Multiple isoforms of the Activin-like receptor baboon differentially regulate proliferation and conversion behaviors of neuroblasts and neuroepithelial cells in the Drosophila larval brain.

In Drosophila coordinated proliferation of two neural stem cells, neuroblasts (NB) and neuroepithelial (NE) cells, is pivotal for proper larval brain growth that ultimately determines the final size and performance of an adult brain. The larval brain growth displays two phases based on behaviors of NB and NEs: the first one in early larval stages, influenced by nutritional status and the second one in the last larval stage, promoted by ecdysone signaling after critical weight checkpoint. Mutations of the baboon (babo) gene that produces three isoforms (BaboA-C), all acting as type-I receptors of Activin-type transforming growth factor ß (TGF-ß) signaling, cause a small brain phenotype due to severely reduced proliferation of the neural stem cells. In this study we show that loss of babo function severely affects proliferation of NBs and NEs as well as conversion of NEs from both phases. By analyzing babo-null and newly generated isoform-specific mutants by CRISPR mutagenesis as well as isoform-specific RNAi knockdowns in a cell- and stage-specific manner, our data support differential contributions of the isoforms for these cellular events with BaboA playing the major role. Stage-specific expression of EcR-B1 in the brain is also regulated primarily by BaboA along with function of the other isoforms. Blocking EcR function in both neural stem cells results in a small brain phenotype that is more severe than baboA-knockdown alone. In summary, our study proposes that the Babo-mediated signaling promotes proper behaviors of the neural stem cells in both phases and achieves this by acting upstream of EcR-B1 expression in the second phase.

Fourth Chromosome Resource Project: a comprehensive resource for genetic analysis in Drosophila that includes humanized stocks.

The fourth chromosome is the final frontier for genetic analysis in Drosophila. Small, heterochromatic, and devoid of recombination the fourth has long been ignored. Nevertheless, its long arm contains 79 protein-coding genes. The Fourth Chromosome Resource Project (FCRP) has a goal of facilitating the investigation of genes on this neglected chromosome. The project has 446 stocks publicly available at the Bloomington and Kyoto stock centers with phenotypic data curated by the FlyBase and FlyPush resources. Four of the five stock sets are nearly complete: (1) UAS.fly cDNAs, (2) UAS.human homolog cDNAs, (3) gene trap mutants and protein traps, and (4) stocks promoting meiotic and mitotic recombination on the fourth. Ongoing is mutagenesis of each fourth gene on a new FRT-bearing chromosome for marked single-cell clones. Beyond flies, FCRP facilitates the creation and analysis of humanized fly stocks. These provide opportunities to apply Drosophila genetics to the analysis of human gene interaction and function. In addition, the FCRP provides investigators with confidence through stock validation and an incentive via phenotyping to tackle genes on the fourth that have never been studied. Taken together, FCRP stocks will facilitate all manner of genetic and molecular studies. The resource is readily available to researchers to enhance our understanding of metazoan biology, including conserved molecular mechanisms underlying health and disease.

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.

Expression of Drosophila VDRC GD RNAi lines in larval skeletal muscle often leads to abnormal proteostasis.

In Drosophila , multiple transgenic RNAi libraries have been generated to facilitate large-scale genetic screens in vivo . Although those libraries have helped generate many new discoveries, certain libraries are associated with technical drawbacks requiring caution in interpreting the results. Here, we report an unexpected effect of VDRC GD lines on proteostasis. When expressed in the larval skeletal muscle, 17 out of 20 GD lines induced protein aggregates enriched around the myonuclei while VDRC KK or TRiP counterparts had no effect. By contrast, the same GD lines failed to induce protein aggregates when expressed in the epidermal cells. Because the GD lines tested in this study target diverse classes of molecules and since the KK or TRiP counterparts exhibited no effect, we conclude that VDRC GD lines, for unknown reasons, tend to interfere with proteostasis in a tissue-specific and target-independent manner.

Autophagy impairment and lifespan reduction caused by Atg1 RNAi or Atg18 RNAi expression in adult fruit flies (Drosophila melanogaster).

Autophagy, an autophagosome and lysosome-based eukaryotic cellular degradation system, has previously been implicated in lifespan regulation in different animal models. In this report, we show that expression of the RNAi transgenes targeting the transcripts of the key autophagy genes Atg1 or Atg18 in adult fly muscle or glia does not affect the overall levels of autophagosomes in those tissues and does not change the lifespan of the tested flies but the lifespan reduction phenotype has become apparent when Atg1 RNAi or Atg18 RNAi is expressed ubiquitously in adult flies or after autophagy is eradicated through the knockdown of Atg1 or Atg18 in adult fly adipocytes. Lifespan reduction was also observed when Atg1 or Atg18 was knocked down in adult fly enteroblasts and midgut stem cells. Overexpression of wild-type Atg1 in adult fly muscle or adipocytes reduces the lifespan and causes accumulation of high levels of ubiquitinated protein aggregates in muscles. Our research data have highlighted the important functions of the key autophagy genes in adult fly adipocytes, enteroblasts, and midgut stem cells and their undetermined roles in adult fly muscle and glia for lifespan regulation.

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.

New aspects of TGF-ß superfamily signaling in development and disease (2022 FASEB meeting review).

The 13th Federation of American Societies for Experimental Biology (FASEB) Summer Research Conference, "TGF-ß superfamily signaling in development and disease" was convened at the Grand Hotel in Malahide, Ireland in July 2022. The Transforming Growth Factor-ß (TGF-ß) family of secreted proteins consists of agents of intercellular communication found in all multicellular animals. Attending the meeting was a diverse group of scholars with shared interests in understanding TGF-ß signaling mechanisms, normal functions, and the diseases associated with misregulation and mutation. Despite intense study over the previous 35 years, new features of TGF-ß activity continue to be discovered. This meeting report offers 21 investigator-provided summaries that illustrate the breadth of the thought-provoking presentations. An emerging theme of the meeting was the power of cross-disciplinary studies, such as one combining immunology, biochemistry, and structural biology, to unravel the secrets of parasitic TGF-ß mimics. Please join us at the next meeting.

OrbNet Denali: A machine learning potential for biological and organic chemistry with semi-empirical cost and DFT accuracy.

We present OrbNet Denali, a machine learning model for an electronic structure that is designed as a drop-in replacement for ground-state density functional theory (DFT) energy calculations. The model is a message-passing graph neural network that uses symmetry-adapted atomic orbital features from a low-cost quantum calculation to predict the energy of a molecule. OrbNet Denali is trained on a vast dataset of 2.3 × 106 DFT calculations on molecules and geometries. This dataset covers the most common elements in biochemistry and organic chemistry (H, Li, B, C, N, O, F, Na, Mg, Si, P, S, Cl, K, Ca, Br, and I) and charged molecules. OrbNet Denali is demonstrated on several well-established benchmark datasets, and we find that it provides accuracy that is on par with modern DFT methods while offering a speedup of up to three orders of magnitude. For the GMTKN55 benchmark set, OrbNet Denali achieves WTMAD-1 and WTMAD-2 scores of 7.19 and 9.84, on par with modern DFT functionals. For several GMTKN55 subsets, which contain chemical problems that are not present in the training set, OrbNet Denali produces a mean absolute error comparable to those of DFT methods. For the Hutchison conformer benchmark set, OrbNet Denali has a median correlation coefficient of R2 = 0.90 compared to the reference DLPNO-CCSD(T) calculation and R2 = 0.97 compared to the method used to generate the training data (ωB97X-D3/def2-TZVP), exceeding the performance of any other method with a similar cost. Similarly, the model reaches chemical accuracy for non-covalent interactions in the S66x10 dataset. For torsional profiles, OrbNet Denali reproduces the torsion profiles of ωB97X-D3/def2-TZVP with an average mean absolute error of 0.12 kcal/mol for the potential energy surfaces of the diverse fragments in the TorsionNet500 dataset.

Coordination among multiple receptor tyrosine kinase signals controls Drosophila developmental timing and body size.

In holometabolous insects, metamorphic timing and body size are controlled by a neuroendocrine axis composed of the ecdysone-producing prothoracic gland (PG) and its presynaptic neurons (PGNs) producing PTTH. Although PTTH/Torso signaling is considered the primary mediator of metamorphic timing, recent studies indicate that other unidentified PGN-derived factors also affect timing. Here, we demonstrate that the receptor tyrosine kinases anaplastic lymphoma kinase (Alk) and PDGF and VEGF receptor-related (Pvr), function in coordination with PTTH/Torso signaling to regulate pupariation timing and body size. Both Alk and Pvr trigger Ras/Erk signaling in the PG to upregulate expression of ecdysone biosynthetic enzymes, while Alk also suppresses autophagy by activating phosphatidylinositol 3-kinase (PI3K)/Akt. The Alk ligand Jelly belly (Jeb) is produced by the PGNs and serves as a second PGN-derived tropic factor, while Pvr activation mainly relies on autocrine signaling by PG-derived Pvf2 and Pvf3. These findings illustrate that a combination of juxtacrine and autocrine signaling regulates metamorphic timing, the defining event of holometabolous development.

Drosophila MOV10 regulates the termination of midgut regeneration.

The molecular mechanisms by which stem cell proliferation is precisely controlled during the course of regeneration are poorly understood. Namely, how a damaged tissue senses when to terminate the regeneration process, inactivates stem cell mitotic activity, and organizes ECM integrity remain fundamental unanswered questions. The Drosophila midgut intestinal stem cell (ISC) offers an excellent model system to study the molecular basis for stem cell inactivation. Here, we show that a novel gene, CG6967 or dMOV10, is induced at the termination stage of midgut regeneration, and shows an inhibitory effect on ISC proliferation. dMOV10 encodes a putative component of the microRNA (miRNA) gene silencing complex (miRISC). Our data, along with previous studies on the mammalian MOV10, suggest that dMOV10 is not a core member of miRISC, but modulates miRISC activity as an additional component. Further analyses identified direct target mRNAs of dMOV10-containing miRISC, including Daughter against Dpp (Dad), a known inhibitor of BMP/TGF-ß signaling. We show that RNAi knockdown of Dad significantly impaired ISC division during regeneration. We also identified six miRNAs that are induced at the termination stage and their potential target transcripts. One of these miRNAs, mir-1, is required for proper termination of ISC division at the end of regeneration. We propose that miRNA-mediated gene regulation contributes to the precise control of Drosophila midgut regeneration.

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.

Control of the insect metamorphic transition by ecdysteroid production and secretion.

Ecdysteroids are a class of steroid hormones that controls molting and metamorphic transitions in Ecdysozoan species including insects, in which ecdysteroid biosynthesis and its regulation have been extensively studied. Insect ecdysteroids are produced from dietary sterols by a series of reduction-oxidation reactions in the prothoracic gland and in Drosophila they are released into the hemolymph via vesicle-mediated secretion at the time of metamorphosis. To initiate precisely controlled ecdysteroid pulses, the prothoracic gland functions as a central node integrating both intrinsic and extrinsic signals to control ecdysteroid biosynthesis and secretion. In this review, we outline recent progress in the characterization of ecdysone biosynthesis and steroid trafficking pathways and the discoveries of novel factors regulating prothoracic gland function.

Drosophila Activin signaling promotes muscle growth through InR/TORC1-dependent and -independent processes.

The Myostatin/Activin branch of the TGF-ß superfamily acts as a negative regulator of vertebrate skeletal muscle size, in part, through downregulation of insulin/insulin-like growth factor 1 (IGF-1) signaling. Surprisingly, recent studies in Drosophila indicate that motoneuron-derived Activin signaling acts as a positive regulator of muscle size. Here we demonstrate that Drosophila Activin signaling promotes the growth of muscle cells along all three axes: width, thickness and length. Activin signaling positively regulates the insulin receptor (InR)/TORC1 pathway and the level of Myosin heavy chain (Mhc), an essential sarcomeric protein, via increased Pdk1 and Akt1 expression. Enhancing InR/TORC1 signaling in the muscle of Activin pathway mutants restores Mhc levels close to those of the wild type, but only increases muscle width. In contrast, hyperactivation of the Activin pathway in muscles increases overall larval body and muscle fiber length, even when Mhc levels are lowered by suppression of TORC1. Together, these results indicate that the Drosophila Activin pathway regulates larval muscle geometry and body size via promoting InR/TORC1-dependent Mhc production and the differential assembly of sarcomeric components into either pre-existing or new sarcomeric units depending on the balance of InR/TORC1 and Activin signals.

Histone Carbonylation Is a Redox-Regulated Epigenomic Mark That Accumulates with Obesity and Aging.

Oxidative stress is a hallmark of metabolic disease, though the mechanisms that define this link are not fully understood. Irreversible modification of proteins by reactive lipid aldehydes (protein carbonylation) is a major consequence of oxidative stress in adipose tissue and the substrates and specificity of this modification are largely unexplored. Here we show that histones are avidly modified by 4-hydroxynonenal (4-HNE) in vitro and in vivo. Carbonylation of histones by 4-HNE increased with age in male flies and visceral fat depots of mice and was potentiated in genetic (ob/ob) and high-fat feeding models of obesity. Proteomic evaluation of in vitro 4-HNE- modified histones led to the identification of both Michael and Schiff base adducts. In contrast, mapping of sites in vivo from obese mice exclusively revealed Michael adducts. In total, we identified 11 sites of 4-hydroxy hexenal (4-HHE) and 10 sites of 4-HNE histone modification in visceral adipose tissue. In summary, these results characterize adipose histone carbonylation as a redox-linked epigenomic mark associated with metabolic disease and aging.

The Role of Muscle in Insect Energy Homeostasis.

Maintaining energy homeostasis is critical for ensuring proper growth and maximizing survival potential of all organisms. Here we review the role of somatic muscle in regulating energy homeostasis in insects. The muscle is not only a large consumer of energy, it also plays a crucial role in regulating metabolic signaling pathways and energy stores of the organism. We examine the metabolic pathways required to supply the muscle with energy, as well as muscle-derived signals that regulate metabolic energy homeostasis.

Training atomic neural networks using fragment-based data generated in virtual reality.

The ability to understand and engineer molecular structures relies on having accurate descriptions of the energy as a function of atomic coordinates. Here, we outline a new paradigm for deriving energy functions of hyperdimensional molecular systems, which involves generating data for low-dimensional systems in virtual reality (VR) to then efficiently train atomic neural networks (ANNs). This generates high-quality data for specific areas of interest within the hyperdimensional space that characterizes a molecule's potential energy surface (PES). We demonstrate the utility of this approach by gathering data within VR to train ANNs on chemical reactions involving fewer than eight heavy atoms. This strategy enables us to predict the energies of much higher-dimensional systems, e.g., containing nearly 100 atoms. Training on datasets containing only 15k geometries, this approach generates mean absolute errors around 2 kcal mol-1. This represents one of the first times that an ANN-PES for a large reactive radical has been generated using such a small dataset. Our results suggest that VR enables the intelligent curation of high-quality data, which accelerates the learning process.