Wnt signaling couples G2 phase control with differentiation during hematopoiesis in Drosophila.

During homeostasis, a critical balance is maintained between myeloid-like progenitors and their differentiated progeny, which function to mitigate stress and innate immune challenges. The molecular mechanisms that help achieve this balance are not fully understood. Using genetic dissection in Drosophila, we show that a Wnt6/EGFR-signaling network simultaneously controls progenitor growth, proliferation, and differentiation. Unlike G1-quiescence of stem cells, hematopoietic progenitors are blocked in G2 phase by a ß-catenin-independent (Wnt/STOP) Wnt6 pathway that restricts Cdc25 nuclear entry and promotes cell growth. Canonical ß-catenin-dependent Wnt6 signaling is spatially confined to mature progenitors through localized activation of the tyrosine kinases EGFR and Abelson kinase (Abl), which promote nuclear entry of ß-catenin and facilitate exit from G2. This strategy combines transcription-dependent and -independent forms of both Wnt6 and EGFR pathways to create a direct link between cell-cycle control and differentiation. This unique combinatorial strategy employing conserved components may underlie homeostatic balance and stress response in mammalian hematopoiesis.

A conserved mechanism for JNK-mediated loss of Notch function in advanced prostate cancer.

Dysregulated Notch signaling is a common feature of cancer; however, its effects on tumor initiation and progression are highly variable, with Notch having either oncogenic or tumor-suppressive functions in various cancers. To better understand the mechanisms that regulate Notch function in cancer, we studied Notch signaling in a Drosophila tumor model, prostate cancer-derived cell lines, and tissue samples from patients with advanced prostate cancer. We demonstrated that increased activity of the Src-JNK pathway in tumors inactivated Notch signaling because of JNK pathway-mediated inhibition of the expression of the gene encoding the Notch S2 cleavage protease, Kuzbanian, which is critical for Notch activity. Consequently, inactive Notch accumulated in cells, where it was unable to transcribe genes encoding its target proteins, many of which have tumor-suppressive activities. These findings suggest that Src-JNK activity in tumors predicts Notch activity status and that suppressing Src-JNK signaling could restore Notch function in tumors, offering opportunities for diagnosis and targeted therapies for a subset of patients with advanced prostate cancer.

Injury-induced inflammatory signaling and hematopoiesis in Drosophila.

Inflammatory response in Drosophila to sterile (axenic) injury in embryos and adults has received some attention in recent years, and most concentrate on the events at the injury site. Here we focus on the effect sterile injury has on the hematopoietic organ, the lymph gland, and the circulating blood cells in the larva, the developmental stage at which major events of hematopoiesis are evident. In mammals, injury activates Toll-like receptor/NF-κB signaling in macrophages, which then express and secrete secondary, proinflammatory cytokines. In Drosophila larvae, distal puncture injury of the body wall epidermis causes a rapid activation of Toll and Jun kinase (JNK) signaling throughout the hematopoietic system and the differentiation of a unique blood cell type, the lamellocyte. Furthermore, we find that Toll and JNK signaling are coupled in their activation. Secondary to this Toll/JNK response, a cytokine, Upd3, is induced as a Toll pathway transcriptional target, which then promotes JAK/STAT signaling within the blood cells. Toll and JAK/STAT signaling are required for the emergence of the injury-induced lamellocytes. This is akin to the derivation of specialized macrophages in mammalian systems. Upstream, at the injury site, a Duox- and peroxide-dependent signal causes the activation of the proteases Grass and SPE, needed for the activation of the Toll-ligand Spz, but microbial sensors or the proteases most closely associated with them during septic injury are not involved in the axenic inflammatory response.

Cardiomyocytes disrupt pyrimidine biosynthesis in nonmyocytes to regulate heart repair.

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.

Intermediate progenitor cells provide a transition between hematopoietic progenitors and their differentiated descendants.

Genetic and genomic analysis in Drosophila suggests that hematopoietic progenitors likely transition into terminal fates via intermediate progenitors (IPs) with some characteristics of either, but perhaps maintaining IP-specific markers. In the past, IPs have not been directly visualized and investigated owing to lack of appropriate genetic tools. Here, we report a Split GAL4 construct, CHIZ-GAL4, that identifies IPs as cells physically juxtaposed between true progenitors and differentiating hemocytes. IPs are a distinct cell type with a unique cell-cycle profile and they remain multipotent for all blood cell fates. In addition, through their dynamic control of the Notch ligand Serrate, IPs specify the fate of direct neighbors. The Ras pathway controls the number of IP cells and promotes their transition into differentiating cells. This study suggests that it would be useful to characterize such intermediate populations of cells in mammalian hematopoietic systems.

Paths and pathways that generate cell-type heterogeneity and developmental progression in hematopoiesis.

Mechanistic studies of Drosophila lymph gland hematopoiesis are limited by the availability of cell-type-specific markers. Using a combination of bulk RNA-Seq of FACS-sorted cells, single-cell RNA-Seq, and genetic dissection, we identify new blood cell subpopulations along a developmental trajectory with multiple paths to mature cell types. This provides functional insights into key developmental processes and signaling pathways. We highlight metabolism as a driver of development, show that graded Pointed expression allows distinct roles in successive developmental steps, and that mature crystal cells specifically express an alternate isoform of Hypoxia-inducible factor (Hif/Sima). Mechanistically, the Musashi-regulated protein Numb facilitates Sima-dependent non-canonical, and inhibits canonical, Notch signaling. Broadly, we find that prior to making a fate choice, a progenitor selects between alternative, biologically relevant, transitory states allowing smooth transitions reflective of combinatorial expressions rather than stepwise binary decisions. Increasingly, this view is gaining support in mammalian hematopoiesis.

The Metabolic Landscape of Thymic T Cell Development In Vivo and In Vitro.

Although metabolic pathways have been shown to control differentiation and activation in peripheral T cells, metabolic studies on thymic T cell development are still lacking, especially in human tissue. In this study, we use transcriptomics and extracellular flux analyses to investigate the metabolic profiles of primary thymic and in vitro-derived mouse and human thymocytes. Core metabolic pathways, specifically glycolysis and oxidative phosphorylation, undergo dramatic changes between the double-negative (DN), double-positive (DP), and mature single-positive (SP) stages in murine and human thymus. Remarkably, despite the absence of the complex multicellular thymic microenvironment, in vitro murine and human T cell development recapitulated the coordinated decrease in glycolytic and oxidative phosphorylation activity between the DN and DP stages seen in primary thymus. Moreover, by inducing in vitro T cell differentiation from Rag1-/- mouse bone marrow, we show that reduced metabolic activity at the DP stage is independent of TCR rearrangement. Thus, our findings suggest that highly conserved metabolic transitions are critical for thymic T cell development.

Metabolic plasticity drives development during mammalian embryogenesis.

Mammalian preimplantation embryos follow a stereotypic pattern of development from zygotes to blastocysts. Here, we use labeled nutrient isotopologue analysis of small numbers of embryos to track downstream metabolites. Combined with transcriptomic analysis, we assess the capacity of the embryo to reprogram its metabolism through development. Early embryonic metabolism is rigid in its nutrient requirements, sensitive to reductive stress and has a marked disequilibrium between two halves of the TCA cycle. Later, loss of maternal LDHB and transcription of zygotic products favors increased activity of bioenergetic shuttles, fatty-acid oxidation and equilibration of the TCA cycle. As metabolic plasticity peaks, blastocysts can develop without external nutrients. Normal developmental metabolism of the early embryo is distinct from cancer metabolism. However, similarities emerge upon reductive stress. Increased metabolic plasticity with maturation is due to changes in redox control mechanisms and to transcriptional reprogramming of later-stage embryos during homeostasis or upon adaptation to environmental changes.

Glycolysis-Independent Glucose Metabolism Distinguishes TE from ICM Fate during Mammalian Embryogenesis.

The mouse embryo undergoes compaction at the 8-cell stage, and its transition to 16 cells generates polarity such that the outer apical cells are trophectoderm (TE) precursors and the inner cell mass (ICM) gives rise to the embryo. Here, we report that this first cell fate specification event is controlled by glucose. Glucose does not fuel mitochondrial ATP generation, and glycolysis is dispensable for blastocyst formation. Furthermore, glucose does not help synthesize amino acids, fatty acids, and nucleobases. Instead, glucose metabolized by the hexosamine biosynthetic pathway (HBP) allows nuclear localization of YAP1. In addition, glucose-dependent nucleotide synthesis by the pentose phosphate pathway (PPP), along with sphingolipid (S1P) signaling, activates mTOR and allows translation of Tfap2c. YAP1, TEAD4, and TFAP2C interact to form a complex that controls TE-specific gene transcription. Glucose signaling has no role in ICM specification, and this process of developmental metabolism specifically controls TE cell fate.

Drosophila as a Genetic Model for Hematopoiesis.

In this FlyBook chapter, we present a survey of the current literature on the development of the hematopoietic system in Drosophila The Drosophila blood system consists entirely of cells that function in innate immunity, tissue integrity, wound healing, and various forms of stress response, and are therefore functionally similar to myeloid cells in mammals. The primary cell types are specialized for phagocytic, melanization, and encapsulation functions. As in mammalian systems, multiple sites of hematopoiesis are evident in Drosophila and the mechanisms involved in this process employ many of the same molecular strategies that exemplify blood development in humans. Drosophila blood progenitors respond to internal and external stress by coopting developmental pathways that involve both local and systemic signals. An important goal of these Drosophila studies is to develop the tools and mechanisms critical to further our understanding of human hematopoiesis during homeostasis and dysfunction.

Extracellular Matrix Remodeling Regulates Glucose Metabolism through TXNIP Destabilization.

The metabolic state of a cell is influenced by cell-extrinsic factors, including nutrient availability and growth factor signaling. Here, we present extracellular matrix (ECM) remodeling as another fundamental node of cell-extrinsic metabolic regulation. Unbiased analysis of glycolytic drivers identified the hyaluronan-mediated motility receptor as being among the most highly correlated with glycolysis in cancer. Confirming a mechanistic link between the ECM component hyaluronan and metabolism, treatment of cells and xenografts with hyaluronidase triggers a robust increase in glycolysis. This is largely achieved through rapid receptor tyrosine kinase-mediated induction of the mRNA decay factor ZFP36, which targets TXNIP transcripts for degradation. Because TXNIP promotes internalization of the glucose transporter GLUT1, its acute decline enriches GLUT1 at the plasma membrane. Functionally, induction of glycolysis by hyaluronidase is required for concomitant acceleration of cell migration. This interconnection between ECM remodeling and metabolism is exhibited in dynamic tissue states, including tumorigenesis and embryogenesis.

Systemic control of immune cell development by integrated carbon dioxide and hypoxia chemosensation in Drosophila.

Drosophila hemocytes are akin to mammalian myeloid blood cells that function in stress and innate immune-related responses. A multi-potent progenitor population responds to local signals and to systemic stress by expanding the number of functional blood cells. Here we show mechanisms that demonstrate an integration of environmental carbon dioxide (CO2) and oxygen (O2) inputs that initiate a cascade of signaling events, involving multiple organs, as a stress response when the levels of these two important respiratory gases fall below a threshold. The CO2 and hypoxia-sensing neurons interact at the synaptic level in the brain sending a systemic signal via the fat body to modulate differentiation of a specific class of immune cells. Our findings establish a link between environmental gas sensation and myeloid cell development in Drosophila. A similar relationship exists in humans, but the underlying mechanisms remain to be established.

Nuclear Localization of Mitochondrial TCA Cycle Enzymes as a Critical Step in Mammalian Zygotic Genome Activation.

Transcriptional control requires epigenetic changes directed by mitochondrial tricarboxylic acid (TCA) cycle metabolites. In the mouse embryo, global epigenetic changes occur during zygotic genome activation (ZGA) at the 2-cell stage. Pyruvate is essential for development beyond this stage, which is at odds with the low activity of mitochondria in this period. We now show that a number of enzymatically active mitochondrial enzymes associated with the TCA cycle are essential for epigenetic remodeling and are transiently and partially localized to the nucleus. Pyruvate is essential for this nuclear localization, and a failure of TCA cycle enzymes to enter the nucleus correlates with loss of specific histone modifications and a block in ZGA. At later stages, however, these enzymes are exclusively mitochondrial. In humans, the enzyme pyruvate dehydrogenase is transiently nuclear at the 4/8-cell stage coincident with timing of human embryonic genome activation, suggesting a conserved metabolic control mechanism underlying early pre-implantation development.

In vivo genetic dissection of tumor growth and the Warburg effect.

A well-characterized metabolic landmark for aggressive cancers is the reprogramming from oxidative phosphorylation to aerobic glycolysis, referred to as the Warburg effect. Models mimicking this process are often incomplete due to genetic complexities of tumors and cell lines containing unmapped collaborating mutations. In order to establish a system where individual components of oncogenic signals and metabolic pathways can be readily elucidated, we induced a glycolytic tumor in the Drosophila wing imaginal disc by activating the oncogene PDGF/VEGF-receptor (Pvr). This causes activation of multiple oncogenic pathways including Ras, PI3K/Akt, Raf/ERK, Src and JNK. Together this network of genes stabilizes Hifα (Sima) that in turn, transcriptionally up-regulates many genes encoding glycolytic enzymes. Collectively, this network of genes also causes inhibition of pyruvate dehydrogenase (PDH) activity resulting in diminished ox-phos levels. The high ROS produced during this process functions as a feedback signal to consolidate this metabolic reprogramming.

Dissection and mounting of Drosophila pupal eye discs.

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Pvr expression regulators in equilibrium signal control and maintenance of Drosophila blood progenitors.

Blood progenitors within the lymph gland, a larval organ that supports hematopoiesis in Drosophila melanogaster, are maintained by integrating signals emanating from niche-like cells and those from differentiating blood cells. We term the signal from differentiating cells the 'equilibrium signal' in order to distinguish it from the 'niche signal'. Earlier we showed that equilibrium signaling utilizes Pvr (the Drosophila PDGF/VEGF receptor), STAT92E, and adenosine deaminase-related growth factor A (ADGF-A) (Mondal et al., 2011). Little is known about how this signal initiates during hematopoietic development. To identify new genes involved in lymph gland blood progenitor maintenance, particularly those involved in equilibrium signaling, we performed a genetic screen that identified bip1 (bric à brac interacting protein 1) and Nucleoporin 98 (Nup98) as additional regulators of the equilibrium signal. We show that the products of these genes along with the Bip1-interacting protein RpS8 (Ribosomal protein S8) are required for the proper expression of Pvr.

Drosophila hematopoiesis: Markers and methods for molecular genetic analysis.

Analyses of the Drosophila hematopoietic system are becoming more and more prevalent as developmental and functional parallels with vertebrate blood cells become more evident. Investigative work on the fly blood system has, out of necessity, led to the identification of new molecular markers for blood cell types and lineages and to the refinement of useful molecular genetic tools and analytical methods. This review briefly describes the Drosophila hematopoietic system at different developmental stages, summarizes the major useful cell markers and tools for each stage, and provides basic protocols for practical analysis of circulating blood cells and of the lymph gland, the larval hematopoietic organ.

Nutritional regulation of stem and progenitor cells in Drosophila.

Stem cells and their progenitors are maintained within a microenvironment, termed the niche, through local cell-cell communication. Systemic signals originating outside the niche also affect stem cell and progenitor behavior. This review summarizes studies that pertain to nutritional effects on stem and progenitor cell maintenance and proliferation in Drosophila. Multiple tissue types are discussed that utilize the insulin-related signaling pathway to convey nutritional information either directly to these progenitors or via other cell types within the niche. The concept of systemic control of these cell types is not limited to Drosophila and may be functional in vertebrate systems, including mammals.

Olfactory control of blood progenitor maintenance.

Drosophila hematopoietic progenitor maintenance involves both near neighbor and systemic interactions. This study shows that olfactory receptor neurons (ORNs) function upstream of a small set of neurosecretory cells that express GABA. Upon olfactory stimulation, GABA from these neurosecretory cells is secreted into the circulating hemolymph and binds to metabotropic GABAB receptors expressed on blood progenitors within the hematopoietic organ, the lymph gland. The resulting GABA signal causes high cytosolic Ca(2+), which is necessary and sufficient for progenitor maintenance. Thus, the activation of an odorant receptor is essential for blood progenitor maintenance, and consequently, larvae raised on minimal odor environments fail to sustain a pool of hematopoietic progenitors. This study links sensory perception and the effects of its deprivation on the integrity of the hematopoietic and innate immune systems in Drosophila. PAPERCLIP: