Steroid hormone ecdysone deficiency stimulates preparation for photoperiodic reproductive diapause.

Diapause, a programmed developmental arrest primarily induced by seasonal environmental changes, is very common in the animal kingdom, and found in vertebrates and invertebrates alike. Diapause provides an adaptive advantage to animals, as it increases the odds of surviving adverse conditions. In insects, individuals perceive photoperiodic cues and modify endocrine signaling to direct reproductive diapause traits, such as ovary arrest and increased fat accumulation. However, it remains unclear as to which endocrine factors are involved in this process and how they regulate the onset of reproductive diapause. Here, we found that the long day-mediated drop in the concentration of the steroid hormone ecdysone is essential for the preparation of photoperiodic reproductive diapause in Colaphellus bowringi, an economically important cabbage beetle. The diapause-inducing long-day condition reduced the expression of ecdysone biosynthetic genes, explaining the drop in the titer of 20-hydroxyecdysone (20E, the active form of ecdysone) in female adults. Application of exogenous 20E induced vitellogenesis and ovarian development but reduced fat accumulation in the diapause-destined females. Knocking down the ecdysone receptor (EcR) in females destined for reproduction blocked reproductive development and induced diapause traits. RNA-seq and hormone measurements indicated that 20E stimulates the production of juvenile hormone (JH), a key endocrine factor in reproductive diapause. To verify this, we depleted three ecdysone biosynthetic enzymes via RNAi, which confirmed that 20E is critical for JH biosynthesis and reproductive diapause. Importantly, impairing Met function, a component of the JH intracellular receptor, partially blocked the 20E-regulated reproductive diapause preparation, indicating that 20E regulates reproductive diapause in both JH-dependent and -independent manners. Finally, we found that 20E deficiency decreased ecdysis-triggering hormone signaling and reduced JH production, thereby inducing diapause. Together, these results suggest that 20E signaling is a pivotal regulator that coordinates reproductive plasticity in response to environmental inputs.

Snail synchronizes endocycling in a TOR-dependent manner to coordinate entry and escape from endoreplication pausing during the Drosophila critical weight checkpoint.

The final body size of any given individual underlies both genetic and environmental constraints. Both mammals and insects use target of rapamycin (TOR) and insulin signaling pathways to coordinate growth with nutrition. In holometabolous insects, the growth period is terminated through a cascade of peptide and steroid hormones that end larval feeding behavior and trigger metamorphosis, a nonfeeding stage during which the larval body plan is remodeled to produce an adult. This irreversible decision, termed the critical weight (CW) checkpoint, ensures that larvae have acquired sufficient nutrients to complete and survive development to adulthood. How insects assess body size via the CW checkpoint is still poorly understood on the molecular level. We show here that the Drosophila transcription factor Snail plays a key role in this process. Before and during the CW checkpoint, snail is highly expressed in the larval prothoracic gland (PG), an endocrine tissue undergoing endoreplication and primarily dedicated to the production of the steroid hormone ecdysone. We observed two Snail peaks in the PG, one before and one after the molt from the second to the third instar. Remarkably, these Snail peaks coincide with two peaks of PG cells entering S phase and a slowing of DNA synthesis between the peaks. Interestingly, the second Snail peak occurs at the exit of the CW checkpoint. Snail levels then decline continuously, and endoreplication becomes nonsynchronized in the PG after the CW checkpoint. This suggests that the synchronization of PG cells into S phase via Snail represents the mechanistic link used to terminate the CW checkpoint. Indeed, PG-specific loss of snail function prior to the CW checkpoint causes larval arrest due to a cessation of endoreplication in PG cells, whereas impairing snail after the CW checkpoint no longer affected endoreplication and further development. During the CW window, starvation or loss of TOR signaling disrupted the formation of Snail peaks and endocycle synchronization, whereas later starvation had no effect on snail expression. Taken together, our data demonstrate that insects use the TOR pathway to assess nutrient status during larval development to regulate Snail in ecdysone-producing cells as an effector protein to coordinate endoreplication and CW attainment.

The Drosophila CCR4-NOT complex is required for cholesterol homeostasis and steroid hormone synthesis.

CCR4-NOT is a highly conserved protein complex that regulates gene expression at multiple levels. In yeast, CCR4-NOT functions in transcriptional initiation, heterochromatin formation, mRNA deadenylation and other processes. The range of functions for Drosophila CCR4-NOT is less clear, except for a well-established role as a deadenylase for maternal mRNAs during early embryogenesis. We report here that CCR4-NOT has an essential function in the Drosophila prothoracic gland (PG), a tissue that predominantly produces the steroid hormone ecdysone. Interfering with the expression of the CCR4-NOT components twin, Pop2, Not1, and Not3 in a PG-specific manner resulted in larval arrest and a failure to initiate metamorphosis. Transcriptome analysis of PG-specific Pop2-RNAi samples revealed that Pop2 is required for the normal expression of ecdysone biosynthetic gene spookier (spok) as well as cholesterol homeostasis genes of the NPC2 family. Interestingly, dietary supplementation with ecdysone and its various sterol precursors showed that 7-dehydrocholesterol and cholesterol completely rescued the larval arrest phenotype, allowing Pop2-RNAi animals to reach pupal stage, and, to a low degree, even survival to adulthood, while the biologically active hormone, 20-Hydroxyecdysone (20E), was significantly less effective. Also, we present genetic evidence that CCR4-NOT has a nuclear function where CCR4-NOT-depleted cells exhibit aberrant chromatin and nucleoli structures. In summary, our findings indicate that the Drosophila CCR4-NOT complex has essential roles in the PG, where it is required for Drosophila steroid hormone production and cholesterol homeostasis, and likely has functions beyond a mere mRNA deadenylase in Drosophila.

The Drosophila DHR96 nuclear receptor binds cholesterol and regulates cholesterol homeostasis.

Cholesterol homeostasis is required to maintain normal cellular function and avoid the deleterious effects of hypercholesterolemia. Here we show that the Drosophila DHR96 nuclear receptor binds cholesterol and is required for the coordinate transcriptional response of genes that are regulated by cholesterol and involved in cholesterol uptake, trafficking, and storage. DHR96 mutants die when grown on low levels of cholesterol and accumulate excess cholesterol when maintained on a high-cholesterol diet. The cholesterol accumulation phenotype can be attributed to misregulation of npc1b, an ortholog of the mammalian Niemann-Pick C1-like 1 gene NPC1L1, which is essential for dietary cholesterol uptake. These studies define DHR96 as a central regulator of cholesterol homeostasis.

Endoplasmic reticulum-localized hepatic lipase decreases triacylglycerol storage and VLDL secretion.

Hepatic triacylglycerol levels are governed through synthesis, degradation and export of this lipid. Here we demonstrate that enforced expression of hepatic lipase in the endoplasmic reticulum in McArdle RH7777 hepatocytes resulted in a significant decrease in the incorporation of fatty acids into cellular triacylglycerol and cholesteryl ester accompanied by attenuation of secretion of apolipoprotein B-containing lipoproteins. Hepatic lipase-mediated depletion of intracellular lipid storage increased the expression of peroxisome proliferator-activated receptor α and its target genes and augmented oxidation of fatty acids. These data show that 1) hepatic lipase is active in the endoplasmic reticulum and 2) intracellular hepatic lipase modulates cellular lipid metabolism and lipoprotein secretion.

Nuclear receptor DHR4 controls the timing of steroid hormone pulses during Drosophila development.

In insects, precisely timed periodic pulses of the molting hormone ecdysone control major developmental transitions such as molts and metamorphosis. The synthesis and release of ecdysone, a steroid hormone, is itself controlled by PTTH (prothoracicotopic hormone). PTTH transcript levels oscillate with an 8 h rhythm, but its significance regarding the timing of ecdysone pulses is unclear. PTTH acts on its target tissue, the prothoracic gland (PG), by activating the Ras/Raf/ERK pathway through its receptor Torso, however direct targets of this pathway have yet to be identified. Here, we demonstrate that Drosophila Hormone Receptor 4 (DHR4), a nuclear receptor, is a key target of the PTTH pathway and establishes temporal boundaries by terminating ecdysone pulses. Specifically, we show that DHR4 oscillates between the nucleus and cytoplasm of PG cells, and that the protein is absent from PG nuclei at developmental times when low titer ecdysone pulses occur. This oscillatory behavior is blocked when PTTH or torso function is abolished, resulting in nuclear accumulation of DHR4, while hyperactivating the PTTH pathway results in cytoplasmic retention of the protein. Increasing DHR4 levels in the PG can delay or arrest development. In contrast, reducing DHR4 function in the PG triggers accelerated development, which is caused by precocious ecdysone signaling due to a failure to repress ecdysone pulses. Finally, we show that DHR4 negatively regulates the expression of a hitherto uncharacterized cytochrome P450 gene, Cyp6t3. Disruption of Cyp6t3 function causes low ecdysteroid titers and results in heterochronic phenotypes and molting defects, indicating a novel role in the ecdysone biosynthesis pathway. We propose a model whereby nuclear DHR4 controls the duration of ecdysone pulses by negatively regulating ecdysone biosynthesis through repression of Cyp6t3, and that this repressive function is temporarily overturned via the PTTH pathway by removing DHR4 from the nuclear compartment.

Drosophila Evi5 is a critical regulator of intracellular iron transport via transferrin and ferritin interactions.

Vesicular transport is essential for delivering cargo to intracellular destinations. Evi5 is a Rab11-GTPase-activating protein involved in endosome recycling. In humans, Evi5 is a high-risk locus for multiple sclerosis, a debilitating disease that also presents with excess iron in the CNS. In insects, the prothoracic gland (PG) requires entry of extracellular iron to synthesize steroidogenic enzyme cofactors. The mechanism of peripheral iron uptake in insect cells remains controversial. We show that Evi5-depletion in the Drosophila PG affected vesicle morphology and density, blocked endosome recycling and impaired trafficking of transferrin-1, thus disrupting heme synthesis due to reduced cellular iron concentrations. We show that ferritin delivers iron to the PG as well, and interacts physically with Evi5. Further, ferritin-injection rescued developmental delays associated with Evi5-depletion. To summarize, our findings show that Evi5 is critical for intracellular iron trafficking via transferrin-1 and ferritin, and implicate altered iron homeostasis in the etiology of multiple sclerosis.

Decapentaplegic retards lipolysis during metamorphosis in Bombyx mori and Drosophila melanogaster.

Insect morphogen decapentaplegic (Dpp) functions as one of the key extracellular ligands of the Bone Morphogenetic Protein (BMP) signaling pathway. Previous studies in insects mainly focused on the roles of Dpp during embryonic development and the formation of adult wings. In this study, we demonstrate a new role for Dpp in retarding lipolysis during metamorphosis in both Bombyx mori and Drosophila melanogaster. CRISPR/Cas9-mediated mutation of Bombyx dpp causes pupal lethality, induces an excessive and premature breakdown of lipids in the fat body, and upregulates the expressions of several lipolytic enzyme genes, including brummer (bmm), lipase 3 (lip3), and hormone-sensitive lipase (hsl), and lipid storage droplet 1 (lsd1), a lipid droplets (LD)-associated protein gene. Further investigation in Drosophila reveals that salivary gland-specific knockdown of the dpp gene and fat body-specific knockdown of Mad involved in Dpp signaling phenocopy the effects of Bombyx dpp mutation on pupal development and lipolysis. Taken together, our data indicate that the Dpp-mediated BMP signaling in the fat body maintains lipid homeostasis by retarding lipolysis, which is necessary for pupa-adult transition during insect metamorphosis.

Strain-specific modifier genes of Cecr2-associated exencephaly in mice: genetic analysis and identification of differentially expressed candidate genes.

Although neural tube defects (NTDs) are common in humans, little is known about their multifactorial genetic causes. While most mouse models involve NTDs caused by a single mutated gene, we have previously described a multigenic system involving susceptibility to NTDs. In mice with a mutation in Cecr2, the cranial NTD exencephaly shows strain-specific differences in penetrance, with 74% penetrance in BALB/cCrl and 0% penetrance in FVB/N. Whole genome linkage analysis showed that a region of chromosome 19 was partially responsible for this difference in penetrance. We now reveal by genetic analysis of three subinterval congenic lines that the chromosome 19 region contains more than one modifier gene. Analysis of embryos showed that although a Cecr2 mutation causes wider neural tubes in both strains, FVB/N embryos overcome this abnormality and close. A microarray analysis comparing neurulating female embryos from both strains identified differentially expressed genes within the chromosome 19 region, including Arhgap19, which is expressed at a lower level in BALB/cCrl due to a stop codon specific to that substrain. Modifier genes in this region are of particular interest because a large portion of this region is syntenic to human chromosome 10q25, the site of a human susceptibility locus.

The DHR96 nuclear receptor regulates xenobiotic responses in Drosophila.

Exposure to xenobiotics such as plant toxins, pollutants, or prescription drugs triggers a defense response, inducing genes that encode key detoxification enzymes. Although xenobiotic responses have been studied in vertebrates, little effort has been made to exploit a simple genetic system for characterizing the molecular basis of this coordinated transcriptional response. We show here that approximately 1000 transcripts are significantly affected by phenobarbital treatment in Drosophila. We also demonstrate that the Drosophila ortholog of the human SXR and CAR xenobiotic receptors, DHR96, plays a role in this response. A DHR96 null mutant displays increased sensitivity to the sedative effects of phenobarbital and the pesticide DDT as well as defects in the expression of many phenobarbital-regulated genes. Metabolic and stress-response genes are also controlled by DHR96, implicating its role in coordinating multiple response pathways. This work establishes a new model system for defining the genetic control of xenobiotic stress responses.

Creatine supplementation prevents the accumulation of fat in the livers of rats fed a high-fat diet.

The aim of the present study was to examine the effects of creatine supplementation on liver fat accumulation induced by a high-fat diet in rats. Rats were fed 1 of 3 different diets for 3 wk: a control liquid diet (C), a high-fat liquid diet (HF), or a high-fat liquid diet supplemented with creatine (HFC). The C and HF diets contained, respectively, 35 and 71% of energy derived from fat. Creatine supplementation involved the addition of 1% (wt:v) of creatine monohydrate to the liquid diet. The HF diet increased total liver fat concentration, liver TG, and liver TBARS and decreased the hepatic S-adenosylmethionine (SAM) concentration. Creatine supplementation normalized all of these perturbations. Creatine supplementation significantly decreased the renal activity of l-arginine:glycine amidinotransferase and plasma guanidinoacetate and prevented the decrease in hepatic SAM concentration in rats fed the HF diet. However, there was no change in either the phosphatidylcholine:phosphatidylethanolamine (PE) ratio or PE N-methyltransferase activity. The HF diet decreased mRNA for PPARα as well as 2 of its targets, carnitine palmitoyltransferase and long-chain acylCoA dehydrogenase. Creatine supplementation normalized these mRNA levels. In conclusion, creatine supplementation prevented the fatty liver induced by feeding rats a HF diet, probably by normalization of the expression of key genes of ß-oxidation.

Krüppel homolog 1 regulates photoperiodic reproductive plasticity in the cabbage beetle Colaphellus bowringi.

Many insects exhibit reproductive plasticity where the photoperiod determines whether the insect becomes reproductively active or enters diapause. Adult reproductive diapause is a strategy that allows insects to survive harsh environmental conditions. A deficiency in juvenile hormone (JH) leads to reproductive diapause. However, little is known about the molecular mechanisms by which JH signaling regulates reproductive diapause. In this study, we used the cabbage beetle Colaphellus bowringi, a serious pest, to investigate the role of Krüppel homolog 1 (Kr-h1) in controlling photoperiodic plasticity of female reproduction. We focused on Kr-h1, since it acts as a key mediator of JH signaling. We show here that JH-Methoprene-tolerant signaling upregulated the expression of Kr-h1 in reproductively active C. bowringi females when reared under short day conditions. In the long day-treated diapausing females, Kr-h1 transcripts decreased dramatically. Interfering with Kr-h1 function repressed reproductive development by blocking vitellogenesis and ovarian growth. Further, Kr-h1 depletion induced other diapause-like traits, including elevated lipid accumulation and high expression of diapause-related genes. RNA-Seq showed that Kr-h1 played both activating and repressive roles, depending on whether downstream genes were acting in reproduction- or diapause pathways, respectively. Finally, we identified the DNA replication gene mini-chromosome maintenance 4 and two triacylglycerol lipase genes as critical downstream factors of Kr-h1 that are critical for reproductive plasticity in C. bowringi. These results reveal that Kr-h1 is a key component of the regulatory pathway that coordinates reproduction and diapause in insects in response to photoperiodic input.

Genome assembly and methylome analysis of the white wax scale insect provides insight into sexual differentiation of metamorphosis in hexapods.

Scale insects are hemimetabolous, showing "incomplete" metamorphosis and no true pupal stage. Ericerus pela, commonly known as the white wax scale insect (hereafter, WWS), is a wax-producing insect found in Asia and Europe. WWS displays dramatic sexual dimorphism, with notably different metamorphic fates in males and females. Males develop into winged adults, while females are neotenic and maintain a nymph-like appearance, which are flightless and remain stationary. Here, we report the de novo assembly of the WWS genome with a size of 638.30 Mbp (69.68 Mbp for scaffold N50) by PacBio sequencing and Hi-C. These data allowed us to perform a robust phylogenetic analysis comprising 24,923 gene orthogroups from 16 representative insect genomes. This analysis indicated that holometabola evolved from insects with incomplete metamorphosis in the Late Carboniferous, about 50 million years earlier than previously thought. To study the distinct developmental fates of males and females, we analysed the methylome landscape in either sex. Surprisingly, WWS displayed high methylation levels (4.42% for males) when compared to other insects. We observed differential methylation patterns in males and females for genes involved in steroid and sesquiterpenoid production as well as genes acting in fatty acid metabolism pathways. We measured titre profiles for ecdysone, the principal insect steroid hormone, and juvenile hormone (a sesquiterpenoid) in both males and females, which suggested that these hormones are the primary drivers of sexually dimorphic development. Our results provide a comprehensive genomic and epigenomic resource of scale insects that provide new insights into the evolution of metamorphosis and sexual dimorphism in insects.

Cecr2 mutations causing exencephaly trigger misregulation of mesenchymal/ectodermal transcription factors.

BACKGROUND: Over 200 mouse genes are associated with neural tube defects (NTDs), including Cecr2, the bromodomain-containing subunit of the CERF chromatin remodeling complex. METHODS: Gene-trap mutation Cecr2(Gt45Bic) results in 74% exencephaly (equivalent of human anencephaly) on the BALB/c strain. Gene expression altered during cranial neural tube closure by the Cecr2 mutation was identified through microarray analysis of 11-14 somites stage Cecr2(Gt45Bic)embryos. RESULTS: Analysis of Affymetrix Mouse 430 2.0 chips detected 60 transcripts up-regulated and 54 transcripts down-regulated in the Cecr2(Gt45Bic) embryos (fold > 1.5, p < 0.05). The Cecr2 transcript was reduced only approximately 7- to 14-fold from normal levels, suggesting the Cecr2(Gt45Bic) is a hypomorphic mutation. We therefore generated a novel Cecr2 null allele (Cecr2 (tm1.1Hemc)). Resulting mutants displayed a stronger penetrance of exencephaly than Cecr2(Gt45Bic) in both BALB/c and FVB/N strains, in addition to midline facial clefts and forebrain encephalocele in the FVB/N strain. The Cecr2 transcript is reduced 260-fold in the Cecr2(tm1.1Hemc) line. Subsequent qRT-PCR using Cecr2 (tm1.1Hemc) mutant heads confirmed downregulation of transcription factors Alx1/Cart1, Dlx5, Eya1, and Six1. CONCLUSIONS: As both Alx1/Cart1 and Dlx5 mouse mutations result in exencephaly, we hypothesize that changes in expression of these mesenchymal/ectodermal transcription factors may contribute to NTDs associated with Cecr2.

Spatial and temporal control of gene manipulation in Drosophila via drug-activated Cas9 nucleases.

Advances in CRISPR/Cas9 have revolutionized molecular biology and greatly facilitated the ability to manipulate gene function through the creation of precisely engineered mutants. We recently reported a collection of modular gateway-compatible Cas9/gRNA Drosophila lines to interfere with gene expression in a tissue-specific manner, including polytene tissues. However, most current in vivo CRISPR/Cas9 tools cannot temporally control the induction of Cas9 or gRNAs via external stimuli such as RU486. A drug-inducible CRISPR/Cas9 system would allow studying genes at later stages where early lethality is an issue. This would be especially useful when combined with tissue-specific expression of Cas9 or gRNAs, allowing for full spatiotemporal control. Here, we present a RU486-inducible version of Cas9 and also show that a Rapamycin-inducible Cas9, previously used in mammalian cell culture, works in Drosophila as well. Both RU486 and rapamycin-inducible Cas9 work in vivo and in Drosophila cell culture. We also present split Cas9 constructs for rapamycin-dependent gene disruption and activation. These approaches establish drug-inducible and thus temporally controlled CRISPR/Cas9 tools for gene disruption and expression in a living model organism. Our CRISPR/Cas9 vector collection can be easily adapted for any tissue and provides higher fidelity compared to RNAi approaches.

A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila.

Advances in CRISPR technology have immensely improved our ability to manipulate nucleic acids, and the recent discovery of the RNA-targeting endonuclease Cas13 adds even further functionality. Here, we show that Cas13 works efficiently in Drosophila, both ex vivo and in vivo. We test 44 different Cas13 variants to identify enzymes with the best overall performance and show that Cas13 could target endogenous Drosophila transcripts in vivo with high efficiency and specificity. We also develop Cas13 applications to edit mRNAs and target mitochondrial transcripts. Our vector collection represents a versatile tool collection to manipulate gene expression at the post-transcriptional level.

Microenvironmental analysis of two alternating hosts and their impact on the ecological adaptation of the horned sumac gall aphid Schlechtendalia chinensis (Hemiptera, Pemphiginae).

The aphid Schlechtendalia chinensis(Bell) induces horned galls on their primary host Rhus chinensis(Mill). These galls serve as closed habitats to support thousands of aphids per gall. Ecological parameters inside a gall are unknown. In this study, we showed that the microclimate inside galls was reltively stable, with nearly 100% humidity and 30-50 lux light regardless of outside environmental conditions. Gall-residing aphids produce waste gas and honeydew. A gall contained 26 organic volatiles inside with acetic acid as the largest component. Honeydew is rich in sugars and may provide nutrients for microbial growth. However, no evidence for pathogenic microorganisms was found inside a gall. The acidic environment in a gall may curb microbial growth. On the secondary host, the moss Plagiomnium maximoviczii (Lindb.) T. J. Kop., the microclimate is unstable and humidity fluctuated at 45~100%, while light ranged from 150 to 500 lux on different environmental conditions. Aphid alternated in two different habitats, the gall generation increased from a single fundatrix to thousands of aphids, however, survival rate of the moss generation is less 3%. A comparison of the environmental traits between gall and moss revealed that a stable habitat with dark and moist is advantageous for aphid reproduction.

A Complex Nutrient Exchange Between a Gall-Forming Aphid and Its Plant Host.

It has been a long-standing question as to whether the interaction between gall-forming insects and their host plants is merely parasitic or whether it may also benefit the host. On its host Rhus chinensis, the aphid Schlechtendalia chinensis induces the formation of closed galls, referred to as horned galls. Typically, mature aphid populations comprise thousands of individuals, which is sufficient to cause the accumulation of high CO2 levels in galls (on average 8-fold higher and up to 16 times than atmospheric levels). Large aphid populations also excrete significant amounts of honeydew, a waste product high in sugars. Based on 13C isotope tracing and genomic analyses, we showed that aphid-derived carbon found in CO2 and honeydew was recycled in gall tissues via photosynthesis and glycometabolism. These results indicated that the aphid-gall system evolved in a manner that allowed nutrient recycling, where the gall provides nutrients to the growing aphid population, and in turn, aphid-derived carbon metabolites provide a resource for the growth of the gall. The metabolic efficiency of this self-circulating system indicates that the input needed from the host plant to maintain aphid population growth less than previously thought and possibly minimal. Aside from the recycling of nutrients, we also found that gall metabolites were transported to other parts of the host plant and is particularly beneficial for leaves growing adjacent to the gall. Taken together, galls in the S. chinensis-Rhus chinensis system are highly specialized structures that serve as a metabolic and nutrient exchange hub that benefits both the aphid and its host plant. As such, host plants provide both shelter and nutrients to protect and sustain aphid populations, and in return, aphid-derived metabolites are channeled back to the host plant and thus provide a certain degree of "metabolic compensation" for their caloric and structural needs.

Glycogen branching enzyme controls cellular iron homeostasis via Iron Regulatory Protein 1 and mitoNEET.

Iron Regulatory Protein 1 (IRP1) is a bifunctional cytosolic iron sensor. When iron levels are normal, IRP1 harbours an iron-sulphur cluster (holo-IRP1), an enzyme with aconitase activity. When iron levels fall, IRP1 loses the cluster (apo-IRP1) and binds to iron-responsive elements (IREs) in messenger RNAs (mRNAs) encoding proteins involved in cellular iron uptake, distribution, and storage. Here we show that mutations in the Drosophila 1,4-Alpha-Glucan Branching Enzyme (AGBE) gene cause porphyria. AGBE was hitherto only linked to glycogen metabolism and a fatal human disorder known as glycogen storage disease type IV. AGBE binds specifically to holo-IRP1 and to mitoNEET, a protein capable of repairing IRP1 iron-sulphur clusters. This interaction ensures nuclear translocation of holo-IRP1 and downregulation of iron-dependent processes, demonstrating that holo-IRP1 functions not just as an aconitase, but throttles target gene expression in anticipation of declining iron requirements.

A Drosophila CRISPR/Cas9 Toolkit for Conditionally Manipulating Gene Expression in the Prothoracic Gland as a Test Case for Polytene Tissues.

Targeting gene function with spatial or temporal specificity is a key goal in molecular genetics. CRISPR-Cas9 has greatly facilitated this strategy, but some standard approaches are problematic. For instance, simple tissue-specific or global overexpression of Cas9 can cause significant lethality or developmental delays even in the absence of gRNAs. In particular, we found that Gal4-mediated expression of UAS-Cas9 in the Drosophila prothoracic gland (PG) was not a suitable strategy to disrupt gene expression, since Cas9 alone caused widespread lethality. The PG is widely used for studying endocrine gland function during animal development, but tools validating PG-specific RNAi phenotypes are lacking. Here, we present a collection of modular gateway-compatible CRISPR-Cas9 tools that allow precise modulation of target gene activity with temporal and spatial specificity. We also demonstrate that Cas9 fused to the progesterone ligand-binding domain can be used to activate gene expression via RU486. Using these approaches, we were able to avoid the lethality associated with simple GAL4-mediated overexpression of Cas9 in the PG. Given that the PG is a polytene tissue, we conclude that these tools work effectively in endoreplicating cells where Cas9 has to target multiple copies of the same locus. Our toolkit can be easily adapted for other tissues and can be used both for gain- and loss-of-function studies.