Ptth regulates lifespan through innate immunity pathway in Drosophila.

The prothoracicotropic hormone (Ptth) is well-known for its important role in controlling insect developmental timing and body size by promoting the biosynthesis and release of ecdysone. However, the role of Ptth in adult physiology is largely unexplored. Here we show that Ptth null mutants (both males and females) show extended lifespan and healthspan, and exhibit increased resistance to oxidative stress. Transcriptomic analysis reveals that age-dependent upregulation of innate immunity pathway is attenuated by Ptth mutants. Intriguingly, we find that Ptth regulates the innate immunity pathway, specifically in fly oenocytes, the homology of mammalian hepatocytes. We further show that oenocyte-specific overexpression of Relish shortens the lifespan, while oenocyte-specific downregulation of ecdysone signaling extends lifespan. Consistently, knocking down torso, the receptor of Ptth in the prothoracic gland also promotes longevity of the flies. Thus, our data reveal a novel function of the insect hormone Ptth in longevity regulation and innate immunity in adult Drosophila.

Endoreplication in the Drosophila melanogaster prothoracic gland is dispensable for the critical weight checkpoint.

Critical weight (CW) attainment is a key life event in the development of holometabolous insects including Drosophila melanogaster. It indicates that sufficient growth has occurred to initiate the juvenile-to-adult transition. The prothoracic gland (PG), the major insect larval endocrine organ, is a polyploid tissue that plays a key role in the determination of CW via release of the steroid hormone ecdysone. Here we show that when the cells of the PG fail to make the mitotic-to-endocycle switch, but instead remain mitotic, the result is more but smaller cells. Nevertheless, they reach the same CW and produce healthy adults after only a moderate developmental delay. We propose that the CW checkpoint can be reached by either an endocycling or mitotic PG and may simply reflect the attainment of sufficient ecdysone biosynthetic capacity to initiate metamorphosis.

The cytochrome P450 Cyp6t3 is not required for ecdysone biosynthesis in Drosophila melanogaster.

The steroid hormone 20-hydroxyecdysone (20E) is essential for proper development and the timing of intermediary stage transitions in insects. As a result, there is intense interest in identifying and defining the roles of the enzymes and signaling pathways that regulate 20E production in the prothoracic gland (PG), the major endocrine organ of juvenile insect phases. Transcriptomics is one powerful tool that has been used to identify novel genes that are up- or down-regulated in the PG which may contribute to 20E regulation. Additional functional characterization of putative regulatory candidate genes typically involves qRT-PCR and/or RNAi mediated knockdown of the candidate mRNA in the PG to assess whether the gene's expression shows temporal regulation in the PG and whether its expression is essential for proper 20E production and the correct timing of developmental transitions. While these methods have proved fruitful for identifying novel regulators of 20E production, characterizing the null phenotype of putative regulatory genes is the gold standard for assigning gene function since RNAi is known to generate various types of "off target" effects. Here we describe the genetic null mutant phenotype of the Drosophila melanogaster Cyp6t3 gene . Cyp6t3 was originally identified as a differentially regulated gene in a PG microarray screen and assigned a place in the "Black Box" step of the E biosynthetic pathway based on RNAi mediated knockdown phenotypes and rescue experiments involving feeding of various intermediate compounds of the E biosynthetic pathway. In contrast, we find that Crispr generated null mutations in Cyp6t3 are viable and have normal developmental timing. Therefore, we conclude that Cyp6t3 is not required for E production under typical lab growth conditions and therefore is not an obligate enzymatic component of the Black Box.

A juxtamembrane basolateral targeting motif regulates signaling through a TGF-ß pathway receptor in Drosophila.

In polarized epithelial cells, receptor-ligand interactions can be restricted by different spatial distributions of the 2 interacting components, giving rise to an underappreciated layer of regulatory complexity. We explored whether such regulation occurs in the Drosophila wing disc, an epithelial tissue featuring the TGF-ß family member Decapentaplegic (Dpp) as a morphogen controlling growth and patterning. Dpp protein has been observed in an extracellular gradient within the columnar cell layer of the disc, but also uniformly in the disc lumen, leading to the question of how graded signaling is achieved in the face of 2 distinctly localized ligand pools. We find the Dpp Type II receptor Punt, but not the Type I receptor Tkv, is enriched at the basolateral membrane and depleted at the junctions and apical surface. Wit, a second Type II receptor, shows a markedly different behavior, with the protein detected on all membrane regions but enriched at the apical side. Mutational studies identified a short juxtamembrane sequence required for basolateral restriction of Punt in both wing discs and mammalian Madin-Darby canine kidney (MDCK) cells. This basolateral targeting (BLT) determinant can dominantly confer basolateral localization on an otherwise apical receptor. Rescue of punt mutants with transgenes altered in the targeting motif showed that flies expressing apicalized Punt due to the lack of a functional BLT displayed developmental defects, female sterility, and significant lethality. We also show that apicalized Punt does not produce an ectopic signal, indicating that the apical pool of Dpp is not a significant signaling source even when presented with Punt. Instead, we find that basolateral presentation of Punt is required for optimal signaling. Finally, we present evidence that the BLT acts through polarized sorting machinery that differs between types of epithelia. This suggests a code whereby each epithelial cell type may differentially traffic common receptors to enable distinctive responses to spatially localized pools of extracellular ligands.

The NDNF-like factor Nord is a Hedgehog-induced extracellular BMP modulator that regulates Drosophila wing patterning and growth.

Hedgehog (Hh) and Bone Morphogenetic Proteins (BMPs) pattern the developing Drosophila wing by functioning as short- and long-range morphogens, respectively. Here, we show that a previously unknown Hh-dependent mechanism fine-tunes the activity of BMPs. Through genome-wide expression profiling of the Drosophila wing imaginal discs, we identify nord as a novel target gene of the Hh signaling pathway. Nord is related to the vertebrate Neuron-Derived Neurotrophic Factor (NDNF) involved in congenital hypogonadotropic hypogonadism and several types of cancer. Loss- and gain-of-function analyses implicate Nord in the regulation of wing growth and proper crossvein patterning. At the molecular level, we present biochemical evidence that Nord is a secreted BMP-binding protein and localizes to the extracellular matrix. Nord binds to Decapentaplegic (Dpp) or the heterodimer Dpp-Glass-bottom boat (Gbb) to modulate their release and activity. Furthermore, we demonstrate that Nord is a dosage-dependent BMP modulator, where low levels of Nord promote and high levels inhibit BMP signaling. Taken together, we propose that Hh-induced Nord expression fine-tunes both the range and strength of BMP signaling in the developing Drosophila wing.

New resources for the Drosophila 4th chromosome: FRT101F enabled mitotic clones and Bloom syndrome helicase enabled meiotic recombination.

Genes on the long arm of the Drosophila melanogaster 4th chromosome are difficult to study because the chromosome lacks mitotic and meiotic recombination. Without recombination numerous standard methods of genetic analysis are impossible. Here, we report new resources for the 4th. For mitotic recombination, we generated a chromosome with an FRT very near the centromere in 101F and a derivative that carries FRT101F with a distal ubiquitously expressed GAL80 transgene. This pair of chromosomes enables both unmarked and MARCM clones. For meiotic recombination, we demonstrate that a Bloom syndrome helicase and recombination defective double mutant genotype can create recombinant 4th chromosomes via female meiosis. All strains will be available to the community via the Bloomington Drosophila Stock Center. Additional resources for studies of the 4th are in preparation and will also be made available. The goal of the 4th Chromosome Resource Project is to accelerate the genetic analysis of protein-coding genes on the 4th, including the 44 genes with no demonstrated function. Studies of these previously inaccessible but largely conserved genes will close longstanding gaps in our knowledge of metazoan development and physiology.

AKH Signaling in D. melanogaster Alters Larval Development in a Nutrient-Dependent Manner That Influences Adult Metabolism.

Metabolism, growth, and development are intrinsically linked, and their coordination is dependent upon inter-organ communication mediated by anabolic, catabolic, and steroid hormones. In Drosophila melanogaster, the corpora cardiaca (CC) influences metabolic homeostasis through adipokinetic hormone (AKH) signaling. AKH has glucagon-like properties and is evolutionarily conserved in mammals as the gonadotropin-releasing hormone, but its role in insect development is unknown. Here we report that AKH signaling alters larval development in a nutrient stress-dependent manner. This activity is regulated by the locus dg2, which encodes a cGMP-dependent protein kinase (PKG). CC-specific downregulation of dg2 expression delayed the developmental transition from larval to pupal life, and altered adult metabolism and behavior. These developmental effects were AKH-dependent, and were observed only in flies that experienced low nutrient stress during larval development. Calcium-mediated vesicle exocytosis regulates ecdysteroid secretion from the prothoracic gland (PG), and we found that AKH signaling increased cytosolic free calcium levels in the PG. We identified a novel pathway through which PKG acts in the CC to communicate metabolic information to the PG via AKH signaling. AKH signaling provides a means whereby larval nutrient stress can alter developmental trajectories into adulthood.

Prothoracicotropic hormone modulates environmental adaptive plasticity through the control of developmental timing.

Adult size and fitness are controlled by a combination of genetics and environmental cues. In Drosophila, growth is confined to the larval phase and final body size is impacted by the duration of this phase, which is under neuroendocrine control. The neuropeptide prothoracicotropic hormone (PTTH) has been proposed to play a central role in controlling the length of the larval phase through regulation of ecdysone production, a steroid hormone that initiates larval molting and metamorphosis. Here, we test this by examining the consequences of null mutations in the Ptth gene for Drosophila development. Loss of Ptth causes several developmental defects, including a delay in developmental timing, increase in critical weight, loss of coordination between body and imaginal disc growth, and reduced adult survival in suboptimal environmental conditions such as nutritional deprivation or high population density. These defects are caused by a decrease in ecdysone production associated with altered transcription of ecdysone biosynthetic genes. Therefore, the PTTH signal contributes to coordination between environmental cues and the developmental program to ensure individual fitness and survival.

Glue protein production can be triggered by steroid hormone signaling independent of the developmental program in Drosophila melanogaster.

Steroid hormones regulate life stage transitions, allowing animals to appropriately follow a developmental timeline. During insect development, the steroid hormone ecdysone is synthesized and released in a regulated manner by the prothoracic gland (PG) and then hydroxylated to the active molting hormone, 20-hydroxyecdysone (20E), in peripheral tissues. We manipulated ecdysteroid titers, through temporally controlled over-expression of the ecdysteroid-inactivating enzyme, CYP18A1, in the PG using the GeneSwitch-GAL4 system in the fruit fly Drosophila melanogaster. We monitored expression of a 20E-inducible glue protein gene, Salivary gland secretion 3 (Sgs3), using a Sgs3:GFP fusion transgene. In wild type larvae, Sgs3-GFP expression is activated at the midpoint of the third larval instar stage in response to the rising endogenous level of 20E. By first knocking down endogenous 20E levels during larval development and then feeding 20E to these larvae at various stages, we found that Sgs3-GFP expression could be triggered at an inappropriate developmental stage after a certain time lag. This stage-precocious activation of Sgs3 required expression of the Broad-complex, similar to normal Sgs3 developmental regulation, and a small level of nutritional input. We suggest that these studies provide evidence for a tissue-autonomic regulatory system for a metamorphic event independent from the primary 20E driven developmental progression.

The Drosophila Zinc Finger Transcription Factor Ouija Board Controls Ecdysteroid Biosynthesis through Specific Regulation of spookier.

Steroid hormones are crucial for many biological events in multicellular organisms. In insects, the principal steroid hormones are ecdysteroids, which play essential roles in regulating molting and metamorphosis. During larval and pupal development, ecdysteroids are synthesized in the prothoracic gland (PG) from dietary cholesterol via a series of hydroxylation and oxidation steps. The expression of all but one of the known ecdysteroid biosynthetic enzymes is restricted to the PG, but the transcriptional regulatory networks responsible for generating such exquisite tissue-specific regulation is only beginning to be elucidated. Here, we report identification and characterization of the C2H2-type zinc finger transcription factor Ouija board (Ouib) necessary for ecdysteroid production in the PG in the fruit fly Drosophila melanogaster. Expression of ouib is predominantly limited to the PG, and genetic null mutants of ouib result in larval developmental arrest that can be rescued by administrating an active ecdysteroid. Interestingly, ouib mutant animals exhibit a strong reduction in the expression of one ecdysteroid biosynthetic enzyme, spookier. Using a cell culture-based luciferase reporter assay, Ouib protein stimulates transcription of spok by binding to a specific ~15 bp response element in the spok PG enhancer element. Most remarkable, the developmental arrest phenotype of ouib mutants is rescued by over-expression of a functionally-equivalent paralog of spookier. These observations imply that the main biological function of Ouib is to specifically regulate spookier transcription during Drosophila development.

UPRT, a suicide-gene therapy candidate in higher eukaryotes, is required for Drosophila larval growth and normal adult lifespan.

Uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage pathway enzyme that catalyzes the conversion of uracil to uridine monophosphate (UMP). The enzyme is highly conserved from prokaryotes to humans and yet phylogenetic evidence suggests that UPRT homologues from higher-eukaryotes, including Drosophila, are incapable of binding uracil. Purified human UPRT also do not show any enzymatic activity in vitro, making microbial UPRT an attractive candidate for anti-microbial drug development, suicide-gene therapy, and cell-specific mRNA labeling techniques. Nevertheless, the enzymatic site of UPRT remains conserved across the animal kingdom indicating an in vivo role for the enzyme. We find that the Drosophila UPRT homologue, krishah (kri), codes for an enzyme that is required for larval growth, pre-pupal/pupal viability and long-term adult lifespan. Our findings suggest that UPRT from all higher eukaryotes is likely enzymatically active in vivo and challenges the previous notion that the enzyme is non-essential in higher eukaryotes and cautions against targeting the enzyme for therapeutic purposes. Our findings also suggest that expression of the endogenous UPRT gene will likely cause background incorporation when using microbial UPRT as a cell-specific mRNA labeling reagent in higher eukaryotes.

R-Smad competition controls activin receptor output in Drosophila.

Animals use TGF-ß superfamily signal transduction pathways during development and tissue maintenance. The superfamily has traditionally been divided into TGF-ß/Activin and BMP branches based on relationships between ligands, receptors, and R-Smads. Several previous reports have shown that, in cell culture systems, "BMP-specific" Smads can be phosphorylated in response to TGF-ß/Activin pathway activation. Using Drosophila cell culture as well as in vivo assays, we find that Baboon, the Drosophila TGF-ß/Activin-specific Type I receptor, can phosphorylate Mad, the BMP-specific R-Smad, in addition to its normal substrate, dSmad2. The Baboon-Mad activation appears direct because it occurs in the absence of canonical BMP Type I receptors. Wing phenotypes generated by Baboon gain-of-function require Mad, and are partially suppressed by over-expression of dSmad2. In the larval wing disc, activated Baboon cell-autonomously causes C-terminal Mad phosphorylation, but only when endogenous dSmad2 protein is depleted. The Baboon-Mad relationship is thus controlled by dSmad2 levels. Elevated P-Mad is seen in several tissues of dSmad2 protein-null mutant larvae, and these levels are normalized in dSmad2; baboon double mutants, indicating that the cross-talk reaction and Smad competition occur with endogenous levels of signaling components in vivo. In addition, we find that high levels of Activin signaling cause substantial turnover in dSmad2 protein, providing a potential cross-pathway signal-switching mechanism. We propose that the dual activity of TGF-ß/Activin receptors is an ancient feature, and we discuss several ways this activity can modulate TGF-ß signaling output.

Prothoracicotropic hormone regulates developmental timing and body size in Drosophila.

In insects, control of body size is intimately linked to nutritional quality as well as environmental and genetic cues that regulate the timing of developmental transitions. Prothoracicotropic hormone (PTTH) has been proposed to play an essential role in regulating the production and/or release of ecdysone, a steroid hormone that stimulates molting and metamorphosis. In this report, we examine the consequences on Drosophila development of ablating the PTTH-producing neurons. Surprisingly, PTTH production is not essential for molting or metamorphosis. Instead, loss of PTTH results in delayed larval development and eclosion of larger flies with more cells. Prolonged feeding, without changing the rate of growth, causes the overgrowth and is a consequence of low ecdysteroid titers. These results indicate that final body size in insects is determined by a balance between growth-rate regulators such as insulin and developmental timing cues such as PTTH that set the duration of the feeding interval.