Interaction between differentiating cell- and niche-derived signals in hematopoietic progenitor maintenance.

Maintenance of a hematopoietic progenitor population requires extensive interaction with cells within a microenvironment or niche. In the Drosophila hematopoietic organ, niche-derived Hedgehog signaling maintains the progenitor population. Here, we show that the hematopoietic progenitors also require a signal mediated by Adenosine deaminase growth factor A (Adgf-A) arising from differentiating cells that regulates extracellular levels of adenosine. The adenosine signal opposes the effects of Hedgehog signaling within the hematopoietic progenitor cells and the magnitude of the adenosine signal is kept in check by the level of Adgf-A secreted from differentiating cells. Our findings reveal signals arising from differentiating cells that are required for maintaining progenitor cell quiescence and that function with the niche-derived signal in maintaining the progenitor state. Similar homeostatic mechanisms are likely to be utilized in other systems that maintain relatively large numbers of progenitors that are not all in direct contact with the cells of the niche.

A conserved nutrient responsive axis mediates autophagic degradation of miRNA-mRNA hybrids in blood cell progenitors.

In animals, microRNAs are amongst the primary non-coding RNAs involved in regulating the gene expression of a cell. Most mRNAs in a cell are targeted by one or many miRNAs. Although several mechanisms can be attributed to the degradation of miRNA and mRNA within a cell, but the involvement of autophagy in the clearance of miRNA and its target mRNA is not known. We discover a leucine-responsive axis in blood cell progenitors that can mediate an autophagy-directed degradation of miRNA-bound mRNA in Drosophila melanogaster and Homo sapiens. This previously unknown miRNA clearance axis is activated upon amino acid deprivation that can traffic miRNA-mRNA-loaded Argonaute for autophagic degradation in a p62-dependent manner. Thus, our research not only reports a novel axis that can address the turnover of a catalytically active miRISC but also elucidates a slicer-independent mechanism through which autophagy can selectively initiate the clearance of target mRNA.

Ubx-Collier signaling cascade maintains blood progenitors in the posterior lobes of the Drosophila larval lymph gland.

Drosophila larval hematopoiesis occurs in a specialized multi-lobed organ called the lymph gland. Extensive characterization of the organ has provided mechanistic insights into events related to developmental hematopoiesis. Spanning from the thoracic to the abdominal segment of the larvae, this organ comprises a pair of primary, secondary, and tertiary lobes. Much of our understanding arises from the studies on the primary lobe, while the secondary and tertiary lobes have remained mostly unexplored. Previous studies have inferred that these lobes are composed of progenitors that differentiate during pupation; however, the mechanistic basis of this extended progenitor state remains unclear. This study shows that posterior lobe progenitors are maintained by a local signaling center defined by Ubx and Collier in the tertiary lobe. This Ubx zone in the tertiary lobe shares several markers with the niche of the primary lobe. Ubx domain regulates the homeostasis of the posterior lobe progenitors in normal development and an immune-challenged scenario. Our study establishes the lymph gland as a model to tease out how the progenitors interface with the dual niches within an organ during development and disorders.

Lar maintains the homeostasis of the hematopoietic organ in Drosophila by regulating insulin signaling in the niche.

Stem cell compartments in metazoa get regulated by systemic factors as well as local stem cell niche-derived factors. However, the mechanisms by which systemic signals integrate with local factors in maintaining tissue homeostasis remain unclear. Employing the Drosophila lymph gland, which harbors differentiated blood cells, and stem-like progenitor cells and their niche, we demonstrate how a systemic signal interacts and harmonizes with local factor/s to achieve cell type-specific tissue homeostasis. Our genetic analyses uncovered a novel function of Lar, a receptor protein tyrosine phosphatase. Niche-specific loss of Lar leads to upregulated insulin signaling, causing increased niche cell proliferation and ectopic progenitor differentiation. Insulin signaling assayed by PI3K activation is downregulated after the second instar larval stage, a time point that coincides with the appearance of Lar in the hematopoietic niche. We further demonstrate that Lar physically associates with InR and serves as a negative regulator for insulin signaling in the Drosophila larval hematopoietic niche. Whether Lar serves as a localized invariable negative regulator of systemic signals such as insulin in other stem cell niches remains to be explored.

High sugar diet-induced fatty acid oxidation potentiates cytokine-dependent cardiac ECM remodeling.

Context-dependent physiological remodeling of the extracellular matrix (ECM) is essential for development and organ homeostasis. On the other hand, consumption of high-caloric diet leverages ECM remodeling to create pathological conditions that impede the functionality of different organs, including the heart. However, the mechanistic basis of high caloric diet-induced ECM remodeling has yet to be elucidated. Employing in vivo molecular genetic analyses in Drosophila, we demonstrate that high dietary sugar triggers ROS-independent activation of JNK signaling to promote fatty acid oxidation (FAO) in the pericardial cells (nephrocytes). An elevated level of FAO, in turn, induces histone acetylation-dependent transcriptional upregulation of the cytokine Unpaired 3 (Upd3). Release of pericardial Upd3 augments fat body-specific expression of the cardiac ECM protein Pericardin, leading to progressive cardiac fibrosis. Importantly, this pathway is quite distinct from the ROS-Ask1-JNK/p38 axis that regulates Upd3 expression under normal physiological conditions. Our results unravel an unknown physiological role of FAO in cytokine-dependent ECM remodeling, bearing implications in diabetic fibrosis.

A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors.

The Drosophila melanogaster lymph gland is a haematopoietic organ in which pluripotent blood cell progenitors proliferate and mature into differentiated haemocytes. Previous work has defined three domains, the medullary zone, the cortical zone and the posterior signalling centre (PSC), within the developing third-instar lymph gland. The medullary zone is populated by a core of undifferentiated, slowly cycling progenitor cells, whereas mature haemocytes comprising plasmatocytes, crystal cells and lamellocytes are peripherally located in the cortical zone. The PSC comprises a third region that was first defined as a small group of cells expressing the Notch ligand Serrate. Here we show that the PSC is specified early in the embryo by the homeotic gene Antennapedia (Antp) and expresses the signalling molecule Hedgehog. In the absence of the PSC or the Hedgehog signal, the precursor population of the medullary zone is lost because cells differentiate prematurely. We conclude that the PSC functions as a haematopoietic niche that is essential for the maintenance of blood cell precursors in Drosophila. Identification of this system allows the opportunity for genetic manipulation and direct in vivo imaging of a haematopoietic niche interacting with blood precursors.

Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta-gonadal-mesonephros mesoderm.

The Drosophila melanogaster lymph gland is a hematopoietic organ and, together with prospective vascular cells (cardioblasts) and excretory cells (pericardial nephrocytes), arises from the cardiogenic mesoderm. Clonal analysis provided evidence for a hemangioblast that can give rise to two daughter cells: one that differentiates into heart or aorta and another that differentiates into blood. In addition, the GATA factor gene pannier (pnr) and the homeobox gene tinman (tin), which are controlled by the convergence of Decapentaplegic (Dpp), fibroblast growth factor (FGF), Wingless (Wg) and Notch signaling, are required for the development of all cardiogenic mesoderm, including the lymph gland. Here we show that an essential genetic switch that differentiates between the blood or nephrocyte and vascular lineages involves the Notch pathway. Further specification occurs through specific expression of the GATA factor Serpent (Srp) in the lymph-gland primordium. Our findings suggest that there is a close parallel between the molecular mechanisms functioning in the D. melanogaster cardiogenic mesoderm and those functioning in the mammalian aorta-gonadal-mesonephros mesoderm.

ISSCR Education Committee syllabus and learning guide for enhancing stem cell literacy.

The rapidly evolving stem cell field puts much stress on developing educational resources. The ISSCR Education Committee has created a flexible stem cell syllabus rooted in core concepts to facilitate stem cell literacy. The free syllabus will be updated regularly to maintain accuracy and relevance.

The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the Drosophila mesoderm.

Blood progenitors arise from a pool of pluripotential cells ("hemangioblasts") within the Drosophila embryonic mesoderm. The fact that the cardiogenic mesoderm consists of only a small number of highly stereotypically patterned cells that can be queried individually regarding their gene expression in normal and mutant embryos is one of the significant advantages that Drosophila offers to dissect the mechanism specifying the fate of these cells. We show in this paper that the expression of the Notch ligand Delta (Dl) reveals segmentally reiterated mesodermal clusters ("cardiogenic clusters") that constitute the cardiogenic mesoderm. These clusters give rise to cardioblasts, blood progenitors and nephrocytes. Cardioblasts emerging from the cardiogenic clusters accumulate high levels of Dl, which is required to prevent more cells from adopting the cardioblast fate. In embryos lacking Dl function, all cells of the cardiogenic clusters become cardioblasts, and blood progenitors are lacking. Concomitant activation of the Mitogen Activated Protein Kinase (MAPK) pathway by Epidermal Growth Factor Receptor (EGFR) and Fibroblast Growth Factor Receptor (FGFR) is required for the specification and maintenance of the cardiogenic mesoderm; in addition, the spatially restricted localization of some of the FGFR ligands may be instrumental in controlling the spatial restriction of the Dl ligand to presumptive cardioblasts.

Combination of Immunofluorescence and Quantitative Fluorescence In-situ Hybridization for Analysing Differential Gene Expression in the Niche Cells of the Drosophila Lymph Gland.

The Drosophila larval haematopoietic organ or lymph gland consists of multiple cell types arranged in zones. The smallest stem cell compartment consists of 40-45 cells that constitute the haematopoietic niche. In order to analyse the haematopoietic niche, it needs to be labelled with a specific antibody to differentiate it from the other cell types. To characterise a phenotype, it is often necessary to investigate the expression of a gene in a particular stem cell compartment within the lymph gland. In such a situation, in-situ hybridization is performed, as it indicates the localization of gene expression. Although chromogenic in-situ hybridization enables us to compare the signal and tissue morphology simultaneously, it fails to harness the information related to the degree of gene expression. Dual immunofluorescence and in-situ hybridization (IF-FISH) serves as the powerful technique that helps to visualize both protein and mRNA expression within the same cell type. This technique also provides reliable quantification regarding mRNA expression levels. When dealing with a few cells within the organ, like the niche of the larval lymph gland, fluorescently labelled riboprobes allows us to localize and assess the magnitude of gene expression within the niche cells, which are also immunolabelled with a niche-specific marker, to distinguish them from the adjoining cell types.

Physiological ROS controls Upd3-dependent modeling of ECM to support cardiac function in Drosophila.

Despite their highly reactive nature, reactive oxygen species (ROS) at the physiological level serve as signaling molecules regulating diverse biological processes. While ROS usually act autonomously, they also function as local paracrine signals by diffusing out of the cells producing them. Using in vivo molecular genetic analyses in Drosophila, we provide evidence for ROS-dependent paracrine signaling that does not entail ROS release. We show that elevated levels of physiological ROS within the pericardial cells activate a signaling cascade transduced by Ask1, c-Jun N-terminal kinase, and p38 to regulate the expression of the cytokine Unpaired 3 (Upd3). Upd3 released by the pericardial cells controls fat body-specific expression of the extracellular matrix (ECM) protein Pericardin, essential for cardiac function and healthy life span. Therefore, our work reveals an unexpected inter-organ communication circuitry wherein high physiological levels of ROS regulate cytokine-dependent modulation of cardiac ECM with implications in normal and pathophysiological conditions.

Hematopoiesis at the onset of metamorphosis: terminal differentiation and dissociation of the Drosophila lymph gland.

The Drosophila melanogaster hematopoietic organ, called lymph gland, proliferates and differentiates throughout the larval period. The lymph gland of the late larva is comprised of a large primary lobe and several smaller secondary lobes. Differentiation into two types of hemocytes, plasmatocytes and crystal cells, is confined to the outer layer (cortical zone) of the primary lobe; the center of the primary lobe (medullary zone), as well as the secondary lobes, contain only proliferating prohemocytes. A small cluster of cells located at the posterior tip of the primary lobe serves as a signaling center (PSC) that inhibits precocious differentiation of the medullary zone. The larval lymph gland is stabilized by layers of extracellular matrix (basement membranes) that surround individual hemocytes, groups of hemocytes, as well as the lymph gland as a whole. In this paper, we investigated the events shaping the lymph gland in the early pupa. The lymph gland dissociates and hemocytes disperse during the first 12 h after puparium formation (APF), leaving behind empty husks of basement membrane. Prior to lymph gland dissociation, cells of the medullary zone differentiate, expressing the early differentiation marker Peroxidasin (Pxn), as well as, in part, the late differentiation marker P1. Cells of the PSC spread throughout the pupal lymph gland prior to their dispersal. Cells of the secondary lobes undergo a rapid phase of proliferation that lasts until 8 h APF, followed by expression of Pxn and dispersal. These hemocytes do not express P1, indicating that they disperse prior to full maturation.

Relish plays a dynamic role in the niche to modulate Drosophila blood progenitor homeostasis in development and infection.

Immune challenges demand the gearing up of basal hematopoiesis to combat infection. Little is known about how during development, this switch is achieved to take care of the insult. Here, we show that the hematopoietic niche of the larval lymph gland of Drosophila senses immune challenge and reacts to it quickly through the nuclear factor-κB (NF-κB), Relish, a component of the immune deficiency (Imd) pathway. During development, Relish is triggered by ecdysone signaling in the hematopoietic niche to maintain the blood progenitors. Loss of Relish causes an alteration in the cytoskeletal architecture of the niche cells in a Jun Kinase-dependent manner, resulting in the trapping of Hh implicated in progenitor maintenance. Notably, during infection, downregulation of Relish in the niche tilts the maintenance program toward precocious differentiation, thereby bolstering the cellular arm of the immune response.

Drosophila metamorphosis involves hemocyte mediated macroendocytosis and efferocytosis.

Drosophila hemocytes are majorly associated with immune responses, but they also undertake several non-immune functions that are crucial during various stages of development. The activity and behaviour of hemocytes are least documented during the metamorphic phase of fly development. Here we describe the activity, form and behaviour of the most abundant type of hemocyte in Drosophila melanogaster, the "plasmatocyte," throughout pupal development. Our study reveals different forms of plasmatocytes laden with varying degrees of histolyzing debris (muscle and fat) which extend beyond the size of the cell itself, highlighting the phagocytic capacity of these plasmatocytes. Interestingly, the engulfment of apoptotic debris by plasmatocytes is an actin-dependent process, and by the end of metamorphosis, clearance is achieved. The uptake of apoptotic debris consisting of muscles and lipids by the plasmatocytes provides us a model that can be employed to dissect out the relevant components of macroendocytosis and lipid-loaded phagocytosis. This understanding, by itself, is crucial for addressing the emerging role of phagocytes in physiology and pathophysiology.

Fatty acid ß-oxidation is required for the differentiation of larval hematopoietic progenitors in Drosophila.

Cell-intrinsic and extrinsic signals regulate the state and fate of stem and progenitor cells. Recent advances in metabolomics illustrate that various metabolic pathways are also important in regulating stem cell fate. However, our understanding of the metabolic control of the state and fate of progenitor cells is in its infancy. Using Drosophila hematopoietic organ: lymph gland, we demonstrate that Fatty Acid Oxidation (FAO) is essential for the differentiation of blood cell progenitors. In the absence of FAO, the progenitors are unable to differentiate and exhibit altered histone acetylation. Interestingly, acetate supplementation rescues both histone acetylation and the differentiation defects. We further show that the CPT1/whd (withered), the rate-limiting enzyme of FAO, is transcriptionally regulated by Jun-Kinase (JNK), which has been previously implicated in progenitor differentiation. Our study thus reveals how the cellular signaling machinery integrates with the metabolic cue to facilitate the differentiation program.

Stem cells are special precursor cells, found in all animals from flies to humans, that can give rise to all the mature cell types in the body. Their job is to generate supplies of new cells wherever these are needed. This is important because it allows damaged or worn-out tissues to be repaired and replaced by fresh, healthy cells. As part of this renewal process, stem cells generate pools of more specialized cells, called progenitor cells. These can be thought of as half-way to maturation and can only develop in a more restricted number of ways. For example, so-called myeloid progenitor cells from humans can only develop into a specific group of blood cell types, collectively termed the myeloid lineage. Fruit flies, like many other animals, also have several different types of blood cells. The fly's repertoire of blood cells is very similar to the human myeloid lineage, and these cells also develop from the fly equivalent of myeloid progenitor cells. These progenitors are found in a specialized organ in fruit fly larvae called the lymph gland, where the blood forms. These similarities between fruit flies and humans mean that flies are a good model to study how myeloid progenitor cells mature. A lot is already known about the molecules that signal to progenitor cells how and when to mature. However, the role of metabolism ­ the chemical reactions that process nutrients and provide energy inside cells ­ is still poorly understood. Tiwari et al. set out to identify which metabolic reactions myeloid progenitor cells require and how these reactions might shape the progenitors' development into mature blood cells. The experiments in this study used fruit fly larvae that had been genetically altered so that they could no longer perform key chemical reactions needed for the breakdown of fats. In these mutant larvae, the progenitors within the lymph gland could not give rise to mature blood cells. This showed that myeloid progenitor cells need to be able to break down fats in order to develop properly. These results highlight a previously unappreciated role for metabolism in controlling the development of progenitor cells. If this effect also occurs in humans, this knowledge could one day help medical researchers engineer replacement tissues in the lab, or even increase our own bodies' ability to regenerate blood, and potentially other organs.

A Forward Genetic Approach to Mapping a P-Element Second Site Mutation Identifies DCP2 as a Novel Tumor Suppressor in Drosophila melanogaster.

The use of transposons to create mutations has been the cornerstone of Drosophila genetics in the past few decades. Second-site mutations caused by transpositions are often devoid of transposons and thereby affect subsequent analyses. In a P-element mutagenesis screen, a second site mutation was identified on chromosome 3, wherein the homozygous mutants exhibit classic hallmarks of tumor suppressor mutants, including brain tumor and lethality; hence the mutant line was initially named as lethal (3) tumorous brain [l(3)tb]. Classical genetic approaches relying on meiotic recombination and subsequent complementation with chromosomal deletions and gene mutations mapped the mutation to CG6169, the mRNA decapping protein 2 (DCP2), on the left arm of the third chromosome (3L). Thus the mutation was renamed as DCP2l(3)tb Fine mapping of the mutation further identified the presence of a Gypsy-LTR like sequence in the 5'UTR coding region of DCP2, along with the expansion of the adjacent upstream intergenic AT-rich sequence. The mutant phenotypes are rescued by the introduction of a functional copy of DCP2 in the mutant background, thereby establishing the causal role of the mutation and providing a genetic validation of the allelism. With the increasing repertoire of genes being associated with tumor biology, this is the first instance of mRNA decapping protein being implicated in Drosophila tumorigenesis. Our findings, therefore, imply a plausible role for the mRNA degradation pathway in tumorigenesis and identify DCP2 as a potential candidate for future explorations of cell cycle regulatory mechanisms.

ROS Inhibits Cell Growth by Regulating 4EBP and S6K, Independent of TOR, during Development.

Reactive oxygen species (ROS), despite having damaging roles, serve as signaling molecules regulating diverse biological and physiological processes. Employing in vivo genetic studies in Drosophila, we show that besides causing G1-S arrest by activation of Dacapo, ROS can simultaneously inhibit cell growth by regulating the expression of 4EBP and S6K. This is achieved by triggering a signaling cascade that includes Ask1, JNK, and FOXO independent of the Tsc-TOR growth regulatory pathway. Qualitative and quantitative differences in the types of ROS molecules generated dictate whether cells undergo G1-S arrest only or experience blocks in both cell proliferation and growth. Importantly, during normal development, this signaling cascade is triggered by ecdysone in late larval fat body cells to restrict their growth prior to pupation by antagonizing insulin signaling. The present work reveals an unexpected role of ROS in systemic control of growth in response to steroid hormone signaling to establish organismal size.

Cell Adhesion-Mediated Actomyosin Assembly Regulates the Activity of Cubitus Interruptus for Hematopoietic Progenitor Maintenance in Drosophila.

The actomyosin network is involved in crucial cellular processes including morphogenesis, cell adhesion, apoptosis, proliferation, differentiation, and collective cell migration in Drosophila, Caenorhabditiselegans, and mammals. Here, we demonstrate that Drosophila larval blood stem-like progenitors require actomyosin activity for their maintenance. Genetic loss of the actomyosin network from progenitors caused a decline in their number. Likewise, the progenitor population increased upon sustained actomyosin activation via phosphorylation by Rho-associated kinase. We show that actomyosin positively regulates larval blood progenitors by controlling the maintenance factor Cubitus interruptus (Ci). Overexpression of the maintenance signal via a constitutively activated construct (ci.HA) failed to sustain Ci-155 in the absence of actomyosin components like Zipper (zip) and Squash (sqh), thus favoring protein kinase A (PKA)-independent regulation of Ci activity. Furthermore, we demonstrate that a change in cortical actomyosin assembly mediated by DE-cadherin modulates Ci activity, thereby determining progenitor status. Thus, loss of cell adhesion and downstream actomyosin activity results in desensitization of the progenitors to Hh signaling, leading to their differentiation. Our data reveal how cell adhesion and the actomyosin network cooperate to influence patterning, morphogenesis, and maintenance of the hematopoietic stem-like progenitor pool in the developing Drosophila hematopoietic organ.

Detecting proliferation of adult hemocytes in Drosophila by BrdU incorporation.

Drosophila and mammalian hematopoiesis share several similarities that ranges from phases to the battery of transcription factors and signaling molecules that execute this process. These resounding similarities along with the rich genetic tools available in fruitfly makes it a popular invertebrate model to study blood cell development both during normal and aberrant conditions. The larval system is the most extensively studied to date. Several studies have shown that these hemocytes just like mammalian counterpart proliferate and get routinely regenerated upon infection. However, employing the same protocol it was concluded that blood cell proliferation although abundant in larval stages is absent in adult fruitfly. The current protocol describes the strategies that can be employed to document the hemocyte proliferation in adulthood. The fact that a subset of blood cells tucked away in the hematopoietic hub are not locked in senescence, rather they still harbour the proliferative capacity to tide over challenges was successfully demonstrated by this method.  Although we have adopted bacterial infection as a bait to evoke this proliferative capacity of the hemocytes, we envision that it can also efficiently characterize the proliferative responses of hemocytes in tumorigenic conditions as well as scenarios of environmental and metabolic stresses during adulthood.

Noncanonical Decapentaplegic Signaling Activates Matrix Metalloproteinase 1 To Restrict Hedgehog Activity and Limit Ectopic Eye Differentiation in Drosophila.

One of the pertinent issues associated with cellular plasticity is to understand how the delicate balance between the determined state of cells and the extent to which they can transdetermine is maintained. Employing the well-established model of generating ectopic eyes in developing wing discs of Drosophila by ectopic eyeless expression, we provide evidence for the genetic basis of this mechanism. By both loss-of-function and gain-of-function genetic analyses, we demonstrate that Matrix metalloproteinase 1 (Mmp1) plays an important role in regulating the extent of ectopic ommatidial differentiation. Transcriptional activation of ectopic Mmp1 by the morphogen Decapentaplegic (Dpp) is not triggered by its canonical signaling pathway which involves Mad. Rather, Dpp activates an alternate cascade involving dTak1 and JNK, to induce ectopic Mmp1 expression. Mutational analyses reveal that Mmp1 negatively regulates ectopic eye differentiation by restricting the rate of proliferation and the levels of expression of retinal-determining genes dachshund and eyes absent This is primarily achieved by restricting the range of Hedgehog (Hh) signaling. Importantly, the increase in proliferation and upregulation of target retinal-determining genes, as observed upon attenuating Mmp1 activity, gets significantly rescued when ectopic eyes are generated in wing discs of hh heterozygous mutants. In conjunction with the previously established instructive and permissive roles of Dpp in facilitating ectopic eye differentiation in wing discs, the outcome of this study sheds light on a mechanism by which Dpp plays a dual role in modulating the delicate balance between the determined state of cells and the extent they can transdetermine.