Production of trus CRISPR/Cas9 mutants

The original trus¹ mutant (toys are us) was isolated from the Zucker EMS mutant collection [32,45]. The mutation caused significant developmental delay throughout development and 3rd instar larvae wandered up to 10 days before pupariating and subsequently dying as pre-pupae [32]. Sequencing revealed that the trus¹ allele carries a point mutation in the start codon, which may result in a failure of translational initiation (Fig 1A) [32]. We found that a small percentage of heterozygous trus¹/Dftrus (Df(3R)BSC847) developed to pharates and a few enclosed as adults that showed notched eyes and thin/short bristles (S1 Fig). These phenotypes, in addition to the prolonged developmental time, resemble the haplo-insufficiency ‘Minute’ syndrome that is often observed in flies carrying a mutation in one of the genes encoding ribosomal proteins [6]. Since no adult escapers were obtained from trus¹ homozygous, we inferred that trus¹ might be an antimorph allele. This could arise from an additional unknown mutation on the trus¹ chromosome or an aberrant translational start at one of the methionine codons downstream of the normal initiation codon which would produce an N-terminally truncated protein (Fig 1A).

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Fig 1. CRISPR/Cas9 induced trus mutations cause developmental delay and defects in tissue growth and cell proliferation during the larval stage.

(A) A diagram showing trus genomic region and trus mutant alleles that are used in this study. Trus CDS is shown in blue. Two guide RNAs were designed to flank the entire exon2 and the following intron (magenta). trus^4-15 has a single bp deletion at 3R:12,456,811 within target A (red). This results in a truncated 42 amino acid peptide with identity up to Arg40, two missense codons, and a premature stop (green+red+brown). Since this peptide is so small even if it is stably expressed, we consider trus^4-15 to be a trus null allele. A second allele, trus^35-2 is a deletion of 1184 bp between 3R:12,456,805 and 3R:12,457,988 starting 3 bp upstream of target A and ending 5 bp downstream of target B (red). This deletion might give rise to a shorter 110 amino acid peptide lacking residues between Trp38 and Phe414 if stable (orange). Since trus¹ allele has a point mutation that alters the initiation codon and the mutant behaves like antimorph, it is possible that the mutation results in an aberrant translational initiation from one of the alternative initiation sites and produces a short fragment. There are two additional candidate initiation sequences which weakly align to the Kozak consensus [90,91] and are in-frame with the Trus protein sequence. Candidate 1 is from +381 bp downstream and candidate 2 is from +579 bp downstream of the original initiation start codon and could produce N-terminally truncated 238 a.a. (~27kDa) or 173 a.a. (~19kDa) Trus fragments, respectively. (B) Pupariation timing of trus mutant and w¹¹¹⁸. Each data point on the graph indicates an average pupariation percentage of two separate plates. The vertical line on each data point indicates the standard deviation. Numbers of 1st instar larvae picked at 1 Day AEL are shown in parentheses after the genotype. Percentage of pupariation at 14 days AEL were 97%, 87%, 69%, 71% for w¹¹¹⁸, trus^4-15/trus^35-2, trus^4-15/Dftrus, and trus^35-2/Dftrus, respectively. After pupariation, most of the trus mutant larvae did not develope further and eventually died (pre-pupal lethal) except for some trus^4-15/trus^35-2 that developed to pharates but did not eclose. More than 95% of the w¹¹¹⁸ animals eclosed as adults. (C) Representative images of brains and wing discs that were dissected from third instar wandering larvae. Genotypes indicated at the top of the panels. DNA was stained with DAPI and mitotic cells are detected with anti-phospho-HistoneH3 (PH3) antibody. Maximum intensity Z-projections are shown. Scale bar: 200μm.

https://doi.org/10.1371/journal.pgen.1011469.g001

Given the potential complications associated with a phenotypic analysis of the trus¹ allele, we generated new trus null alleles using CRISPR/Cas9 targeted mutagenesis and isolated two alleles, trus^4-15 and trus^35-2, that are used in this study (Fig 1A) [46]. Western blot analysis using a polyclonal antibody raised against full-length recombinant Trus showed a band migrating around 60 kDa, corresponding to the full-length Trus protein (predicted Trus molecular weight is 53.2 kDa), that is missing in trus^4-15/trus^4-15, trus^35-2/trus^35-2, and trus¹/trus¹ larval extracts, while the protein level was reduced in heterozygous trus^4-15/TM6, trus^35-2/TM6, and trus¹/TM6 larval extracts (S2 Fig). We detected no additional truncated fragments that reacted to the anti-Trus antibody on our Western blot (S2 Fig); however, it remains possible that the anti-Trus antibody that was raised against full length of Trus does not react to N-terminally truncated shorter fragments and since trus^35-2 and/or trus¹ could produce truncated fragments, these may be responsible for their hypomorphic or antimorphic phenotypes, respectively (described below).

trus mutants show developmental delay and pre-pupal lethality

We examined the phenotype of the CRISPR/Cas9 trus mutants and first noticed that the mutant animals displayed higher embryonic lethality compared to the control animals (Table 1). We determined the hatch rate of trus^4-15/Dftrus embryos to be 35% (n = 101), whereas the hatch rate of Dfd-GMR-nvYPF positive embryos was 88% (n = 91) (Table 1). Transheterozygous trus^4-15/Dftrus, trus^35-2/Dftrus, or trus^4-15/trus^35-2 mutants that hatched exhibited extensive developmental delay and pupariated at 10–12 days AEL (After Egg Lay), which was 5–7 days later than w¹¹¹⁸ (Fig 1B). Some of the larvae remained in the wandering stage for up to 7 days, consistent with the phenotype observed with trus¹/Dftrus mutants. We noticed that trus^4-15/Dftrus and trus^35-2/Dftrus larvae did not actively crawl to a high position on the vial wall during the 3rd instar stage but instead stayed near the food surface. Although trus^4-15/Dftrus, trus^35-2/Dftrus, and trus^4-15/ trus^35-2 produced significant embryonic lethality, those that hatched to become 1st instar larvae largely survived through the larval stages and the pupariation rate of the different mutant allele combinations reached 70–85%, although only after substantial developmental delay. (Fig 1B). After pupariation, most of the larvae died without becoming pupae. Noticeably, the pupariation rate of trus^4-15/ trus^35-2 animals were 15% higher than trus^4-15/Dftrus and trus^35-2/Dftrus (Fig 1B), and some of these pupated and developed to pharate adults, although none eclosed.

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Table 1. trus mutant displays higher embryonic lethality.

https://doi.org/10.1371/journal.pgen.1011469.t001

The trus^4-15/Dftrus trans-heterozygous combination is a protein null based on genomic sequencing and Western blotting results (S2 Fig), and it produces consistent pre-pupal lethality and developmental delay. We used this genotype as representative of the ‘trus zygotic null mutant phenotype’ in most of our subsequent experiments.

trus mutants show defects in tissue growth and cell proliferation

To determine what causes the developmental delay and lethality in trus mutants, we first dissected third instar wandering larvae just before pupariation and performed immuno-fluorescent staining of imaginal discs and brains with anti-phospho-HistoneH3 (anti-PH3) antibody and 4’,6-diamidino-2-phenylindole (DAPI). Fig 1C shows representative confocal images of brains (top two rows) and wing discs (bottom two rows) from wild type (Fig 1C, a–d: w¹¹¹⁸) and trus mutants (Fig 1C, e–m). We first noticed that in trus^4-15/trus^35-2 and trus^4-15/Dftrus larvae, brains were significantly smaller than wild-type, and the brain lobes looked unstructured, meaning there was no characteristic ring-structure in the optic lobes as normally appears in wild-type brain lobes (Fig 1C, e and i, compared to a). Their ventral nerve cords (VNC) were narrower and elongated, and the surface of the entire brain appeared disrupted (Fig 1C, e and i).

We also examined tissues from Zfrp8^P/DfZfrp8 (Df(2R)BSC356) larvae to determine whether the phenotype of Zfrp8 mutant larvae is similar to that of the trus mutant (Fig 1C, n–q). Zfrp8^P is a P-element insertion in the 5’-UTR of the Zfrp8 gene, and it is not a protein null, therefore it is expected to show a mild phenotype [47]. Df(2R)BSC356B is a deficiency chromosome that lacks 42 genes including Zfrp8. Brains from Zfrp8^P/DfZfrp8 (Df(2R)BSC356) larvae (Fig 1C, n) were smaller than wild type (Fig 1C, a) but not as small as trus mutants (Fig 1C, e and i). The Zfrp8 null allele Df(SM)206 combined with the Zfrp8^M-1-1 allele that deletes part of the Zfrp8 gene (Df(SM)206/Zfrp8^M-1-1) is lethal during the early larval stage; therefore we were unable to obtain 3rd instar larval tissue to analyze [31].

We next examined the imaginal discs including leg, haltere, and wing discs from trus^4-15/Dftrus larvae (Fig 1C, i and k) and found that they were smaller than wild type (Fig 1C, c), with wing discs showing the most severe growth impairment and morphological defects. The discs were under-developed and too small to dissect out from early wandering larvae; however, after additional development during the prolonged wandering stage, the wing discs reached a size that could be dissected and further examined. Intriguingly, wing discs from trus^4-15/trus^35-2 larvae developed inconsistently during the late wandering stage. Some were larger than wild type and often showed excessive folds, whereas the others were smaller than wild type, so the overall size distribution was diverse. Wing discs from Zfrp8^P/DfZfrp8 were less affected than trus^4-15/Dftrus but were still smaller than wild type (Fig 1C, c and p). Fig 2B shows quantification of PH3 positive cells (number/mm²) and area (mm²) of brain lobe, ventral nerve cord, and wing pouch plus hinge as indicated with yellow-line enclosed areas in Fig 2A. In brain lobes from trus^4-15/Dftrus and trus^4-15/trus^35-2 larvae, PH3 positive (mitotic) cell numbers were significantly less than w¹¹¹⁸ control, and the area (size) of brain lobes was also significantly smaller than w¹¹¹⁸. We observed less significant changes of PH3 count of ventral nerve cords from trus^4-15/Dftrus and trus^4-15/trus^35-2, however, area reductions were still significant for the trus mutants comparing to wild type. We saw significant decrease of PH3 positive cells in brain lobes from Zfrp8^P/DfZfrp8. The quantification confirmed a significant reduction of area of wing pouch + hinge from trus^4-15/Dftrus compared to w¹¹¹⁸ (Fig 2B, and additional information in the accompanying figure legend).

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Fig 2. trus mutation caused a significant reduction of mitotic cell number and size of brain and wing disc.

(A) Diagrams showing the larval brain and wing-disc. The areas surrounded by yellow lines indicates brain lobe, ventral nerve cord, and wing pouch plus hinge areas that are quantified in B. (B) Quantification of PH3 positive cells/mm² (left column) and area in mm² (right column) for brain lobe, ventral nerve cord, and [wing pouch + hinge] are shown. Genotypes and number of tissues measured for each genotype are indicated in parenthesis under the graphs. The x in the boxes indicates the mean value and the line inside the box indicates the median. Box-and-whisker plots were generated using Microsoft Excel (Redmond, WA) and ANOVA analysis followed by Dunnett’s test to compare each mutant to wild-type (w¹¹¹⁸) were performed using Prism 10 (GraphPad Software, INC., Boston MA). When the P-value in Dunnett’s test indicates that the pair variance is statistically significant, it is shown as a horizontal line across the w¹¹¹⁸ and the mutant with * (P<=0.05), ** (P<=0.01), *** (P<=0.001), or **** (P<=0.0001) (data in S2 File). PH3 positive cells and area measurements of [wing pouch+hinge] from trus^4-15/trus^35-2 larvae show significant variations within the group, some wing discs are small, and others are overgrown, suggesting that the trus^35-2 allele that possibly produces a 110 a.a. Trus fragment is causing hypo/hyper-morphic effects in combination with the protein null trus^4-15 allele. The observed variation is not likely to be caused by experimental errors but is merely indicative of the trus^4-15/trus^35-2 mutant phenotype. When we removed the intrinsically variable trus^4-15/trus^35-2 group from ANOVA analysis of [wing pouch+hinge], the results for PH3 positive cell count and area measurement showed P < 0.05 (*) and P < 0.01 (**), respectively. Following Dunnett’s tests indicated P < 0.01 (**) for both PH3 count and area comparisons between trus^4-15/Dftrus vs. w¹¹¹⁸ (see ANOVA analysis 2 in S2 File). This indicates that wing disc growth is clearly inhibited in trus null mutant (trus^4-15/Dftrus) compared to w¹¹¹⁸. The Dunnett’s test also showed that area of [wing pouch+hinge] from Zfrp8^P/DfZfrp8 larvae compared to w¹¹¹⁸ was significantly smaller with P < 0.05 (*).

https://doi.org/10.1371/journal.pgen.1011469.g002

In contrast to the absolute pre-pupal lethality of trus^4-15/Dftrus larvae, we observed rare escapers from trus^4-15/trus^35-2 larvae that developed to pharate adults but did not eclose. The size and cell proliferation variances observed in wing discs from trus^4-15/trus^35-2 animals are consistent with the variable penetrance in the pre-pupal/pharate lethality of the mutant. We speculate that the trus^35-2 allele may produce a truncated Trus protein product that has partial function and appears to trigger metamorphosis of a limited number of mutant larvae allowing them to progress into the pupal-pharate stage.

Given the observed structural abnormalities and extensive reduction of brain and wing disc size in trus mutants (Fig 1C), we investigated whether apoptosis contributes to the tissue growth defects in the mutants and whether this ultimately leads to developmental delay and lethality. As we could not detect an increase in apoptosis in trus mutant brain and wing discs by anti-cleaved caspase3 antibody staining or TUNEL assay, we further examined whether inhibiting apoptosis by ubiquitously overexpressing baculovirus p35, a caspase inhibitor [48,49], with daughterless-GAL4 could rescue the defects in tissue growth, cell proliferation, and developmental timing of trus mutants, and found that it could not (S4 Fig). Our results suggest that loss of Trus does not trigger widespread apoptosis, rather the developmental and tissue growth abnormalities likely reflect a deficiency in the control of cellular proliferation.

RNAi mediated trus knockdown in wing discs causes growth and cell proliferation defects

To help distinguish autonomous verses non-autonomous effects of trus loss on imaginal disc growth, we used RNAi knockdown mediated by different imaginal disc Gal4 driver lines including engrailed-GAL4 (en-GAL4), en-GAL4 and cubitus-interruptus-GAL4 (ci-GAL4) simultaneously, or nubbin-GAL4 (nub-GAL4). Fig 3A shows resulting wing phenotypes, in which nub-GAL4 caused an overall wing size reduction (nub > UAS-trusRNAi), and en-GAL4 (en > UAS-trusRNAi) or en-GAL4 and ci-GAL4 (en,ci > UAS-trusRNAi) caused disruption of wing veins and reduction of wing area, especially in the posterior part of wings compared to the control (UAS-trusRNAi without a driver) (Fig 3A). Quantification of the wing area using ImageJ indicates that the reduction in wing size is significant with either wing driver (Fig 3B). Consistent with the reduced size, we found that trusRNAi caused a reduction of proliferation in larval wing discs specific to the area where trusRNAi was expressed (Fig 3C). The expression pattern of each GAL4 driver used in the trusRNAi experiments and later used in trus mutant rescue experiments can be found in S3 Fig.

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Fig 3. trusRNAi induced with various wing disc drivers affects wing size and morphology and decreases cellular proliferation.

(A) Representative images of wings from female adult flies that had Trus RNAi induced with wing disc drivers. Adult wings from flies carrying UAS-trusRNAi without any driver, en-GAL4 driven UAS-trusRNAi, en and ci-GAL4 driven UAS-trusRNAi, or nub-GAL4 driven UAS-trusRNAi are shown. (B) Quantification of wing area calculated in mm². An example area is represented as magenta dashed outline in the top-left in A. ImageJ/Fiji (https://imagej.net) was used for area measurement. Box-and-whisker plots were generated using Microsoft Excel. The x in the boxes indicates the mean value and the line inside the box indicates the median. Sample numbers are indicated at the bottom of the graph (ex. n = 29 for UAS-trusRNAi). ANOVA analysis followed by Dunnett’s test to compare wing area of each RNAi samples to w¹¹¹⁸ were performed using Prism 10 (GraphPad Software, INC.). **** indicates P value <=0.0001 (S3 File). (C) Reduction of anti-PH3 stained foci is observed in the pouch area of wing and haltere discs in nub>trusRNAi, UAS-dicer2 larvae (a-d) and the posterior half of wing discs and one half of the leg disc in en>trusRNAi, UAS-dicer2 larvae (e-h). In a and c, green: DAPI staining and magenta: anti-PH3 staining. Yellow dashed lines indicate pouch of wing and haltere discs. In e and g, blue: DAPI staining and yellow: anti-PH3 staining. Arrows in f and h indicate the anterior (A)- posterior (P) axis of wing discs. For each genotype, more than 12 wing discs were dissected and they showed the similar phenotypes. Maximum intensity Z-projections are shown. Bar: 200μm.

https://doi.org/10.1371/journal.pgen.1011469.g003

To analyze expression patterns in detail, we chose a fast-maturing nuclear reporter UAS-RedStinger (DsRed.T4.NLS) [50]. We confirmed that nub-GAL4 is only expressed in the pouch area of wing and haltere discs, en-GAL4 is expressed only in the posterior half of wing and haltere discs, and ci-GAL4 is expressed in the anterior half of wing and haltere discs, as reported previously [51–53] (S3 Fig, c, d, k, m, p, and q). We observed that nub-GAL4 driven trusRNAi leads to a reduction of mitotic cell number detected by anti-PH3 antibody only in the pouch of wing and haltere discs, resulting in a reduced pouch size (Fig 3C, a–d). Knockdown of trus using en-GAL4 reduced the mitotic cell number only in the posterior half of the wing disc and the leg disc, resulting in considerable reduction of the posterior half of the wing disc (Fig 3C, e–h). Our observations indicate that trus knockdown inhibits cellular proliferation in a tissue and cell-autonomous manner. We note that trusRNAi driven by a combination of en-GAL4 and ci-GAL4 affects the posterior part of the wing more than the anterior (en,ci > UAS-trusRNAi) (Fig 3A). This can be explained by the fact that when both en-GAL4 and ci-GAL4 are used simultaneously, en-GAL4 expression is much stronger than ci-GAL4 for unknown reasons (S3 Fig, r-u).

Loss of trus in wing discs causes developmental delay and lethality

When we enhanced trusRNAi knockdown using UAS-dicer2, together with nub>trusRNAi or pdm2 > trusRNAi, both of which express primarily in the wing pouch, (S3 Fig, c-d, g-h), we noted a complete loss of wing blades leaving a small hinge in adult flies, haltere defects, and extra bristles (Fig 4A). Interestingly, dicer2-enhanced nub>trusRNAi, pdm2 > trusRNAi, and en>trusRNAi also resulted in partial lethality during the pre-pupal or pupal stages. The pupariation rate was high with all the drivers; however, the adult eclosion rate of pdm2 > trusRNAi decreased to ~10% and nub>trusRNAi to ~30%, while the eclosion rate without a driver averaged 79% (Table 2). Notably, en>trusRNAi resulted in a high rate of embryonic lethality, and the surviving larvae were 100% pre-pupal lethal (Table 2). In addition, many larvae showed a “Tubby-like” phenotype resulting in significantly smaller pre-pupae than w¹¹¹⁸ or pdm2 > trusRNAi pupae (Fig 4B). This observation is in line with reports that engrailed mutant embryos are Tubby-like and lethal during the embryonic stage [53]. We suspect that trusRNAi driven with en-GAL4 causes inhibition of cell proliferation, specifically in posterior compartments, leading to a failure of posterior segment establishment during embryogenesis similar to engrailed mutants, and this likely contributes to the high rate of embryonic lethality.

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Table 2. Percentage of pupariation and eclosion of larvae that were induced trus RNAi in wing discs.

https://doi.org/10.1371/journal.pgen.1011469.t002

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Fig 4. trusRNAi induced with wing disc drivers delay pupariation timing.

(A) trusRNAi induced with either nub-GAL4 or pdm2-GAL4 in the presence of UAS-dicer2 show a complete loss of wing blade (magenta arrows and magenta dotted-circle), morphological defects in halteres (blue arrows), and extra/disorganized bristles (green arrows). (B) (left) pdm2 > trusRNAi pharates with wing defects. (right) en>trusRNAi larvae pupariate precociously resulting in smaller pre-pupae that never become pupae. (middle) w¹¹¹⁸ (control) pupa shown for size and structural comparison. (C) Pupariation timing of trusRNAi larvae induced with nub-GAL4 (red), pdm2-GAL4 (green), or en-GAL4 (purple). Pupariation timing of w¹¹¹⁸ larvae (blue) and larvae carrying UAS-trusRNAi and UAS-dicer2 without driver (light blue) are shown as controls. Each data point represents an average pupariation percentage from multiple plates. The vertical line on each data point indicates the standard deviation. Numbers of 1st instar larvae picked at 1 Day AEL are shown in parentheses after the genotype.

https://doi.org/10.1371/journal.pgen.1011469.g004

In addition to these morphological defects, we found that dicer2-enhanced trusRNAi also caused significant larval developmental delay. Without a driver (UAS-trusRNAi), pupariation occurred at 5–6 days AEL similar to the w¹¹¹⁸ control (Fig 4C light blue and blue). In contrast, nub>Trus RNAi larvae pupariated at 5–8 days AEL (delayed 1–2 days compared to the control, Fig 4C red), pdm2 > trusRNAi larvae pupariated at 7–10 days AEL (delayed 2–5 days, Fig 4C green), and en>trusRNAi pupariated at 9–11 days AEL (delayed 4–6 days, Fig 4C purple).

Since the disc-specific RNAi knockdown of trus results largely recapitulates the cell proliferation and developmental delay phenotypes exhibited by genetic null mutants, we infer that it may be the reduced imaginal disc cell proliferation that is key to understanding the developmental delay.

The Xrp1-Dilp8 stress response pathway is largely responsible for the developmental delay of trus mutants

The Xrp1-Dilp8 signaling pathway has been shown to coordinate Drosophila tissue growth with developmental timing [15,54,55]. Xrp1 is a stress response transcription factor and is activated in growth impaired tissues promoting production and release of the peptide hormone Dilp8. Dilp8 then acts remotely on Lgr3 positive neurons in the central brain to inhibit the release of PTTH thereby blocking the production of molting hormone ecdysone [56]. Since trus mutants show growth and cell proliferation defects in imaginal discs, and are developmentally delayed, we hypothesized that the Xrp1-Dilp8 pathway is the link between tissue growth impairment and slow development in trus mutants. To test this hypothesis, we recombined a genomic GFP insertion of dilp8 (dilp8-GFP) [57] onto the trus^4-15 chromosome. We found that Dilp8-GFP protein is expressed in wing, leg, and genital discs in trus^4-15/Dftrus larvae, while Dilp8-GFP could not be detected in the control larvae (Fig 5A). As described previously, wing discs and, to some extent, leg discs dissected from these late wandering larvae are small and disorganized but show strong patches of Dilp8-GFP (Fig 5B Dilp8-GFP). We also induced trusRNAi with nub-GAL4 and found that Dilp8-GFP was expressed in the pouch area of wing and haltere discs (magenta and yellow allows in Fig 5C), suggesting that dilp8 expression is involved in the developmental delay observed with trusRNAi described above (Fig 4C).

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Fig 5. Xrp1-Dilp8 pathway is activated leading to developmental delay in trus mutants.

(A) (left) Dilp8-GFP expression in third instar wandering trus ^4-15/ Dftrus larvae. (right) Dilp8-GFP expression in 3rd instar wandering trus^4-15/ TM6B P[Dfd-GMR-nvYPF]Sb or Dftrus/ TM6B P[Dfd-GMR-nvYPF]Sb larvae. Two bright GFP dots are Dfd-GMR-nvYFP signals on eyes. (B) Fixed and dissected wing (first and second rows) and leg (bottom row) discs from trus ^4-15/ Dftrus third instar wandering larvae show Dilp8-GFP expression (green). Rhodamine-Phalloidin and DAPI staining reveal significant reduction in size and abnormal morphologies of the discs. 16 larvae were dissected, and representative images are shown. Maximum intensity Z-projections are shown. Scale bar: 200μm. (C) (left) Expression of Dilp8-GFP in the pouch region of the wing disc (magenta arrows) and haltere disc (yellow arrow) from nub> trusRNAi larvae. (right) A dissected nub>trus RNAi larval wing discs are marked with a magenta dashed line and show GFP fluorescence in the middle area of the wing discs (wing pouch). (D) Developmental timing curves show that da>dilp8RNAi (red) or da > Xrp1RNAi (green) significantly rescue the developmental delay of trus^4-15/Dftrus larvae. da > UAS-trus rescues the developmental delay and lethality of trus^4-15/Dftrus to the wild-type level and is shown as the positive control (purple). trus^4-15/Dftrus larvae with the UAS-dilp8RNAi transgene without driver serve as the negative control (blue).

https://doi.org/10.1371/journal.pgen.1011469.g005

To examine directly whether the Xrp1-Dilp8 signaling pathway is involved in the developmental delay of trus mutants, we individually knocked down Xrp1 and dilp8 in trus^4-15/Dftrus mutants by RNAi induced with daughterless-GAL4, a ubiquitous driver. We found that Xrp1RNAi and dilp8RNAi accelerated the trus mutant’s pupariation timing by 4 and 5 days, respectively, compared to the control (Fig 5D). With both Xrp1RNAi and dilp8RNAi, however, the majority of animals were pre-pupal lethal the same as trus^4-15/Dftrus mutants without RNAi.

We next asked if in addition to rescue of developmental delay, whether knockdown of Xrp1 or dilp8 could also rescue the CNS and imaginal disc cell proliferation defects. We noted that when the Xrp1RNAi rescue crosses were kept on vial food, some of the da > Xrp1RNAi; trus^4-15/Dftrus pre-pupae wandered out of the food and developed to pupae, and a few became pharate adults that did not eclose. This indicates some rescue of the metamorphosis defect since normally trus null mutants are 100% pre-pupal lethal and remain close to the food. When we examined the brain and imaginal discs from da > Xrp1RNAi; trus^4-15/Dftrus third instar wandering larvae with anti-phospho-Histone H3 and DAPI staining, surprisingly, in 10 dissected larvae, cell proliferation in all the brains was significantly rescued compared to the original trus^4-15/Dftrus brains (S5A Fig), although wing disc defects were only moderately rescued (S5B Fig). We also looked at brain and discs of Dilp8RNAi induced trus^4-15/Dftrus third instar wandering larvae. Again, cell proliferation in the brain lobes appeared to be moderately rescued comparing to original trus^4-15/Dftrus mutant (S6A Fig) but not as much as the Xrp1RNAi induced larvae (S5A Fig). In addition, wing discs defects were not rescued at all by dilp8RNAi (S6B Fig). These results suggest that the both the tissue growth/cell proliferation defect and the developmental delay are caused to a large extent by the activation of the Xrp1-Dilp8 stress response pathway.

Developmental delay of trus mutants is rescued by ecdysone feeding

To demonstrate directly that Xrp1-Dilp8 stress response signal induced in trus mutants is causing developmental delay by inhibiting ecdysone production, we fed trus mutant larvae with 20-hydroxyecdysone (20E) or its precursor ecdysone. The feeding scheme is outlined in Fig 6A. We found that feeding 20E to w¹¹¹⁸ larvae did not affect the pupariation timing (green in Fig 6B) compared to the controls fed with water (blue) or ethanol (red), while feeding the precursor ecdysone slightly accelerated pupariation and reduced overall pupariation rate by 10% (purple) compared to the control or 20E-fed larvae (Fig 6B). This data is consistent with the results reported previously by Ono (2014) where it was shown that feeding ecdysone to wild type larvae after L3 ecdysis accelerated pupariation by 6–12 hours and increased larval lethality [58].

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Fig 6. Ecdysone feeding to trus mutant larvae accelerates pupariation timing but causes precocious pupariation and does not rescue the pre-pupal lethality.

(A) Schedule of the ecdysone-feeding experiment. The larvae were fed mashed regular cornmeal fly food that was mixed with 20E or ecdysone dissolved in solvent (ethanol) beginning at the 1st instar stage (1 day AEL). For the controls, fly food was mixed with either water or ethanol only. (B) Pupariation timing of w¹¹¹⁸ larvae fed cornmeal fly food mixed with either ecdysone (purple), 20-hydroxyecdysone (20E) (green), dH₂O (blue), or ethanol (red). (C) Pupariation timing of trus^4-15/Dftrus larvae fed cornmeal food mixed with either ecdysone (purple), 20E (green), dH₂O (blue), or ethanol (red). Pupariation timing of w¹¹¹⁸ fed cornmeal food mixed with dH₂O (light blue), or ethanol (orange) are from the same data set shown in B. Each circle represents the average percentage of pupariated larvae from multiple dishes of the same genotype. Pupariated larvae were counted every 24 hours. Standard deviations are shown as vertical lines for each data point in B and C. (D) Pre-pupae that were fed cornmeal food mixed with either dH₂O, ethanol, 20E, or ecdysone with the time of pupariation indicated below. w¹¹¹⁸ pupa is shown on the left for size comparison.

https://doi.org/10.1371/journal.pgen.1011469.g006

When we examined trus^4-15/Dftrus larvae fed with regular cornmeal food supplied with water (blue) or ethanol (red), they pupariated at 9–12 days AEL, while w¹¹¹⁸ larvae pupariated at 5–6 days AEL. 20E-fed larvae pupariated at 7–11 days AEL, and ecdysone-fed larvae pupariated at 6–7 days AEL (purple) (Fig 6C). Interestingly, feeding ecdysone was much more effective in rescuing the developmental delay of trus mutants than feeding 20E, and as suggested by Ono (2014), this may indicate that ecdysone has an unknown function in regulating developmental timing separately from 20E. Despite rescue of the developmental delay, 100% of the trus^4-15/Dftrus animals that were fed 20E or ecdysone died as pre-pupae without developing into pupae. Notably, however those 20E-fed or ecdysone-fed pre-pupae were significantly smaller than the control mutant larvae (Fig 6D) likely due to accelerated development, as has been noted previously in ecdysone feeding experiments [58]. We conclude that the developmental delay in trus mutant larvae is caused by ecdysone deficiency as a result of induction of the Xrp1-Dilp8 stress response pathway.

The core structure of Trus protein and its orthologs are conserved

Fig 7A shows the domain structures of Drosophila Trus and Zfrp8 together with several vertebrate and yeast orthologs. Trus and Zfrp8 have highly conserved N-terminal (shown in green and light blue) and C-terminal (shown in magenta) globular domains. The C-terminal conserved domain was previously relegated to the PDCD2L/PDCD2 protein superfamily and named PDCD2_C in the Pfam protein database [59]. We call the N-terminal conserved domain PDCD2_N in this paper. AlphaFold prediction of the Drosophila Trus 3D structure shown in Fig 7B [60,61] reveals a core module that includes two β-sheets facing each other, one consists of three β-strands from PDCD2_N domain (green) and the other consists of four β-strands from PDCD2_C domain (magenta), and a pair of interacting β-strands (light blue and blue) (Fig 7B and 7C).

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Fig 7. Predicted 3D Structure of Trus and its paralog Zfrp8 share a core module that is conserved through evolution.

(A) Domain structure comparison of Drosophila Trus, its paralog Zfrp8, and their orthologs from different organisms. D. melanogaster Trus (Accession number: Q9VG62; Dmel\CG5333), Homo sapiens_PDCD2L (Q9BRP1), Danio retio_PDCD2L (Q5RGB3), S. cerevisiae_Tsr4 (P87156), D. melanogaster_Zfrp8 (Q9W1A3), Homo sapiens_PDCD2 (Q16342), and Danio retio_PDCD2 (Q1MTH6) are shown. Green: PDCD2_N, magenta: PDCD2_C, red: MYND-type zinc finger domain (Znf-MYND), light blue: first β-strand in PDCD2_N domain, blue: β-strand that is predicted to interact with the first β-strand, gray: unstructured loop. (B) D. melanogaster Trus protein 3D structure predicted by AlphaFold (https://alphafold.ebi.ac.uk/). Color-coding of domains are the same as shown in A. The PDCD2_N consists of four β-strands from which three β-strands form a β-sheet structure immediately followed by an α-helix (shown in green). In addition, the first β-strand in the PDCD2_N domain (residues 9-16, shown in light blue) is predicted to form hydrogen bonds with another β-strand (residues 307-317, shown in blue) in the middle of the loop region between the domain PDCD2_N and the domain PDCD2_C. (C) Predicted Aligned Error (PAE) of Drosophila Trus 3-D structure calculated by the AlphaFold. Color-coded bars representing the Trus protein domains shown in B are placed on upper and right sides of the panel. The PAE indicates high confidence in the relative position of scored residues 1-109 (PDCD2_N; shown in light blue and green) when aligned with residues 382-485 (PDCD2_C; shown in magenta), supporting the packing between these regions that form a structural module despite the large unstructured loops (gray) that separate the PDCD2_N and PDCD2_C domains. The PAE of the blue β−strands (residues 307-317) against both the N-terminal domain (PDCD2_N, residues 1-109) and the C-terminal domain (PDCD2_C, residues 382-485) shows high confidence, indicating packing of a core module that includes the PDCD2_N (light blue and green, residues 1-109), the PDCD2_C (magenta, residue 382-485), and a β-strand in-between (blue, residues 307-317). (D) Structural alignment of Dm Trus and Hs PDCD2L shows evolutional conservation of the core module in the PDCD2L protein family. (E) Structural alignment of Dm Zfrp9 and Hs PDCD2 shows evolutional conservation of the core module in the PDCD2 protein family. 3-D structural alignments were performed with PyMOL (Schrödinger LLC., NY).

https://doi.org/10.1371/journal.pgen.1011469.g007

Notably, structural alignments of Drosophila Trus against human PDCD2L (Fig 7D), zebrafish PDCD2L (S7C Fig), and Saccharomyces cerevisiae TSR4 (S7D Fig) show striking conservations of the core module through evolution. Structural alignment of Drosophila Zfrp8 against human PDCD2 indicates that the core structural module is also conserved through evolution in the Zfrp8/PDCD2 orthologs (Fig 7E). We also note that AlphaFold predictions demonstrate that the core structural module is well aligned between Trus/PDCD2L and Zfrp8/PDCD2 paralogs (S7B Fig), with the latter lacking the α-helix at the end of the PDCD2_N domain having instead a MYND-type zinc finger domain in the loop region outside of the core module that was previously shown to be involved in protein-protein interactions [62] (shown in red in Figs 7E, and S7A and S7B). The core module of the PDCD2L/PDCD2 protein superfamily has been previously annotated as a TYPP domain by Burroughs and Aravind (2014) using extensive comparative genome sequence and structure analytical techniques [39]. The conservation of the core module among TSR4, PDCD2L and PDCD2 orthologs suggests that these proteins exhibit similar biochemical properties, whereas the structural divergence between PDCD2L and PDCD2 paralogs suggests that PDCD2L and PDCD2 may control specific functions or steps within the highly complex process of ribosomal biogenesis (Figs 7 and S7).

Trus is expressed at high levels in larval mitotic tissues

We performed in situ hybridization with an anti-sense trus RNA probe to examine trus mRNA expression in 3rd instar wandering stage larval tissues. As shown in Fig 8, we detected trus expression at high levels in larval gut, ovary, brain lobe, wing disc, and lymph gland, where active cell proliferation is happening [63–66]. We also detected trus expression to a lesser extent in the salivary and prothoracic glands where cells are non-mitotic [67,68].

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Fig 8. trus expresses in mitotic tissues.

In situ hybridization with anti-sense RNA that hybridizes with trus mRNA reveals high level expression of trus in larval lymph gland, ovary, wing disc, gut, and brain lobe. Low expression is detected in ring and salivary glands.

https://doi.org/10.1371/journal.pgen.1011469.g008

Trus localizes to the cytoplasm and is exported from the nucleus in a CRM1/XPO1-dependent manner

Landry-Voyer et al. [37] reported that PDCD2L, the human ortholog of Trus, primarily localizes to the cytoplasm but shuttles between the nucleus and the cytoplasm, and the exportation from the nucleus is dependent on CRM1/XPO1, the RanGTP-binding exportin, which recognizes a leucine-rich nuclear export signal (NES) sequence. They showed that while PDCD2L primarily localizes to the cytoplasm, inhibiting CRM1 with Leptomycin B (LMB) or mutating the predicted NES sequence leads to retention of the protein in the nucleus [37]. We investigated Trus protein localization in S2 cells and in vivo and found that Drosophila Trus protein localizes in the same way as the human PDCD2L. As shown in Fig 9A, EGFP-tagged Trus protein expressed in Drosophila S2 cells primarily localized to the cytoplasm (No LMB); however, after Leptomycin B treatment, EGFP-Trus became more concentrated in the nucleus, and longer LMB treatment was more effective (LMB 15min vs. LMB 115min), indicating that Trus shuttles between the nucleus and the cytoplasm in CRM1-dependent manner. We searched for a putative NES in Trus, based on NES consensus sequences reported in Kosugi et al.(2008) [69], and found at least 6 candidates in the loop region between the PDCD2_N and the PDCD2_C domains, which is much longer in Trus (273 a.a.) than human PDCD2L (132 a.a.) (shown in gray in Fig 7A and 7B). Further investigations by mutating the candidates individually or in combinations are necessary to determine which NES candidate(s) is the Trus NES(s).

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Fig 9. Trus localizes to the cytoplasm in cultured cells and in vivo.

(A) Drosophila Trus localizes to the cytoplasm in S2 cells and shuttles between the nucleus and the cytoplasm in a CRM1/XPO1-dependent manner. Leptomycin B (LMB) is an inhibitor of CRM1/XPO1. Without LMB treatment (top row), EGFP-Trus expressed in S2 cells localizes primarily to the cytoplasm, and after treatment of the cells with LMB (middle and bottom rows), EGFP-Trus accumulates in the nucleus (LMB 15min, LMB 115min). Maximum intensity Z-projections are shown. Bar: 20μm. (B) da-GAL4 induced UAS-EGFP-trus expression in larval tissues. da-GAL4 expression is ubiquitous in most of the tissues. EGFP-Trus primarily localizes in the cytoplasm of cells (b, f, and j). Representative images of a wing disc (a-d), brain (e-h), and prothoracic grand (i-k) are shown. (b-d) and (f-h) are magnified images of the area marked with a yellow square in a and e, respectively. Each image is a single Z slice from multiple Z series confocal scanning data. Scale bar: 200μm.

https://doi.org/10.1371/journal.pgen.1011469.g009

To examine cellular localization of Trus in vivo, we over-expressed EGFP-tagged Trus using UAS-EGFP-trus driven by da-GAL4. EGFP-Trus was expressed ubiquitously in larval tissues, the over-expression did not affect viability of the flies and did not cause any defects in larval tissues. As shown in Fig 9B, EGFP-Trus localizes primarily to the cytoplasm within the epithelial cells in wing discs (Fig 9B, a–d), neurons in the brain (e-h), and cells in prothoracic gland (i-k). We confirmed that da > UAS-EGFP-trus rescues trus mutant lethality and developmental delay similar to da > UAS-trus, indicating that EGFP-Trus is functional (S3 Table). Our results suggest that the PDCD2L protein family including Drosophila Trus may work as a nucleus to cytoplasm shuttling transporter within the cell [29,37].

Identifying the tissues that require Trus protein activity during development

To determine which tissue(s) require Trus function during development, we expressed Trus protein in trus mutant (trus^4-15/Dftrus) animals from a UAS-trus transgene using various GAL4 drivers and examined whether trus developmental delay or lethality was rescued (Fig 10A and Table 3). Trus expression with a ubiquitously expressed daughterless-GAL4 (da-GAL4) rescued both developmental delay and lethality of trus^4-15/Dftrus animals (Fig 10A shown in blue). The eclosed adult flies had no defects and were fully fertile.

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Table 3. Rescue summary of trus^4-15/Dftrus developmental timing and lethality.

https://doi.org/10.1371/journal.pgen.1011469.t003

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Fig 10. Rescue of lethality and developmental delay of the trus mutant can be achieved by Trus expression induced with specific drivers.

(A) Developmental timing of trus mutants (trus^4-15/Dftrus) with trus expression driven by various drivers display varying degrees of rescue. Compared to the w¹¹¹⁸ control (orange), da-Gal4 (dark blue) rescues the best, with en-Gal4 and ci-Gal4 (aqua), en-Gal4 (green), and ci-Gal4 (purple) showing progressively lower degrees of rescue. ci-Gal4 without UAS-trus (red) serves as the negative control. (B) Adult and pupal phenotypes of rescued lines. ci > UAS-trus rescued trus^4-15/Dftrus pre-pupal lethality with significant defects in wings, halteres, legs, and bristles (upper panels). Many flies eclose only half-way from the pupal case and die (lower left). trus^4-15/Dftrus mutants without the UAS-trus transgene arrest and die during the pre-pupal stage (lower right). (C) (a-f, c’, and f’) Ovariole phenotypes of rescued lines confirms that da > UAS-trus rescue of trus^4-15/Dftrus mutant females. They are fertile and show no defects in oogenesis. The yellow star in d indicates a mature egg produced. (g-m, i’, and m’) en,ci > UAS-trus in combination with trus^4-15/Dftrus mutants, while being able to rescue mutant lethality, give rise to females that are sterile. Their ovarioles produce no mature eggs because egg chambers degrade at mid-oogenesis. (green) Rhodamine-Phalloidin, (blue) DAPI. For each genotype, 10 ovaries from 10 individual female were dissected and all showed the similar phenotypes. Maximum intensity Z-projections are shown. Scale bar: 200μm.

https://doi.org/10.1371/journal.pgen.1011469.g010

Trus expression driven by ci-GAL4 or en-GAL4 accelerated pupariation timing by up to 3 or 5 days, respectively, compared to the control, and a combination of en-GAL4 and ci-GAL4 accelerated pupariation timing by 6 days (Fig 10A). Since en-GAL4 and ci-GAL4 alone express in complementary halves of each imaginal disc, rescue of developmental timing with Trus expression using both drivers may work in a synergetic way (Fig 10A). We also found that nub-GAL4 and pdm2-GAL4, which are strongly expressed in the wing pouch but not in a major part of the leg discs or the hinge/notum of wing discs, rescued neither the developmental delay nor lethality of the trus mutant (Table 3). This is not surprising since Dilp8 is likely to be strongly expressed in the notum and hinge area of wing discs, leg discs, and other discs in trus mutants when Trus is expressed only in wing pouch using nub-GAL4 or pdm2-GAL4.

Importantly, while either en-GAL4 and ci-GAL4 driven Trus expression could individually rescue the developmental delay of trus mutants, the lethality of trus mutants was rescued only by ci-GAL4. Most of the trus mutant larvae with ci > UAS-trus pupated and reached the pharate stage. 18% of the animals eclosed or halfway-eclosed as adult flies with severe defects in legs and wings (Fig 10B and Table 3). We examined larval brain and wing disc for cell proliferation and tissue structures. As shown in S8A Fig, mitotic cells (PH3 foci) in brain lobes and VNC as well as the size and ring-structure of the brain lobes from ci > UAS-trus; trus^4-15/Dftrus larvae look similar to those from w¹¹¹⁸. Cell proliferation and tissue growth of wing/haltere/leg discs also appeared to be substantially rescued and were similar in size to those of w¹¹¹⁸ larvae (S8B Fig). We noticed that PH3 foci are enriched in the half of each wing/haltere/leg disc, suggesting a cell-autonomous induction of mitosis as ci-GAL is expressed only in the anterior half of the discs therefore could induce Trus expression primally in the anterior half of the discs (S3 and S8B Figs).

As we have shown (S3 Fig), expression of en-GAL4 and ci-GAL4 complement each other in wing and leg discs (i-m and n-q in S3 Fig); however, their brain expression patterns are distinctly different. Notably, ci-GAL4 is strongly expressed in a large number of cells in brain lobes including differentiating neural epithelia cells in optic lobes (n and o in S3 Fig), whereas en-GAL4 expression in brain is limited to some cells in the VNC (i and j in S3 Fig). We speculate that it is this difference in central nervous system (CNS) expression of ci-GAL4 vs en-GAL4 that accounts for the rescue of trus lethality by one and not the other.

Trus is essential for oogenesis

In our rescue experiments, we expressed Trus using a combination of en-GAL and ci-GAL4 together. This double driver further accelerated pupariation timing up to 6 days (light blue in Fig 10A) and significantly rescued trus mutant lethality. 41% of 1st instar larvae eclosed as adult flies with no morphological defects (Table 3). However, we found that the eclosed females were completely sterile and laid no eggs. To determine when the block occurs in oogenesis, we dissected their ovaries and stained them with Rhodamine-Phalloidin and DAPI, As shown in Fig 10C, in en,ci > UAS-trus rescued ovaries, a normal number of egg chambers seemed to form, but nurse cell nuclei started to show abnormal morphologies at early stages and severe aggregation and fragmentation of the nuclear DNA by mid-oogenesis (stage 5/6) (Fig 10C, g-m, i’, and m’). Additionally, follicle cells that normally surround the developing nurse cells and oocyte allow the egg chamber to hold its structure and change its shape from spheroid to elliptical as seen in the control (Fig 10C, a–f, c’, and f’), also deteriorate and degrade by stage 5/6 in en,ci > UAS-trus rescue (see Fig 10C, i’ and m’ comparing to c’ and f’). Oogenesis seemed to arrest and egg chambers degraded completely after Stage5/6. Mature eggs always seen at the end of the egg chambers in the control ovaries (Fig 10C, d, an egg shown with yellow star) did not exist in ovaries derived from en,ci > UAS-trus rescued females. High-throughput transcriptome analyses have reported that both engrailed (en) and cubitus interruptus (ci) are not expressed or have very low expression in ovaries (http://flybase.org). Therefore, we infer that, although en-GAL4 and ci-GAL4 zygotically expressed UAS-trus and rescued trus mutant (trus^4-15/Dftrus) lethality, the drivers did not express UAS-trus in ovaries, and thus did not rescue the Trus function that is essential for oogenesis.

Trus is found in a complex with SOP/RpS2 and eEF1α1

To search for possible Trus interactors, we used the Tap-tagging system described by Veraksa et al. (2005) [73], which identifies at least transiently stable protein complexes. Drosophila Kc cultured cells expressing a Tap-Trus fusion were lysed, and interacting proteins were isolated by affinity chromatography against the tag. The final eluate from the affinity column had two major bands and one band of somewhat lower intensity that were not found in controls (S9 Fig). One of these bands was (as expected) Trus itself; this band assignment was verified both by Western blotting with anti-Trus antibody and by mass spectrometry. Using mass-spectrometry, the other major band (band 3 on S9 Fig) is identified as String of pearls (Sop), protein S2 of the 40S ribosomal subunit (RpS2) [30], while band 2 is identified as eukaryotic translation Elongation Factor 1 alpha 1 (eEF1α1: CG8280), a factor that plays a role in shuttling tRNAs to the ribosome during translation [74]. To the best of our knowledge eEF1α1 has not been previously associated with either PDCD2L or PDCD2 containing complexes. RpS2/Sop has been reported as a binding partner of Zfrp8 [42]. However, we did not find Zfrp8 in our Trus pulldown, suggesting Trus and Zfrp8 are not in a same protein complex supporting the idea that they play different roles in ribosome biogenesis and development [29].

Fly lines

Detailed genotype and sources of fly stocks that were used in this study are listed in S1 Table. Flies were reared on standard cornmeal fly medium at either 18, 25°C, or room temperature depending on the purpose.

Production of trus CRISPR/Cas9 mutants

trus mutants were produced using the CRISPR/Cas9 system. Two target sequences in the trus gene (CG5333) that are unlikely to have off-target binding were identified using CRISPR Optimal Target Finder (http://targetfinder.flycrispr.neuro.brown.edu/) [80]. Sense and antisense double strands of the targets A and B (see S2 Table) were cloned into pU6-BbsI-chiRNA (Addgene #45946) [46] for production of single stranded guide RNAs (sgRNAs) and were co-injected into vas-Cas9 embryos (BDSC_51323) by BestGene Inc. (Chino Hills CA). Single G0 adults were crossed to w¹¹¹⁸ and then 10 F1 males were crossed to w^-; Bl/CyOGFP; TM2/TM6B P[Dfd-GMR-nvYPF]SbTb. From the established lines balanced over TM6B P[Dfd-GMR-nvYPF]SbTb, homozygous lethal lines were selected as trus null mutant candidates. Genomic DNA from balanced adults or homozygous larvae from each candidate line was prepared [81] and a 1.7kb trus genomic region was amplified by PCR (Expand High Fidelity PCR System, Millipore Sigma, Burlington, MA) with two primers, Trus2_for and Trus1_rev (S2 Table). Of the 5 pupal lethal lines from G0 #35, 4 had the same deletion of 1184 bp. Line 35–2 was selected and is hereafter called trus^35-2. Of the 5 pupal lethal lines from G0 #4, 2 had the same 1 bp deletion at target A and were designed trus^4-15. Genomic DNA from trus¹/ TM6B P[Dfd-GMR-nvYPF]SbTb adults was also prepared and sequenced as described above with Trus1_for, Trus3_rev, TrusB_for, and TrusC_for primers.

Trus antibody production

Full length Trus protein N-terminally tagged with 6xHis-EGFP (His-EGFP-Trus) was expressed in Sf9 cultured insect cells using the Bac-to-Bac Baculovirus Expression System (Thermo Fisher Scientific, Waltham MA). The trus coding sequence, codon-optimized for insect cell expression and N-terminally tagged with 6xHis-EGFP, was cloned into pFastBac1 (pFB-His-EGFP- HRV3C -trus) using NEBuilder High-Fidelity DNA Assembly (New England Biolabs, Ipswich MA). A HRV3C cleavage target sequence (3C) was inserted between EGFP and Trus. The plasmid was transformed into DH10Bac E. coli. cells and recombinant bacmids were isolated. Sf9 cells were seeded in a 6-well plate and transfected with the His-EGFP- HRV3C -Trus bacmid using CellFectinII (Thermo Fischer Scientific). The transfected cells were cultured at 28°C for 72 hrs. The supernatant (P0 virus stock) was collected and subsequently used to infect 10mL Sf9 cells to produce a P1 virus stock; the P1 was used to produce a 100mL P2 virus stock, and the P2 was used to produce a 300mL P3 virus stock. Virus infection of each amplification stage was confirmed under an optical microscope and His-EGFP-3C -Trus protein production was monitored by EGFP expression under a fluorescent microscope. Virus titer was measured for the P3 virus stock by Expression Systems (Davis CA) and used for subsequent His-EGFP-Trus protein expression. His-EGFP-3C-Trus recombinant protein was expressed in 2L of Sf9 cells, and the cell lysate was applied to a TALON CellThru metal affinity resin column (Takada Bio USA Inc., San Jose CA). The column was washed, and His-EGFP-3C-Trus protein was eluted with 150mM imidazole. The protein elution was monitored with EGFP fluorescence, and EGFP- positive fractions were collected and combined. The combined elution was incubated with HisGST-HRV3C protease to cut the His-EGFP tag at the 3C site, concentrated using Amicon Ultra-15 (MilliporeSigma, Burlington, MA), and incubated overnight at 4°C. It was then buffer-exchanged to lower imidazole concentration to 10mM using a Sephadex G-25/PD-10 desalting column (Cytiva, Marlborough MA) and combined with HisPur Ni-NTA resin (Thermo Fisher Scientific) and incubated at 4°C for 3 hours. The protein-resin solution was centrifuged, and the supernatant that contained Trus protein was collected, concentrated with Amicon Ultra-15, and loaded onto a Superdex-200 gel filtration column (MilliporeSigma). Trus protein containing fractions were combined, buffer-exchanged to Phosphate Buffer Saline (PBS, pH7.4) and concentrated. This concentrated purified Trus fraction was used to inject two rabbits to produce polyclonal anti-Trus antibody by Thermo Fisher Scientific. Anti-Trus antibody (AB2866) was affinity-purified from serum obtained from one of the rabbits with bacterially expressed recombinant Trus protein using AminoLink Plus Coupling Resin (Thermo Fisher Scientific) and was used for Western blotting (WB) at 1:2000.

Other antibodies and cell staining reagents

Phospho-histone H3 (Ser10) antibody (Cell Signaling Technology, Danvers MA) was used for IF at 1:1000 and α-Tubulin antibody (DM1A) (MilliporeSigma, T9026) was used for WB at 1:5000. Alexa 488-Goat anti-Rabbit IgG (H + L) and Alexa 555-Goat anti-Mouse IgG (H + L) Secondary Antibodies were used at 1:500–1:1000 for IF (Cell Signaling Technology). DyLight 680-Goat anti-Mouse IgG (H + L) and DyLight 800-Goat anti-Rabbit IgG(H + L) Secondary Antibodies were used at 1:10000 for WB (Thermo Fisher Scientific). For DNA staining, 4’,6-diamidino-2-phenylindole (DAPI) was used at 1.25mg/ml (MilliporeSigma), for F-actin staining, Rhodamine-Phalloidin was used (Thermo Fisher Scientific).

Western blotting

Wandering 3rd instar larvae of w¹¹¹⁸ and trus mutants were collected and homogenized in SDS-PAGE sample buffer, boiled, and centrifuged to remove debris. Samples were run on 4–12% NuPAGE Bis-Tris Mini Protein gels (Thermo Fisher Scientific) together with the purified recombinant Trus protein fraction and the separated proteins were transferred to Polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with x1/10 1xPBS1%Casein Blocker (BIO-RAD, Hercules CA), probed with purified rabbit anti-Trus antibody (this study) and mouse anti-α-Tubulin antibody (MilliporeSigma, T9026), washed, and then detected with DyLight secondary antibodies (Thermo Fisher Scientific). Finally, the membrane was washed and scanned with the Odyssey M imaging system (LICORbio, Lincoln NE).

Immuno-fluorescence staining, imaging, and image analysis

Third instar wandering stage larvae were roughly, turned inside-out and removed most of the gut and the fat tissues removed in PBS, fixed with 3.2% EM grade Paraformaldehyde (Electron Microscopy Sciences, Hatfield PA) in PBS for 25 minutes, washed multiple times with PBST (0.3% Triton-X100, 1x PBS) on rotary shaker at room temperature. They were then incubated with primary antibody solution overnight at 4°C, washed with PBST and incubated with Alexa Fluor conjugated secondary antibody solution (Thermo Fisher Scientific, Waltham MA) overnight at 4°C. Next day, they were rinsed with PBST, incubated with DAPI and/or Rhodamine-phalloidin for 5min. at RT, washed again multiple times with PBST. Brain and imaginal discs were dissected and mounted on a slide glass with anti-fading 1,4-Diazabicyclo[2.2.2]octane (DABCO) containing glycerin/PBS solution. Images were acquired with a Zeiss LSM 710 laser scanning confocal microscope using Zen imaging software (Zeiss, Jena Germany). To scan Drosophila tissues, 20x Plan-Apochromat/0.8 objective was used. An Argon laser, a HeNe laser, or a 405nm Diode laser were used to detect Alexa Fluor 488 and EGFP, Alexa Fluor 555 and mRFP, or DAPI, respectively. Typically, 25–40 Z slices with 1.20μm interval covering the entire thickness/stack of brain or disc (pressed to 25–45μm) were scanned and ImageJ/Fiji [82] (https://imagej.net/software/fiji/) was used to construct a maximum intensity Z-projection from the Z slices. Further image analysis and quantifications were performed using ImageJ/Fiji as well. Based on the image analyses, box-and-whisker plots were generated and ANOVA analysis followed by Dunnett’s test to compare each mutant to qwild-type (w1118) were preformed using Prism10 (Graph Pad software, Inc., Boston, MA.

In situ hybridization

To make the trus antisense in situ probe, the pFLC-1-trus cDNA clone (BDGP RE69372) was linearized with Eco53K1 and transcribed using DIG RNA Labeling Mix with T3 RNA polymerase (Roche, Indianapolis IN) according to manufacturer’s instructions. Wandering larvae of w¹¹¹⁸ and trus¹ were collected, dissected, and fixed. Hybridization using the digoxigenin-labeled antisense trus RNA probe and detection were performed as described previously [83,84].

Generating UAS-trus and UAS-EGFP-trus transgenics

To generate UAS-trus and UAS-EGFP-trus transgenics, the Trus coding sequence from an EST plasmid (Berkeley Drosophila Genome Project: RE69372) [85] and EGFP coding sequence from pEGFP-c1 (Clontech) were cloned into pUAST.attB [86] using NEBuilder HiFi DNA Assembly (New England Biolabs) and injected into embryos derived from a cross between flies with the attP recombination site inserted at 2L-VK00037 (BDSC_9752) and the dPhiC31 integrase source (PhiC31 integrase source; BDSC_40161) for phiC31-mediated site-specific recombination [86] and screened for w+ transgenics that integrated UAS-trus or UAS-EGFP-trus (BestGene Inc., Chino Hills CA).

Developmental timing assay

trus^4-15, trus^35-2, and Dftrus chromosomes were balanced over TM6B P[Dfd-GMR-nvYPF]Sb. As the Dfd-GMR-nvYPF fluorescent marker shows distinctive eye expression from embryonic stage 13 to adulthood, it is easy to score at the early larval stage [87]. These flies were crossed in different combinations. Embryos were collected on apple juice plates for 12–24 hours. 30–50 1st instar larvae (24–36 hours AEL) were screened for Dfd-GMR-nvYFP negative under a fluorescent dissecting microscope to obtain 1st instar larvae of trus^4-15/Dftrus, trus^35-2/Dftrus, trus^4-15/ trus^35-2, trus^4-15 homozygous, or trus^35-2 homozygous and transferred to a standard cornmeal-agar fly medium in a 60mm culture dish. Another 60mm culture dish with a 20mm hole in the center covered with nylon mesh was placed as a lid and tightly taped together. 3–4 plates (total of at least 150 larvae) for each genotype were prepared and placed in humidified chambers at 25°C. Each plate was observed under a dissecting microscope throughout the subsequent development, and pupariation, pupation, and adult eclosion were assessed once every 24 hours for 10–18 days.

Ecdysone feeding assay

α-Ecdysone (MilliporeSigma E9004) and 20-Hydroxyecdysone (MilliporeSigma H5142) were dissolved in ethanol at 10mg/mL and used as stock solutions. 50μL of either (1) water, (2) ethanol, (3) α-Ecdysone stock solution, or (4) 20-Hydroxyecdysone stock solution were mixed with 2g of mashed standard cornmeal/agar fly medium and placed in the center of 60mm plastic culture dish. The supplemented fly medium was air-dried for 2–3 hours to allow ethanol to evaporate. Cages were set up for w¹¹¹⁸ and a cross of trus^4-15/TM6SbTbHuYFP x Dftrus/TM6SbTbHuYFP, and embryos were collected on apple juice agar plates for 12–24 hours. 50 first instar larvae of w¹¹¹⁸ or trus mutant (nvYFP negative) were transferred to the prepared supplemented or control media for following developmental timing (Fig 6A).

Trus rescue experiment

To perform Trus expression rescue experiments, the UAS-trus transgene on the second chromosome was combined with Dftrus (Df(3R)BSC847) on the third chromosome that was balanced with TM6B P[Dfd-GMR-nvYPF] Sb [87]. On the other hand, each of the GAL4 lines were combined with the trus^4-15 allele that was maintained over TM6B P[Dfd-GMR-nvYPF] Sb (ex. w¹¹¹⁸; ***-GAL4; trus^4-15/ TM6B P[Dfd-GMR-nvYPF]SbTb or w¹¹¹⁸;; ***-GAL4, trus^4-15/ TM6B P[Dfd-GMR-nvYPF] Sb). Then, w¹¹¹⁸; UAS-trus; Dftrus/ TM6B P[Dfd-GMR-nvYPF] Sb flies were crossed with each of the GAL4 fly line bearing the trus ^4-15 allele. Embryos were collected on an apple juice plate, 1st instar larvae without the nvYFP marker (***-GAL4 > UAS-trus in the trus^4-15/Dftrus background) were collected, transferred to regular cornmeal fly food in a 60mm dish and monitoring for developmental timing.

RNAi

To knockdown dilp8 or Xrp1 in a trus mutant, UAS-dilp8RNAi or UAS-Xrp1RNAi on the second chromosome was combined with Dftrus on TM6B P[Dfd-GMR-nvYPF]SbTb balancer chromosome to make w¹¹¹⁸; UAS-dilp8RNAi; Dftrus/ TM6B P[Dfd-GMR-nvYPF]SbTb and w¹¹¹⁸; UAS-Xrp1RNAi; Dftrus/ TM6B P[Dfd-GMR-nvYPF]SbTb. Those flies were crossed with w¹¹¹⁸;; da-GAL4, trus^4-15/ TM6B P[Dfd-GMR-nvYPF]SbTb to ubiquitously induce the RNAi. 1st instar larvae negative for nvYFP were transferred to 60mm culture dishes that contain regular fly food at 24–36 hours AEL for following developmental timing. To knockdown trus, each GAL4 driver line was crossed with a fly line bearing the UAS-trusRNAi and UAS-dicer2 transgenes. 250–400 1st instar larvae of nub-GAL4 > UAS-trusRNAi, pdm2-GAL4 > UAS-trusRNAi, or en-GAL4 > UAS-trusRNAi were collected along with controls (w¹¹¹⁸ and UAS-trusRNAi without a driver) and monitored for developmental timing.

Trus localization in S2 cells

To express N-terminal EGFP-tagged Trus protein in S2 cells, the 6xHis-EGFP-HRV3C and Trus coding sequences (BDGP: RE69372) were assembled together on the pMT-puro vector using NEBuilder HiFi DNA Assembly (New England Biolabs). EGFP-tagged Trus expression induced with da-GAL4 in vivo completely rescued the trus mutant (trus^4-15/Dftrus) phenotypes similarly to un-tagged Trus (S3 Table), indicating that N-terminal EGFP tagging does not disrupt Trus function. S2 cells were transfected with the pMT-HisEGFP-Trus-puro plasmid and a stably transfected cell line was established by a few weeks of puromycin selection. The stably transfected S2 cells were cultured in 12-well plates and induced EGFP-Trus expression was induced with 0.7mM CuSO4. At day3, the cells were suspended in flesh media and a Concanavalin A-coated cover glass was placed in each well and left for 30 min. The wells were treated with or without 10nM Leptomycin B (InvivoGen, San Diego CA). The cover glasses were removed from the well at either 15 mins. or 115 mins., and S2 cells on each cover glass were immediately fixed with 3.2% paraformaldehyde/PBS, washed, stained with DAPI, and rinsed. Each cover glass was mounted on a slide glass facing up-side-down with DABCO/glycerol/PBS anti-fade mounting media. Edges of the cover glass were sealed with nail polish. EGFP and DAPI signals were scanned with the LSM710 laser confocal scanning microscope (Zeiss).

Purification of protein complexes containing Trus

The coding sequence of trus from cDNA clone RE69372 was cloned into pMK33-NTAP [73], and the resulting constructs were stably transfected into Drosophila Kc cells using CellFectin (Thermo Fischer Scientific). The TAP-Trus fusion protein was assayed on Western blots using HRP-conjugated anti-Protein A antibody (Rockland, Gilbertsville PA) and by immunofluorescence of fixed cells using goat anti-Protein A antibody at 1:1000 followed by TRITC-conjugated anti-goat antibody (Jackson Laboratories, Bar Harbor ME) at a dilution of 1:500. Protein complexes from one liter of TAP- Trus-expressing cells were isolated following Puig et al., 2001 [88], using a lysis buffer for making Drosophila extracts [73]. After purification using IgG-Sepharose and Calmodulin-Sepharose beads (Thermo Fisher Scientific - Invitrogen), the final eluate was precipitated with trichloroacetic acid (TCA), resolubilized in Laemmli sample buffer (BIO-RAD) and subjected to SDS-PAGE. Bands were excised, trypsinized and analyzed by MALDI (Matrix-assisted laser desorption/ionization) mass spectrometry (Cornell Institute of Biotechnology) [89].