Locomotion is the most significant behavior that drives organismal design. However, the mechanisms that connect musculoskeletal morphology to diverse anatomical features along the body axis are poorly understood. Using Drosophilabody wall muscles as a model of position-dependent morphological diversification, we show the Hox transcription factor Ultrabithorax (Ubx) activates expression of the zinc-finger transcription factor Spalt major (Salm) to regulate muscle shape. Hox genes in the Antennapedia-complex (ANT-C) are expressed in the anterior of the embryo and Hox genes inthe Bithorax-complex (BX-C), including Ubx, are expressed in the posterior. Body wall muscle morphology abruptly changes at ANT-C/BX-C expression boundary in the mesoderm and shifting the ANT-C/BX-C boundary concomitantly shifted muscle morphology. We identified a salm embryonic enhancer that is regulated by Hox genes in the BX-C but not by Hox genes in the ANT-C. Salm in turn activates a cytoskeletal regulatory module that governs muscle shape but not muscle identity. A Hox-Salm morphogenetic gene regulatory network therefore links region-specific muscle morphology to the body plan, providing a mechanism to couple locomotion and organismal design.

Abstract Coagulation is related to inflammation, but the key pathway, especially innate immune system and coagulation regulation, is not well understood and need to be further explored. Here, we demonstrated that neutrophil gelatinase-associated lipocalin (NGAL), an innate immune inflammatory mediator, is upregulated in patients with thrombosis. Furthermore, it contributes to the initiation and amplification of coagulation, hemostasis, and thrombosis. This occurs by enhancing tissue factor expression on the cell surface, potentiating various clotting factors such as thrombin, kallikrein, factor XIa (FXIa), and FVIIa, promoting thrombin-induced platelet aggregation, and inhibiting antithrombin. NGAL knockout led to strikingly prolonged clot reaction time and kinetic time in thromboelastography analysis, along with reduced thrombus generation angle and lower thrombus maximum amplitude, which were in line with remarkably prolonged activated partial thromboplastin time and prothrombin time. In several mouse hemostasis and thrombosis models, NGAL overexpression or IV administration promoted coagulation and hemostasis and aggravated thrombosis, whereas NGAL knockout or treatment with anti-NGAL monoclonal antibody significantly prolonged bleeding time and alleviated thrombus formation. Notably, NGAL knockout prolonged mouse tail bleeding time or artery occlusion time to over 40 or 60 minutes, respectively, resembling uncontrollable bleeding and clotting disorder seen in hemophilic mice. Furthermore, anti-NGAL monoclonal antibody treatment markedly reduced the formation of blood clots in inflammation-induced thrombosis models. Collectively, these findings unveil a previously unidentified role of NGAL in the processes of coagulation, hemostasis, and thrombosis, as well as the cross talk between innate immunity, inflammation, and coagulation. Thus, modulating NGAL levels could potentially help balance thrombotic and hemorrhagic risks.

Mitotic exit is a necessary step for highly specialized cells to terminally differentiate and acquire unique functions. The FUCCI system can be used to visualize mitotic and post-mitotic cells during development and regeneration in both live organisms and fixed tissues. Here we describe a Fly-FUCCI protocol for assaying mitotic exit in Drosophila embryos.

Infections and neurodegenerative diseases induce neuroinflammation, but affected individuals often show nonneural symptoms including muscle pain and muscle fatigue. The molecular pathways by which neuroinflammation causes pathologies outside the central nervous system (CNS) are poorly understood. We developed multiple models to investigate the impact of CNS stressors on motor function and found that Escherichia coli infections and SARS-CoV-2 protein expression caused reactive oxygen species (ROS) to accumulate in the brain. ROS induced expression of the cytokine Unpaired 3 (Upd3) in Drosophila and its ortholog, IL-6, in mice. CNS-derived Upd3/IL-6 activated the JAK-STAT pathway in skeletal muscle, which caused muscle mitochondrial dysfunction and impaired motor function. We observed similar phenotypes after expressing toxic amyloid-β (Aβ42) in the CNS. Infection and chronic disease therefore activate a systemic brain-muscle signaling axis in which CNS-derived cytokines bypass the connectome and directly regulate muscle physiology, highlighting IL-6 as a therapeutic target to treat disease-associated muscle dysfunction.

Rare genetic disease discovery efforts typically lead to the identification of new disease genes. PreMIER ( Pre cision M edicine Integrated E xperimental R esources) is a collaborative platform designed to facilitate functional evaluation of human genetic variants in model systems, and to date the PreMIER Consortium has evaluated over 50 variants in patients with genetic disorders. To understand if Drosophila could be used to identify pathogenic disease loci as part of the PreMIER Consortium, we used tissue-specific gene knockdown in the fly as a proof of principle experiment. Tissue-specific knockdown of seven conserved disease genes caused significant changes in viability, longevity, behavior, motor function, and neuronal survival arguing a set of defined assays can be used to determine if a gene of uncertain significance (GUS) regulates specific physiological processes. This study highlights the utility of a tissue-specific knockdown platform in Drosophila to characterize GUS, which may provide the first genephenotype correlations for patients with idiopathic genetic disorders.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been detected in almost all organs of coronavirus disease-19 patients, although some organs do not express angiotensin-converting enzyme-2 (ACE2), a known receptor of SARS-CoV-2, implying the presence of alternative receptors and/or co-receptors. Here, we show that the ubiquitously distributed human transferrin receptor (TfR), which binds to diferric transferrin to traffic between membrane and endosome for the iron delivery cycle, can ACE2-independently mediate SARS-CoV-2 infection. Human, not mouse TfR, interacts with Spike protein with a high affinity (KD ~2.95 nM) to mediate SARS-CoV-2 endocytosis. TfR knock-down (TfR-deficiency is lethal) and overexpression inhibit and promote SARS-CoV-2 infection, respectively. Humanized TfR expression enables SARS-CoV-2 infection in baby hamster kidney cells and C57 mice, which are known to be insusceptible to the virus infection. Soluble TfR, Tf, designed peptides blocking TfR-Spike interaction and anti-TfR antibody show significant anti-COVID-19 effects in cell and monkey models. Collectively, this report indicates that TfR is a receptor/co-receptor of SARS-CoV-2 mediating SARS-CoV-2 entry and infectivity by likely using the TfR trafficking pathway.

SummaryRare genetic disease discovery efforts typically lead to the identification of new disease genes. PreMIER (PrecisionMedicine IntegratedExperimentalResources) is a collaborative platform designed to facilitate functional evaluation of human genetic variants in model systems, and to date the PreMIER Consortium has evaluated over 50 variants in patients with genetic disorders. To understand ifDrosophilacould be used to identify pathogenic disease loci as part of the PreMIER Consortium, we used tissue-specific gene knockdown in the fly as a proof of principle experiment. Tissue-specific knockdown of seven conserved disease genes caused significant changes in viability, longevity, behavior, motor function, and neuronal survival arguing a set of defined assays can be used to determine if a gene of uncertain significance (GUS) regulates specific physiological processes. This study highlights the utility of a tissue-specific knockdown platform inDrosophilato characterize GUS, which may provide the first genephenotype correlations for patients with idiopathic genetic disorders

Rare genetic disease discovery efforts typically lead to the identification of new disease genes. While there is inherent value in uncovering the genetic basis of disease for diagnosis, there is also significant opportunity to gain deep mechanistic or biological insights into disease pathogenesis. PreMIER (Precision Medicine Integrated Experimental Resources) is a collaborative platform among Washington University faculty designed to facilitate functional evaluation of human genetic variants in model systems. The PreMIER platform has identified seven cases could be effectively modeled in fruit flies. A survey of recent, high-impact disease-modeling studies in Drosophila identified six commonly used physiological assays that assess viability, longevity, behavior, and motor function. We knocked down each of the seven genes in neurons and in muscle throughout development and into adulthood, and assessed physiological phenotypes in adults. Tissue-specific knockdown of each gene caused significant changes in adult physiology in multiple assays, arguing that a set of six physiological assays can be used to show a candidate GUS is required for normal viability, longevity, behavior, or motor function. This study lays the foundation for our ongoing GUS screens, which may provide the first genephenotype correlations for patients with idiopathic genetic disorders.

Embryogenesis depends on a tightly regulated balance between mitosis, differentiation, and morphogenesis. Understanding how the embryo uses a relatively small number of proteins to transition between growth and morphogenesis is a central question of developmental biology, but the mechanisms controlling mitosis and differentiation are considered to be fundamentally distinct. Here we show the mitotic kinase Polo, which regulates all steps of mitosis in Drosophila , also directs cellular morphogenesis after cell cycle exit. In mitotic cells, the Aurora kinases activate Polo to control a cytoskeletal regulatory module that directs cytokinesis. We show that in the post-mitotic mesoderm, the control of Polo activity transitions from the Aurora kinases to the uncharacterized kinase Back Seat Driver (Bsd), where Bsd and Polo cooperate to regulate muscle morphogenesis. Polo and its effectors therefore direct mitosis and cellular morphogenesis, but the transition from growth to morphogenesis is determined by the spatiotemporal expression of upstream activating kinases.

Infections and neurodegenerative diseases induce neuroinflammation, but affected individuals often show a number of non-neural symptoms including muscle pain and muscle fatigue. The molecular pathways by which neuroinflammation causes pathologies outside the central nervous system (CNS) are poorly understood, so we developed three models to investigate the impact of neuroinflammation on muscle performance. We found that bacterial infection, COVID-like viral infection, and expression of a neurotoxic protein associated with Alzheimer′ s disease promoted the accumulation of reactive oxygen species (ROS) in the brain. Excessive ROS induces the expression of the cytokine Unpaired 3 (Upd3) in insects, or its orthologue IL-6 in mammals, and CNS-derived Upd3/IL-6 activates the JAK/Stat pathway in skeletal muscle. In response to JAK/Stat signaling, mitochondrial function is impaired and muscle performance is reduced. Our work uncovers a brain-muscle signaling axis in which infections and chronic diseases induce cytokine-dependent changes in muscle performance, suggesting IL-6 could be a therapeutic target to treat muscle weakness caused by neuroinflammation.

Summary Nemaline Myopathy (NM) is the most common congenital myopathy, characterized by extreme weakness of the respiratory, limb, and facial muscles. Pathogenic variants in Tropomyosin 2 ( TPM2 ), which encodes a skeletal muscle specific actin binding protein essential for sarcomere function, cause a spectrum of musculoskeletal disorders that include NM as well as Cap Myopathy, congenital fiber type disproportion, and distal arthrogrypsosis (DA). TPM2 -related disorders have not been modeled in vivo , so we expressed a series of dominant, pathogenic TPM2 variants in Drosophila embryos and found two variants, K49Del and E122K, significantly affected muscle morphogenesis and muscle function, in part by disrupting myotube guidance. Transient overexpression of K49Del and E122K also disrupted the morphogenesis of immortalized mouse myoblasts in vitro , and negatively affected zebrafish muscle development and function in vivo . We used our transient overexpression assays in zebrafish to characterize two novel TPM2 variants that we identified in DA patients (V129A and E139K), and found these variants caused musculoskeletal defects similar to those of the known pathogenic variants. In addition, the severity of musculoskeletal phenotypes in zebrafish expressing TPM2 variants correlated with the severity of clinical phenotypes observed in DA patients. Our study establishes transient overexpression in zebrafish as an efficient platform to characterize variants of uncertain significance in TPM2 in vivo , and suggests that this method can be used to predict the clinical severity of variants associated with DA and congenital myopathies.

Nemaline Myopathy (NM) is the most common congenital myopathy, characterized by extreme weakness of the respiratory, limb, and facial muscles. Pathogenic variants in Tropomyosin 2 ( TPM2 ), which encodes a skeletal muscle specific actin binding protein essential for sarcomere function, cause a spectrum of musculoskeletal disorders that include NM as well as Cap Myopathy, congenital fiber type disproportion, and distal arthrogrypsosis (DA). TPM2 -related disorders have not been modeled in vivo , so we expressed a series of dominant, pathogenic TPM2 variants in Drosophila embryos and found two variants, K49Del and E122K, significantly affected muscle morphogenesis and muscle function, in part by disrupting myotube guidance. Transient overexpression of K49Del and E122K also disrupted the morphogenesis of immortalized mouse myoblasts in vitro , and negatively affected zebrafish muscle development and function in vivo . We used our transient overexpression assays in zebrafish to characterize two novel TPM2 variants that we identified in DA patients (V129A and E139K), and found these variants caused musculoskeletal defects similar to those of the known pathogenic variants. In addition, the severity of musculoskeletal phenotypes in zebrafish expressing TPM2 variants correlated with the severity of clinical phenotypes observed in DA patients. Our study establishes transient overexpression in zebrafish as an efficient platform to characterize variants of uncertain significance in TPM2 in vivo , and suggests that this method can be used to predict the clinical severity of variants associated with DA and congenital myopathies.

Nascent myotubes undergo a dramatic morphological transformation during myogenesis, in which the myotubes elongate over several cell diameters and are directed to the correct muscle attachment sites. Although this process of myotube guidance is essential to pattern the musculoskeletal system, the mechanisms that control myotube guidance remain poorly understood. Using transcriptomics, we found that components of the Fibroblast Growth Factor (FGF) signaling pathway were enriched in nascent myotubes in Drosophila embryos. Null mutations in the FGF receptor heartless (htl), or its ligands, caused significant myotube guidance defects. The FGF ligand Pyramus is expressed broadly in the ectoderm, and ectopic Pyramus expression disrupted muscle patterning. Mechanistically, Htl regulates the activity of Rho/Rac GTPases in nascent myotubes and effects changes in the actin cytoskeleton. FGF signals are thus essential regulators of myotube guidance that act through cytoskeletal regulatory proteins to pattern the musculoskeletal system.

Interorgan immunological communication is critical to connect the local-systemic innate immune response and orchestrate a homeostatic host defense. However, the factors and their roles in this process remain unclear. We find Drosophila IMD response in guts can sequentially trigger a systemic IMD reaction in the fat body. Sugar alcohols of the polyol pathway are essential for the spatiotemporal regulation of gut-fat body immunological communication (GFIC). IMD activation in guts causes elevated levels of sorbitol and galactitol in hemolymph. Aldose reductase (AR) in hemocytes, the rate-limiting enzyme of the polyol pathway, is necessary and sufficient for the increase of plasma sugar alcohols. Sorbitol relays GFIC by subsequent activation of Metalloprotease 2, which cleaves PGRP-LC to activate IMD response in fat bodies. Thus, this work unveils how GFIC relies on the intermediate activation of the polyol pathway in hemolymph and demonstrates that AR provides a critical metabolic checkpoint in the global inflammatory response.

Nascent myotubes undergo a dramatic morphological transformation during myogenesis in which the myotubes elongate over several cell diameters and choose the correct muscle attachment sites. Although this process of myotube guidance is essential to pattern the musculoskeletal system, the mechanisms that control myotube guidance remain poorly understood. Using transcriptomics, we found that components of the Fibroblast Growth Factor (FGF) signaling pathway were enriched in nascent myotubes in Drosophila embryos. Null mutations in the FGF receptor heartless ( htl ), or its ligands, caused significant myotube guidance defects. Mechanistically, paracrine FGF signals to Htl in the mesoderm regulate the activity of Rho/Rac GTPases in nascent myotubes to effect changes in the actin cytoskeleton. FGF signals are thus essential regulators of myotube guidance that act through cytoskeletal regulatory proteins to pattern the musculoskeletal system.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

In order to establish productive infection and dissemination, viruses usually evolve a number of strategies to hijack and/or subvert the host defense systems. However, host factors utilized by the virus to facilitate infection remain poorly characterized. In this work, we found that Drosophila melanogaster deficient in budding uninhibited by benzimidazoles 1 (bub1), a highly conserved subunit of the kinetochore complex regulating chromosome congression (1), became resistant to Drosophila C virus (DCV) infection, evidenced in increased survival rates and reduced viral loads, compared to the wild-type control. Mechanistic analysis further showed that Bub1 also functioned in the cytoplasm and was essentially involved in clathrin-dependent endocytosis of DCV and other pathogens, thus limiting pathogen entry. DCV infection potentially had strengthened the interaction between Bub1 and the clathrin adaptor on the cell membrane. Furthermore, the conserved function of Bub1 was also verified in a mammalian cell line. Thus, our data demonstrated a previously unknown function of Bub1 that could be hijacked by pathogens to facilitate their infection and spread.IMPORTANCE In this work, we identify for the first time that the nuclear protein Bub1 (budding uninhibited by benzimidazoles 1), a highly conserved subunit of the kinetochore complex regulating chromosome congression, has a novel and important function on the cell membrane to facilitate the virus to enter host cells. Bub1 deficiency empowers the host to have the ability to resist viral infection in Drosophila and a human cell line. Bub1 is involved in the virus entry step through regulating endocytosis. The DCV capsid protein can recruit Bub1, and DCV infection can strengthen the interaction between Bub1 and a clathrin-dependent endocytosis component. The restricted entry of vesicular stomatitis virus (VSV) and Listeria monocytogenes in bub1-deficient flies and cell lines was also observed. Therefore, our data implicate a previously unknown function of Bub1 that can be hijacked by pathogens to facilitate their entry, and Bub1 may serve as a potential antiviral therapy target for limiting viral entry.

The metabolic intermediate of acetaminophen (APAP) can cause severe hepatocyte necrosis, which triggers aberrant immune activation of liver non-parenchymal cells (NPC). Overzealous hepatic inflammation determines the morbidity and mortality of APAP-induced liver injury (AILI). Interleukin-1 receptor (IL-1R) signaling has been shown to play a critical role in various inflammatory conditions, but its precise role and underlying mechanism in AILI remain debatable. Herein, we show that NLRP3 inflammasome activation of IL-1β is dispensable to AILI, whereas IL-1α, the other ligand of IL-1R1, accounts for hepatic injury by a lethal dose of APAP. Furthermore, Kupffer cells function as a major source of activated IL-1α in the liver, which is activated by damaged hepatocytes through TLR4/MyD88 signaling. Finally, IL-1α is able to chemoattract and activate CD11b+Gr-1+ myeloid cells, mostly neutrophils and inflammatory monocytes, to amplify deteriorated inflammation in the lesion. Therefore, this work identifies that MyD88-dependent activation of IL-1α in Kupffer cells plays a central role in the immunopathogenesis of AILI and implicates that IL-1α is a promising therapeutic target for AILI treatment.

Immune homeostasis is a prerequisite to protective immunity against gastrointestinal infections. In Drosophila, immune deficiency (IMD) signalling (tumour necrosis factor receptor/interleukin-1 receptor, TNFR/IL-1R in mammals) is indispensable for intestinal immunity against invading bacteria. However, how this local antimicrobial immune response contributes to inflammatory regulation remains poorly defined. Here, we show that flies lacking intestinal Bap180 (a subunit of the chromatin-remodelling switch/sucrose non-fermentable (SWI/SNF) complex) are susceptible to infection as a result of hyper-inflammation rather than bacterial overload. Detailed analysis shows that Bap180 is induced by the IMD-Relish response to both enteropathogenic and commensal bacteria. Upregulated Bap180 can feed back to restrain overreactive IMD signalling, as well as to repress the expression of the pro-inflammatory gene eiger (TNF), a critical step to prevent excessive tissue damage and elongate the lifespan of flies, under pathological and physiological conditions, respectively. Furthermore, intestinal targeting of Baf180 renders mice susceptible to a more aggressive infectious colitis caused by Citrobacter rodentium. Together, Bap180 and Baf180 serve as a conserved transcriptional repressor that is critical for the maintenance of innate immune homeostasis in the intestines.