control (Fig. 3B). Furthermore, our immunofluorescence staining also revealed that the ubiquitin and Ref (2)p punctate structures had accumulated in the mutant brain, suggesting the formation of protein aggregates in the central nervous system of Atg101 6h mutants (Fig. 3, C-DЈ and G-HЈ, quantified in F and J). Partial co-localization between Ref(2)p and ubiquitin was also observed in Atg101 6h mutants (Fig. S3, A-BЉ). These protein aggregate defects were rescued by the overexpression of Atg101 (Fig. 3, E, EЈ, I, and IЈ, quantified in F and J). To further determine whether Ref (2)p accumulation occurs in neurons or glial cells, we double-stained Atg101 6h mutant adult brains with anti-Ref (2)p and antibodies against Elav or Repo, which mark neurons or glia, respectively. Colocalization of Ref (2)p-positive cells with Elav and Repo in Atg101 6h mutant brains showed Ref (2)p accumulation in both neurons and glial cells (Fig. 3, K-LЈ). Taken together, these data indicate that Atg101 function is important for the elimination of protein aggregates and neuron homeostasis in the adult brain.

Atg101 maintains midgut homeostasis
Defects in the maintenance of intestinal stem cell homeostasis could result in a short lifespan in adult flies (36 -39). During our study, we noticed that the Atg101 6h mutant abdomen was enlarged as compared with the WT animals, which indicates a possible defect in the midgut tissue (Fig. S4, A and B). This defect was also rescued by overexpressing Atg101 (Fig. S4C). We therefore extended our phenotypical analysis to the adult midgut tissue in Atg101 6h mutants. Interestingly, the midgut in Atg101 6h mutants was significantly shorter and thicker than that in WT controls (Fig. 4A, quantified in B). This defect was suppressed in the rescued flies, confirming that the midgut phenotype was specifically caused by the loss of Atg101 (Fig. 4A, quantified in B). To begin understanding the basis for these defects, we then focused on the posterior midgut and stained the tissue with phalloidin and DAPI to examine the posterior midgut morphology. Our phalloidin staining revealed that the regular organization of the visceral muscles was disrupted in Atg101 6h mutant midguts, which suggests that the peristalsis was less efficient in the mutants (Fig. 4, C-DЉ). Cells with different sized nuclei are present in the posterior midgut. The number of polyploid enterocytes with large nuclei was reduced, whereas the number of diploid cells with small nuclei increased ( Fig. 4, C-DЉ, quantified in E). We also noticed that the nuclear size of enterocyte cells was larger in the mutant midguts compared with the control (Fig. 4, CЈ and DЈ, quantified in F). Consistently, the enterocyte cell size was enlarged in Atg101 6h mutant midguts (Fig. 4, G and H). In addition, the midgut epithelium was thickened and the lumen size was increased in the Atg101 mutants (Fig. 4, I and J). Overexpression of Atg101 largely rescued all of these posterior midgut defects (Fig. 4, C-M). It is likely that food digestion and nutrient absorption were less efficient in the mutant midgut because of the irregular organization of the visceral muscles and reduced number of polyploid enterocytes. Together, these data demonstrate that Atg101 is required for the maintenance of adult midgut homeostasis.

Atg101 functions to promote intestinal stem cell differentiation
To examine the posterior midgut defect in more detail, we next performed immunofluorescence staining with antibodies against various markers for different types of posterior midgut cells. Escargot (esg)-GFP is specifically expressed in intestinal stem cells (ISC) and enteroblasts (EB), referred to collectively as midgut precursor cells (40). In Atg101 6h mutant midguts, the

Atg101 regulates both neuron and midgut homeostasis
number of esg-GFP-expressing cells was increased and cells often clustered (Fig. 5, A-BЈ, quantified in C and D). The increasing number of esg-GFP-positive cells could be a result of overproliferation of intestinal stem cell, a blockage of stem cell differentiation, or both. To further discriminate among these possibilities, we used anti-phospho-Ser10-Histone H3 (PH3) antibodies to label cells in mitosis. In Atg101 6h mutant midguts, the number of cells labeling for PH3 was comparable with the controls, indicating that the expansion of esg-GFP-positive cells was not likely to be attributable to the increase in ISC proliferation (Fig. 5, E-FЈ, quantified in G). We then considered the possibility that cell differentiation was blocked in Atg101 6h mutants. To test this possibility, we used anti-Prospero and anti-Pdm1 antibodies to stain for enteroendocrine (EE) and enterocyte (EC) cells, respectively, and found a significant reduction in the number of cells for EC cell types in Atg101 mutant midguts but not for the EE cell types (Fig. 5, H-IЈ and K-LЈ, quantified in J and M) (40 -42). Altogether, these analyses reveals that Atg101 is required for intestinal stem cell differentiation, especially for the differentiation of EC cell lineage in adult midguts.

Atg101 acts in ECs to limit cell growth
Having shown that the loss of Atg101 causes defects in adult midgut homeostasis, including enlarged EC cell size and reduced intestinal stem cell differentiation, we next sought to identify the cell types in which Atg101 might function. For this purpose, we performed a rescue experiment with the UAS-Atg101 transgene using several cell type-specific Gal4 drivers in adult midguts. First, esg-Gal4 combined with a temperaturesensitive GAL80 was used to overexpress Atg101 specifically in the adult ISC and EB populations of Atg101 6h mutant flies. To activate transgene expression, adult flies were shifted to the nonpermissive temperature. The results showed that there was no rescue effect on the enterocyte size when expressing UAS-Atg101 by esg-Gal4 as compared with Atg101 6h mutant alone (Fig. 6, A-C). We then used the EC-specific Myo1A-Gal4 combined with Gal80 ts to restrict the expression of UAS-Atg101 to ECs in the adult midgut. Interestingly, Myo1A-Gal4-driven Atg101 expression in Atg101 6h mutant flies displayed a rescue of the enlarged enterocyte size normally seen in Atg101 6h mutants (Fig. 6, D-F). Furthermore, no obvious rescue was

Atg101 regulates both neuron and midgut homeostasis
observed when an EE-specific pros-Gal4 was used to drive Atg101 expression in Atg101 6h mutant midguts (Fig. 6, G-I).
Thus, we concluded that Atg101 has a cell autonomous effect in ECs to limit cell growth.

Discussion
Here we generated and characterized an Atg101 loss-offunction mutant in Drosophila. The Atg101 mutant showed reduced viability in the embryonic, larval, and pupal stages. Most mutant animals can survive to adult stages but have a short lifespan. Our study also provides genetic evidence that Atg101 has a key role in maintaining neuron and midgut homeostasis.
Atg101 is a core subunit of the Atg1 complex, which is essential for autophagosome formation (13,23). Studies in mammalian cells have identified the role of Atg101 in autophagy initiation (14,15). Knockdown of Atg101 by RNAi in Drosophila also leads to autophagy defects (17). Consistent with this, the lack of Atg101 function causes defects in both starvation-in-duced and developmental autophagy in Drosophila third instar larval fat body tissues. In addition, we observed a variety of phenotypes in adult flies, such as reduced lifespan, impaired locomotion, accumulation of ubiquitinated proteins, and blockage of intestinal stem cell differentiation.
Autophagy has been implicated in the process of aging (43). Suppression of autophagy disrupts age-dependent tissue homeostasis in various organs (43). In flies, the loss of Atg7, Atg8a, or Atg17 in the entire organism leads to a reduced lifespan as well as the accumulation of ubiquitinated protein aggregates in the brains (29, 31-33). It has been proposed that the basal level of autophagy in the nervous system is required for the clearance of toxic proteins or damaged organelles (43,44). Similar to other Atg mutants, Atg101 mutant flies also have a shorter lifespan. Protein aggregates were evident in Atg101 mutant fly brains. The decline of locomotion ability in Atg101 mutants during aging further demonstrates a neurondegeneration defect in the absence of autophagy function.

Atg101 regulates both neuron and midgut homeostasis
Recently, several reports indicate that autophagy-related genes also regulate intestine homeostasis. A core autophagy gene, Atg16L1, has been shown to be associated with Crohn disease (45). Later studies performed in mice show that Paneth cells are abnormal in Atg16L1 or Atg5 knockout mutant animals (46). In flies, Atg9 has been shown to be required for Jun N-terminal kinase (JNK)-mediated intestinal stem cell proliferation (47). Interestingly, the induction of autophagy can block stress-induced ISC proliferation (47). In addition, Atg9 is also required for midgut homeostasis under normal physiological condition, and it specifically acts in enterocytes to control cell growth by limiting TOR signaling (48). Analysis of various Atg genes in the Drosophila midgut has demonstrated that the Atg1 complex components, including Atg1, Atg13, and Atg17, play crucial roles in controlling enterocytes cell growth (48). Furthermore, it appears that the Atg1 complex and Atg9 regulate TOR signaling via different mechanisms during enterocyte cell growth (48). Interestingly, other core autophagy-related genes, such as Atg7, Atg12, Atg16, Atg18, and Vps32, are not required for enterocyte cell growth in Drosophila adult midguts, indicating that the role of the Atg1 complex and Atg9 in maintaining midgut homeostasis might be specific (48). Consistent with the reported phenotype, upon knocking down Atg1, Atg13, and Atg17, we found that loss of Atg101 caused defects in adult midgut homeostasis and resulted in abnormal midgut mor- E. An unpaired t test was used for statistical analysis. K-LЈ, Z-projection confocal images of WT and Atg101 6h mutant posterior midguts stained for anti-Pdm1, which labels EC cells. M, quantification of Pdm1-positive cells. Quantification was performed as described in C. Cells in ten defined regions from ten WT midguts and nine defined regions from nine Atg101 6h mutant midguts were counted. Data are presented as mean Ϯ S.E. An unpaired t test was used for statistical analysis. **, p Ͻ 0.01.

Atg101 regulates both neuron and midgut homeostasis
phology with enlarged enterocytes. Interestingly, the number of intestinal progenitor cells was increased in Atg101 mutant midguts. However, the number of dividing intestinal stem cells when stained with anti-PH3 remained the same as in the control. In contrast, we observed reduced numbers of differentiating enterocyte cells. These data strongly indicate that Atg101 plays an important role in intestinal stem cell differentiation but not proliferation. In addition, our cell type-specific rescue experiments revealed that Atg101 functions in ECs to limit cell growth autonomously. It has been reported that apoptotic enterocytes promote intestinal stem cell division nonautonomously (49). It remains unclear whether the overgrowth of ECs has an effect on intestinal stem cell function. In summary, these findings indicate that Atg101 plays essential roles in maintaining neuron and midgut homeostasis, both of which may affect the aging process. A recent study also reveals that the activation of autophagy in the adult brain by expression of either AMPK or Atg1 induces autophagy in the intestine and leads to an increased lifespan (50). Further studies on the connection between the brain and midgut homeostasis in Drosophila Atg101 mutant animals will likely provide novel insights into the cross-talk between the brain and the midgut.

Generation of Atg101 mutant and transgenic fly lines
CRISPR-mediated mutagenesis was performed according to a previous report (51). Briefly, the Atg101 target sequence and PAM site were determined using http://tools.flycrispr.molbio. wisc.edu/targetFinder/ (52). 3 After identifying the target region, two primers (TAATACGACTCACTATAGGAGGTG-TGGACGGTGCACCGTTTTAGAGCTAGAAATAGC and AAAAAAAGCACCGACTCGGTGCCAC) were used to amplify the DNA fragment from the pMD19-T gRNA scaffold vector. The PCR products were used for gRNA in vitro transcription with the RiboMAX Large Scale RNA Production Systems T7 kit. To synthesize Cas9 mRNA, the pSP6 -2sNLS-sp-Cas9 plasmid was first cut by XbaI and then purified. Transcription was performed using the Sp6 mMESSAGE mMACHINE kit (Ambion). The poly(A) tails were added to the 3Ј-end of Cas9 mRNAs using Escherichia coli poly(A) polymerase kit (New England Biolabs). Cas9 mRNAs and Atg101 gRNA were then mixed and injected into w 1118 fly embryos. Genomic DNA from dead embryos were used for PCR amplification and sequencing to determine the efficiency and usefulness of gRNA. The primers used for amplifying the target region were 5Ј-TTTCACACCGTCCTCTTCCAC-3Ј and 5Ј-ATGATGGGA-GGATTTGCGTTC-3Ј. The detection of a string of "double peaks" in the sequencing chromatogram indicates the mismatched region and the usefulness of gRNA. Single flies were selected and balanced over FM6B. Exact deletions were determined by PCR screening and sequencing for individual flies. Atg101 6h mutants have a 13-bp deletion (from ChX 18792261 to ChX 18792273).
For the generation of UAS-Atg101-HA transgenic flies, an Atg101 cDNA fragment was amplified using the primers 5Ј-CGGCGGCCGCATGAACGCGCGTTCGCAGGT-3Ј and 5Ј-CGCTCGAGCATTGCGAGCGTTTCCTTGA-3Ј and cloned into a modified pUAST vector with a 3HA tag at the C-terminal. The pUAST-Atg101-HA construct was then injected into the ZF-25C landing site on chromosome II using standard methods.

Immunostaining and microscopy
Drosophila adult midguts were dissected in SD medium and fixed with 4% paraformaldehyde in PBS for 40 min with rocking at room temperature. Midguts were washed three times in PBST (0.1% Triton X-100 in PBS) before blocking for 1 h in PBST plus 3% BSA buffer at room temperature. Next, midguts were incubated with the primary antibodies overnight at 4°C. After four washes in PBST, the midguts were incubated with secondary antibodies for 2 h at room temperature with rocking. DAPI was added for the last 20 min. After four further washes with PBST, the midguts were mounted in Vectashield mounting medium. 7-day-old adult males were used in all analyses. The posterior region of the midgut was chosen for imaging. Drosophila adult brains and larval NMJs were dissected in SD medium. Samples were then fixed with 4% paraformaldehyde in PBS for 20 min and stained as described above. The following primary antibodies were used: . Phalloidin (phalloidin 568, Invitrogen A12380, 1033926) was used in a 1:1000 dilution. DNA was labeled with DAPI (1 g/ml, Sigma). To induce starvation, middle L3 stage larvae were collected and transferred to a 20% sucrose solution for 4 h. For live imaging, larval fat body tissues were dissected in SD medium. Imaging was performed on an Olympus FV1000 confocal microscope, and images were processed using ImageJ and Adobe Photoshop.

Quantification and statistical analysis
In Fig. 1, D, E, H, and I, the number of RFP-Atg8a spots was counted manually, and the size of the RFP-Atg8a spots was measured with NIS-Elements. In Fig. 3, F and J, the number of ubiquitin-positive spots and the area of the Ref(2)p-positive spots were measured with ImageJ. In Fig. 4B, midgut length and width were measured with NIS-Elements from images of whole midguts acquired with a Nikon Eclipse 80i microscope. In Fig.  4, F and G, the number of nuclei was counted manually, and the size of the nuclei was measured with NIS-Elements. Posterior midgut regions R4a and R4b were chosen. In Fig. 5, C, J, and M, the number of esg-positive, pros-positive, and pdm1-positive cells were counted manually. Posterior midgut regions R4a and R4b were chosen. In Fig. 5D, the number of esg-positive cell clusters was counted manually for the entire frame. In Fig. 5G, the number of PH3-positive cells was counted manually for each midgut. In Fig. S2B, the gastric caeca size was measured with NIS-Elements. The sample size for the quantification analysis is indicated in the legends for Figs. 1, 3, 4, 5, S1 and S2. Statistical analysis was performed using GraphPad Prism 5.
Hatching rate, pupation rate, and eclosion rate 3-4-day-old flies were collected and put in cages. Embryos were collected every 2 h. To measure the hatching rate, 200 embryos were transferred to a fresh plate, and the number of hatched first instar larvae were counted. To monitor the rate and timing of pupation and eclosion, 50 first instar larvae were collected and cultured in a vial. The number of pupae and adults were counted every 12 h. The pupation rate was calculated as the percentage of the number of pupae versus the number of first instar larvae contained in each vial. The eclosion rate was calculated as the percentage of the number of eclosed adults versus the total number of pupae contained in each vial.

Climbing assay
For negative geotaxis assay, aged flies were separated by gender and grouped in cohorts of 20 animals. Before testing, the flies were transferred to a tube made by two vertically joined empty vials and allowed to rest for 1 h before the assay. After tapping the flies down to the bottom of the vial, we measured the number of flies that could climb above the 15-cm mark within 15 s. A climbing index was calculated as the percentage of the number of flies above the mark versus the total number of flies in the tube. Six replicate sets of experiments were performed for each genotype.

Lifespan assay
For the lifespan analysis, groups of 20 newly eclosed males or females were collected and transferred to vials with fresh food every 2-3 days. The number of dead flies was counted. Survival rates were calculated as the percentage of the number of surviving flies versus the total number of flies. Three replicate sets of experiments were performed for each genotype.