The Interactive Fly

Genes involved in tissue and organ development

Development of Haemocytes and the Lymph Gland

A haematopoietic organ that produces plasmatocytes, crystal cells and lamellocytes, with functions reminiscent of the vertebrate myeloid lineage

  • Haemocyte development in Drosophila melanogaster - from Nature Reviews (Wood, 2007)
  • Subdivision and developmental fate of the head mesoderm in Drosophila
  • The two origins of hemocytes in Drosophila
  • The peripheral nervous system supports blood cell homing and survival in the Drosophila larva
  • The Drosophila lymph gland as a developmental model of hematopoiesis
  • Hematopoietic progenitors and hemocyte lineages in the Drosophila lymph gland
  • A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors
  • Serpent, suppressor of hairless and U-shaped are crucial regulators of hedgehog niche expression and prohemocyte maintenance during Drosophila larval hematopoiesis
  • Active hematopoietic hubs in Drosophila adults generate hemocytes and contribute to immune response
  • Extracellular matrix-modulated Heartless signaling in Drosophila blood progenitors regulates their differentiation via a Ras/ETS/FOG pathway and target of rapamycin function
  • Reactive oxygen species prime Drosophila haematopoietic progenitors for differentiation
  • Oxidative stress in the haematopoietic niche regulates the cellular immune response in Drosophila
  • An unexpected link between Notch signaling and ROS in restricting the differentiation of hematopoietic progenitors in Drosophila
  • Drosophila Rabex-5 restricts Notch activity in hematopoietic cells and maintains hematopoietic homeostasis
  • Sumoylation is tumor-suppressive and confers proliferative quiescence to hematopoietic progenitors in Drosophila melanogaster larvae
  • The Hippo pathway regulates hematopoiesis in Drosophila melanogaster
  • Screening and analysis of Janelia FlyLight project enhancer-Gal4 strains identifies multiple gene enhancers active during hematopoiesis in normal and wasp-challenged Drosophila larvae
  • Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila
  • A glutamate-dependent redox system in blood cells is integral for phagocytosis in Drosophila melanogaster
  • Pvr expression regulators in equilibrium signal control and maintenance of Drosophila blood progenitors
  • Haemocytes control stem cell activity in the Drosophila intestine
  • Active JNK-dependent secretion of Drosophila Tyrosyl-tRNA synthetase by loser cells recruits haemocytes during cell competition
  • Extracellular reactive oxygen species drive apoptosis-induced proliferation via Drosophila macrophages
  • Genetic screen in Drosophila larvae links ird1 function to Toll signaling in the fat body and hemocyte motility
  • The role of variant histone H2AV in D. melanogaster larval hematopoiesis
  • Regulation of Drosophila hematopoietic sites by Activin-β from active sensory neurons
  • Control of RUNX-induced repression of Notch signaling by MLF and its partner DnaJ-1 during Drosophila hematopoiesis

    Lymph Glands

  • Transcription Factors
  • Others

  • Embryonic origin of hemocytes

    Hemocytes are derived exclusively from the mesoderm of the head and disperse along several invariant migratory paths throughout the embryo. The notion of the head as the origin of hemocytes is supported by the finding that hemocytes do not form in Bicaudal D, a mutation lacking all head structures, and in twist-snail double mutants, where no mesoderm develops. All embryonic hemocytes behave like a homogenous population with respect to their potential for phagocytosis. Thus, in the wild type, about 80-90% of hemocytes become macrophages during late development. In mutations with an increased amount of cell death (knirps, stardust, fork head), this figure approaches 100% (Tepass, 1994).

    The two origins of hemocytes in Drosophila

    As in many other organisms, the blood of Drosophila consists of several types of hemocytes, which originate from the mesoderm. By lineage analyses of transplanted cells, two separate anlagen have been defined that give rise to different populations of hemocytes: embryonic hemocytes and lymph gland hemocytes. The anlage of the embryonic hemocytes is restricted to a region within the head mesoderm between 70% and 80% egg length. In contrast to all other mesodermal cells, the cells of this anlage are already determined as hemocytes at the blastoderm stage. Unexpectedly, these hemocytes do not degenerate during late larval stages, but have the capacity to persist through metamorphosis and are still detectable in the adult fly. A second anlage, which gives rise to additional hemocytes at the onset of metamorphosis, is located within the thoracic mesoderm at 50% to 53% egg length. After transplantation within this region, clones were detected in the larval lymph glands. Labeled hemocytes are released by the lymph glands not before the late third larval instar. The anlage of these lymph gland-derived hemocytes is not determined at the blastoderm stage, as indicated by the overlap of clones with other tissues. These analyses reveal that the hemocytes of pupae and adult flies consist of a mixture of embryonic hemocytes and lymph gland-derived hemocytes, originating from two distinct anlagen that are determined at different stages of development (Holz, 2003).

    The origin of the embryonic hemocytes (EH) can be traced back to the head mesoderm of late stage 11 embryos by morphological criteria. Owing to the fact that srp is expressed in a narrow stripe within the cephalic mesoderm at the blastoderm stage and that a loss of srp function leads to a complete loss of embryonic hemocytes, this domain is considered to be the primordium of the EH. By homotopic single-cell transplantations it was possible to restrict the anlage to a sharply delimitated region located at 70% to 80% EL within the mesoderm, exactly corresponding to the cephalic expression domain of srp. The fact that none of the EH clones overlapped with other tissues indicates that the hemocytes are already determined at the blastoderm stage. This was confirmed by heterotopic transplantations from the EH anlage into the abdominal mesoderm; these transplanted cells give rise to hemocytes. Since mesodermal cells transplanted into the EH anlage do not transform into embryonic hemocytes, the determining factor is not able to induce a hemocyte fate within these cells and seems to function cell-autonomously. A good candidate for such a factor is Srp. However, since srp is also expressed in many other tissues that do not give rise to hemocytes, there must be additional genes that lead to a determination of the EH at the blastoderm stage. The early determination of the EH is quite unusual, since all other mesodermal tissues analyzed to date -- including the anlage of the lymph gland-derived hemocytes -- are not restricted to a tissue-specific fate prior to the second postblastodermal mitoses. This might be a developmental adaptation of the EH, which at stage 12 are already differentiated into functional macrophages and are responsible for the removal of apoptotic cells within developing tissues (Holz, 2003).

    It is commonly believed that in Drosophila during larval development the EH population is entirely replaced by hemocytes that have been released by the larval lymph glands. However, it is possible to trace hemocytes originating from the head mesoderm through all stages of development until 14-day-old adult flies. The number of hemocytes progressively rises during larval life, from less than 200 to more than 5000 per individual. Cell lineage analyses unambiguously demonstrate that this increase is due to postembryonic proliferation of the EH. The contribution of the lymph glands to the hemocyte population was determined by means of cell lineage analyses. These studies reveal that the lymph glands do not release blood cells into the hemocoel during all larval stages but exclusively at the end of the third larval instar (Holz, 2003).

    With the onset of metamorphosis, additional hemocytes are released from the lymph glands. Although the lymph glands do not persist through metamorphosis, the marked hemocytes released by the labeled lymph glands are still detectable in adult flies. Hence, all hemocytes found throughout larval life originate solely from the EH anlage, whereas the pupal and imaginal blood is made up of two different populations: EH and LGH (Holz, 2003).

    The two populations of hemocytes share many functional, morphological and genetic similarities. In both cases, the determination of hemocytes depends on srp, while the specification towards the distinct blood cell types is induced by the expression of lozenge (lz) glia cells missing (gcm) and the gcm homolog gcm2. Both EH and LGH differentiate into podocytes, crystal cells and plasmatocytes. Hemocytes of both populations have the capability to adopt macrophage characteristics. However, despite all similarities, the history of the two populations is quite different, since they originate from two different mesodermal regions and are determined at different developmental stages. In view of the fact that the lymph glands do not release hemocytes before the onset of metamorphosis under nonimmune conditions, all hemocytes found in the larval hemocoel represent EH (Holz, 2003).

    The many similarities between EG and LGH raise the question why there are two populations at all. A massive release of hemocytes by the lymph glands is seen just at the onset of pupation. The lymph glands additionally have the capacity to differentiate and release a special type of hemocytes, the lamellocytes, under immune conditions even before the onset of metamorphosis. Thus, because under nonimmune conditions the lymph glands do not release any cells before the onset of pupation, it might be their primary role to provide a reservoir of immune defensive hemocytes. The massive apoptosis and accumulation of cell debris might be a secondary trigger to stimulate proliferation and release of the lymph gland hemocytes (Holz, 2003).

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

    The Drosophila 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. An essential genetic switch 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. These findings suggest that there is a close parallel between the molecular mechanisms functioning in the Drosophila cardiogenic mesoderm and those functioning in the mammalian aorta-gonadal-mesonephros mesoderm (Mandal, 2004).

    Blood and vascular cells in the vertebrate embryo are thought to derive from oligopotent progenitor cells, called hemangioblasts, that arise in the yolk sac and in the aorta-gonadal-mesonephros (AGM) mesenchyme. A close relationship between blood and vascular progenitors is well established, but in vivo evidence that a single cell can divide to produce a blood cell and an endothelial cell is lacking in vertebrate systems. Similarly, the molecular mechanism that distinguishes between the two lineages is not well understood. To address these issues in a simple, genetically amenable system, the genetic control of hematopoiesis was analyzed in Drosophila. The results show that there are close lineage relationships between hematopoietic and vascular cells, similar to those present in the AGM of mammalian systems. Evidence is provided for conserved cassettes of transcription factors and signaling cascades that limit the pool of hemangioblastic cells and promote the blood versus vascular fate (Mandal, 2004).

    In the mature Drosophila embryo, the lymph gland is formed by a paired cluster of ~20 cells flanking the aorta. The aorta and heart represent a contractile tube lined by a layer of myoepithelial vascular cells called cardioblasts. The cells flanking the aorta and heart posterior to the lymph gland are the pericardial cells, which function as excretory cells (nephrocytes). Lymph gland progenitors express the prohemocyte marker Srp and ultrastructurally resemble prohemocytes that develop at an earlier stage from the head mesoderm. Monitoring expression of the zinc-finger protein Odd-skipped (Odd) shows that the lymph gland originates from the dorsal thoracic mesoderm. Odd is expressed in segmental clusters in the dorsal mesoderm of segments T1-A6. The three thoracic Odd-positive clusters coalesce to form the lymph gland, whereas the abdominal clusters formed the pericardial nephrocytes (Mandal, 2004).

    Lymph-gland progenitors, cardioblasts and pericardial cells are closely related by lineage. Labeled 'flipout' (FLP/FRT) clones were induced in embryos aged 3-4 h such that the clones contained only 2-4 cells. Of the two-cell clones, ~50% contained cardioblast and lymph-gland cells; the other clones comprised either cardioblasts or lymph-gland cells alone. Mixed clones were recovered at the late third larval stage. The finding of mixed clones indicates that the cardiogenic mesoderm of D. melanogaster contains oligopotent progenitors that, up to the final division, can give rise both to Srp-positive blood-cell progenitors that form the lymph gland and to vascular cells (Mandal, 2004).

    The cardiogenic mesoderm forms part of the dorsal mesoderm, which requires the homeobox protein Tin and the GATA factor Pnr. In embryos with mutations in tin or pnr, the lymph gland was absent. Maintenance of Tin expression in the dorsal mesoderm requires the activity of at least two signaling pathways regulated by Dpp (the Drosophila homolog of transforming growth factor-ß) and Heartless (Htl; one of the D. melanogaster homologs of the FGF receptor); the dependence of cardioblast and pericardial nephrocyte development on these signaling pathways has been documented. Lymph-gland progenitors did not develop in loss-of-function dpp and htl mutants (Mandal, 2004).

    Between 6 h and 8 h of development, the dorsal mesoderm splits into the cardiogenic mesoderm and the visceral mesoderm. The cardiogenic mesoderm is regulated positively by Wg and negatively by Notch. Lack of Wg signaling results in the absence of all cardiogenic lineages including lymph gland. Notch signaling has the opposite effect and restricts cardiogenic mesodermal fate. Notch is active in the dorsal mesoderm from 6 h to 10 h of development. Eliminating Notch during the first half of this interval by raising embryos homozygous with respect to the temperature-sensitive allele Nts1 at the restrictive temperature resulted in substantially more cardioblasts, pericardial cells and lymph-gland progenitors (Mandal, 2004).

    Lymph-gland progenitors, cardioblasts and pericardial nephrocytes are specified in the cardiogenic mesoderm around the phase of germband retraction 8-10 h after fertilization. At this stage, Tin, which was initially expressed in the whole cardiogenic mesoderm, becomes restricted to a narrow medial compartment containing the cardioblasts. Pnr follows the same restriction. Cells located at a more lateral level in the cardiogenic mesoderm give rise to lymph-gland progenitors (in the thoracic domain) and pericardial nephrocytes (in the abdominal domain) and activate the gene odd. Slightly later, Srp is expressed in lymph-gland progenitors. As reported for the early hemocytes derived from the embryonic head, srp is centrally involved in lymph-gland specification. In srp-null embryos, Odd-expressing cells still formed a lymph gland-shaped cluster flanking the aorta, but these cells also express the pericardial marker pericardin (Prc), suggesting that they lose some aspects of hemocyte precursor identity or gain properties of nephrocytes. As a countercorrelate, ectopic expression of Srp in the whole cardiogenic mesoderm directed by mef2-Gal4 induces pericardial cells to adopt lymph-gland fate (Mandal, 2004).

    Downregulation of tin and pnr in cells in the lateral domain of the cardiogenic mesoderm is essential for lymph-gland specification. Ectopic expression of tin or pnr by twist-Gal4 (or mef2-Gal4) causes a marked reduction in the number of lymph-gland and pericardial cells. The antagonistic effect of tin on lymph-gland progenitors resembles its earlier role in the head mesoderm that gives rise to the larval blood cells; here too, ectopic expression of tin causes a reduction in the number of hemocytes (Mandal, 2004).

    Inhibiting tin and upregulating odd and srp requires input from the Notch signaling pathway. A function of Notch at 6-8 h in specification of the cardiogenic mesoderm is described. Reducing Notch function between 8 h and 10 h causes an increase in the number of cardioblasts and a concomitant loss of pericardial and lymph-gland cells. Overexpressing an activated Notch construct causes a marked increase in lymph-gland size. This late requirement for Notch signaling is separable from the earlier role of Notch in restricting the overall size of the cardiogenic mesoderm. Thus, the sum total of cardioblasts and pericardial or lymph-gland cells in Nts1 embryos shifts between 8 h and 10 h and does not differ substantially from that in wild type, whereas a combined effect on cell number and cell fate is seen in embryos with a Notch deletion. In these embryos, the cardiogenic mesoderm is hyperplasic and develops as cardioblasts at the expense of lymph-gland progenitors and pericardial nephrocytes. The dual role of Notch in restricting the numbers of a pluripotent progenitor pool and in distinguishing between the progeny of these progenitors is reminiscent of the function of Notch in sense-organ development (Mandal, 2004).

    Lymph-gland formation is restricted to the thoracic region by positional cues that are provided by expression of the homeobox proteins of the Antennapedia and Bithorax complex. Specifically, Ultrabithorax (Ubx), which is expressed in segments A2-A5 of the cardiogenic mesoderm, inhibits lymph-gland formation. Loss of Ubx results in the expansion of the lymph-gland fate into the abdominal segments. Conversely, overexpression of Ubx driven by mef2-Gal4 causes the transformation of lymph-gland progenitors into pericardial nephrocytes (Mandal, 2004).

    These findings are suggestive of a model of lymph-gland development in Drosophila that is similar to mammalian hematopoiesis. Lymph-gland progenitors develop as part of the cardiogenic mesoderm that also gives rise to the vascular cells (aorta and heart) and to excretory cells. Similarly, progenitor cells of the blood, aorta and excretory system are closely related both molecularly and developmentally in mammals, where they form part of the AGM. Specification of the cardiogenic mesoderm requires the input of FGF and Wg signaling, as in vertebrate hematopoiesis, where the AGM region is induced in response to several converging signaling pathways including FGF, BMP and Wnt (Mandal, 2004).

    The cardiogenic mesoderm in Drosophila evolves from the dorsal mesoderm and requires input from the Htl, Dpp, Wg and Notch (N) signaling pathways. The cardiogenic mesoderm then differentiates into lymph gland, vascular cells (cardioblasts) and excretory cells (pericardial nephrocytes). A subpopulation of cardioblasts and lymph-gland cells is derived from one progenitor (hemangioblast; HB). Essential for the differentiation of the cardiogenic mesoderm is the Notch-Delta (Dl)-dependent restriction of Tin and Pnr to cardioblasts and the expression of Srp in the lymph gland. In vertebrates, similar cell types are derived from a mesodermal domain called the AGM, which also requires the input of FGF, BMP and Wnt signaling. A subset of AGM-derived cells has been proposed to constitute hemangioblasts, which produce blood progenitors and endothelial cells (Mandal, 2004).

    These findings show that in Drosophila, the cardiovascular and blood-cell lineages are differentiated by an antagonistic relationship between Tin or Pnr expression in the cardioblasts and Srp expression in the lymph-gland progenitors. In vertebrates, GATA factors also have a pivotal role in specifying different lineages among blood-cell progenitors, although not much is known about what differentiates between blood progenitors as a group and endothelial progenitors. The results indicate that this step is driven by input from the Notch signaling pathway. In the thoracic cardiogenic mesoderm, Notch antagonizes tin and pnr expression and aortic cardioblast formation, and promotes srp expression and the development of lymph-gland progenitors. In vertebrates, Notch signaling is also involved in both blood and vascular development. The role of Notch during AGM morphogenesis remains to be investigated (Mandal, 2004).

    Cardioblasts and lymph-gland cells can arise from the division of a single cardiogenic mesodermal cell, which should be called a hemangioblast. A previous study induced clones in the cardiogenic mesoderm but used only Tin as a marker. This study also yielded mixed two-cell clones comprising a cardioblast and a nonlabeled cell, which, in light of the current findings, must be interpreted as a lymph-gland cell. Hemangioblasts have been proposed in vertebrates, although the definitive experiment in which a precursor is marked and its lineage is tracked has not been done. Blast colony-forming cells that give rise to both lineages in vitro and common markers that belong to both cell types in vivo have been identified, but direct evidence for the existence of a common precursor has not yet been found. This study, using genetic analysis of two-cell clones, establishes the existence of such a population in Drosophila. On the basis of these results, and given the conservation of the signaling and transcriptional components described here, the prediction is that many cells of the AGM in vertebrates may give rise to only blood or only vascular cells, but a number of intermixed hemangioblasts may give rise to mixed lineages. Future genetic screens aimed at finding components in early lymph-gland development will probably identify additional pathways and strategies important for vertebrate hematopoiesis (Mandal, 2004).

    The Drosophila lymph gland as a developmental model of hematopoiesis

    Drosophila hematopoiesis occurs in a specialized organ called the lymph gland. In this systematic analysis of lymph gland structure and gene expression, the developmental steps in the maturation of blood cells (hemocytes) from their precursors are defined. In particular, distinct zones of hemocyte maturation, signaling and proliferation in the lymph gland during hematopoietic progression are described. Different stages of hemocyte development have been classified according to marker expression and placed within developmental niches: a medullary zone for quiescent prohemocytes, a cortical zone for maturing hemocytes and a zone called the posterior signaling center for specialized signaling hemocytes. This establishes a framework for the identification of Drosophila blood cells, at various stages of maturation, and provides a genetic basis for spatial and temporal events that govern hemocyte development. The cellular events identified in this analysis further establish Drosophila as a model system for hematopoiesis (Jung, 2005).

    In the late embryo, the lymph gland consists of a single pair of lobes containing ~20 cells each. These express the transcription factors Srp and Odd skipped (Odd), and each cluster of hemocyte precursors is followed by a string of Odd-expressing pericardial cells that are proposed to have nephrocyte function. These lymph gland lobes are arranged bilaterally such that they flank the dorsal vessel, the simple aorta/heart tube of the open circulatory system, at the midline. By the second larval instar, lymph gland morphology is distinctly different in that two or three new pairs of posterior lobes have formed and the primary lobes have increased in size approximately tenfold (to ~200 cells. By the late third instar, the lymph gland has grown significantly in size (approximately another tenfold) but the arrangement of the lobes and pericardial cells has remained the same. The cells of the third instar lymph gland continue to express Srp (Jung, 2005).

    The third instar lymph gland also exhibits a strong, branching network of extracellular matrix (ECM) throughout the primary lobe. This network was visualized using several GFP-trap lines in which GFP is fused to endogenous proteins. For example, line G454 represents an insertion into the viking locus, which encodes a Collagen IV component of the extracellular matrix. The hemocytes in the primary lobes of G454 (expressing Viking-GFP) appear to be clustered into small populations within pockets or chambers bounded by GFP-labeled branches of various sizes. Other lines, such as the uncharacterized GFP-trap line ZCL2867, also highlight this branching pattern. What role this intricate ECM network plays in hematopoiesis, as well as why multiple cells cluster within these ECM chambers, remains to be determined (Jung, 2005).

    Careful examination of dissected, late third-instar lymph glands by differential interference contrast (DIC) microscopy revealed the presence of two structurally distinct regions within the primary lymph gland lobes that have not been previously described. The periphery of the primary lobe generally exhibits a granular appearance, whereas the medial region looks smooth and compact. These characteristics were examined further with confocal microscopy using a GFP-trap line G147, in which GFP is fused to a microtubule-associated protein. The G147 line is expressed throughout the lymph gland but, in contrast to nuclear markers such as Srp and Odd, distinguishes morphological differences among cells because the GFP-fusion protein is expressed in the cytoplasm in association with the microtubule network. Cells in the periphery of the lymph gland make relatively few cell-cell contacts, thereby giving rise to gaps and voids among the cells within this region. This cellular individualization is consistent with the granularity of the peripheral region observed by DIC microscopy. By contrast, cells in the medial region were relatively compact with minimal intercellular space, which is also consistent with the smoother appearance of this region by DIC microscopy. Thus, in the late third instar, the lymph gland primary lobes consist of two physically distinct regions: a medial region consisting of compactly arranged cells, which was termed the medullary zone; and a peripheral region of loosely arranged cells, termed the cortical zone (Jung, 2005).

    Mature hemocytes have been shown to express several markers, including collagens, Hemolectin, Lozenge, Peroxidasin and P1 antigen. The expression of the reporter Collagen-gal4 (Cg-gal4), which is expressed by both plasmatocytes and crystal cells, is restricted to the periphery of the primary lymph gland lobe. Comparison of Cg-gal4 expression in G147 lymph glands, in which the medullary zone and cortical zone can be distinguished, reveals that maturing hemocytes are restricted to the cortical zone. In fact, the expression of each of the maturation markers mentioned above is found to be restricted to the cortical zone. The reporter hml-gal4 and Pxn, which are expressed by the plasmatocyte and crystal cell lineages, are extensively expressed in this region. Likewise, the expression of the crystal cell lineage marker Lozenge is restricted in this manner. The spatial restriction of maturing crystal cells to the cortical zone was verified by several means, including the distribution of melanized lymph gland crystal cells in the Black cells background and analysis of the terminal marker ProPOA1. The cortical zone is also the site of P1 antigen expression, a marker of the plasmatocyte lineage. The uncharacterized GFP fusion line ZCL2826 also exhibits preferential expression in the cortical zone. Last, it was found that the homeobox transcription factor Cut is preferentially expressed in the cortical zone of the primary lobe. Although the role of Cut in Drosophila hematopoiesis is currently unknown, homologs of Cut are known to be regulators of the myeloid hematopoietic lineage in both mice and humans. Cells of the rare third cell type, lamellocytes, are also restricted to the cortical zone, based upon cell morphology and the expression of a msn-lacZ reporter (msn06946). In summary, based on the expression patterns of several genetic markers that identify the three major blood cell lineages, it is proposed that the cortical zone is a specific site for hemocyte maturation (Jung, 2005).

    The medullary zone was initially defined by structural characteristics and subsequently by the lack of expression of mature hemocyte markers. However, several markers have been identified that are exclusively expressed in the medullary zone at high levels but not the cortical zone. Consistent with the compact arrangement of cells in the medullary zone, it was found that Drosophila E-cadherin (DE-cadherin or Shotgun) is highly expressed in this region. No significant expression of DE-cadherin was observed among maturing cells in the cortical zone. E-cadherin, in both vertebrates and Drosophila, is a Ca2+-dependent, homotypic adhesion molecule often expressed by epithelial cells and is a crucial component of adherens junctions. Attempts to study DE-cadherin mutant clones in the medullary zone where the protein is expressed were unsuccessful since no clones were recoverable. The reporter lines domeless-gal4 and unpaired3-gal4 are preferentially expressed in the medullary zone. The gene domeless (dome) encodes a receptor molecule known to mediate the activation of the JAK/STAT pathway upon binding of the ligand Unpaired. The unpaired3 (upd3) gene encodes a protein with homology to Unpaired and has been associated with innate immune function. These gal4 lines are in this study only as markers that correlate with the medullary zone and, at the present time, there is no evidence that their associated proteins have a role in lymph gland hematopoiesis. Other markers of interest with preferential expression in the medullary zone include the molecularly uncharacterized GFP-trap line ZCL2897 and actin5C-GFP. Cells expressing hemocyte maturation markers are not seen in the medullary zone. It is therefore reasonable to propose that this zone is largely populated by prohemocytes that will later mature in the cortical zone. Prohemocytes are characterized by their lack of maturation markers, as well as their expression of several markers described as expressed in the medullary zone (Jung, 2005).

    The posterior signaling center (PSC), a small cluster of cells at the posterior tip of each of the primary (anterior-most) lymph gland lobes, is defined by its expression of the Notch ligand Serrate and the transcription factor Collier. During this analysis, several additional markers were identified that exhibit specific or preferential expression in the PSC region. For example, it was found that the reporter Dorothy-gal4 is strongly expressed in this zone. The Dorothy gene encodes a UDP-glycosyltransferase, which belongs to a class of enzymes that function in the detoxification of metabolites. The upd3-gal4 reporter, which has preferential expression in the medullary zone, is also strongly expressed among cells of the PSC. Last, three uncharacterized GFP-gene trap lines, ZCL2375, ZCL2856 and ZCL0611 were found, that are preferentially expressed in the PSC. This analysis has made it clear that the PSC is a distinct zone of cells that can be defined by the expression of multiple gene products (Jung, 2005).

    The PSC can be defined just as definitively by the characteristic absence of several markers. For example, the RTK receptor Pvr, which is expressed throughout the lymph gland, is notably absent from the PSC. Likewise, dome-gal4 is not expressed in the PSC, further suggesting that this population of cells is biased toward the production of ligands rather than receptor proteins. Maturation markers such as Cg-gal4, which are expressed throughout the cortical zone, are not expressed by PSC cells. Additionally, the expression levels of the hemocyte marker Hemese and the Friend-of-GATA protein U-shaped are dramatically reduced in the PSC when compared with other hemocytes of the lymph gland. Taken together, both the expression and lack of expression of a number of genetic markers defines the cells of the PSC as a unique hemocyte population (Jung, 2005).

    In contrast to primary lobes of the third instar, maturing hemocytes are generally not seen in the secondary lobes. Correspondingly, secondary lobes often have a smooth and compact appearance, much like the medullary zone of the primary lobe. Consistent with this appearance, secondary lymph gland lobes also express high levels of DE-cadherin. The size of the secondary lobe, however, varies from animal to animal and this correlates with the presence or absence of maturation markers. Smaller secondary lobes contain a few or no cells expressing maturation markers, whereas larger secondary lobes usually exhibit groups of differentiating cells. Direct comparison of DE-cadherin expression in secondary lobes with that of Cg-gal4, hml-gal4 or Lz revealed that the expression of these maturation markers occurs only in areas in which DE-cadherin is downregulated. Therefore, although there is no apparent distinction between cortical and medullary zones in differentiating secondary lobes, there is a significant correlation between the expression of maturation markers and the downregulation of DE-cadherin, as is observed in primary lobes (Jung, 2005).

    The relatively late 'snapshot' of lymph gland development in the third larval instar establishes the existence of spatial zones within the lymph gland that are characterized by differences in structure as well as gene expression. In order to understand how these zones form over time, lymph glands of second instar larvae, the earliest time at which it was possible to dissect and stain, were examined for the expression of hematopoietic markers. As expected, Srp and Odd are expressed throughout the lymph gland during the second instar since they are in the late embryo and third instar lymph gland. Likewise, the hemocyte-specific marker Hemese is expressed throughout the lymph gland at this stage, although it is not present in the embryonic lymph gland (Jung, 2005).

    To determine whether the cortical zone is already formed or forming in second instar lymph glands, the expression of various maturation markers were examined in a pair-wise manner to establish their temporal order. Of the markers examined, hml-gal4 and Pxn are the earliest to be expressed. The majority of maturing cells were found to be double-positive for hml-gal4 and Pxn expression, although a few cells were found to express either hml-gal4 or Pxn alone. This indicates that the expression of these markers is initiated at approximately the same time, although probably independently, during lymph gland development. The marker Cg-gal4 is next to be expressed since it was found among a subpopulation of Pxn-expressing cells. Finally, P1 antigen expression is initiated late, usually in the early third instar. Interestingly, the early expression of each of these maturation markers is restricted to the periphery of the primary lymph gland lobe, indicating that the cortical zone begins to form in this position in the second instar. Whenever possible, each genetic marker was directly compared with other pertinent markers in double-labeling experiments, except in cases such as the comparison of two different gal4 reporter lines or when available antibodies were generated in the same animal. In such cases, the relationship between the two markers, for example dome-gal4 and hml-gal4, was inferred from independent comparison with a third marker such as Pxn (Jung, 2005).

    By studying the temporal sequence of expression of hemocyte-specific markers, one can describe stages in the maturation of a hemocyte. It should be noted, however, that not all hemocytes of a particular lineage are identical. For example, in the late third instar lymph gland, the large majority of mature plasmatocytes (~80%) expresses both Pxn and hml-gal4, but the remainder express only Pxn (~15%) or hml-gal4 (~5%) alone. Thus, while plasmatocytes as a group can be characterized by the expression of representative markers, populations expressing subsets of these markers indeed exist. It remains unclear at this time whether this heterogeneity in the hemocyte population is reflective of specific functional differences (Jung, 2005).

    In the third instar, Pxn is a prototypical hemocyte maturation marker, while immature cells of the medullary zone express dome-gal4. Comparing the expression of these two markers in the second instar reveals an interesting developmental progression. A group of cells along the peripheral edge of these early lymph glands already express Pxn. These developing hemocytes downregulate the expression of dome-gal4, as they do in the third instar. Next to these developing hemocytes is a group of cells that expresses dome-gal4 but not Pxn; these cells are most similar to medullary zone cells of the third instar and are therefore prohemocytes. Interestingly, there also exists a group of cells in the second instar that expresses neither Pxn nor dome-gal4. This population is most easily seen in the medial parts of the gland, close to the centrally placed dorsal. These cells resemble earlier precursors in the embryo, except they express the marker Hemese. These cells are called pre-prohemocytes. Interpretation of the expression data is that pre-prohemocytes upregulate dome-gal4 to become prohemocytes. As prohemocytes begin to mature into hemocytes, dome-gal4 expression is downregulated, while the expression of maturation markers is initiated. The prohemocyte and hemocyte populations continue to be represented in the third instar as components of the medullary and cortical zones, respectively (Jung, 2005).

    The cells of the PSC are already distinguishable in the late embryo by their expression of collier. It was found that the canonical PSC marker Ser-lacZ is not expressed in the embryonic lymph gland and is only expressed in a small number of cells in the second instar. This relatively late onset of expression is consistent with collier acting genetically upstream of Ser. Another finding was that the earliest expression of upd3-gal4 parallels the expression of Ser-lacZ and is restricted to the PSC region. Finally, Pvr and dome-gal4 are excluded from the PSC in the second instar, similar to what is seen in the third instar (Jung, 2005).

    To determine whether maturing cortical zone cells are indeed derived from medullary zone prohemocytes, a lineage-tracing experiment was performed in which dome-gal4 was used to initiate the permanent marking of all daughter cell lineages. In this system, the dome-gal4 reporter expresses both UAS-GFP and UAS-FLP. The FLP recombinase excises an intervening FRT-flanked 'STOP cassette', allowing constitutive expression of lacZ under the control of the actin5C promoter. At any developmental time point, GFP is expressed in cells where dome-gal4 is active, while lacZ is expressed in all subsequent daughter cells regardless of whether they continue to express dome-gal4. In this experiment, cortical zone cells are permanently marked with ß-galactosidase despite not expressing dome-gal4 (as assessed by GFP), indicating that these cells are derived from a dome-gal4-positive precursor. This result is consistent with and further supports independent marker analysis that shows that dome-gal4-positive prohemocytes downregulate dome-gal4 expression as they initiate expression of maturation markers representative of cortical zone cells. As controls to the above experiment, the expression patterns of two other gal4 lines, twist-gal4 and Serrate-gal4 were determined. The reporter twist-gal4 is expressed throughout the embryonic mesoderm from which the lymph gland is derived. Accordingly, the entire lymph gland is permanently marked by ß-galactosidase despite a lack of twist-gal4 expression (GFP) in the third instar lymph gland. Analysis of Ser-gal4 reveals that PSC cells remain a distinct population of signaling cells that do not contribute to the cortical zone (Jung, 2005).

    Genetic manipulation of Pvr function provides valuable insight into its involvement in the regulation of temporal events of lymph gland development. To analyze Pvr function, FLP/FRT-based Pvr-mutant clones were generated in the lymph gland early in the first instar and then examined during the third instar for the expression of maturation markers. It was found that loss of Pvr function abolishes P1 antigen and Pxn expression, but not Hemese expression. The crystal cell markers Lz and ProPOA1 are also expressed normally in Pvr-mutant clones, consistent with the observation that mature crystal cells lack or downregulate Pvr. The fact that Pvr-mutant cells express Hemese and can differentiate into crystal cells suggests that Pvr specifically controls plasmatocyte differentiation. Pvr-mutant cells do not become TUNEL positive but do express the hemocyte marker Hemese and can differentiate into crystal cells, all suggesting that the observed block in plasmatocyte differentiation within the mutant clone is not due to cell death. Additionally, Pvr-mutant clones were large and not significantly different in size from their wild-type twin spots. Thus, the primary role of Pvr is not in the control of cell proliferation. Targeting Pvr by RNA interference (RNAi) revealed the same phenotypic features, confirming that Pvr controls the transition of Hemese-positive cells to plasmatocyte fate (Jung, 2005).

    Entry into S phase was monitored using BrdU incorporation and distinct proliferative phases were identified that occur during lymph gland hematopoiesis. In the second instar, proliferating cells are evenly distributed throughout the lymph gland. By the third instar, however, the distribution of proliferating cells is no longer uniform; S-phase cells are largely restricted to the cortical zone. This is particularly evident when BrdU-labeled lymph glands are co-stained with Pxn. Medullary zone cells, which can be identified by the expression of dome-gal4, rarely incorporate BrdU. Therefore, the rapidly cycling prohemocytes of the second instar lymph gland quiesce as they populate the medullary zone of the third instar. As prohemocytes transition into hemocyte fates in the cortical zone, they once again begin to expand in number. This is supported by the observation that the medullary zone in white pre-pupae does not appear diminished in size, suggesting that the primary mechanism for the expansion of the cortical zone prior to this stage is through cell division within the zone. Proliferating cells in the secondary lobes continue to be distributed uniformly in the third instar, suggesting that secondary-lobe prohemocytes do not reach a state of quiescence as do the cells of the medullary zone. These results indicate that cells of the lymph gland go through distinct proliferative phases as hematopoietic development proceeds (Jung, 2005).

    This analysis of the lymph gland revealed three key features that arise during development. The first feature is the presence of three distinct zones in the primary lymph gland lobe of third instar larvae. Two of these zones, termed the cortical and medullary zones, exhibit structural characteristics that make them morphologically distinct. These zones, as well as the third zone, the PSC, are also distinguishable by the expression of specific markers. The second key feature is that cells expressing maturation markers such as Lz, ProPOA1, Pxn, hml-gal4 and Cg-gal4 are restricted to the cortical zone. The medullary zone is consistently devoid of maturation marker expression and is therefore defined as a region composed of immature hemocytes (prohemocytes). The finding of different developmental populations within the lymph gland (prohemoctyes and their derived hemocytes) is similar to the situation in vertebrates where it is known that hematopoietic stem cells and other blood precursors give rise to various mature cell types. Additionally, Drosophila hemocyte maturation is akin to the progressive maturation of myeloid and lymphoid lineages in vertebrate hematopoiesis. The third key feature of lymph gland hematopoiesis is the dynamic pattern of cellular proliferation observed in the third instar. At this stage, the vast majority of S-phase cells in the primary lobe are located in the cortical zone, suggesting a strong correlation between proliferation and hemocyte differentiation. Compared with earlier developmental stages, cell proliferation in the medullary zone actually decreases by the late third instar, suggesting that these cells have entered a quiescent state. Thus, proliferation in the lymph gland appears to be regulated such that growth, quiescence and expansion phases are evident throughout its development (Jung, 2005).

    Drosophila blood cell precursors, prohemocytes and maturing hemocytes each exhibit extensive phases of proliferation. The competence of these cells to proliferate seems to be a distinct cellular characteristic that is superimposed upon the intrinsic maturation program. Based on the patterns of BrdU incorporation in developing primary and secondary lymph gland lobes, it is possible to envision at least two levels of proliferation control during hematopoiesis. It is proposed that the widespread cell proliferation observed in second instar lymph glands and in secondary lobes of third instar lymph glands occurs in response to a growth requirement that provides a sufficient number of prohemocytes for subsequent differentiation. The mechanisms promoting differentiation in the cortical zone also trigger cell proliferation, which accounts for the observed BrdU incorporation in this zone and serves to expand the effector hemocyte population. The quiescent cells of the medullary zone represent a pluripotent precursor population because they, similar to vertebrate hematopoietic precursors, rarely divide and give rise to multiple lineages and cell types (Jung, 2005).

    Based on this analysis a model is proposed by which hemocytes mature in the lymph gland. Hematopoietic precursors that populate the early lymph gland are first distinguishable as Srp+, Odd+ (S+O+) cells. These will eventually give rise to a primary lymph gland lobe where the steps of hemocyte maturation are most apparent. During the first or early second instar, these S+O+ cells begin to express the hemocyte-specific marker Hemese (He) and the tyrosine kinase receptor Pvr. Such cells can be called pre-prohemocytes and, in the second instar, cells expressing only these markers occupy a narrow region near the dorsal vessel. Subsequently, a subset of these Srp+, Odd+, He+, Pvr+ (S+O+H+Pv+) pre-prohemocytes initiate the expression of dome-gal4 (dg4), thereby maturing into prohemocytes. The prohemocyte population (S+O+H+Pv+dg4+) can be subdivided into two developmental stages. Stage 1 prohemocytes, which are abundantly seen in the second instar, are proliferative, whereas stage 2 prohemocytes, exemplified by the cells of the medullary zone, are quiescent. As development continues, prohemocytes begin to downregulate dome-gal4 and express maturation markers (M; becoming S+O+H+Pv+dg4lowM+). Eventually, dome-gal4 expression is lost entirely in these cells (becoming S+O+H+Pv+dg4-M+), found generally in the cortical zone. Thus, the maturing hemocytes of the cortical zone are derived from prohemocytes previously belonging to the medullary zone. This is supported by lineage-tracing experiments that show cells expressing medullary zone markers can indeed give rise to cells of the cortical zone. In turn, the medullary zone is derived from the earlier, pre-prohemocytes. Early cortical zone cells continue to express successive maturation markers (M) as they proceed towards terminal differentiation. Depending on the hemocyte type, examples of expressed maturation markers are Pxn, P1, Lz, L1, msn-lacZ, etc. These studies have shown that differentiation of the plasmatocyte lineage requires Pvr, while previous work has shown that the Notch pathway is crucial for the crystal cell fate. Both the JAK/STAT and Notch pathways have been implicated in lamellocyte production (Jung, 2005).

    Previous investigations have demonstrated that similar transcription factors and signal transduction pathways are used in the specification of blood lineages in both vertebrates and Drosophila. Given this relationship, Drosophila represents a powerful system for identifying genes crucial to the hematopoietic process that are conserved in the vertebrate system. The work presented here provides an analysis of hematopoietic development in the Drosophila lymph gland that not only identifies stage-specific markers, but also reveals developmental mechanisms underlying hemocyte specification and maturation. The prohemocyte population in Drosophila becomes mitotically quiescent, much as their multipotent precursor counterparts in mammalian systems. These conserved mechanisms further establish Drosophila as an excellent genetic model for the study of hematopoiesis (Jung, 2005).

    Subdivision and developmental fate of the head mesoderm in Drosophila

    This paper defines temporal and spatial subdivisions of the embryonic head mesoderm and describes the fate of the main lineages derived from this tissue. During gastrulation, only a fraction of the head mesoderm (primary head mesoderm; PHM) invaginates as the anterior part of the ventral furrow. The PHM can be subdivided into four linearly arranged domains, based on the expression of different combinations of genetic markers (tinman, heartless, snail, serpent, mef-2, zfh-1). The anterior domain (PHMA) produces a variety of cell types, among them the neuroendocrine gland (corpus cardiacum). PHMB, forming much of the'T-bar' of the ventral furrow, migrates anteriorly and dorsally and gives rise to the dorsal pharyngeal musculature. PHMC is located behind the T-bar and forms part of the anterior endoderm, besides contributing to hemocytes. The most posterior domain, PHMD, belongs to the anterior gnathal segments and gives rise to a few somatic muscles, but also to hemocytes. The procephalic region flanking the ventral furrow also contributes to head mesoderm (secondary head mesoderm, SHM) that segregates from the surface after the ventral furrow has invaginated, indicating that gastrulation in the procephalon is much more protracted than in the trunk. This study distinguishes between an early SHM (eSHM) that is located on either side of the anterior endoderm and is the major source of hemocytes, including crystal cells. The eSHM is followed by the late SHM (lSHM), which consists of an anterior and posterior component (lSHMa, lSHMp). The lSHMa, flanking the stomodeum anteriorly and laterally, produces the visceral musculature of the esophagus, as well as a population of tinman-positive cells that is interpreted as a rudimentary cephalic aorta ('cephalic vascular rudiment'). The lSHM contributes hemocytes, as well as the nephrocytes forming the subesophageal body, also called garland cells (de Velasco, 2005).

    The mesoderm is a morphologically distinct cell layer that can be recognized in early embryos of most bilaterian phyla and that gives rise to tissues interposed between ectodermal and endodermal epithelia, including muscle, connective, blood, vascular, and excretory tissue. Besides the differentiative fate of tissues derived from it, the mesoderm shares several common properties in regard to its formation during gastrulation. The anlage of the mesoderm is sandwiched in between the anlage of the endoderm and the neurectoderm. This has been documented in most detail in anamniote vertebrates, where signals from the vegetal blastomeres (the anlage of the endoderm) act on the adjacent marginal zone of the future ectoderm to induce mesoderm. Although gastrulation proceeds quite differently in arthropods from the way it does in chordates, the proximity of the mesodermal anlage to future endoderm and neurectoderm is conserved, and numerous signaling pathways and transcriptional regulators that share similar function and expression patterns in arthropods and chordates have been identified (de Velasco, 2005 and references therein).

    Following gastrulation, the mesoderm is subdivided along the dorso-ventral axis into several subdivisions laid out in a distinct dorso-ventral order. In vertebrates, cells located in the dorsal part of the mesoderm anlage give rise to notochord and somites, which in turn produce muscular, skeletal, and connective tissue. Next to the somitic mesoderm is the intermediate mesoderm that will form the excretory and reproductive system. The ventral mesoderm (lateral plate) gives rise to blood, vascular system, visceral musculature, and coelomic cavity. In arthropods, fundamentally similar mesodermal subdivisions can be recognized, and similarities extend to the relative positions these domains obtain relative to each other and relative to the adjacent neurectoderm. For example, precursors of visceral muscles, vascular system, and blood are at the edge of the mesoderm facing away from the neural primordium (ventral in vertebrates, dorsal in arthropods (de Velasco, 2005 and references therein).

    The subdivision of the vertebrate mesoderm into distinct longitudinal tissue columns with different fates is seen throughout the trunk and head of the embryo. However, several significant differences between the head and the trunk are immediately apparent. For example, cells derived from the anterior neurectoderm form the neural crest that migrates laterally and gives rise to many of the tissues that are produced by mesoderm in the trunk. As a result, the fates taken over by the head mesoderm are more limited than those of the trunk mesoderm. In contrast, the head mesoderm produces several unique lineages, such as the heart (cardiac mesoderm) and a population of early differentiating macrophages. Moreover, some of the signaling pathways responsible for inducing different mesodermal fates in the trunk appear to operate in a different manner in the head. A recently described example is the Wnt signal that induces somatic musculature in the trunk, but inhibits the same fate in the head (de Velasco, 2005 and references therein).

    The head mesoderm of arthropods, like that of vertebrates, also appears to deviate in many ways from the trunk mesoderm. For example, specialized lineages like embryonic blood cells and nephrocytes forming the subesophageal body (also called garland cells) arise exclusively in the head. That being said, very little is known about how the arthropod head mesoderm arises and what types of tissues derive from it. The existing literature mainly uses histology, which severely limits the possibilities of following different cell types forward or backward in time. In this paper, several molecular markers have been used to initiate more detailed studies of the head mesoderm in Drosophila. The goal was to establish temporal and spatial subdivisions of the head mesoderm and, using molecular markers expressed from early stages onward, to follow the fate of the lineages derived from this embryonic tissue. Besides hemocytes and pharyngeal muscles described earlier, the head mesoderm also gives rise to several other lineages, including visceral muscle, putative vascular cells, nephrocytes, and neuroendocrine cells. The development of the head mesoderm is discussed in comparison with the trunk mesoderm and in the broader context of insect embryology (de Velasco, 2005).

    The Drosophila head mesoderm, as traditionally defined, includes all mesoderm cells originating anterior to the cephalic furrow. The formation of the head mesoderm is complicated by the fact that (unlike the mesoderm of the trunk) only part of it invaginates with the ventral furrow; by far, the majority of head mesoderm cells, recognizable in a stage 10 or 11 embryo, segregate from the surface epithelium of the head after the ventral furrow has formed. Another complicating factor is that head mesoderm cells derived from different antero-posterior levels adopt very different fates, unlike the situation in the trunk where mesodermal fates within different segments along the AP axis are fairly homogenous, with obvious exceptions such as the gonadal mesoderm that is derived exclusively from a subset of abdominal segments. Using several different markers, this study has followed the origin, migration pathways, and later, fates of head mesoderm cells (de Velasco, 2005).

    The anterior part of the ventral furrow, called primary head mesoderm (PHM) in the following, includes cells that will contribute to diverse tissues, including muscle, hemocytes, endoderm, and several ill-defined cell populations closely associated with the brain and neuroendocrine system. For clarification, the anterior ventral furrow will be divided into the following domains:

    • Domain A (PHMA): the anterior lip of the T-bar, defined by the expression of sine oculis (so), giant (gt), and a reduced level of heartless (htl).
    • Domain B (PHMB): the posterior lip of the T-bar, defined by high levels of htl and tinman (tin) expression and absence of serpent (srp).
    • Domain C (PHMC): the anterior part of the procephalic ventral furrow proper, defined by the expression of srp and other endoderm markers and by low levels of htl or tin.
    • Domain D (PHMD): the posterior part of the procephalic ventral furrow (anterior gnathal mesoderm), which initially resembles the trunk mesoderm in expressing high levels of htl and tin. Secondarily, much of the gnathal mesoderm switches to srp expression as it adopts the fate of blood cell precursors (de Velasco, 2005).

    The anterior lip of the T-bar (PHMA) is the source of the corpus cardiacum, as well as other gt-positive cells that at least in part end up as nerve cells flanking the frontal connective and frontal ganglion. These cells continue the expression of giant throughout late embryonic development; they represent a hitherto unknown class of nonneuroblast-derived neurons (de Velasco, 2005).

    The posterior lip of the T-bar (PHMB) can be followed towards later stages by its continued expression of htl. These cells, called the procephalic somatic mesoderm, form a bilateral cluster that moves dorso-anteriorly into the labrum and becomes the dorsal pharyngeal musculature. Htl expression almost disappears in these cells around late stage 11, but is reinitiated at stage 12 and stays strong until stage 14, when the dorsal pharyngeal muscles differentiate. Many of the genes expressed in the somatic musculature of the trunk and its precursors (Dmef2, beta-3-tubulin) are also expressed in the procephalic somatic mesoderm (de Velasco, 2005).

    The part of the ventral furrow posteriorly adjacent to the T-bar (PHMC) expresses srp, forkhead (fkh), and other endoderm/hemocyte markers. After the ventral furrow closes in the ventral midline (stage 7/8), these cells form a compact median mass, most of which represents part of the anterior endoderm that gives rise to the midgut epithelium. Starting at around this stage, the lateral part of the hemocyte-forming 'secondary head mesoderm' ingresses in between the endoderm and the surface ectoderm. It is likely that some of the PHMC cells invaginating already with the ventral furrow, along with the cells that form the anterior endoderm, also give rise to hemocytes. Precursors of hemocytes and midgut are difficult to distinguish during and shortly after ventral furrow invagination since both express srp and other markers shared between hemocytes and midgut precursors. At around stage 9, the two populations of precursors disengage. The endoderm remains a compact mesenchyme attached to the invaginating stomodeum; hemocyte precursors move dorsally and take on the shape of expanding vertical plates interposed in between endoderm and ectoderm (de Velasco, 2005).

    Domain PHMD, the short portion of the ventral furrow situated posterior to the endoderm, along with a considerable portion of the mesoderm behind the cephalic furrow, forms the mesoderm of the three gnathal segments (mandible, maxilla, labium). The gnathal mesoderm in many ways behaves like the mesoderm of thoracic and abdominal segments. It gives rise to somatic muscle (the lateral pharyngeal muscles), visceral muscle, and fat body. Unlike trunk mesoderm, gnathal mesoderm does not produce cardioblasts and pericardial cells. Instead, a large proportion of gnathal mesoderm cells, joining the anteriorly adjacent secondary procephalic mesoderm, adopt the fate of hemocytes (de Velasco, 2005).

    Besides the ventral furrow, other parts of the ventral procephalon produce head mesoderm in a complex succession of delamination and ingression events. The head mesoderm that forms from outside the ventral furrow will be called 'secondary mesoderm' (SHM) in the following. Based on the time of formation and the position relative to the stomodeum, the following phases and domains of secondary head mesoderm development can be distinguished (de Velasco, 2005).

    • The early secondary head mesoderm (eSHM) ingresses and delaminates during stages 7-8 from the ventral epithelium flanking the endoderm.
    • The posterior late secondary head mesoderm (lSHMp) ingresses during stages 9-11 from a similar position as the eSHM. Its cells give rise to blood cells and nephrocytes (the subesophageal body or garland cells).
    • The anterior late secondary head mesoderm cells (lSHMa) delaminate lateral and anterior to the stomodeum. Coming from within the anlagen of the esophagus and epipharynx, these cells produce esophageal visceral muscle cells, as well as a novel tin-positive cell type that is interpreted as a rudimentary cephalic vascular structure (de Velasco, 2005).

    Following the obliteration of the ventral furrow at stage 8, the eSHM delaminates from the ventral surface 'meso-ectoderm' (considering that this epithelium still contains mesodermal progenitors!) flanking the endodermal mass. The eSHM forms two monolayered sheets that gradually move dorsally and posteriorly; by stage 9, the eSHM cells line the basal surface of the emerging head neuroblasts. An undefined number of primary head mesoderm cells derived from domain PHMC of the ventral furrow are mingled together with the eSHM cells. The ultimate fate of the eSHM is that of hemocytes: they express srp, followed slightly later by other blood cell markers (e.g., peroxidasin and asrij). A subset of hemocytes, called crystal cells, derive from precursors that form a morphologically conspicuous cluster at the dorsal edge of the eSHM, identifiable from early stage 10 onward by the expression of lz. The mechanism by which at least part of the eSHM delaminates is unique. Thus, it is formed by the vertically oriented division of the surface epithelium, whereby the inner daughters will become eSHMe and the outer ones ectoderm. The focus of vertical mitosis has named the procephalic domain in which it occurs 'mitotic domain #9' (de Velasco, 2005).

    From late stage 9 onward, the early SHMs are followed inside the embryo by the closely adjacent posterior late SHMs. One cluster of posterior late secondary head mesoderm (lSHMp) cells delaminates from the surface epithelium flanking the posterior lip of the stomodeum; a second lSHMp cluster appears at the same stage at a slightly more posterior level. The first cluster seems to contribute to the hemocyte population; the posterior cluster gives rise to the nephrocytes forming the subesophageal body (also called garland cells; labeled by CG32094). Garland cell precursors are initially arranged as a paired cluster latero-ventrally of the esophagus primordium; subsequently, the clusters fuse in the midline and form a crescent underneath the esophagus. Garland cells are distinguished from crystal cells by their size, location, and arrangement: crystal cells are large, round cells grouped in an oblong cloud dorso-anterior to the proventriculus. Garland cells are smaller, closely attached to each other, and lie ventral of the esophagus (de Velasco, 2005).

    During stages 10 and 11, cells delaminate beside and anterior to the stomodeum, originating from the anlage of the esophagus and the epipharynx (labrum). These cells, called anterior late secondary head mesoderm cells (lSHMa), can be followed by their expression of tin. Two groups can be distinguished. The tin-positive cells delaminating from the esophageal anlage (es) give rise to the visceral musculature (vm) surrounding the esophagus. These cells lose tin expression soon after their segregation, but can be recognized by other visceral mesoderm markers such as anti-Connectin. More dorsally, in the anlage of the clypeolabrum (cl) delaminate, the dorsal subpopulation of the lSHMas, which rapidly migrates posteriorly on either side and slightly dorsal of the esophagus, can be found. These cells retain expression of tin into the late embryo. They assemble into two longitudinal rows stretching alongside the roof of the esophagus primordium. During late embryogenesis, they move posteriorly along with the esophagus towards a position behind the brain commissure. Many of the tin-positive SHMs apparently undergo apoptosis: initially counting approximately 25 on either side, they decrease to 12-15 at stage 14 to finally form a single, irregular row of about 15 cells total in the late embryo. These cells come into contact with the anterior tip of the dorsal vessel. This formation of previously undescribed cells, for which the term 'procephalic vascular cells', is proposed, is interpreted as a rudiment of the head aorta, which forms a prominent part of the dorsal vessel in many insect groups (de Velasco, 2005).

    On the basis of additional molecular markers, the tin-positive procephalic vascular cells are further subdivided into two populations. The first subpopulation expresses the muscle and cardioblast-specific marker Dmef2; the second type is Dmef2-negative. In the dorsal vessel of the trunk, tin-positive cells also fall into a Dmef2-positive and a Dmef2-negative population. Dmef2-positive cells of the trunk represent the cardioblasts, myoendothelial cells lining the lumen of the dorsal vessel. Dmef2-negative/tin-positive cells form a somewhat irregular double row of cells attached to the ventral wall of the dorsal vessel. The ultimate fate of these cells has not been explored yet. However, preliminary data suggest that they develop into a muscle band that runs alongside the larval dorsal vessel. This would correspond to the situation in other insects in which such a ventral cardiac muscle band has been described (de Velasco, 2005).

    The role of tinman in the formation of the procephalic vascular rudiment was investigated by assaying tin-mutant embryos for the expression of Dmef2. Similar to the cardioblasts of the trunk, the Dmef2-positive cells of the procephalic vascular rudiment are absent in tin mutants. It is quite likely that the (Dmef2-negative) remainder of the procephalic vascular rudiment is affected as well by loss of tin, but in the absence of appropriate markers (besides tin itself, which is not expressed in the mutant), it was not possible to substantiate this proposal (de Velasco, 2005).

    At the time of appearance of the ventral furrow, segmental markers such as hh do not allow the distinction between distinct 'preoral' segments. Thus, hh is expressed in a wide procephalic stripe in front of the regularly sized mandibular stripe. During stage 7, the procephalic hh stripe splits into an anterior, antennal stripe and a posterior, short, intercalary stripe. The anterior lip of the ventral furrow (domain PHMA) coincides with the anterior boundary of the antenno-intercalary stripe. Thus, the primary head mesoderm and endoderm originating from within the anterior ventral furrow can be considered a derivative of the antennal and intercalary segments. This interpretation is supported by the expression of the homeobox gene labial (lab) found in the intercalary segment. The labial domain covers much of the anterior ventral furrow, including domains PHMB-C (de Velasco, 2005).

    Morphogenetic movements in the ventral head, associated with the closure of the ventral furrow, the formation of the stomodeal placode, and the subsequent invagination of the stomodeum result in a shift of head segmental boundaries. The antennal segment tilts backward, as can be seen from the orientation of the antennal hh stripe that from stage 8 onward forms an almost horizontal line, connecting the cephalic furrow with the sides of the stomodeal invagination (which falls within the ventral realm of the antennal segment, in Drosophila as well as other insects). Since the expression of hh, like that of engrailed (en), coincides with the posterior boundary of a segment, the territory located ventral to the antennal hh stripe falls within the intercalary segment. This implies that most, if not all, of the posterior late SHM, is intercalary in origin. It is further plausible to consider that the anterior lSHM belongs to the intercalary and antennal segment. The vascular cells of the head, a conspicuous derivative of the anterior lSHM in Drosophila, are derived from the antennal mesoderm in other insects. The labrum, with which much of the anterior lSHM is associated, represents a structure that has always been difficult to integrate in the segmental organization of the head. Most likely the labrum represents part of the intercalary segment; this would help explain some of the unusual characteristics of the head mesoderm (de Velasco, 2005).

    In conclusion, several fundamental similarities are found between the mesoderm of the head and that of the trunk regarding the tissues they give rise to, and possibly the signaling pathways deciding over these fates. After an initial phase of structural and molecular homogeneity, the trunk mesoderm becomes subdivided into a dorsal and a ventral domain by a Dpp-signaling event that emanates from the dorsal ectoderm. The dorsal domain, characterized by the Dpp-dependent continued expression of tinman, becomes the source of visceral and cardiogenic mesoderm, among other cell types. A role of Dpp/BMP signaling in cardiogenesis seems to be conserved among insects and vertebrates. Subsequent signaling steps, involving both Wingless and Notch/Delta, separate between these two fates and further subdivide the cardiogenic mesoderm into several distinct lineages, such as cardioblast, pericardial cells, and secondary hemocyte precursors (lymph gland). As a result of these signaling events, Tinman and several other fate-determining transcription factors become restricted to their respective lineages: tin to the cardioblasts, odd to pericardial cells and hemocyte precursors, zfh1 and srp to hemocyte precursors and fat body. Dmef2 and several other transcription factors become restricted to various combinations of muscle types (somatic, visceral, cardiac) (de Velasco, 2005).

    In the head mesoderm, the above genes are associated with similar fates. Tin and Dmef2 appear widely in the procephalic ventral furrow and the anterior lSHM before getting restricted to the procephalic vascular rudiment and/or the pharyngeal musculature, respectively. In contrast with the initially ubiquitous expression of Tin and Dmef2 in the trunk mesoderm, those parts of the head mesoderm giving rise to hemocytes (PHMC, posterior lSHM) never express these mesodermal genes. Previous work has shown that the head gap gene buttonhead (btd) is responsible for the early repression of tin in the above mentioned domains of the head mesoderm. The early absence of Tin and Dmef2 in the head mesodermal hemocyte precursors is paralleled by the presence of Srp and Zfh1 in these cells. Interestingly, Srp/Zfh-positive cells of the head produce only hemocytes and no fat body, suggesting that an as-yet-uncharacterized signaling step prevents the formation of fat body in the head. It is tempting to speculate that there exists within the mesoderm a 'blood/fat body equivalence group'. Blood cells and fat body share not only the expression of fate-determining genes such as srp and zfh1, but also, later, functional properties that have to do with immunity. In the trunk, the blood/fat body equivalence group gives rise mostly to fat body, producing only a limited number of hemocyte precursors in the dorsal mesoderm of the thoracic segments. In the head, on the other hand, all cells of the equivalence group become hemocytes (de Velasco, 2005).

    Attention is drawn to another mesodermal lineage that produces related, yet not identical, cell types in the trunk and the head: the nephrocytes. Nephrocytes are defined by their characteristic ultrastructure (membrane invaginations sealed off by junctions) that attests to their excretory function. In the trunk, nephrocytes are represented by the pericardial cells that settle beside the cardioblasts; a newly discovered nephrocyte population ('star cells') invading the Malpighian tubules is derived from the mesoderm of the tail segments. In the head, nephrocytes aggregate near the junction between esophagus and proventriculus as the subesophageal body, also called garland cells. The fact that from the early stages of development onward different transcription factors are expressed in garland cells and pericardial cells suggests that these cells perform similar, yet not fully overlapping, functions (de Velasco, 2005).

    The two origins of hemocytes in Drosophila

    As in many other organisms, the blood of Drosophila consists of several types of hemocytes, which originate from the mesoderm. By lineage analyses of transplanted cells, two separate anlagen have been defined that give rise to different populations of hemocytes: embryonic hemocytes and lymph gland hemocytes. The anlage of the embryonic hemocytes is restricted to a region within the head mesoderm between 70% and 80% egg length. In contrast to all other mesodermal cells, the cells of this anlage are already determined as hemocytes at the blastoderm stage. Unexpectedly, these hemocytes do not degenerate during late larval stages, but have the capacity to persist through metamorphosis and are still detectable in the adult fly. A second anlage, which gives rise to additional hemocytes at the onset of metamorphosis, is located within the thoracic mesoderm at 50% to 53% egg length. After transplantation within this region, clones were detected in the larval lymph glands. Labeled hemocytes are released by the lymph glands not before the late third larval instar. The anlage of these lymph gland-derived hemocytes is not determined at the blastoderm stage, as indicated by the overlap of clones with other tissues. These analyses reveal that the hemocytes of pupae and adult flies consist of a mixture of embryonic hemocytes and lymph gland-derived hemocytes, originating from two distinct anlagen that are determined at different stages of development (Holz, 2003).

    The origin of the embryonic hemocytes (EH) can be traced back to the head mesoderm of late stage 11 embryos by morphological criteria. Owing to the fact that srp is expressed in a narrow stripe within the cephalic mesoderm at the blastoderm stage and that a loss of srp function leads to a complete loss of embryonic hemocytes, this domain is considered to be the primordium of the EH. By homotopic single-cell transplantations it was possible to restrict the anlage to a sharply delimitated region located at 70% to 80% EL within the mesoderm, exactly corresponding to the cephalic expression domain of srp. The fact that none of the EH clones overlapped with other tissues indicates that the hemocytes are already determined at the blastoderm stage. This was confirmed by heterotopic transplantations from the EH anlage into the abdominal mesoderm; these transplanted cells give rise to hemocytes. Since mesodermal cells transplanted into the EH anlage do not transform into embryonic hemocytes, the determining factor is not able to induce a hemocyte fate within these cells and seems to function cell-autonomously. A good candidate for such a factor is Srp. However, since srp is also expressed in many other tissues that do not give rise to hemocytes, there must be additional genes that lead to a determination of the EH at the blastoderm stage. The early determination of the EH is quite unusual, since all other mesodermal tissues analyzed to date -- including the anlage of the lymph gland-derived hemocytes -- are not restricted to a tissue-specific fate prior to the second postblastodermal mitoses. This might be a developmental adaptation of the EH, which at stage 12 are already differentiated into functional macrophages and are responsible for the removal of apoptotic cells within developing tissues (Holz, 2003).

    It is commonly believed that in Drosophila during larval development the EH population is entirely replaced by hemocytes that have been released by the larval lymph glands. However, it is possible to trace hemocytes originating from the head mesoderm through all stages of development until 14-day-old adult flies. The number of hemocytes progressively rises during larval life, from less than 200 to more than 5000 per individual. Cell lineage analyses unambiguously demonstrate that this increase is due to postembryonic proliferation of the EH. The contribution of the lymph glands to the hemocyte population was determined by means of cell lineage analyses. These studies reveal that the lymph glands do not release blood cells into the hemocoel during all larval stages but exclusively at the end of the third larval instar (Holz, 2003).

    With the onset of metamorphosis, additional hemocytes are released from the lymph glands. Although the lymph glands do not persist through metamorphosis, the marked hemocytes released by the labeled lymph glands are still detectable in adult flies. Hence, all hemocytes found throughout larval life originate solely from the EH anlage, whereas the pupal and imaginal blood is made up of two different populations: EH and LGH (Holz, 2003).

    The two populations of hemocytes share many functional, morphological and genetic similarities. In both cases, the determination of hemocytes depends on srp, while the specification towards the distinct blood cell types is induced by the expression of lozenge (lz) glia cells missing (gcm) and the gcm homolog gcm2. Both EH and LGH differentiate into podocytes, crystal cells and plasmatocytes. Hemocytes of both populations have the capability to adopt macrophage characteristics. However, despite all similarities, the history of the two populations is quite different, since they originate from two different mesodermal regions and are determined at different developmental stages. In view of the fact that the lymph glands do not release hemocytes before the onset of metamorphosis under nonimmune conditions, all hemocytes found in the larval hemocoel represent EH (Holz, 2003).

    The many similarities between EG and LGH raise the question why there are two populations at all. A massive release of hemocytes by the lymph glands is seen just at the onset of pupation. The lymph glands additionally have the capacity to differentiate and release a special type of hemocytes, the lamellocytes, under immune conditions even before the onset of metamorphosis. Thus, because under nonimmune conditions the lymph glands do not release any cells before the onset of pupation, it might be their primary role to provide a reservoir of immune defensive hemocytes. The massive apoptosis and accumulation of cell debris might be a secondary trigger to stimulate proliferation and release of the lymph gland hemocytes (Holz, 2003).

    The peripheral nervous system supports blood cell homing and survival in the Drosophila larva

    Interactions of hematopoietic cells with their microenvironment control blood cell colonization, homing and hematopoiesis. This study introduces larval hematopoiesis as the first Drosophila model for hematopoietic colonization and the role of the peripheral nervous system (PNS) as a microenvironment in hematopoiesis. The Drosophila larval hematopoietic system is founded by differentiated hemocytes of the embryo, which colonize segmentally repeated epidermal-muscular pockets and proliferate in these locations. Importantly, these resident hemocytes tightly colocalize with peripheral neurons, and it was demonstrated that larval hemocytes depend on the PNS as an attractive and trophic microenvironment. atonal (ato) mutant or genetically ablated larvae, which are deficient for subsets of peripheral neurons, show a progressive apoptotic decline in hemocytes and an incomplete resident hemocyte pattern, whereas supernumerary peripheral neurons induced by ectopic expression of the proneural gene scute (sc) misdirect hemocytes to these ectopic locations. This PNS-hematopoietic connection in Drosophila parallels the emerging role of the PNS in hematopoiesis and immune functions in vertebrates, and provides the basis for the systematic genetic dissection of the PNS-hematopoietic axis in the future (Makhijani, 2011).

    Previous reports suggested that embryonic hemocytes persist into postembryonic stages, and that larval hemocyte numbers increase over time. However, the identity of the founders of the larval hematopoietic system, and their lineage during expansion, remained unclear. This study demonstrates that it is the differentiated plasmatocytes of the embryo that persist into larval stages and proliferate to constitute the population of larval hemocytes. Embryonic plasmatocytes comprise 80-90% of a population of 600-700 hemocytes that are BrdU-negative in the late embryo and that do not expand in number, even upon experimental stimulation of their phagocytic function, suggesting their exit from the cell cycle. Thus, proliferation of these hemocytes in the larva implies re-entry into (or progression in) the cell cycle, and expansion by self-renewal in the differentiated state. This finding contrasts with the common mechanism of cell expansion, in which undifferentiated prohemocytes expand by proliferation, which ceases once cell differentiation ensues. In Drosophila, another case of self-renewing differentiated cells has been described in the developing adult tracheal system, and expression of oncogenes such as RasV12 triggers expansion of differentiated larval hemocytes. In vertebrates, differentiated cell populations that self-renew and expand are known for hematopoietic and solid, 'self-duplicating' or 'static', tissues, and neoplasias such as leukemias can develop from differentiated cells that re-gain the ability to expand. Controlling the proliferation of differentiated cells is pivotal in regenerative medicine and cancer biology, and Drosophila larval hemocytes may be an attractive system to study this phenomenon in the future (Makhijani, 2011).

    Previous publications reported dorsal-vessel-associated hemocyte clusters as a 'larval posterior hematopoietic organ' that plays a role in larval immunity. This study now reveals that the earliest compartmentalization of the larval hematopoietic system is based on epidermal-muscular pockets that persist throughout larval development. The retreat of larval hemocytes to secluded hematopoietic environments parallels the vertebrate seeding of hematopoietic sites by hematopoietic stem cells (HSCs) or committed progenitors, which occur at multiple times during development (Makhijani, 2011).

    Correlation of hemocyte residency with elevated proliferation levels and anti-apoptotic cell survival are consistent with the idea that inductive and trophic local microenvironments support hemocytes in epidermal-muscular pockets. Using gain- and loss-of-function analyses, the PNS was identified as such a functional hematopoietic microenvironment. Correspondingly, in vertebrates, HSCs or committed progenitors typically require an appropriate microenvironment, or niche, that provides signals to ensure the survival, maintenance and controlled proliferation and differentiation of these cells. Examples include the bone marrow niche, and inducible peripheral niches in tissue repair, revascularization and tumorigenesis (Makhijani, 2011).

    Larval resident hemocytes are in a dynamic equilibrium, showing at least partial exchange between various resident locations. Based on real-time and time-lapse studies, and consistent with the previously reported adhesion-based recruitment of circulating hemocytes to wound sites, and hemocyte dynamics in the terminal cluster, some of this exchange may be attributed to the detachment, circulation and subsequent re-attachment of hemocytes to resident sites. However, lateral movement of hemocytes during re-formation of the resident pattern suggests that hemocytes can also travel continuously, presumably within the epidermal-muscular layer. This idea is further supported by the elevated hemocyte exchange in young larvae, in which most of the hemocytes reside in epidermal-muscular pockets. The (re-)colonization of resident sites is defined as hemocyte 'homing', which might be based on active processes such as cell migration, and/or passive processes that might involve circulation of the hemolymph or undulation. Negative effects of dominant-negative Rho1 on the resident hemocyte pattern suggest a role for active cytoskeletal processes. These findings show intriguing parallels with vertebrates, in which hematopoietic stem and progenitor cells cycle between defined microenvironments and the peripheral blood (Makhijani, 2011).

    The PNS was identified as a microenvironment that supports hemocyte attraction and trophic survival. Resident hemocytes colocalize with lateral ch and other lateral and dorsal PNS neurons such as md, and loss of ch neurons in ato1 mutants results in distinct hemocyte pattern and number defects. Likewise, genetic ablation of ch and other peripheral neurons strongly affects larval hemocytes regarding their resident pattern and trophic survival. Overexpression of the proneural gene sc induces supernumerary ectopic neurons that effectively attract hemocytes in 3rd instar larvae, providing evidence for a direct role of peripheral neurons or their recruited and closely associated glia or support cells in hemocyte attraction. This, together with the direct or indirect trophic dependence of hemocytes on the PNS, clearly distinguishes these findings from a previously reported role of hemocytes in dendrite and axon pruning, which typically is initiated at the onset of metamorphosis. A functional connection of the PNS with the hematopoietic system might be of fundamental importance across species: in vertebrates, PNS activity governs regulation of HSC egress from the bone marrow and proliferation, and immune responses in lymphocytes and myeloid cells. Indeed, all hematopoietic tissues, such as bone marrow, thymus, spleen and lymph nodes, are highly innervated by the sympathetic and, in some cases in addition, the sensory nervous system. However, since in Drosophila the PNS largely comprises sensory neurons rather than autonomic neurons, future studies will determine mechanistic parallels in the use of these distinct subsets of the PNS with respect to hematopoiesis in different phyla. As direct sensory innervation is present in the mammalian bone marrow and lymph nodes, this work in Drosophila provides important precedence for a role of the sensory nervous system in hematopoiesis (Makhijani, 2011).

    In Drosophila larva, hemocyte attraction to specific PNS locations is developmentally regulated: although the abdominal PNS clusters are maintained from embryonic stages onward, they do not associate with hemocytes in the embryo. In the larva, attraction of resident hemocytes to PNS clusters proceeds in several steps, starting with the lateral PNS cluster (lateral patch) and posterior sensory organs (terminal cluster), and expanding at ~72 hours AEL to the dorsal PNS cluster (dorsal stripe). Only late during larval development, from ~110 hours AEL, can hemocytes be found in ventral locations. This suggests differential upregulation of certain factors that attract hemocytes in otherwise similar classes of neurons or their associated cells, and/or changes in the responsiveness of hemocytes over time (Makhijani, 2011).

    In all backgrounds examined, PNS-dependent hemocyte phenotypes become most apparent from mid-larval development onwards, coincident with the developmental emergence of dorsal hemocyte stripes. An increasing limitation of trophic factors or a developmental loss of redundancy is hypothesized in directional and/or trophic support. The observed phenotypes might be direct or indirect, e.g. involving glia or other closely associated cells. Likewise, sc misexpression experiments show potent attraction of hemocytes by ectopic neurons predominantly in late 3rd instar larvae, suggesting the need for some level of anatomical or molecular differentiation or maturation. All PNS manipulations showed only mild effects on lateral hemocyte patches, suggesting redundant signals of a larger group of neurons or glia, which could not be manipulated in aggregate without inducing embryonic lethality. Also, resident hemocyte homing and induction might involve complex combinations of attractive and/or repulsive signals, similar to the cues operating in axon guidance and directed cell migrations in Drosophila and vertebrates. Alternatively, attraction of hemocytes to the lateral patches might rely on additional, yet to be identified, microenvironments. Dorsal-vessel-associated hemocyte clusters do not colocalize with peripheral neurons and are not affected by manipulations of the PNS. As these clusters build up quickly after resident hemocyte disturbance, it is speculated that their formation might relate to the accumulation of circulating hemocytes, consistent with previous observations (Makhijani, 2011).

    In vertebrates, efforts to characterize at a molecular level the emerging connection between the PNS and the hematopoietic system are ongoing. Both indirect effects, via PNS signals to stromal cells of the bone marrow niche that engage in SDF-1/CXCR4 signaling, and direct effects through stimulation of HSCs with neurotransmitters have been reported. Drosophila larval hematopoiesis will allow the systematic dissection of the cellular and molecular factors that govern PNS-hematopoietic regulation. Future studies will reveal molecular evolutionary parallels and inform the understanding of PNS-controlled hematopoiesis in vertebrates. Furthermore, the system will allow investigation of the mechanisms of self-renewal of differentiated cells in a simple, genetically tractable model organism (Makhijani, 2011).

    The Drosophila lymph gland as a developmental model of hematopoiesis

    Drosophila hematopoiesis occurs in a specialized organ called the lymph gland. In this systematic analysis of lymph gland structure and gene expression, the developmental steps in the maturation of blood cells (hemocytes) from their precursors are defined. In particular, distinct zones of hemocyte maturation, signaling and proliferation in the lymph gland during hematopoietic progression are described. Different stages of hemocyte development have been classified according to marker expression and placed within developmental niches: a medullary zone for quiescent prohemocytes, a cortical zone for maturing hemocytes and a zone called the posterior signaling center for specialized signaling hemocytes. This establishes a framework for the identification of Drosophila blood cells, at various stages of maturation, and provides a genetic basis for spatial and temporal events that govern hemocyte development. The cellular events identified in this analysis further establish Drosophila as a model system for hematopoiesis (Jung, 2005).

    In the late embryo, the lymph gland consists of a single pair of lobes containing ~20 cells each. These express the transcription factors Srp and Odd skipped (Odd), and each cluster of hemocyte precursors is followed by a string of Odd-expressing pericardial cells that are proposed to have nephrocyte function. These lymph gland lobes are arranged bilaterally such that they flank the dorsal vessel, the simple aorta/heart tube of the open circulatory system, at the midline. By the second larval instar, lymph gland morphology is distinctly different in that two or three new pairs of posterior lobes have formed and the primary lobes have increased in size approximately tenfold (to ~200 cells. By the late third instar, the lymph gland has grown significantly in size (approximately another tenfold) but the arrangement of the lobes and pericardial cells has remained the same. The cells of the third instar lymph gland continue to express Srp (Jung, 2005).

    The third instar lymph gland also exhibits a strong, branching network of extracellular matrix (ECM) throughout the primary lobe. This network was visualized using several GFP-trap lines in which GFP is fused to endogenous proteins. For example, line G454 represents an insertion into the viking locus, which encodes a Collagen IV component of the extracellular matrix. The hemocytes in the primary lobes of G454 (expressing Viking-GFP) appear to be clustered into small populations within pockets or chambers bounded by GFP-labeled branches of various sizes. Other lines, such as the uncharacterized GFP-trap line ZCL2867, also highlight this branching pattern. What role this intricate ECM network plays in hematopoiesis, as well as why multiple cells cluster within these ECM chambers, remains to be determined (Jung, 2005).

    Careful examination of dissected, late third-instar lymph glands by differential interference contrast (DIC) microscopy revealed the presence of two structurally distinct regions within the primary lymph gland lobes that have not been previously described. The periphery of the primary lobe generally exhibits a granular appearance, whereas the medial region looks smooth and compact. These characteristics were examined further with confocal microscopy using a GFP-trap line G147, in which GFP is fused to a microtubule-associated protein. The G147 line is expressed throughout the lymph gland but, in contrast to nuclear markers such as Srp and Odd, distinguishes morphological differences among cells because the GFP-fusion protein is expressed in the cytoplasm in association with the microtubule network. Cells in the periphery of the lymph gland make relatively few cell-cell contacts, thereby giving rise to gaps and voids among the cells within this region. This cellular individualization is consistent with the granularity of the peripheral region observed by DIC microscopy. By contrast, cells in the medial region were relatively compact with minimal intercellular space, which is also consistent with the smoother appearance of this region by DIC microscopy. Thus, in the late third instar, the lymph gland primary lobes consist of two physically distinct regions: a medial region consisting of compactly arranged cells, which was termed the medullary zone; and a peripheral region of loosely arranged cells, termed the cortical zone (Jung, 2005).

    Mature hemocytes have been shown to express several markers, including collagens, Hemolectin, Lozenge, Peroxidasin and P1 antigen. The expression of the reporter Collagen-gal4 (Cg-gal4), which is expressed by both plasmatocytes and crystal cells, is restricted to the periphery of the primary lymph gland lobe. Comparison of Cg-gal4 expression in G147 lymph glands, in which the medullary zone and cortical zone can be distinguished, reveals that maturing hemocytes are restricted to the cortical zone. In fact, the expression of each of the maturation markers mentioned above is found to be restricted to the cortical zone. The reporter hml-gal4 and Pxn, which are expressed by the plasmatocyte and crystal cell lineages, are extensively expressed in this region. Likewise, the expression of the crystal cell lineage marker Lozenge is restricted in this manner. The spatial restriction of maturing crystal cells to the cortical zone was verified by several means, including the distribution of melanized lymph gland crystal cells in the Black cells background and analysis of the terminal marker ProPOA1. The cortical zone is also the site of P1 antigen expression, a marker of the plasmatocyte lineage. The uncharacterized GFP fusion line ZCL2826 also exhibits preferential expression in the cortical zone. Last, it was found that the homeobox transcription factor Cut is preferentially expressed in the cortical zone of the primary lobe. Although the role of Cut in Drosophila hematopoiesis is currently unknown, homologs of Cut are known to be regulators of the myeloid hematopoietic lineage in both mice and humans. Cells of the rare third cell type, lamellocytes, are also restricted to the cortical zone, based upon cell morphology and the expression of a msn-lacZ reporter (msn06946). In summary, based on the expression patterns of several genetic markers that identify the three major blood cell lineages, it is proposed that the cortical zone is a specific site for hemocyte maturation (Jung, 2005).

    The medullary zone was initially defined by structural characteristics and subsequently by the lack of expression of mature hemocyte markers. However, several markers have been identified that are exclusively expressed in the medullary zone at high levels but not the cortical zone. Consistent with the compact arrangement of cells in the medullary zone, it was found that Drosophila E-cadherin (DE-cadherin or Shotgun) is highly expressed in this region. No significant expression of DE-cadherin was observed among maturing cells in the cortical zone. E-cadherin, in both vertebrates and Drosophila, is a Ca2+-dependent, homotypic adhesion molecule often expressed by epithelial cells and is a crucial component of adherens junctions. Attempts to study DE-cadherin mutant clones in the medullary zone where the protein is expressed were unsuccessful since no clones were recoverable. The reporter lines domeless-gal4 and unpaired3-gal4 are preferentially expressed in the medullary zone. The gene domeless (dome) encodes a receptor molecule known to mediate the activation of the JAK/STAT pathway upon binding of the ligand Unpaired. The unpaired3 (upd3) gene encodes a protein with homology to Unpaired and has been associated with innate immune function. These gal4 lines are in this study only as markers that correlate with the medullary zone and, at the present time, there is no evidence that their associated proteins have a role in lymph gland hematopoiesis. Other markers of interest with preferential expression in the medullary zone include the molecularly uncharacterized GFP-trap line ZCL2897 and actin5C-GFP. Cells expressing hemocyte maturation markers are not seen in the medullary zone. It is therefore reasonable to propose that this zone is largely populated by prohemocytes that will later mature in the cortical zone. Prohemocytes are characterized by their lack of maturation markers, as well as their expression of several markers described as expressed in the medullary zone (Jung, 2005).

    The posterior signaling center (PSC), a small cluster of cells at the posterior tip of each of the primary (anterior-most) lymph gland lobes, is defined by its expression of the Notch ligand Serrate and the transcription factor Collier. During this analysis, several additional markers were identified that exhibit specific or preferential expression in the PSC region. For example, it was found that the reporter Dorothy-gal4 is strongly expressed in this zone. The Dorothy gene encodes a UDP-glycosyltransferase, which belongs to a class of enzymes that function in the detoxification of metabolites. The upd3-gal4 reporter, which has preferential expression in the medullary zone, is also strongly expressed among cells of the PSC. Last, three uncharacterized GFP-gene trap lines, ZCL2375, ZCL2856 and ZCL0611 were found, that are preferentially expressed in the PSC. This analysis has made it clear that the PSC is a distinct zone of cells that can be defined by the expression of multiple gene products (Jung, 2005).

    The PSC can be defined just as definitively by the characteristic absence of several markers. For example, the RTK receptor Pvr, which is expressed throughout the lymph gland, is notably absent from the PSC. Likewise, dome-gal4 is not expressed in the PSC, further suggesting that this population of cells is biased toward the production of ligands rather than receptor proteins. Maturation markers such as Cg-gal4, which are expressed throughout the cortical zone, are not expressed by PSC cells. Additionally, the expression levels of the hemocyte marker Hemese and the Friend-of-GATA protein U-shaped are dramatically reduced in the PSC when compared with other hemocytes of the lymph gland. Taken together, both the expression and lack of expression of a number of genetic markers defines the cells of the PSC as a unique hemocyte population (Jung, 2005).

    In contrast to primary lobes of the third instar, maturing hemocytes are generally not seen in the secondary lobes. Correspondingly, secondary lobes often have a smooth and compact appearance, much like the medullary zone of the primary lobe. Consistent with this appearance, secondary lymph gland lobes also express high levels of DE-cadherin. The size of the secondary lobe, however, varies from animal to animal and this correlates with the presence or absence of maturation markers. Smaller secondary lobes contain a few or no cells expressing maturation markers, whereas larger secondary lobes usually exhibit groups of differentiating cells. Direct comparison of DE-cadherin expression in secondary lobes with that of Cg-gal4, hml-gal4 or Lz revealed that the expression of these maturation markers occurs only in areas in which DE-cadherin is downregulated. Therefore, although there is no apparent distinction between cortical and medullary zones in differentiating secondary lobes, there is a significant correlation between the expression of maturation markers and the downregulation of DE-cadherin, as is observed in primary lobes (Jung, 2005).

    The relatively late 'snapshot' of lymph gland development in the third larval instar establishes the existence of spatial zones within the lymph gland that are characterized by differences in structure as well as gene expression. In order to understand how these zones form over time, lymph glands of second instar larvae, the earliest time at which it was possible to dissect and stain, were examined for the expression of hematopoietic markers. As expected, Srp and Odd are expressed throughout the lymph gland during the second instar since they are in the late embryo and third instar lymph gland. Likewise, the hemocyte-specific marker Hemese is expressed throughout the lymph gland at this stage, although it is not present in the embryonic lymph gland (Jung, 2005).

    To determine whether the cortical zone is already formed or forming in second instar lymph glands, the expression of various maturation markers were examined in a pair-wise manner to establish their temporal order. Of the markers examined, hml-gal4 and Pxn are the earliest to be expressed. The majority of maturing cells were found to be double-positive for hml-gal4 and Pxn expression, although a few cells were found to express either hml-gal4 or Pxn alone. This indicates that the expression of these markers is initiated at approximately the same time, although probably independently, during lymph gland development. The marker Cg-gal4 is next to be expressed since it was found among a subpopulation of Pxn-expressing cells. Finally, P1 antigen expression is initiated late, usually in the early third instar. Interestingly, the early expression of each of these maturation markers is restricted to the periphery of the primary lymph gland lobe, indicating that the cortical zone begins to form in this position in the second instar. Whenever possible, each genetic marker was directly compared with other pertinent markers in double-labeling experiments, except in cases such as the comparison of two different gal4 reporter lines or when available antibodies were generated in the same animal. In such cases, the relationship between the two markers, for example dome-gal4 and hml-gal4, was inferred from independent comparison with a third marker such as Pxn (Jung, 2005).

    By studying the temporal sequence of expression of hemocyte-specific markers, one can describe stages in the maturation of a hemocyte. It should be noted, however, that not all hemocytes of a particular lineage are identical. For example, in the late third instar lymph gland, the large majority of mature plasmatocytes (~80%) expresses both Pxn and hml-gal4, but the remainder express only Pxn (~15%) or hml-gal4 (~5%) alone. Thus, while plasmatocytes as a group can be characterized by the expression of representative markers, populations expressing subsets of these markers indeed exist. It remains unclear at this time whether this heterogeneity in the hemocyte population is reflective of specific functional differences (Jung, 2005).

    In the third instar, Pxn is a prototypical hemocyte maturation marker, while immature cells of the medullary zone express dome-gal4. Comparing the expression of these two markers in the second instar reveals an interesting developmental progression. A group of cells along the peripheral edge of these early lymph glands already express Pxn. These developing hemocytes downregulate the expression of dome-gal4, as they do in the third instar. Next to these developing hemocytes is a group of cells that expresses dome-gal4 but not Pxn; these cells are most similar to medullary zone cells of the third instar and are therefore prohemocytes. Interestingly, there also exists a group of cells in the second instar that expresses neither Pxn nor dome-gal4. This population is most easily seen in the medial parts of the gland, close to the centrally placed dorsal. These cells resemble earlier precursors in the embryo, except they express the marker Hemese. These cells are called pre-prohemocytes. Interpretation of the expression data is that pre-prohemocytes upregulate dome-gal4 to become prohemocytes. As prohemocytes begin to mature into hemocytes, dome-gal4 expression is downregulated, while the expression of maturation markers is initiated. The prohemocyte and hemocyte populations continue to be represented in the third instar as components of the medullary and cortical zones, respectively (Jung, 2005).

    The cells of the PSC are already distinguishable in the late embryo by their expression of collier. It was found that the canonical PSC marker Ser-lacZ is not expressed in the embryonic lymph gland and is only expressed in a small number of cells in the second instar. This relatively late onset of expression is consistent with collier acting genetically upstream of Ser. Another finding was that the earliest expression of upd3-gal4 parallels the expression of Ser-lacZ and is restricted to the PSC region. Finally, Pvr and dome-gal4 are excluded from the PSC in the second instar, similar to what is seen in the third instar (Jung, 2005).

    To determine whether maturing cortical zone cells are indeed derived from medullary zone prohemocytes, a lineage-tracing experiment was performed in which dome-gal4 was used to initiate the permanent marking of all daughter cell lineages. In this system, the dome-gal4 reporter expresses both UAS-GFP and UAS-FLP. The FLP recombinase excises an intervening FRT-flanked 'STOP cassette', allowing constitutive expression of lacZ under the control of the actin5C promoter. At any developmental time point, GFP is expressed in cells where dome-gal4 is active, while lacZ is expressed in all subsequent daughter cells regardless of whether they continue to express dome-gal4. In this experiment, cortical zone cells are permanently marked with ß-galactosidase despite not expressing dome-gal4 (as assessed by GFP), indicating that these cells are derived from a dome-gal4-positive precursor. This result is consistent with and further supports independent marker analysis that shows that dome-gal4-positive prohemocytes downregulate dome-gal4 expression as they initiate expression of maturation markers representative of cortical zone cells. As controls to the above experiment, the expression patterns of two other gal4 lines, twist-gal4 and Serrate-gal4 were determined. The reporter twist-gal4 is expressed throughout the embryonic mesoderm from which the lymph gland is derived. Accordingly, the entire lymph gland is permanently marked by ß-galactosidase despite a lack of twist-gal4 expression (GFP) in the third instar lymph gland. Analysis of Ser-gal4 reveals that PSC cells remain a distinct population of signaling cells that do not contribute to the cortical zone (Jung, 2005).

    Genetic manipulation of Pvr function provides valuable insight into its involvement in the regulation of temporal events of lymph gland development. To analyze Pvr function, FLP/FRT-based Pvr-mutant clones were generated in the lymph gland early in the first instar and then examined during the third instar for the expression of maturation markers. It was found that loss of Pvr function abolishes P1 antigen and Pxn expression, but not Hemese expression. The crystal cell markers Lz and ProPOA1 are also expressed normally in Pvr-mutant clones, consistent with the observation that mature crystal cells lack or downregulate Pvr. The fact that Pvr-mutant cells express Hemese and can differentiate into crystal cells suggests that Pvr specifically controls plasmatocyte differentiation. Pvr-mutant cells do not become TUNEL positive but do express the hemocyte marker Hemese and can differentiate into crystal cells, all suggesting that the observed block in plasmatocyte differentiation within the mutant clone is not due to cell death. Additionally, Pvr-mutant clones were large and not significantly different in size from their wild-type twin spots. Thus, the primary role of Pvr is not in the control of cell proliferation. Targeting Pvr by RNA interference (RNAi) revealed the same phenotypic features, confirming that Pvr controls the transition of Hemese-positive cells to plasmatocyte fate (Jung, 2005).

    Entry into S phase was monitored using BrdU incorporation and distinct proliferative phases were identified that occur during lymph gland hematopoiesis. In the second instar, proliferating cells are evenly distributed throughout the lymph gland. By the third instar, however, the distribution of proliferating cells is no longer uniform; S-phase cells are largely restricted to the cortical zone. This is particularly evident when BrdU-labeled lymph glands are co-stained with Pxn. Medullary zone cells, which can be identified by the expression of dome-gal4, rarely incorporate BrdU. Therefore, the rapidly cycling prohemocytes of the second instar lymph gland quiesce as they populate the medullary zone of the third instar. As prohemocytes transition into hemocyte fates in the cortical zone, they once again begin to expand in number. This is supported by the observation that the medullary zone in white pre-pupae does not appear diminished in size, suggesting that the primary mechanism for the expansion of the cortical zone prior to this stage is through cell division within the zone. Proliferating cells in the secondary lobes continue to be distributed uniformly in the third instar, suggesting that secondary-lobe prohemocytes do not reach a state of quiescence as do the cells of the medullary zone. These results indicate that cells of the lymph gland go through distinct proliferative phases as hematopoietic development proceeds (Jung, 2005).

    This analysis of the lymph gland revealed three key features that arise during development. The first feature is the presence of three distinct zones in the primary lymph gland lobe of third instar larvae. Two of these zones, termed the cortical and medullary zones, exhibit structural characteristics that make them morphologically distinct. These zones, as well as the third zone, the PSC, are also distinguishable by the expression of specific markers. The second key feature is that cells expressing maturation markers such as Lz, ProPOA1, Pxn, hml-gal4 and Cg-gal4 are restricted to the cortical zone. The medullary zone is consistently devoid of maturation marker expression and is therefore defined as a region composed of immature hemocytes (prohemocytes). The finding of different developmental populations within the lymph gland (prohemoctyes and their derived hemocytes) is similar to the situation in vertebrates where it is known that hematopoietic stem cells and other blood precursors give rise to various mature cell types. Additionally, Drosophila hemocyte maturation is akin to the progressive maturation of myeloid and lymphoid lineages in vertebrate hematopoiesis. The third key feature of lymph gland hematopoiesis is the dynamic pattern of cellular proliferation observed in the third instar. At this stage, the vast majority of S-phase cells in the primary lobe are located in the cortical zone, suggesting a strong correlation between proliferation and hemocyte differentiation. Compared with earlier developmental stages, cell proliferation in the medullary zone actually decreases by the late third instar, suggesting that these cells have entered a quiescent state. Thus, proliferation in the lymph gland appears to be regulated such that growth, quiescence and expansion phases are evident throughout its development (Jung, 2005).

    Drosophila blood cell precursors, prohemocytes and maturing hemocytes each exhibit extensive phases of proliferation. The competence of these cells to proliferate seems to be a distinct cellular characteristic that is superimposed upon the intrinsic maturation program. Based on the patterns of BrdU incorporation in developing primary and secondary lymph gland lobes, it is possible to envision at least two levels of proliferation control during hematopoiesis. It is proposed that the widespread cell proliferation observed in second instar lymph glands and in secondary lobes of third instar lymph glands occurs in response to a growth requirement that provides a sufficient number of prohemocytes for subsequent differentiation. The mechanisms promoting differentiation in the cortical zone also trigger cell proliferation, which accounts for the observed BrdU incorporation in this zone and serves to expand the effector hemocyte population. The quiescent cells of the medullary zone represent a pluripotent precursor population because they, similar to vertebrate hematopoietic precursors, rarely divide and give rise to multiple lineages and cell types (Jung, 2005).

    Based on this analysis a model is proposed by which hemocytes mature in the lymph gland. Hematopoietic precursors that populate the early lymph gland are first distinguishable as Srp+, Odd+ (S+O+) cells. These will eventually give rise to a primary lymph gland lobe where the steps of hemocyte maturation are most apparent. During the first or early second instar, these S+O+ cells begin to express the hemocyte-specific marker Hemese (He) and the tyrosine kinase receptor Pvr. Such cells can be called pre-prohemocytes and, in the second instar, cells expressing only these markers occupy a narrow region near the dorsal vessel. Subsequently, a subset of these Srp+, Odd+, He+, Pvr+ (S+O+H+Pv+) pre-prohemocytes initiate the expression of dome-gal4 (dg4), thereby maturing into prohemocytes. The prohemocyte population (S+O+H+Pv+dg4+) can be subdivided into two developmental stages. Stage 1 prohemocytes, which are abundantly seen in the second instar, are proliferative, whereas stage 2 prohemocytes, exemplified by the cells of the medullary zone, are quiescent. As development continues, prohemocytes begin to downregulate dome-gal4 and express maturation markers (M; becoming S+O+H+Pv+dg4lowM+). Eventually, dome-gal4 expression is lost entirely in these cells (becoming S+O+H+Pv+dg4-M+), found generally in the cortical zone. Thus, the maturing hemocytes of the cortical zone are derived from prohemocytes previously belonging to the medullary zone. This is supported by lineage-tracing experiments that show cells expressing medullary zone markers can indeed give rise to cells of the cortical zone. In turn, the medullary zone is derived from the earlier, pre-prohemocytes. Early cortical zone cells continue to express successive maturation markers (M) as they proceed towards terminal differentiation. Depending on the hemocyte type, examples of expressed maturation markers are Pxn, P1, Lz, L1, msn-lacZ, etc. These studies have shown that differentiation of the plasmatocyte lineage requires Pvr, while previous work has shown that the Notch pathway is crucial for the crystal cell fate. Both the JAK/STAT and Notch pathways have been implicated in lamellocyte production (Jung, 2005).

    Previous investigations have demonstrated that similar transcription factors and signal transduction pathways are used in the specification of blood lineages in both vertebrates and Drosophila. Given this relationship, Drosophila represents a powerful system for identifying genes crucial to the hematopoietic process that are conserved in the vertebrate system. The work presented here provides an analysis of hematopoietic development in the Drosophila lymph gland that not only identifies stage-specific markers, but also reveals developmental mechanisms underlying hemocyte specification and maturation. The prohemocyte population in Drosophila becomes mitotically quiescent, much as their multipotent precursor counterparts in mammalian systems. These conserved mechanisms further establish Drosophila as an excellent genetic model for the study of hematopoiesis (Jung, 2005).

    Hematopoietic progenitors and hemocyte lineages in the Drosophila lymph gland

    The Drosophila lymph gland (LG) is a model system for studying hematopoiesis and blood cell homeostasis. This study investigated the patterns of division and differentiation of pro-hemocytes in normal developmental conditions and response to wasp parasitism, by combining lineage analyses and molecular markers for each of the three hemocyte types. The results show that the embryonic LG contains primordial hematopoietic cells which actively divide to give rise to a pool of pro-hemocytes. No evidence was found for the existence of bona fide stem cells and rather suggest that Drosophila pro-hemocytes are regulated as a group of cells, rather than individual stem cells. The fate-restriction of plasmatocyte and crystal cell progenitors occurs between the end of embryogenesis and the end of the first larval instar, while Notch activity is required for the differentiation of crystal cells in third instar larvae only. Upon parasitism, lamellocyte differentiation prevents crystal cell differentiation and lowers plasmatocyte production. It was also found that a new population of intermediate progenitors appears at the onset of hemocyte differentiation and accounts for the increasing number of differentiated hemocytes in the third larval instar. These findings provide a new framework to identify parameters of developmental plasticity of the Drosophila lymph gland and hemocyte homeostasis in physiological conditions and in response to immunological cues (Krzemien, 2011).

    The posterior signaling center, PSC, initially identified as a small cluster of posterior LG cells expressing the Notch (N) ligand Serrate (Ser) has been shown to play a key function in controlling the balance between multipotent pro-hemocytes and differentiating hemocytes in the larval LG. In L3 larvae, PSC cells act, in a non-cell autonomous manner, to maintain JAK/STAT signalling activity in pro-hemocytes, thereby preserving the multipotent character necessary for these cells to adopt a lamellocyte fate in response to parasitism. PSC cells are specified in the embryo by expression of Antennapedia (Antp) and Collier (Col). The morphogen Hedgehog (Hh) starts to be expressed in PSC cells in second instar (L2) larvae and is required for hemocyte homeostasis in L3 larvae. Wg and its receptor are also expressed in the PSC where their activity controls the number of PSC cells. How Hh and Wg activity provided by PSC could be connected to JAK-STAT signalling in pro-hemocytes remains, however, unknown (Krzemien, 2011 and references therein).

    The key role of the PSC in the maintenance of hematopoietic progenitors is reminiscent of the vertebrate hematopoietic stem cell (HSC) niche, a term coined more than 30 years ago to describe the structural and regulatory micro-environment sustaining long-term renewal of HSC in the bone marrow. Cell 'stemness' refers to the potential to self renew and at the same time produce daughter cells that can commit to lineage-specific differentiation. Mouse HSCs isolated from the bone marrow were operationally defined as able to reconstitute long-term, multilineage hematopoiesis after transplantation in a recipient individual. Unfortunately, a similar reconstitution assay is not available in Drosophila (Krzemien, 2011 and references therein).

    Drosophila larval hematopoiesis relies upon the early specification of two cell lineages in the lymph gland, primordial hematopoietic cells at the origin of the three types of hemocytes and PSC cells. PSC cells divide rarely, remain clustered and act as a niche in third instar larvae to control hemocyte homeostasis. Primordial hematopoietic cells actively divide to generate a large pool of progenitors before hemocytes start to differentiate in early L3 larvae. No evidence for the existence of 'classical' stem cells. Based on clonal analyses, it is proposed that larval hematopoietic pro-hemocytes are regulated as a population, rather than as individual stem cells. Lamellocyte differentiation in response to wasp parasitism is preceded by a wave of mitosis and takes place at the complete expense of crystal cell differentiation and part of plasmatocyte differentiation. Finally, evidence was found for a pool of mitotic undifferentiated cells interspersed with differentiated hemocytes which were designated as intermediate progenitors and account for the increase in hemocyte numbers observed throughout the 3rd instar. Overall, the Drosophila hematopoietic organ shows a striking developmental plasticity since the size and number of LG lobes and the extent of hemocyte differentiation may vary from one larva to the other. The findings provide a useful framework to identify the parameters of this plasticity and more broadly how the communications between the niche and hematopoietic progenitors integrate physiological and immunological cues (Krzemien, 2011).

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

    The Drosophila lymph gland is a haematopoietic organ in which pluripotent blood cell progenitors proliferate and mature into differentiated haemocytes. Previous work (Jung, 2005) 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. This study shows 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. It is concluded 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 (Mandal, 2007).

    The Drosophila lymph gland primordium is formed by the coalescence of three paired clusters of cells that express Odd-skipped (Odd) and arise within segments T1-T3 of the embryonic cardiogenic mesoderm. At developmental stages 11-12, mesodermal expression of Antp is restricted to the T3 segment. A fraction of these Antp-expressing cells will contribute to the formation of the dorsal vessel, whereas the remainder, which also express Odd, give rise to the PSC. By stages 13-16, the clusters coalesce and Antp is observed in 5-6 cells at the posterior boundary of the lymph gland. The expression of Antp is subsequently maintained in the PSC through the third larval instar. The embryonic stage 16 PSC can also be distinguished by Fasciclin III expression and at stage 17 these are the only cells in the lymph gland that incorporate BrdU (Mandal, 2007).

    Previous studies have identified the transcription factor Collier (Col) as an essential component regulating PSC function. The gene for this protein is initially expressed in the entire embryonic lymph gland anlagen and by stage 16 is refined to the PSC. In col mutants, the PSC is initially specified, but is entirely lost by the third larval instar. To address further the role of Antp and Col in embryonic lymph gland development, the expression of each gene was investigated in the loss-of-function mutant background of the other. It was found that loss of col does not affect embryonic Antp expression. In contrast, col expression is absent in the PSC of Antp mutant embryos, establishing that Antp functions genetically upstream of Col in the PSC (Mandal, 2007).

    In imaginal discs, the expression of Antp is related to that of the homeodomain cofactor Homothorax (Hth). In the embryonic lymph gland, Hth is initially expressed ubiquitously but is subsequently downregulated in PSC cells, which become Antp-positive. In hth loss-of-function mutants, the lymph gland is largely missing, whereas misexpression of hth causes loss of PSC and the size of the embryonic lymph gland remains relatively normal. It is concluded that a mutually exclusive functional relationship exists between Antp and Hth in the lymph gland such that Antp specifies the PSC, whereas Hth specifies the rest of the lymph gland tissue. Interestingly, knocking out the mouse homologue of Hth, Meis1, eliminates definitive haematopoiesis (Hisa, 2004; Azcoitia, 2005). Meis1 is also required for the leukaemic transformation of myeloid precursors overexpressing HoxB9 (Mandal, 2007).

    Although lymph gland development is initiated in the embryo, the establishment of zones and the majority of haemocyte maturation takes place in the third larval instar. At this stage, Antp continues to be expressed in the wild-type PSC. To investigate how the loss of PSC cells affects haematopoiesis, Antp expression was examined in third instar col mutant lymph glands. In this background, all Antp-positive PSC cells are missing, consistent with the previously described role for col in PSC maintenance. Overexpression of Antp within the PSC increases the size of PSC from the usual 30-45 cells to 100-200 cells. These PSC cells are scattered over a larger volume, often forming two or three large cell clusters rather than the single, dense population seen in wild type (Mandal, 2007).

    To determine the role of PSC in haematopoiesis, the expression pattern of various markers was investigated in lymph glands of larvae of the above genotypes, which either lack a PSC or have an enlarged PSC. The status of blood cell progenitors was directly assessed using the medullary-zone-specific markers ZCL2897, DE-cadherin (Shotgun) and domeless-gal4. In col mutant lymph glands, expression of these markers is absent or severely reduced and when the PSC is expanded, the medullary zone is greatly enlarged. Previous work demonstrated that medullary zone precursors are relatively quiescent, a characteristic similar to the slowly cycling stem cell or progenitor populations in other systems. BrdU incorporation in the wild-type lymph gland is largely restricted to the cortical zone, but in third-instar col mutants incorporation of BrdU is increased relative to wild type and becomes distributed throughout the lymph gland, suggesting that the quiescence of the medullary zone haematopoietic precursors is no longer maintained in the absence of the PSC. Similarly, when the PSC domain is expanded, BrdU incorporation is significantly suppressed throughout the lymph gland (Mandal, 2007).

    P1 and ProPO were used as markers for plasmatocytes and crystal cells, respectively, to assess the extent of haemocyte differentiation within lymph glands of the above genotypes. Loss of the PSC does not compromise haemocyte differentiation; rather, mature plasmatocytes and crystal cells are found abundantly within the lymph gland. Furthermore, the distribution of these differentiating cells is not restricted to the peripheral region that normally constitutes the cortical zone and many cells expressing ProPO and P1 can be observed medially throughout the region normally occupied by the medullary zone. Increasing the PSC domain causes a concomitant reduction in the differentiation of haemocytes (Mandal, 2007).

    In summary, loss of the PSC causes a loss of medullary zone markers, a loss of the quiescence normally observed in the wild-type precursor population and an increase in cellular differentiation throughout the lymph gland. Similarly, increased PSC size leads to an increase in the medullary zone, a decrease in BrdU incorporation and a decrease in the expression of maturation markers. It is concluded that the PSC functions as a haematopoietic niche that maintains the population of multipotent blood cell progenitors within the lymph gland. The observed abundance of mature cells in the absence of the PSC suggests that the early blood cell precursors generated during the normal course of development will differentiate in the absence of a PSC-dependent mechanism that normally maintains progenitors as a population. This situation is reminiscent of the Drosophila and C. elegans germ lines in which disruption of the niche does not block differentiation per se, but lesser numbers of differentiated cells are generated as a result of the failure to maintain stem cells. It is also interesting to note that col mutant larvae are unable to mount a lamellocyte response to immune challenge. It is speculated that this could be because of the loss of precursor cells that are necessary as a reserve to differentiate during infestation (Mandal, 2007).

    Recent work on several vertebrate and invertebrate developmental systems has highlighted the importance of niches as unique microenvironments in the maintenance of precursor cell populations. Examples include haematopoietic, germline and epidermal stem cell niches that provide, through complex signalling interactions, stem cells with the ability to self-renew and persist in a non-differentiated state. The work presented in this report demonstrates that the PSC is required for the maintenance of medullary zone haematopoietic progenitors. The medullary zone represents a group of cells within the lymph gland that are compactly arranged and express the homotypic cell-adhesion molecule, DE-cadherin. These cells are pluripotent, slowly cycling and undifferentiated and are capable of self-renewal. It is presently uncertain whether Drosophila has blood stem cells capable of long-term repopulation as haematopoietic stem cells are in vertebrates. Nevertheless, it is clear that the maintenance of medullary zone cells as precursors is niche dependent (Mandal, 2007).

    In order for the PSC to function as a haematopoietic niche there should exist a means by which the PSC can communicate with precursors. As such, a signal emanating from the PSC and sensed by the medullary zone represents an attractive model of how this might occur. Although it has been reported that Ser and Upd3 are expressed in the PSC, preliminary analysis suggests that elimination of either of these ligands alone will not cause the phenotype seen for Antp and col mutants. Therefore the haematopoietic role of several signalling pathways was investigated and the hedgehog (hh) signalling pathway was identified as a putative regulator in the maintenance of blood cell progenitors. The hhts2 lymph gland is remarkably similar in its phenotype to that seen for Antp hypomorphic or col loss-of-function mutants. Blocking Hh signalling in the lymph gland through the expression of a dominant-negative form of the downstream activator Cubitus interruptus (Ci, the Drosophila homologue of Gli) also causes a phenotype similar to that observed in Antp and col loss-of-function backgrounds. This is true when expressed either specifically in the medullary zone or throughout the lymph gland (Mandal, 2007).

    Consistent with the above functional results, Hh protein is expressed in the second instar PSC and continues to be expressed in third instar PSC cells. In the hhts2 mutant background, the PSC cells continue to express Antp at the restrictive temperature indicating that, unlike col and Antp, Hh is not essential for the specification of the PSC. Rather, Hh constitutes a component of the signalling network that allows the PSC to maintain the precursor population of the medullary zone. Consistent with this notion, downstream components of the Hh pathway, the receptor Patched (Ptc) and activated Ci, are found in the medullary zone. On the basis of both functional and expression data, it is proposed that Hh in the PSC signals through activated Ci in medullary zone cells, thereby keeping them in a quiescent precursor state (Mandal, 2007).

    The Hh pathway has been studied extensively in the context of animal development. Although the Hh signal does not disperse widely on secretion, many studies have shown that this signal can be transmitted over long distances. The mechanism by which this occurs is not fully clear and this is also true of how the PSC delivers Hh to medullary zone progenitors. However, when labelled with green fluorescent protein (GFP), it was found that PSC cells extend numerous thin processes over many cell diameters. The morphology of the PSC cells, taken together with the long-range function of Hh revealed by the mutant phenotype, indicates that the long cellular extensions may deliver Hh to receiving cells not immediately adjacent to the PSC. In this respect, the Drosophila haematopoietic system shows remarkable similarity to the C. elegans germline. In both cases, precursors are maintained as a population over some distance from the niche and in both instances, the niche cells extend long processes when interacting with the precursors (Mandal, 2007).

    Several studies have highlighted the importance of homeodomain proteins in stem cell development and leukaemias. Likewise, the role of Hh in vertebrate and invertebrate stem cell maintenance has recently received much attention. This study describes direct roles for Antp in the specification and Hh in the functioning of a haematopoietic niche. The medullary zone cells are blood progenitors that are maintained in the lymph gland at later larval stages by Hh, a signal that originates in the PSC. The maintenance of these progenitors provides the ability to respond to additional developmental or immune-based haematopoietic signals. On the basis of these findings, understanding the specific roles of Hh signalling and Hox genes in the establishment and function of vertebrate haematopoietic niches warrants further investigation. The identification of a haematopoietic niche in Drosophila will allow future investigation of in vivo niche/precursor interactions in a haematopoietic system that allows direct observation, histological studies and extensive genetic analysis (Mandal, 2007).

    Serpent, suppressor of hairless and U-shaped are crucial regulators of hedgehog niche expression and prohemocyte maintenance during Drosophila larval hematopoiesis

    The lymph gland is a specialized organ for hematopoiesis, utilized during larval development in Drosophila. This tissue is composed of distinct cellular domains populated by blood cell progenitors (the medullary zone), niche cells that regulate the choice between progenitor quiescence and hemocyte differentiation [the posterior signaling center (PSC)], and mature blood cells of distinct lineages (the cortical zone). Cells of the PSC express the Hedgehog (Hh) signaling molecule, which instructs cells within the neighboring medullary zone to maintain a hematopoietic precursor state while preventing hemocyte differentiation. As a means to understand the regulatory mechanisms controlling Hh production, a PSC-active transcriptional enhancer was characterized that drives hh expression in supportive niche cells. The findings indicate that a combination of positive and negative transcriptional inputs program the precise PSC expression of the instructive Hh signal. The GATA factor Serpent (Srp) is essential for hh activation in niche cells, whereas the Suppressor of Hairless [Su(H)] and U-shaped (Ush) transcriptional regulators prevent hh expression in blood cell progenitors and differentiated hemocytes. Furthermore, Srp function is required for the proper differentiation of niche cells. Phenotypic analyses also indicated that the normal activity of all three transcriptional regulators is essential for maintaining the progenitor population and preventing premature hemocyte differentiation. Together, these studies provide mechanistic insights into hh transcriptional regulation in hematopoietic progenitor niche cells, and demonstrate the requirement of the Srp, Su(H) and Ush proteins in the control of niche cell differentiation and blood cell precursor maintenance (Tokusumi, 2010).

    The lymph gland hematopoietic organ is formed near the end of embryogenesis from two clusters of cells derived from anterior cardiogenic mesoderm (Crozatier, 2004; Mandal, 2004). About 20 pairs of hemangioblast-like cells give rise to three distinct lineages that will form the lymph glands and anterior part of the dorsal vessel. Notch (N) pathway signaling serves as the genetic switch that differentially programs these progenitors towards cell fates that generate the lymph glands (blood lineage), heart tube (vascular lineage), or heart tube-associated pericardial cells (nephrocytic lineage). An essential requirement has also been proven for Tailup (Islet1) in lymph gland formation, in which it functions as an early-acting regulator of serpent, odd-skipped and Hand hematopoietic transcription factor gene expression (Tokusumi, 2010).

    By the end of the third larval instar, each anterior lymph gland is composed of three morphologically and molecularly distinct regions (Jung, 2005). The posterior signaling center (PSC) is a cellular domain formed during late embryogenesis due to the specification function of the homeotic gene Antennapedia (Antp) (Mandal, 2007) and the maintenance function of Collier, the Drosophila ortholog of the vertebrate transcription factor early B-cell factor. PSC cells selectively express the Hedgehog (Hh) and Serrate (Ser) signaling molecules and extend numerous thin filopodia into the neighboring medullary zone. This latter lymph gland domain is populated by undifferentiated and slowly proliferating blood cell progenitors (Mandal, 2007). Prohemocytes within the medullary zone express the Hh receptor Patched (Ptc) and the Hh pathway transcriptional effector Cubitus interruptus (Ci). Medullary zone cells also express components of the Jak/Stat signaling pathway. By contrast, the third lymph gland domain -- the cortical zone -- solely contains differentiating and mature hemocytes, such as plasmatocytes and crystal cells. Upon wasp parasitization, or in certain altered genetic backgrounds, lamellocytes will also appear in the cortical zone as a third type of differentiated hemocyte (Tokusumi, 2010).

    Two independent studies have provided compelling data to support the contention that the PSC functions as a hematopoietic progenitor niche within the lymph gland, with this cellular domain being essential for maintaining normal hemocyte homeostasis (Krzemien, 2007; Mandal, 2007). These investigations showed that communication between the PSC and prohemocytes present in the medullary zone is crucial for the preservation of the progenitor population and to prevent these cells from becoming abnormally programmed to differentiate into mature hemocytes. Seminal findings from these studies can be summarized as follows: Col expression must be restricted to the PSC by the localized expression of Ser; Hh must be expressed selectively in the PSC, coupled with the non-autonomous activation of the Hh signaling pathway in prohemocytes of the medullary zone; and the PSC triggers activation of the Jak/Stat pathway within cells of the medullary zone. With the perturbation of any of these molecular events, the precursor population of the medullary zone is lost owing to the premature differentiation of hemocytes, which swell the cortical zone. Although the exact interrelationship of Ser, Hh and Jak/Stat signaling within the lymph gland is currently unknown, the cytoplasmic extensions emanating from PSC cells might facilitate instructive signaling between these niche cells and hematopoietic progenitors present in the medullary zone (Krzemien, 2007; Mandal, 2007). A more recent study showed that components of the Wingless (Wg) signaling pathway are expressed in the stem-like prohemocytes to reciprocally regulate the proliferation and maintenance of cells within the supportive PSC niche (Sinenko, 2009). The cellular organization and molecular signaling of the Drosophila lymph gland are remarkably similar to those of the hematopoietic stem cell niches of vertebrate animals, including several mammals (Tokusumi, 2010 and references therein).

    Through detailed molecular and gene expression analyses this study has identified the PSC-active transcriptional enhancer within hh intron 1 and delimited its location to a minimal 190 bp region. The hh enhancer-GFP transgene faithfully recapitulates the niche cell expression of Hh derived from the endogenous gene, as double-labeling experiments with the GFP marker and Antp or Hh show a clear co-expression in PSC domain cells. Appropriately, GFP expression is not detected in Antp loss-of-function or TCFDN genetic backgrounds, which culminate in an absence of niche cells from the lymph gland. The hematopoietic GATA factor Srp serves as a positive activator of hh PSC expression, as mutation of two evolutionarily conserved GATA elements in the enhancer abrogates its function and Srp functional knockdown via srp RNAi results in hh enhancer-GFP transgene inactivity and the absence of Hh protein expression. An additional intriguing phenotype was observed in lymph glands expressing the srp RNAi transgene, that being a strong reduction in the number of filopodial extensions emerging from cells of the PSC. This phenotype suggests a functional role for Srp in the correct differentiation of niche cells, via a requirement for normal Hh presentation from these cells and/or the transcriptional regulation by Srp of additional genes needed for the formation of filopodia (Tokusumi, 2010).

    As Srp accumulates in all cells of the lymph gland, a question arose as to how hh expression is restricted to cells of the PSC. This paradox could be explained by a mechanism in which hh expression is also under some means of negative transcriptional control in non-PSC cells of the lymph gland. This possibility proved to be correct, with the analyses identifying two negative regulators of hh lymph gland expression. The first is Su(H). Mutation of the evolutionarily conserved GTGGGAA element, a predicted recognition sequence for this transcriptional repressor, resulted in an expanded activity of the hh PSC enhancer-GFP transgene; that is, the de novo appearance of GFP was observed in prohemocytes of the medullary zone. Likewise, ectopic medullary zone expression of the wild-type PSC enhancer-GFP transgene and of Hh protein was seen in lymph glands mutant for Su(H). These findings, coupled with the detection of Su(H) in blood cell progenitors, strongly implicate this factor as a transcriptional repressor of the hh PSC enhancer, restricting its expression to niche cells (Tokusumi, 2010).

    Additional studies identified Ush as a second negative regulator of hh expression. Ush is expressed in most cells of the lymph gland, with the exception of those cells resident within the PSC domain. Previous research demonstrated that ush expression in the lymph gland is under the positive control of both Srp. Why Ush protein fails to be expressed in the PSC remains to be determined. Forced expression of ush in niche cells resulted in inactivation of the hh PSC enhancer and reduced the formation of filopodia. It was hypothesized that Ush might be forming an inhibitory complex with the SrpNC protein, changing Srp from a positive transcriptional activator to a negative regulator of hh lymph gland expression. Such a mechanism has been demonstrated previously in the negative regulation by Ush of crystal cell lineage commitment. The expansion of wild-type hh enhancer-GFP transgene and Hh protein expression to prohemocytes within the medullary zone and to differentiated hemocytes within the cortical zone in lymph glands mutant for ush is also supportive of Ush functioning as a negative regulator of hh expression (Tokusumi, 2010).

    Bringing these results together, a model can be proposed for the regulatory events that culminate in the precise expression of the vital Hh signaling molecule in niche cells. Srp is a direct transcriptional activator of hh in the lymph gland and Hh protein is detected in niche cells due to this activity. hh expression is inhibited in prohemocytes of the medullary zone by Su(H) action, while a repressive SrpNC-Ush transcriptional complex prevents Srp from activating hh expression in prohemocytes and in differentiated hemocytes of the medullary zone and cortical zone. Together, these positive and negative modes of regulation would allow for the niche cell-specific expression of Hh and facilitate the localized presentation of this crucial signaling molecule to neighboring hematopoietic progenitors (Tokusumi, 2010).

    The identification of Srp and Su(H) as key regulators of Hh expression in the larval hematopoietic organ prompted an investigation into the functional requirement of these proteins in the control of blood cell homeostasis. Since Srp knockdown by RNAi leads to an absence of the crucial Hh signal, it was not surprising to find that normal Srp function is required for prohemocyte maintenance and the control of hemocyte differentiation within the lymph gland; that is, a severe reduction of Ptc-positive hematopoietic progenitors and a strong increase in differentiated plasmatocytes and crystal cells was observed in srp mutant tissue (Tokusumi, 2010).

    Likewise, Ptc-positive prohemocytes were lost and large numbers of plasmatocytes were prematurely formed in Su(H) mutant lymph glands. This disruption of prohemocyte maintenance occurred even though Hh protein expression was expanded throughout the medullary zone. This raised the question as to why expanded Hh protein and possible Hh pathway activation did not increase the progenitor population in Su(H) mutant lymph glands, instead of the observed loss of prohemocytes and appearance of differentiated plasmatocytes. One explanation might be that the PSC niche is not expanded in Su(H) mutant lymph glands and Hh might only function in promoting blood cell precursor maintenance within the context of the highly ordered progenitor-niche microenvironment. It has been hypothesized that the filopodial extensions that emanate from differentiated niche cells are crucial for Hh signal transduction from the PSC to progenitor cells of the medullary zone. The possibility exists that ectopic Hh protein, which is not produced or presented by niche cells, is unable to positively regulate prohemocyte homeostasis. An experimental result consistent with this hypothesis is that expression of UAS-hh under the control of the medullary zone-specific tepIV-Gal4 driver failed to expand the blood cell progenitor population. A second possibility is that the Hh pathway transcriptional effector Ci might require the co-function of Su(H) in its control of prohemocyte maintenance. This model would predict that, in the absence of Su(H) function, Hh signaling would be less (or non) effective in controlling the genetic and cellular events needed for the maintenance of the prohemocyte state. Third, Su(H) might regulate additional target genes, the expression (or repression) of which is crucial for normal blood cell precursor maintenance and the prevention of premature hemocyte differentiation. Finally, it cannot be ruled out that the expression of ectopic Hh in medullary zone cells, in the context of the adverse effects of Su(H) loss of function in these cells, culminates in the disruption of normal Hh pathway signaling due to an unforeseen dominant-negative effect (Tokusumi, 2010).

    In summary, these findings add significantly to knowledge of hematopoietic transcription factors that function to control stem-like progenitor maintenance and blood cell differentiation in the lymph gland. An additional conclusion from these studies is that the hh enhancer-GFP transgene can serve as a beneficial reagent to identify and characterize genes and physiological conditions that control the cellular organization of the hematopoietic progenitor-niche cell microenvironment. RNAi-based genetic screens could be undertaken using this high-precision marker to determine signaling pathways and/or environmental stress conditions that might alter niche cell number and function, leading to an alteration in hematopoietic progenitor maintenance coupled with the robust production of differentiated blood cells. Much remains to be determined about the regulated control of these critical hematopoietic changes and their likely relevance to hematopoietic stem cell-niche interactions in mammals (Tokusumi, 2010).

    Active hematopoietic hubs in Drosophila adults generate hemocytes and contribute to immune response

    Blood cell development in Drosophila shares significant similarities with vertebrate. The conservation ranges from biphasic mode of hematopoiesis to signaling molecules crucial for progenitor cell formation, maintenance, and differentiation. Primitive hematopoiesis in Drosophila ensues in embryonic head mesoderm, whereas definitive hematopoiesis happens in larval hematopoietic organ, the lymph gland. This organ, with the onset of pupation, ruptures to release hemocytes into circulation. It is believed that the adult lacks a hematopoietic organ and survives on the contribution of both embryonic and larval hematopoiesis. However, these studies revealed a surge of blood cell development in the dorsal abdominal hemocyte clusters of adult fly. These active hematopoietic hubs are capable of blood cell specification and can respond to bacterial challenges. The presence of progenitors and differentiated hemocytes embedded in a functional network of Laminin A and Pericardin within this hematopoietic hub projects it as a simple version of the vertebrate bone marrow (Ghosh, 2015).

    Employing hemolectin-Gal4, UAS-GFP, this study has identified four hematopoietic blood cell clusters along the dorsal midline in the abdominal segments A1-A4 of adult flies. Of the four clusters, the one in the abdominal segment A1 has the maximum aggregation of cells that occupies the area that spans the lateral and dorsal sides of the heart. Located just below the dorsal cuticle of the abdominal cavity, the cells are assembled in a groove defined by transverse heart muscles and body wall muscles. The longitudinal heart muscle forming the dorsal diaphragm separates the heart and the cluster from abdominal cavity. With respect to the pericardial diaphragm formed by pericardial cells present on either side of the cardiac tube, these hemocytes are located dorsally. Thus, these clusters remain secluded from rest of the abdominal cavity by the dorsal and the pericardial diaphragm (Ghosh, 2015).

    The hemocytes within the clusters are embedded in an extensive network of extracellular matrix proteins surrounding the heart and the pericardial cells. One of the important components of this network is the type IV collagen-like protein, Pericardin<. In homozygous mutant for lonely heart (loh) , a gene encoding a secreted receptor of Pericardin (Drechsler, 2013), the hemocytes fail to form the cluster, as this network gets significantly affected. Similar result is observed upon knocking down the expression of Laminin A, another important component of the network, by driving UAS-laminin A RNAi in the cardiac tube by mef2-Gal4. Based on expression and functional analyses, it is concluded that both Pericardin and Laminin A function in maintaining adhesive interaction with the hemocytes aiding in formation of the clusters. Interestingly, Laminin A polypeptides and collagen IV are also prevalent in vertebrate bone marrow. The finding that the blood cells are fenestrated in a functional network of Laminin A and Pericardin raised the speculation that these sites might function as bone marrow-like tissues in adult flies and thereby demanded an in-depth analysis of the cell types present therein (Ghosh, 2015).

    For detailed characterization of the cell types, this study focused on the largest aggregation present in the segment A1. Primarily based on the expression of peroxidasin-GFP (pxn-GFP) and NimC1/ P1, the cluster was found to house a large number of plasmatocytes, the most predominant differentiated blood cell. Interestingly, the cells in the cluster express croquemort (crq), an embryonic marker for plasmatocytes. The embryonic origin of the plasmatocytes was further validated by using G-TRACE construct that enables detection of cells that had once expressed any particular gene prior to the time of investigation (lineage traced) as well as those in which the gene is expressed at the time of observation (live expression). Activation of G-TRACE system by a Gal4 for glial cell missing (gcm), a gene known to express exclusively in embryonic plasmatocytes, results in the detection of few P1-positive gcm lineage traced (enhanced Green Fluorescent Protein [EGFP]) cells, thereby confirming that the cluster harbors plasmatocytes of embryonic origin. In addition to these markers, the cells in the cluster express several lymph gland hemocyte-specific markers like ZCL2897, and are Serpent (Srp) and dorothy-GFP positive, even some of them are lineage traced for collier. Thus, the hemocyte clusters is a medley of embryonic and larval lineages (Ghosh, 2015).

    Despite one report that suggests the presence of C4 expressing crystal cells in adult circulation, it is considered that crystal cells are not present in adults. Primarily, this is due to the absence of any Prophenoloxidase (proPO) expressing crystal cell in circulation. This study, however, observed that 5-days post-eclosion (dpe), there are some Hindsight (Hnt)-positive crystal cells present within the cluster. Co-localization of lozenge-GFP (lz) with proPO further supports these findings. To have a functional correlate, activation of proPO was heat induced in crystal cells. This results in formation of melanized crystal cells on dorsal side of the abdomen, corresponding to the position of first cluster. These results clearly establish the presence of resident functional crystal cells in the clusters (Ghosh, 2015).

    GATA factor Serpent (Srp) is expressed in low levels in all hemocytes, including plasmatocytes and crystal cells. However, the hemocyte precursor cell can be identified by the presence of high levels of Srp expression. Analysis of developing cluster at 2 dpe reveals the presence of cells positive for both Srp and Hemolectin (plasmatocytes), and a small subset of cells exclusively expressing Srp. No crystal cells (Hnt) are present in the cluster. In contrast, at 5 dpe, along with the two cell types mentioned above, some Srp- and Hnt-positive crystal cells are seen. Quantitative analysis of the above observations clearly demonstrate an increase in the number of differentiated cells (plasmatocytes and crystal cells) with a concomitant decline in the number of cells exclusively expressing Srp at 5 versus 2 dpe. These results also indicate that the Srp-positive cells within the cluster that do not express either hml or Hnt might be the precursor cells, yet to turn on differentiation (Ghosh, 2015).

    The crystal cell development was followed in the cluster. Since activation of Notch (N) pathway precedes Lz expression in crystal cells, a recombinant fly line was generated with 12XSu(H)lacZ in the background of lz-GFP. Su(H) lacZ-positive cells are first seen in the cluster 2 dpe, whereas the expression of lz-GFP is observed only on 3 dpe. Interestingly, some of these lzGFP-positive cells still have low levels of Su(H)lacZ expression. By 5 dpe, an increase was observed in the number of cells that are either expressing lz-GFP or have low levels of Su(H)lacZ expression along with lz-GFP expression. However, at 7 dpe, while an increase in number of lz-GFP cells can be seen, there is a decrease in the number of double-positive cells. The number of cells expressing only Su(H)lacZ that remain more or less unaltered till 5 dpe demonstrates a sharp decline on day 7. Quantitative analysis of the cell types present in the cluster based on the expressions of Su(H)lacZ and lz-GFP further ascertains the above observations. These results, therefore, clearly demonstrate de novo origin of lz-GFP-positive crystal cells from Su(H)lacZ-positive cells within the cluster (Ghosh, 2015).

    To determine whether Su(H)lacZ-positive cells originate from the Srp positive precursors, the the expression of both Srp and Su(H)lacZ within the cluster was monitored. Initially, while some cells that turn on Su(H)lacZ have high levels of Srp expression in subsequent days, as the Su(H)lacZ expression gets stabilized, a reduction in Srp expression is observed. This result establishes that crystal cells develop in adult cluster from high Srp-positive precursor cells, and this process requires N signaling. As a functional correlate to establish the presence of precursor cells in the cluster, N signaling was tweaked to determine its effect on differentiation of crystal cells. Since the onset of Su(H)lacZ and lz-GFP expression in the cluster is observed at 2 and 3 dpe, respectively, N signaling was impaired in the precursors by driving UAS-N RNAi using hemese-Gal4 from 2 dpe. This resulted in complete loss of crystal cells compared with that observed in WT clusters. Interestingly, the marginal increase in the number of plasmatocytes observed by knocking down N correlates with the number of crystal cells missing in this genetic background when compared to control. Likewise, overexpressing N in these cells results in almost 7-fold increase in the number of crystal cells with a significant drop in the number of plasmatocytes (Ghosh, 2015).

    It is therefore quite evident from the results that the clusters of blood cells on dorsal side of adult fly are not a mere aggregation of hemocytes of embryonic and larval origin but also houses true progenitors. The very fact that they house blood cell precursors and exhibit dynamicity as de novo crystal cells get differentiated within them qualifies them to be considered as active hubs of hematopoiesis in adult (Ghosh, 2015).

    Upon identifying the hemocyte precursors, attempts were made to define their origin. The results demonstrate that collier lineage traced progenitors in the hub originate from the hemocyte precursors present in the tertiary and quaternary lobes of larval lymph gland and that they can give rise to both plasmatocytes and crystal cells (Ghosh, 2015).

    In summary this study unravels the presence of active hematopoietic hubs in Drosophila adults. Refuting the existing notion that adults rely on long-lived hemocytes originating from embryonic and larval stages, this study was successful in establishing that a surge of hematopoiesis happens in these hubs as the precursors present within differentiate into both crystal cells and plasmatocytes. The functionality of the hub gets further validated, since it was observed that besides exhibiting phagocytic activity the otherwise quiescent cells re-enter into proliferative mode in response to bacterial infection. These findings bring about a paradigm shift in understanding of the process of hematopoiesis in Drosophila. With its well-characterized embryonic and larval hematopoietic activities, Drosophila has been serving as a powerful model for hematopoietic studies. In spite of that, the system seemed to be incomplete due to lack of detailed developmental analysis of hematopoiesis in adults. This effort in establishing that the process of definitive hematopoiesis extends to adults expands the scope of exploiting this model system (Ghosh, 2015).

    Extracellular matrix-modulated Heartless signaling in Drosophila blood progenitors regulates their differentiation via a Ras/ETS/FOG pathway and target of rapamycin function

    Maintenance of hematopoietic progenitors ensures a continuous supply of blood cells during the lifespan of an organism. Thus, understanding the molecular basis for progenitor maintenance is a continued focus of investigation. A large pool of undifferentiated blood progenitors are maintained in the Drosophila hematopoietic organ, the larval lymph gland, by a complex network of signaling pathways that are mediated by niche-, progenitor-, or differentiated hemocyte-derived signals. This study examined the function of the Drosophila fibroblast growth factor receptor (FGFR), Heartless, a critical regulator of early lymph gland progenitor specification in the late embryo, during larval lymph gland hematopoiesis. Activation of Heartless signaling in hemocyte progenitors by its two ligands, Pyramus and Thisbe, is both required and sufficient to induce progenitor differentiation and formation of the plasmatocyte-rich lymph gland cortical zone. Two transcriptional regulators were identified that function downstream of Heartless signaling in lymph gland progenitors, the ETS protein, Pointed, and the Friend-of-GATA (FOG) protein, U-shaped, which are required for this Heartless-induced differentiation response. Furthermore, cross-talk of Heartless and target of rapamycin signaling in hemocyte progenitors is required for lamellocyte differentiation downstream of Thisbe-mediated Heartless activation. Finally, the Drosophila heparan sulfate proteoglycan, Trol, was identified as a critical negative regulator of Heartless ligand signaling in the lymph gland, demonstrating that sequestration of differentiation signals by the extracellular matrix is a unique mechanism employed in blood progenitor maintenance that is of potential relevance to many other stem cell niches (Dragojlovic-Munther, 2013).

    Reactive oxygen species prime Drosophila haematopoietic progenitors for differentiation

    Reactive oxygen species (ROS), produced during various electron transfer reactions in vivo, are generally considered to be deleterious to cells. In the mammalian haematopoietic system, haematopoietic stem cells contain low levels of ROS. However, unexpectedly, the common myeloid progenitors (CMPs) produce significantly increased levels of ROS. The functional significance of this difference in ROS level in the two progenitor types remains unresolved. This study shows that Drosophila multipotent haematopoietic progenitors, which are largely akin to the mammalian myeloid progenitors, display increased levels of ROS under in vivo physiological conditions, which are downregulated on differentiation. Scavenging the ROS from these haematopoietic progenitors by using in vivo genetic tools retards their differentiation into mature blood cells. Conversely, increasing the haematopoietic progenitor ROS beyond their basal level triggers precocious differentiation into all three mature blood cell types found in Drosophila, through a signalling pathway that involves JNK and FoxO activation as well as Polycomb downregulation. It is concluded that the developmentally regulated, moderately high ROS level in the progenitor population sensitizes them to differentiation, and establishes a signalling role for ROS in the regulation of haematopoietic cell fate. These results lead to a model that could be extended to reveal a probable signalling role for ROS in the differentiation of CMPs in mammalian haematopoietic development and oxidative stress response (Owusu-Ansah, 2009).

    The Drosophila lymph gland is a specialized haematopoietic organ which produces three blood cell types -- plasmatocytes, crystal cells and lamellocytes -- with functions reminiscent of the vertebrate myeloid lineage. During the first and early second larval instars, the lymph gland comprises only the progenitor population. However, by late third instar, multipotent stem-like progenitor cells become restricted to the medial region of the primary lymph gland lobe, in an area referred to as the medullary zone; whereas a peripheral zone, referred to as the cortical zone, contains differentiated blood cells. By late third instar, the progenitors within the medullary zone are essentially quiescent, whereas the mature, differentiated population in the cortical zone proliferates extensively. The posterior signalling centre is a group of about 30 cells that secretes several signalling molecules and serves as a stem-cell niche regulating the balance between cells that maintain 'stemness' and those that differentiate (Owusu-Ansah, 2009).

    Although several studies have identified factors that regulate the differentiation and maintenance of Drosophila blood cells and the stem-like progenitor population that generates them, intrinsic factors within the stem-like progenitors are less explored. Interrogation of these intrinsic factors is the central theme of this investigation. It was observed that by the third instar, the progenitor population in the normal wild-type lymph gland medullary zone contains significantly increased ROS levels compared with their neighbouring differentiated progeny that express mature blood cell markers in the cortical zone. ROS are not increased during the earlier larval instars but increase as the progenitor cells become quiescent and subside as they differentiate. This first suggested that the rise in ROS primes the relatively quiescent stem-like progenitor cells for differentiation. ROS was reduced by expressing antioxidant scavenger proteins GTPx-1 or catalase, specifically in the progenitor cell compartment using the GAL4/UAS system, and it was found that suppressing increased ROS levels in haematopoietic progenitors significantly retards their differentiation into plasmatocytes. As a corollary, mutating the gene encoding the antioxidant scavenger protein superoxide dismutase (Sod2) led to a significant increase in differentiated cells and decrease in progenitors (Owusu-Ansah, 2009).

    ROS levels in cells can be increased by the genetic disruption of complex I proteins of the mitochondrial electron transport chain, such as ND75 and ND42. Unlike in wild type, where early second-instar lymph glands exclusively comprise undifferentiated cells, mitochondrial complex I depletion triggers premature differentiation of the progenitor population. This defect is even more evident in the third instar, where a complete depletion of the progenitors is seen as primary lobes are populated with differentiated plasmatocytes and crystal cells. The third differentiated cell type, the lamellocyte, defined by the expression of the antigen L1, is rarely observed in the wild-type lymph gland but is abundantly seen in the mutant. Finally, the secondary and tertiary lobes, largely undifferentiated in wild type, also embark on a robust program of differentiation upon complex I depletion. Importantly, the phenotype resulting from ND75 disruption can be suppressed by the co-expression of the ROS scavenger protein GTPx-1, which provides a causal link between increased ROS and the premature differentiation phenotype. It is concluded that the normally increased ROS levels in the stem-like progenitors serve as an intrinsic factor that sensitizes the progenitors to differentiation into all three mature cell types. Any further increase or decrease in the level of ROS away from the wild-type level enhances or suppresses differentiation respectively (Owusu-Ansah, 2009).

    In unrelated systems, increased ROS levels have been demonstrated to activate the JNK signal transduction pathway. Consequently, it was tested whether the mechanism by which the progenitors in the medullary zone differentiate when ROS levels increase could involve this pathway. The gene puckered (puc) is a downstream target of JNK signalling and its expression has been used extensively to monitor JNK activity. Although puc transcripts are detectable by reverse transcriptase PCR (RT- PCR), the puc-lacZ reporter is very weakly expressed in wild type. After disruption of ND75, however, a robust transcriptional upregulation of puc-lacZ expression can be seen, indicating that JNK signalling is induced in these cells in response to high ROS levels. The precocious progenitor cell differentiation caused by mitochondrial disruption is suppressed upon expressing a dominant negative version of basket (bsk), the sole Drosophila homologue of JNK. This suppression is associated with a decrease in the level of expression of the stress response gene encoding phosphoenol pyruvate carboxykinase; quantitatively a 68% suppression of the ND75 crystal cell phenotype was observed when JNK function was removed as well. Although disrupting JNK signalling suppressed differentiation, ROS levels remain increased in the mutant cells, as would be expected from JNK functioning downstream of ROS (Owusu-Ansah, 2009).

    In several systems and organisms, JNK function can be mediated by activation of FoxO as well as through repression of Polycomb activity. FoxO activation can be monitored by the expression of its downstream target Thor, using Thor-lacZ as a transcriptional read-out. This reporter is undetectable in wild-type lymph glands although Thor transcripts are detectable by RT-PCR; however, the reporter is robustly induced when complex I is disrupted, suggesting that the increase in ROS that is mediated by loss of complex I activates FoxO. To monitor Polycomb de-repression, a Polycomb reporter was used that expresses lacZ when Polycomb proteins are downregulated. Although undetectable in wild-type lymph glands, disrupting ND75 leads to lacZ expression suggesting that Polycomb activity is downregulated by the altered ROS and resulting JNK activation. Direct FoxO overexpression causes a remarkable advancement in differentiation to a time as early as the second instar, never seen in wild type. By early third instar, the entire primary and secondary lobes stained for plasmatocyte and crystal cell markers when FoxO is expressed in the progenitor population. Unlike with ROS increase, no a significant increase in lamellocytes was found upon FoxO overexpression. However, downregulating the expression of two polycomb proteins, Polyhomeotic Proximal (Php-x) and Enhancer of Polycomb [E(Pc)], that function downstream of JNK, markedly increased lamellocyte number without affecting plasmatocytes and crystal cells. When FoxO and a transgenic RNA interference (RNAi) construct against E(Pc) are expressed together in the progenitor cell population, differentiation to all three cell types is evident. It is concluded that FoxO activation and Polycomb downregulation act combinatorially downstream of JNK to trigger the full differentiation phenotype: an increase in plasmatocytes and crystal cells due to FoxO activation, and an increase in lamellocytes primarily due to Polycomb downregulation (Owusu-Ansah, 2009).

    This analysis of ROS in the wild-type lymph gland highlights a previously unappreciated role for ROS as an intrinsic factor that regulates the differentiation of multipotent haematopoietic progenitors in Drosophila. Any further increase in ROS beyond the developmentally regulated levels, owing to oxidative stress, will cause the progenitors to differentiate into one of three myeloid cell types. It has been reported that the ROS levels in mammalian haematopoietic stem cells is low but that in the CMPs is relatively high. The Drosophila haematopoietic progenitors give rise entirely to a myeloid lineage and therefore are functionally more similar to CMPs than they are to haematopoietic stem cells. It is therefore a remarkable example of conservation to find that they too have high ROS levels. The genetic analysis makes it clear that the high ROS in Drosophila haematopoietic progenitors primes them towards differentiation. It will be interesting to determine whether such a mechanism operates in mammalian CMPs. In mice, as in flies, a function of FoxO is to activate antioxidant scavenger proteins. Consequently, deletion of FoxO increases ROS levels in the mouse haematopoietic stem cell and drives myeloid differentiation. However, even in the mouse haematopoietic system, FoxO function is dose and context dependent, as ROS levels in CMPs are independent of FoxO. Thus, although the basic logic of increased ROS in myeloid progenitors is conserved between flies and mice, the exact function of FoxO in this context may have diverged (Owusu-Ansah, 2009).

    Past work has hinted that ROS can function as signalling molecules at physiologically moderate levels. This work supports and further extends this notion. Although excessive ROS is damaging to cells, developmentally regulated ROS production can be beneficial. The finding that ROS levels are moderately high in normal Drosophila haematopoietic progenitors and mammalian CMPs raises the possibility that wanton overdose of antioxidant products may in fact inhibit the formation of cells participating in the innate immune response (Owusu-Ansah, 2009).

    Oxidative stress in the haematopoietic niche regulates the cellular immune response in Drosophila

    Oxidative stress induced by high levels of reactive oxygen species (ROS) is associated with the development of different pathological conditions, including cancers and autoimmune diseases. This study analysed whether oxidatively challenged tissue can have systemic effects on the development of cellular immune responses using Drosophila as a model system. Indeed, the haematopoietic niche that normally maintains blood progenitors can sense oxidative stress and regulate the cellular immune response. Pathogen infection induces ROS in the niche cells, resulting in the secretion of an epidermal growth factor-like cytokine signal that leads to the differentiation of specialized cells involved in innate immune responses (Sinenko, 2011).

    Abnormal metabolism is often associated with oxidative stress that results in increased production of ROS by mitochondria. Different concentrations of ROS and their derivatives are required for proper maintenance, proliferation, differentiation and apoptosis of stem cells and their committed progenitors. In Drosophila, developmentally regulated levels of ROS are critical for maintenance of haematopoietic progenitors within the medullary zone (MZ) of the lymph gland. In contrast, under normal growth conditions, posterior signaling center (PSC) cells in wild-type larvae had very low levels of ROS expression compared with that in the progenitor population of cells within the MZ. To induce oxidative stress in the PSC ND75, a component of complex I of the electron transport chain (ETC), was inactivated with double-stranded RNA (dsRNA) using the Gal4/UAS misexpression system and the PSC-specific Antp-Gal4 driver. ND75 inactivation causes a readily detectable increase in ROS in the PSC cells, rising to levels similar to those seen in the progenitor cells of the MZ. The phenotypic consequence of inducing oxidative stress in the cells of the PSC was a remarkably robust increase in numbers of circulating lamellocytes. Such an elevated number of lamellocytes was usually observed in wild-type larvae only if they were infested by parasitic wasps. Although Antp-Gal4 is not expressed anywhere in the blood system, except the PSC, this driver is also expressed in other larval tissues. To exclude the possibility that the effect was due to a non-PSC expression of Antp-Gal4, the function of ND75 was also eliminated using the Dot-Gal4 driver normally expressed at high levels in the PSC, and this resulted in an identical lamellocyte response. In contrast, oxidative challenge to various other larval tissues, including the fat body (LSP2-GaI4), the epidermis (A58-GaI4), the neurons (C127-GaI4), the dorsal vessel (Hand-GaI4), the ring gland (5015-GaI4), the wing imaginal disc (ap-Gal4) or the trachea (btl-GaI4), did not have a significant effect on lamellocyte differentiation. Furthermore, high ROS levels generated within the progenitor cells (dome-GaI4) of the lymph gland, which causes autonomous differentiation of this population, also did not have any significant effect on the non-autonomous differentiation of lamellocytes in the circulation. In contrast, oxidative challenge of the PSC caused non-autonomous lamellocyte response in circulation as well as within the lymph gland. The PSC-mediated effect was due to mitochondrial dysfunction and not specifically linked to the product of the ND75 gene, because attenuation of PDSW (another complex I component), cytochrome-c oxidase, subunit Va (CoVa, a component of ETC complex IV) or Marf (mitochondrial assembly regulatory factor) function in the PSC, all induced increases in lamellocyte differentiation. The strength of the lamellocyte response to complex I inactivation depended on the strength of the dsRNA construct used in the experiment. Temporally, induction of the mutation in the second-larval instar caused the lamellocyte response to be seen in the third instar. This correlates well with the timescale of response to parasitic wasp infection. Finally, this oxidative stress elicited a cell-specific response; for example, no significant effect was seen on the differentiation of crystal cells and plasmatocytes in circulation. These results establish that the oxidative status of the PSC has a specific and non-autonomous role in lamellocyte differentiation as an immune response to parasitic invasion (Sinenko, 2011).

    The status of the PSC cells on oxidative stress conditions was further analysed in some detail. ND75 dysfunction does not affect proliferation or maintenance of the PSC, because the number of PSC cells, which maintain expression of Antp, remains intact in this mutant background. In addition, no apoptosis is detected in ND75-deficient PSC cells, and also, apoptosis in the PSC alone, specifically induced by overexpression of Hid/Rpr, has no effect on lamellocyte differentiation (Sinenko, 2011).

    Overexpression of superoxide dismutase-2 (SOD2) as a scavenger for ROS in ND75-deficient PSC is able to suppress the lamellocyte response significantly. Furthermore, activation of the Forkhead box O (FoxO) transcription factor that positively regulates expression of antioxidant enzymes, including SOD2, completely suppresses the dsND75-induced lamellocyte response. Inactivation of the Akt1 protein kinase in PSC also results in a near-complete suppression of the dsND75-induced lamellocyte response, suggesting a role for the PI3K/Akt pathway in the regulation of FoxO. This is an important issue because FoxO activity can also be controlled by the Jun N-terminal kinase (JNK) pathway, but in the PSC the AKT pathway mediates this effect. The JNK reporter (puc69-lacZ) is not expressed in the PSC, and inactivation of JNK (encoded by the basket gene) using the dominant-negative form (bskDN) does not suppress dsND75-induced lamellocyte response. The FoxO reporter (4E-BP-lacZ) is robustly activated in the ND75-deficient PSC; however, loss of translational inhibition mediated by 4E-BP does not mimic this effect. It is important to point out that under wild-type non-stressed conditions, the PSC has relatively low levels of ROS, and therefore inactivation of either Foxo or SOD2 has no phenotypic consequence. These data are interpreted to indicate that metabolic dysfunction induces an oxidatively stressed PSC that causes the activation of this pathway and the lamellocyte response (Sinenko, 2011).

    Differentiation of lamellocytes has been associated with the JAK/STAT, JNK and Ras/Erk signalling pathways. These pathways were genetically altered in an ND75-deficient PSC background to identify which, if any, is involved in the lamellocyte response. Inactivation of the unpaired ligands (upd3, upd2 or upd) that activate the JAK/STAT pathway or of eiger (egr), which activates JNK signalling, did not suppress the lamellocyte phenotype. This strongly suggests that these pathways are not involved in the process downstream of ROS in the PSC and is consistent with previous studies showing that components of the JAK/STAT pathway (upd3, dome and Tep4) and JNK (puc69-lacZ reporter) are not involved in the functioning of the PSC. However, these pathways are likely to be involved in direct regulation of lamellocyte differentiation independently of the PSC function. In contrast, inactivation of spitz (spi), encoding the ligand for epidermal growth factor receptor (EGFR), in the context of ND75-deficient PSC significantly suppresses the lamellocyte response. Furthermore, overexpression of the secreted form of Spi (s.Spi), but not the alternative EGFR ligand, Vein (Vn) in the PSC, causes increased differentiation of circulating lamellocytes in an otherwise wild-type larva. EGFR mutant EgfrTS/Egfr18 lymph glands develop normally, suggesting that EGFR signalling is not required for normal lymph gland development but rather is involved in the regulation of a cellular immune response as a signalling event from the PSC only when the latter is oxidatively stressed (Sinenko, 2011).

    The PSC-dependent parasitic challenge induced by wasp egg infestation and the mechanism described above both give rise to the same cellular response. Therefore, whether parasitization causes oxidative stress to the PSC was examined. Immune challenge caused by wasp infestation was found to induce high levels of ROS in the PSC cells as seen 12 h after invasion. The most prominent effect is on superoxide radicals detected with dihydroethidium staining; a smaller but detectable elevation of peroxide radicals revealed by RedoxSensor staining is also apparent in PSC cells on this immune challenge. Scavenging these ROS types in the PSC by overexpressing SOD2 or catalase (Cat) but not glutathione peroxidase (GPx), which reduces thioredoxin-mediated effects, significantly suppresses the lamellocyte response caused by wasp infestation. These genetic results are consistent with a model in which parasitic infection by wasp eggs raises ROS levels in the PSC, which then causes lamellocyte induction by expressing Spitz. To test this model, spi within the PSC was inactivated in larvae infected by parasitic wasps. This caused a strong suppression of the lamellocyte response; the few remaining L1 marker-positive cells are immature, as indicated by their relatively small cell size and their morphology. In addition, melanotic capsules that are indicative of extensive cellular immune response to parasitic infection do not develop in a spi mutant background during wasp infestation. Inactivation of spitz in the PSC did not affect the increase in ROS triggered by wasp infeststion. Thus spi does not regulate the ROS levels in the PSC; rather, wasp infection raises ROS levels, which leads to release of the s.Spi. Previous studies have shown that s.Spi production requires the function of the trafficking protein Star (S), and the protease Rhomboid (Rho1). This study found that the wasp-induced lamellocyte response and melanotic capsule formation are robustly suppressed on the loss of a single copy of Star. More importantly, parasite-induced immune challenge specifically upregulates Rho1 in the PSC by an as yet unidentified mechanism. These data establish that S and Rho1 are canonically required for processing and releasing the Spitz from the PSC (Sinenko, 2011).

    Secreted Spitz is known to bind to EGFR and activate the Ras/Erk pathway. A dominant-negative form of EGFR (EgfrDN) strongly suppresses the lamellocyte response induced by wasp infestation when it is expressed in the lymph gland and the circulating haemocytes using the pan-haemocyte HHLT Gal4 driver. This phenotype is virtually identical to that seen when spiRNAi is expressed in the PSC using Antp-Gal4. In addition, compartment-specific drivers were used, and inactivation of the receptor in the cortical zone of the lymph gland and in circulating haemocytes (using lineage-traced HmlΔ-Gal4 line) was found to prevent Hml-positive cells from becoming lamellocytes on wasp infestation. Importantly, it was also found that a small subset of lamellocytes does not express Hml in the wild-type background and consequently EgfrDN is not expressed in these cells when HmlΔ-Gal4 is used as a driver. These Hml,L1+ lamellocytes are easily detectable in this genetic background and act as an internal control. Expression of an activated form of EGFR (EgfrAct) in Hml+ haemocytes causes a robust increase in lamellocyte differentiaion. This is also consistent with previous work, which showed that activated Ras induces an increase in the total number of haemocytes, including lamellocytes. Finally, both loss of ND75 in the PSC and wasp infestation cause robust activation of Erk as evident by an increase in dpErk staining in circulating haemocytes including lamellocytes. This indicates that lamellocytes in circulation differentiate from precursor cells on activation of Spi/EGFR/Erk signalling (Sinenko, 2011).

    PSC cells have two independent functions: they serve as a haematopoietic niche in the lymph gland, where they orchestrate the maintenance and proper differentiation of haematopoietic progenitors, and they regulate the cellular immune response by controlling lamellocyte differentiation in response to infection. The results presented in this study establish the mechanism for this latter function. Changes in oxidative status, caused by events of parasite invasion or ETC dysfunction, initiates a signal within this immunocompetent compartment causing the secretion of a cytokine ligand, Spitz, that induces differentiation of lamellocyte precursors in the circulatory system of the larva. The identified mechanism is consistent with previously reported studies in mammals, which have shown that mitochondrial ROS can trigger systemic signals that reinforce the innate immune response. These studies raise the possibility that specific populations of cells also exist in mammalian systems that sense oxidative stress due to infection and non-autonomously signal myeloid progenitors to initiate differentiation and enhance the immune response. Whether such populations are to be found within the haematopoietic niche as in Drosophila remains a speculation that can be tested in future studies (Sinenko, 2011).

    An unexpected link between Notch signaling and ROS in restricting the differentiation of hematopoietic progenitors in Drosophila

    A fundamental question in hematopoietic development is how multipotent progenitors achieve precise identities, while the progenitors themselves maintain quiescence. In Drosophila larvae, multipotent hematopoietic progenitors support the production of three lineages, exhibit quiescence in response to cues from a niche, and from their differentiated progeny. Infection by parasitic wasps alters the course of hematopoiesis. This study addresses the role of Notch (N) signaling in lamellocyte differentiation in response to wasp infection. Notch activity is moderately high and ubiquitous in all cells of the lymph gland lobes, with crystal cells exhibiting the highest levels. Wasp infection reduces Notch activity, which results in fewer crystal cells and more lamellocytes. Robust lamellocyte differentiation is induced even in N mutants. Using RNA interference-knockdown of N, Serrate, and Neuralized, and twin clone analysis of a N null allele, this study shows that all three genes inhibit lamellocyte differentiation. However, unlike its cell-autonomous function in crystal cell development, Notch's inhibitory influence on lamellocyte differentiation is not cell-autonomous. High levels of reactive oxygen species in the lymph gland lobes, but not in the niche, accompany NRNAi-induced lamellocyte differentiation and lobe dispersal. These results define a novel dual role for Notch signaling in maintaining competence for basal hematopoiesis: while crystal cell development is encouraged, lamellocytic fate remains repressed. Repression of Notch signaling in fly hematopoiesis is important for host defense against natural parasitic wasp infections. These findings can serve as a model to understand how reactive oxygen species and Notch signals are integrated and interpreted in vivo (Small, 2013).

    Drosophila Rabex-5 restricts Notch activity in hematopoietic cells and maintains hematopoietic homeostasis

    Hematopoietic homeostasis requires the maintenance of a reservoir of undifferentiated blood cell progenitors and the ability to replace or expand differentiated blood cell lineages when necessary. Multiple signaling pathways function in these processes, but how their spatiotemporal control is established and their activity is coordinated in the context of the entire hematopoietic network are still poorly understood. This study reports that loss of the gene Rabex-5 in Drosophila causes several hematopoietic abnormalities including blood cell (hemocyte) overproliferation, increased size of the hematopoietic organ (the lymph gland), lamellocyte differentiation, and melanotic mass formation. Hemocyte-specific Rabex-5 knockdown was sufficient to increase hemocyte populations, increase lymph gland size, and induce melanotic masses. Rabex-5 negatively regulates Ras, and Ras activity was shown to be responsible for specific Rabex-5 hematopoietic phenotypes. Surprisingly, Ras-independent Notch protein accumulation and transcriptional activity in the lymph gland underlie multiple distinct hematopoietic phenotypes of Rabex-5 loss. Thus, Rabex-5 plays an important role in Drosophila hematopoiesis and may serve as an axis coordinating Ras and Notch signaling in the lymph gland (Reimels, 2015).

    Sumoylation is tumor-suppressive and confers proliferative quiescence to hematopoietic progenitors in Drosophila melanogaster larvae

    How cell-intrinsic regulation of the cell cycle and the extrinsic influence of the niche converge to provide proliferative quiescence, safeguard tissue integrity, and provide avenues to stop stem cells from giving rise to tumors is a major challenge in gene therapy and tissue engineering. This question was explored in sumoylation-deficient mutants of Drosophila. In wild type third instar larval lymph glands, a group of hematopoietic stem/progenitor cells acquires quiescence; a multicellular niche supports their undifferentiated state. However, how proliferative quiescence is instilled in this population is not understood. This study showed that Ubc9 protein is nuclear in this population. Loss of the SUMO-activating E1 enzyme, Aos1/Uba2, the conjugating E2 enzyme, Ubc9, or the E3 SUMO ligase, PIAS, results in a failure of progenitors to quiesce; progenitors become hyperplastic, misdifferentiate, and develop into microtumors that eventually detach from the dorsal vessel. Significantly, dysplasia and lethality of Ubc9 mutants are rescued when Ubc9(wt) is provided specifically in the progenitor populations, but not when it is provided in the niche or in the differentiated cortex. While normal progenitors express high levels of the Drosophila cyclin-dependent kinase inhibitor p21 homolog, Dacapo, the corresponding overgrown mutant population exhibits a marked reduction in Dacapo. Forced expression of either Dacapo or human p21 in progenitors shrinks this population. The selective expression of either protein in mutant progenitor cells, but not in other hematopoietic populations, limits overgrowth, blocks tumorogenesis, and restores organ integrity. An essential and complex role for sumoylation in preserving the hematopoietic progenitor states for stress response and in the context of normal development of the fly is discussed (Kalamarz, 2012).

    In a quest to identify the source of microtumors in Ubc9 mutants, this study discovered that even though Ubc9 protein is ubiquitously expressed, it plays a specific and essential, niche-independent function in maintaining proliferative quiescence within progenitors of the medullary and transition zones. Reduction of sumoylation via knockdown of any of the other core enzymes of the pathway also leads to progenitor dysplasia and tumorogenesis. Once detached from the dorsal vessel, the microtumors float in the hemolymph (Kalamarz, 2012).

    The progenitor population that serves as the source of microtumors is heterogeneous with respect to Dome>GFP and ZCL2897 expression. One of the earliest detectable effects of the mutation is on the differential expression of Dome>GFP and ZCL2897 or 76B>GFP in the expanding population. The onset of the effects of Ubc9 mutation coincides with the period when the progenitors in the medulla of the anterior lobes undergoes proliferative restraint. At the same time, cells of the posterior lobes lag behind; they continue to divide and follow a defined heterochronic developmental pattern. It is somewhat surprising that even though the Ubc9 mutation has differential effects on cells of the anterior versus posterior lobes, the overproliferation defects in both are largely rescued by ectopic expression of p21/Dap. This observation suggests a fundamental role for the enzyme in inhibiting cell cycle progression and conferring quiescence to progenitors. Since the decline in Dome>GFP expression precedes overproliferation in mutant lobes and each defect can be rescued by the expression of wild type Ubc9, it is possible that Dome>GFP expression marks the quiescent cell state. The inability of p21 or Dap to restore normal Dome>GFP expression attests to the notion that the sequential series of events, even at the earliest stages of tumorogenesis, can be genetically teased out in vivo (Kalamarz, 2012).

    While the changes in cell identities in mutant lobes are complex, the discovery of heterogeneity in the medullary zone populations of anterior and first posterior lobes is consistent with recent reports that this population has distinct fate-restricted cell populations. The current results suggest that lymph gland progenitors are similar to mammalian transit amplifying cells or those in the Drosophila testis, that have limited proliferative capacity and possess a restricted differentiation potential relative to their multipotent stem cells. With an appropriate immune or developmental cue, Drosophila hematopoietic progenitors may re-enter the cell cycle to produce differentiated progeny (Kalamarz, 2012).

    What is the physiological significance of retaining some cells in quiescence at this stage in larval life? One possibility is that mitotic exit shelters progenitors from precocious development and provides a mechanism that determines the number of times they must divide before they differentiate. Additionally, a reserve of progenitors, ready to divide and differentiate rapidly guards larvae against natural enemies such as parasitic wasps that attack them at this stage of the life cycle. This tactic parallels mitotic exit of hematopoietic stem cells (HSCs) in mice about three weeks after birth, or in humans, at about four years of age, when they become adult HSCs. The dormant adult HSCs are activated as the organism recovers from injury (Kalamarz, 2012).

    This similarity in strategies between flies and humans in normal hematopoiesis is further reinforced even when the process becomes aberrant. Like in dUbc9 mutants, uncontrolled proliferation of progenitors in human leukemias can occur independently of the signals from the niche. It is intriguing that Antp, a niche marker, is also expressed in the dorsal vessel. Furthermore, Dome>GFP expression, undetectable in normal cells, is strongly activated in mutant cells of the dorsal vessel. Thus, it is possible that cues from the cells of the dorsal vessel influence the state of the hematopoietic progenitors and integrity of the lobes. Conversely, the status of the progenitors themselves may determine the association of the lobes to the dorsal vessel. Further analysis of Ubc9 mutants will clarify the role of the microenvironment in supporting progenitor quiescence and maintaining tissue integrity (Kalamarz, 2012).

    A key mechanism by which sumoylation maintains proliferative quiescence in larval hematopoiesis is cell cycle regulation through Dacapo/p21. In the embryo, Dap/p21 binds to cyclin E/Cdk2 complexes to block the G1/S transition in cell cycle. Furthermore, the human p21 protein can block mitosis in the Drosophila eye. This function of Dap/p21 in larval hematopoiesis is similar to the roles of p27KIP1 or p21CIP1/WAF1 in enforcing HSC quiescence (Kalamarz, 2012).

    Dap is expressed in Dome>GFP progenitors in wild type and mutant glands, and is reduced shortly after Dome>GFP is downregulated in mutant glands. Overexpression of Dap/p21 in these cells leads to decrease in progenitor number. It is noteworthy that dap mutants do not exhibit apparent tumorous overgrowth, a trait that is similar to young p21 null mice. However, with age, or in the presence of other mutations (e.g., oncogenic Ras), p21 null mice are prone to developing tumors. It is therefore very likely that tumorogenesis in Ubc9 mutants is supported not only by loss of Dap/p21 but also by the activation of other oncogenic and pro-inflammatory proteins (Kalamarz, 2012).

    The mechanism by which Ubc9 controls Dap protein levels is not known. dap transcription has been studied in embryonic development where it regulates mitotic exit. High dap transcript levels in stage 16 embryonic central and peripheral nervous system, or in differentiating postmitotic cells of a developing eye disc, correlate with exit from mitosis. These observations suggest that regulation of dap transcription is coupled with mitotic exit, and it is therefore possible that its transcription in the lymph gland progenitors is similarly synchronized. Microarray experiments of whole Ubc9 larvae compared to their heterozygous siblings indicate dap transcript downregulation. An intriguing possibility is that Dacapo itself, or another protein in complex with Dap, is a sumoylation target. In high throughput yeast two-hybrid assay, Dap was found to physically interact with Ubc9. Future experiments including biochemical analyses of Dap and interacting proteins are required to test this idea (Kalamarz, 2012).

    The causal relationship between cancer and inflammation is now widely accepted, even though the mechanisms that establish and sustain this relationship remain unresolved . Drosophila Toll-Dorsal pathway not only manages immunity, but also governs hematopoietic development. Ubc9 microtumor development requires Rel/NF-kappa B family transcription factors Dorsal and Dif. Aberrant activation of NF-kappa B signaling in Ubc9 mutants resembles hematopoieitic malignancies in vertebrates that arise due to ectopic germline or somatic disruption of the pathway (Kalamarz, 2012).

    It has recently been discovered that sumoylation provides a homeostatic mechanism to restrain systemic inflammation in the fly larva, where it keeps the Toll/Dorsal-dependent immune response in check. Ubc9 controls the 'set point' by maintaining normal levels of IkappaB/Cactus protein in immune tissues (Paddibhatla, 2010). The Ubc9 cancer-inflammation model offers novel opportunities to examine the dynamics of tumor growth, its relationship to metastasis, and the links between cancer and inflammation. Ubc9 tumors are sensitive to aspirin. This model is well-suited for identifying and testing drugs that target highly-conserved biochemical mechanisms, such as sumoylation, which oversee self-renewal pathways in progenitor populations (Kalamarz, 2012).

    The Hippo pathway regulates hematopoiesis in Drosophila melanogaster

    The Salvador-Warts-Hippo (Hippo) pathway is an evolutionarily conserved regulator of organ growth and cell fate. It performs these functions in epithelial and neural tissues of both insects and mammals, as well as in mammalian organs such as the liver and heart. Despite rapid advances in Hippo pathway research, a definitive role for this pathway in hematopoiesis has remained enigmatic. The hematopoietic compartments of Drosophila melanogaster and mammals possess several conserved features. D. melanogaster possess three types of hematopoietic cells that most closely resemble mammalian myeloid cells: plasmatocytes (macrophage-like cells), crystal cells (involved in wound healing), and lamellocytes (which encapsulate parasites). The proteins that control differentiation of these cells also control important blood lineage decisions in mammals. This study defines the Hippo pathway as a key mediator of hematopoiesis by showing that it controls differentiation and proliferation of the two major types of D. melanogaster blood cells, plasmatocytes and crystal cells. In animals lacking the downstream Hippo pathway kinase Warts, lymph gland cells overproliferated, differentiated prematurely, and often adopted a mixed lineage fate. The Hippo pathway regulated crystal cell numbers by both cell-autonomous and non-cell-autonomous mechanisms. Yorkie and its partner transcription factor Scalloped were found to regulate transcription of the Runx family transcription factor Lozenge, which is a key regulator of crystal cell fate. Further, Yorkie or Scalloped hyperactivation induced ectopic crystal cells in a non-cell-autonomous and Notch-pathway-dependent fashion (Milton, 2014).

    Screening and analysis of Janelia FlyLight project enhancer-Gal4 strains identifies multiple gene enhancers active during hematopoiesis in normal and wasp-challenged Drosophila larvae

    A GFP expression screen has been conducted on greater than one thousand Janelia FlyLight Project enhancer-Gal4 lines to identify transcriptional enhancers active in the larval hematopoietic system. A total of 190 enhancers associated with 87 distinct genes showed activity in cells of the third instar larval lymph gland and hemolymph. That is, gene enhancers were active in cells of the lymph gland posterior signaling center (PSC), medullary zone (MZ), and/or cortical zone (CZ), while certain of the transcriptional control regions were active in circulating hemocytes. Phenotypic analyses were undertaken on 81 of these hematopoietic-expressed genes with nine genes characterized in detail as to gain- and loss-of-function phenotypes in larval hematopoietic tissues and blood cells. These studies demonstrated the functional requirement of the cut gene for proper PSC niche formation, the hairy, Btk29A, and E2F1 genes for blood cell progenitor production in the MZ domain, and the longitudinals lacking, dFOXO, kayak, cap-n-collar, and Delilah genes for lamellocyte induction and/or differentiation in response to parasitic wasp challenge and infestation of larvae. Together, these findings contribute substantial information to knowledge of genes expressed during the larval stage of Drosophila hematopoiesis and newly identify multiple genes required for this developmental process (Tokusumi, 2016).

    Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila

    Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. This syudy shows that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils) is still not well understood. In a simpler model system, this study used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. In response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of immunity in vivo in Drosophila, and paves the way for genetic dissection of the mechanisms at work behind steroid regulation of innate immune cells (Regan, 2013).

    Using an in vivo genetic approach to block EcR signaling specifically in hemocytes, this study has shown that ecdysone directly regulates their cell shape. Moreover, the data indicates that ecdysone regulates the onset of hemocyte motility and dispersal at metamorphosis, reflecting its function in border cell motility during oogenesis. Microarray data reveal that EcR up-regulates the expression of several genes functioning in cell motility or cell shape regulation, which could account for these phenotypes. Arguably, migration of hemocytes between tissues is required for clearing dying larval tissues during the pupal period. Hemocytes expressing the EcRDN construct do not engulf dead cells, which is potentially a consequence of impaired phagocytosis, motility, or a combination of both, although it is not possible to distinguish between these possibilities. Ecdysone has previously been shown to induce the expression in the hemocyte-derived mbn2 cell line of croquemort (crq), a gene encoding a receptor for apoptotic cells in the embryo. crq was identified in the microarray analysis as showing EcR-dependent up-regulation at metamorphosis, and this was confirmed by qPCR, where crq expression is almost completely suppressed in EcRDN-expressing pupal hemocytes. The impaired expression of crq in EcRDN hemocytes likely contributes to their deficiency in apoptotic cell phagocytosis. Functionally, the regulation of hemocytes by ecdysone, which is the coordinator of larval tissue apoptosis, may be a smart way for the fly to synchronize its macrophage scavenging activity with the moment it is most needed, at metamorphosis. Surprisingly, no gross developmental consequences were observed of the loss of this function, whereby HmlΔGal4>EcRB1DN individuals completed metamorphosis without delay. This is in agreement with studies showing that under sterile conditions, pupae lacking hemocytes altogether progress normally through metamorphosis. It suggests that dead cells might be engulfed by other, non-professional phagocytes (e.g. neighbor cells as reported for tumorigenesis), cleared up by other unidentified means, or simply tolerated, in the absence of functional hemocytes (Regan, 2013).

    Furthermore, it was show that the activation of hemocyte motility at metamorphosis also correlates with a change in their response to induced epithelial damage. While in the larva hemocytes are passively recruited to wounds from circulation, this study demonstrates that in the pupa they actively migrate to damaged tissues. Induction of epithelial wounds at different times APF demonstrated that active wound responsiveness is progressively acquired at metamorphosis. In agreement with previous ex vivo analysis, the current data highlights an intriguing plasticity of hemocytes to adapt their migratory activity and their response to wounds throughout development: chemotaxis in embryos and pupae versus passive circulation and ‘capture’ to wounds in larvae. This correlates with the observation that, although the heart is beating in a 20 h APF-old pupa, hemocytes are not propelled in the hemolymph by the heartbeat, but maintain a slow, steady, active migration on tissues (Regan, 2013).

    Most importantly, this study provides the first in vivo evidence of hormonal regulation of the Drosophila cellular response to bacterial challenge. With both ex vivo and in vivo data, this study has demonstrated an important role for EcR in the up-regulation of hemocyte phagocytic activity at metamorphosis. How does ecdysone signaling regulate phagocytosis? Previous studies in hemocyte-derived cell lines have shown that ecdysone treatment increases the transcription of some immune-related genes encoding AMPs and immune receptors such as Crq. Using a tissue-specific, whole genome transcriptomic approach, this study demonstrates that many genes are regulated by ecdysone signaling in hemocytes at metamorphosis. This analysis reveals the molecular regulation behind the observed phenotypes and allows for the identification of candidate effector genes. For example, 35 genes up-regulated by EcR at metamorphosis have been previously attributed a function in phagocytosis. These genes encode proteins involved in different steps of the phagocytosis process, such as recognition (e.g. the receptors PGRP-LC, croquemort, and Nimrod family members, Dscam and scab), or cytoskeletal rearrangements required for the engulfment step (e.g., RhoGAP71E, Rac2, Arpc5 and SCAR). Interestingly, PGRP-LC (FC 1.8 by microarray, 3.9 by qPCR) was recently shown to be induced in ecdysone-treated S2 cells. It appears that ecdysone can regulate the phagocytosis process at different levels, which may be necessary to co-ordinate the ability of hemocytes to recognize and engulf their target. Moreover, genes regulated by ecdysone signaling can be implicated in more than one process, for example phagocytosis and AMP expression (e.g. PGRP-LC), or phagocytosis and cell migration (e.g. SCAR); this may contribute to synchronisation of different hemocyte immune functions (Regan, 2013).

    The functional relevance of increased cellular immune activity at metamorphosis is an intriguing question. Recent studies of the contribution of cellular immunity to Drosophila defenses have revealed that flies in which hemocytes are genetically ablated present a high lethality at metamorphosis. This is likely the result of opportunistic bacterial infections, as feeding antibiotics was sufficient to restore wild-type viability. No such lethality was observed under normal conditions when expressing EcRDN in hemocytes; Phagoless lethality in absence of infection is also lower than that previously described . This suggests that the fly strains and fly food used in this study do not harbor the same bacterial types as those used in previous studies, leading to distinct opportunistic infection scenarios. Nevertheless, these data indicate a significant lethality of HmlΔ>EcRDN pupae not only after septic injury with E. faecalis or E. carotovora, but also after oral infection at larval stages with E. carotovora, a bacterium that is not usually lethal in wild-type individuals. This lethality is quite dramatic considering only hemocytes express the transgene, and is similar to the lethality in hemocyte-ablated individuals . It indicates that ecdysone regulation is essential for hemocyte immune functions and survival after infection (Regan, 2013).

    Metamorphosis may represent a stage of predisposition to opportunistic oral infection, as the larval midgut is replaced by the adult intestinal epithelium. It is speculated that histolysis of the gut could release bacteria from the lumen into the body cavity; active hemocytes may be required to limit the spreading of bacteria from temporary weak points in the epithelium. HmlΔ>EcRDN prepupae induce a normal intestinal and systemic humoral immune response after being orally infected at larval stage. In the case of both septic injury and oral infection, it is therefore likely that the main cause of decreased survival in HmlΔ>EcRDN pupae is their striking hemocyte phagocytosis phenotype, possibly in combination with lack of motility, inability to chemotax to damaged tissue or other potential uncharacterized hemocyte defects (Regan, 2013).

    The synchronization of multiple processes is a fundamental requirement for successful development, and likely to rely on hormonal signaling. Altogether, the current data reveal the importance of steroid hormone signaling in the synchronization of development and immunity in Drosophila, by ecdysone-dependent activation of hemocytes at pupariation. it has been have recently shown that ecdysone signaling affects the humoral response through regulation of PGRP-LC expression. Interestingly, an impact of this regulation was obsered on the ability of adult flies to survive infection, indicating that ecdysone regulation of immunity extends beyond metamorphosis. In humans, hormonal activation of macrophages underpins various cancer pathologies and is therefore highly relevant in clinical terms. It is also generally accepted that steroid hormones impact immunity in mammals. For example, glucocorticoids are commonly used in pharmacology for their anti-inflammatory properties. However, their regulation of the immune response is complex, as they can also enhance the immune response. More generally, steroid hormones' specific action on monocytes is still not very well documented, mainly due to the complexity of mammalian systems and experimental limitations. Elucidating mechanisms for steroid hormone regulation of cellular immunity will be essential for a full understanding of sex differences in immunity and inflammation (Regan, 2013).

    A glutamate-dependent redox system in blood cells is integral for phagocytosis in Drosophila melanogaster

    Glutamate transport is highly regulated as glutamate directly acts as a neurotransmitter and indirectly regulates the synthesis of antioxidants. Although glutamate deregulation has been repeatedly linked to serious human diseases such as HIV infection and Alzheimer's, glutamate's role in the immune system is still poorly understood. A putative glutamate transporter in Drosophila melanogaster, polyphemus (polyph), was found to play an integral part in the fly's immune response. Flies with a disrupted polyph gene exhibit decreased phagocytosis of microbial-derived bioparticles. When infected with S. aureus, polyph flies show an increase in both susceptibility and bacterial growth. Additionally, the expression of two known glutamate transporters, genderblind and excitatory amino acid transporter 1, in blood cells affects the flies' ability to phagocytose and survive after an infection. Consistent with previous data showing a regulatory role for glutamate transport in the synthesis of the major antioxidant glutathione, polyph flies produce more reactive oxygen species (ROS) as compared to wild-type flies when exposed to S. aureus. In conclusion, this study has demonstrated that a polyph-dependent redox system in blood cells is necessary to maintain the cells' immune-related functions. Furthermore, the model provides insight into how deregulation of glutamate transport may play a role in disease (Gonzales, 2013).

    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. The signal from differentiating cells has been termed the 'equilibrium signal' in order to distinguish it from the 'niche signal'. Earlier work showed that Equilibrium signaling utilizes Pvr (the Drosophila PDGF/VEGF receptor), STAT92E, and Adenosine deaminase-related growth factor A (ADGF-A). 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, a genetic screen was performed that identified bip1 (bric a brac interacting protein 1) and Nucleoporin 98 (Nup98) as additional regulators of the equilibrium signal. The products of these genes along with the Bip1-interacting protein RpS8 (Ribosomal protein S8) are required for the proper expression of Pvr (Mondal, 2014: PubMed).

    Haemocytes control stem cell activity in the Drosophila intestine

    Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes were recruited to the intestine and secreted the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switched their response to DPP by inducing expression of Thickveins, a second type I receptor that had previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promoted infection resistance, but also contributed to the development of intestinal dysplasia in ageing flies. The study proposes that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans (Ayyaz, 2015).

    Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes are recruited to the intestine and secrete the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switch their response to DPP by inducing expression of Thickveins, a second type I receptor that has previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promotes infection resistance, but also contributes to the development of intestinal dysplasia in ageing flies. It is proposed that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans (Ayyaz, 2015).

    The results extend the current model for the control of epithelial regeneration in the wake of acute infections in the Drosophila intestine. It is proposed that the control of ISC proliferation by haemocyte-derived DPP integrates with the previously described regulation of ISC proliferation by local signals from the epithelium and the visceral muscle, allowing precise temporal control of ISC proliferation in response to tissue damage, inflammation and infection (Ayyaz, 2015).

    The association of haemocytes with the intestine is extensive, and can be dynamically increased on infection or damage. In this respect, the current observations parallel the invasion of subepithelial layers of the vertebrate intestine by blood cells that induce proliferative responses of crypt stem cells during infection. A role for macrophages and myeloid cells in promoting tissue repair and regeneration has been described in adult salamanders and in mammals, where TGFβ ligands secreted by these immune cells can inhibit ISC proliferation, but can also contribute to tumour progression. The results provide a conceptual framework for immune cell/stem cell interactions in these contexts (Ayyaz, 2015).

    The observation that DPP/SAX/SMOX signalling is required for UPD-induced proliferation of ISCs suggests that SAX/SMOX signalling cooperates with JAK/STAT and EGFR signalling in the induction of ISC proliferation. Accordingly, while constitutive activation of EGFR/RAS or JAK/STAT signalling in ISCs is sufficient to promote ISC proliferation cell autonomously, this study found that this partially depends on Smox. Even in these gain-of-function conditions, ISC proliferation can thus be fully induced only in the presence of basal SMOX activity. As short-term overexpression of DPP in haemocytes does not induce ISC proliferation, it is further proposed that DPP/SAX/SMOX signalling can activate ISCs only when JAK/STAT and/or EGFR signalling are activated in parallel. However, long-term overexpression of DPP in haemocytes results in increased ISC proliferation, suggesting that chronic activation of immune cells disrupts normal signalling mechanisms and results in ISC activation even in the absence of tissue damage (Ayyaz, 2015).

    BMP TGFβ signalling pathways are critical for metazoan growth and development and have been well characterized in flies. Multiple ligands, receptors and transcription factors with highly context-dependent interactions and function have been described. This complexity is reflected by the sometimes conflicting studies exploring DPP/TKV/SAX signalling in the adult intestine. These studies consistently highlight two important aspects of BMP signalling in the adult Drosophila gut: ISCs can undergo opposite proliferative responses to BMP signals; and there are various sources of DPP that differentially influence ISC function in specific conditions. By characterizing the temporal regulation of BMP signalling activity in ISCs, the results resolve some of these conflicts: it is proposed that early in the regenerative response, haemocyte-derived DPP triggers ISC proliferation by activating SAX/SMOX signalling, and ISC quiescence is re-established by muscle-derived DPP as soon as TKV becomes expressed. Of note, some of the conflicting conclusions described in the literature may have originated from problems with the genetic tools used in some studies. This study have used two independent RNAi lines (BL25782 and BL33618) that effectively decrease dpp mRNA levels in haemocytes when expressed using HmlΔ::Gal4 (Ayyaz, 2015).

    The close association of haemocytes with the type IV collagen Viking suggests that the stimulation of ISC proliferation by haemocyte-derived DPP may also be controlled at the level of ligand availability, as suggested previously for DPP from other sources. The regulation of SAX/SMOX signalling by DPP observed in this study is surprising, but consistent with earlier reports showing that SAX can respond to DPP in certain contexts. Biochemical studies have suggested that heterotetrameric complexes between the type II receptor PUNT and the type I receptors SAX and TKV can bind DPP, and complexes with TKV/TKV homodimers preferentially bind DPP, and complexes with SAX/SAX homodimers preferentially bind GBB. In the absence of TKV, SAX has been proposed to sequester GBB, shaping the GBB activity gradient, but to fail to signal effectively. Expression of GBB in the midgut epithelium has recently been described, and ligand heterodimers between GBB and DPP are well established. Consistent with earlier reports, this study found that GBB knockdown in ECs significantly reduces ISC proliferation in response to infection. Complex interactions between haemocyte-derived DPP, epithelial GBB, and ISC-expressed SAX, PUNT and TKV thus probably shape the response of ISCs to damage, and will be an interesting area of further study (Ayyaz, 2015).

    Similar complexities exist in the regulation of transcription factors by SAX and TKV. Canonically, SMOX is regulated by Activin ligands (Activin, Dawdle, Myoglianin and maybe more), and the type I receptor Baboon. This study has tested the role of Activin and Dawdle in ISC regulation, and, in contrast to DPP, this study could not detect a requirement for these factors in the induction of ISC proliferation after Ecc15 infection. Furthermore, the data establish a requirement for haemocyte-derived DPP as well as for SAX expression in ISCs in the nuclear translocation of SMOX after a challenge. This study thus indicates that in this context, SAX responds to DPP and regulates SMOX. Regulation of SMOX by SAX has been described before, yet SAX is also known to promote MAD phosphorylation, but only in the presence of TKV. Consistent with such observations, this study has detected MAD phosphorylation in ISCs only in the late recovery phase on bacterial infection, when TKV is simultaneously induced in ISCs. During this recovery phase, ISCs maintain high SAX expression, but SMOX nuclear localization is not detected anymore, suggesting that SAX cannot activate SMOX in the presence of TKV, and might actually divert signals towards MAD instead. The data also suggest that Medea (the Drosophila SMAD4 homologue) is not required for SMOX activity. Although surprising, this observation is consistent with recent reports that SMAD proteins in mammals can translocate into the nucleus and activate target genes in a SMAD4-independent manner. The specific signalling readouts in ISCs when these cells are exposed to various BMP ligands and are expressing different combinations of receptors are thus likely to be complex (Ayyaz, 2015).

    The current findings demonstrate that the control of ISC proliferation by haemocyte-derived DPP is critical for tolerance against enteropathogens, but contributes to ageing-associated epithelial dysfunction, highlighting the importance of tightly controlled interactions between blood cells and stem cells in this tissue. Nevertheless, where haemocytes themselves are required for normal lifespan, loss of haemocyte-derived DPP does not impact lifespan. One interpretation of this finding is that beneficial (improved gut homeostasis) and deleterious (for example, reduced immune competence of the gut epithelium) consequences of reduced haemocyte-derived DPP cancel each other out over the lifespan of the animal. It will be interesting to test this hypothesis in future studies. Ageing is associated with systemic inflammation, and a role for immune cells in promoting inflammation in ageing vertebrates has been proposed. In humans, recruitment of immune cells to the gut is required for proper stem cell proliferation in response to luminal microbes, and prolonged inflammatory bowel disease further contributes to cancer development. It is thus anticipated that conserved macrophage/stem cell interactions influence the aetiology and progression of such diseases. The data confirm a role for haemocytes in age-related intestinal dysplasia in the fly intestine, and provide mechanistic insight into the causes for this deregulation. It can be anticipated that similar interactions between macrophages and intestinal stem cells may contribute to the development of IBDs, intestinal cancers, and general loss of homeostasis in the ageing human intestine (Ayyaz, 2015).

    Active JNK-dependent secretion of Drosophila Tyrosyl-tRNA synthetase by loser cells recruits haemocytes during cell competition

    Cell competition is a process by which the slow dividing cells (losers) are recognized and eliminated from growing tissues. Loser cells are extruded from the epithelium and engulfed by the haemocytes, the Drosophila macrophages. However, how macrophages identify the dying loser cells is unclear. This study shows that apoptotic loser cells secrete Tyrosyl-tRNA synthetase (TyrRS), which is best known as a core component of the translational machinery. Secreted TyrRS is cleaved by matrix metalloproteinases generating MiniTyr and EMAP fragments. EMAP acts as a guiding cue for macrophage migration in the Drosophila larvae, as it attracts the haemocytes to the apoptotic loser cells. JNK signalling and Kish, a component of the secretory pathway, are autonomously required for the active secretion of TyrRS by the loser cells. Altogether, this mechanism guarantees effective removal of unfit cells from the growing tissue (Casas-Tinto, 2015).

    Extracellular reactive oxygen species drive apoptosis-induced proliferation via Drosophila macrophages

    Apoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial cells of Drosophila imaginal discs, the Caspase-9 ortholog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. This study shows that caspase-induced activation of JNK during AiP depends on an inflammatory response. This is mediated by extracellular reactive oxygen species (ROSs) generated by the NADPH oxidase Duox in epithelial disc cells. Extracellular ROSs activate Drosophila macrophages (hemocytes), which in turn trigger JNK activity in epithelial cells by signaling through the tumor necrosis factor (TNF) ortholog Eiger. It is proposed that in an immortalized ('undead') model of AiP, in which the activity of the effector caspases is blocked, signaling back and forth between epithelial disc cells and hemocytes by extracellular ROSs and TNF/Eiger drives overgrowth of the disc epithelium. These data illustrate a bidirectional cell-cell communication pathway with implication for tissue repair, regeneration, and cancer (Fogarty, 2016).

    The role of ROSs as a regulated form of redox signaling in damage detection and damage response is becoming increasingly clear. This study has shown that in Drosophila, extracellular ROSs generated by the NADPH oxidase Duox drive compensatory proliferation and overgrowth following hid-induced activation of the initiator caspase Dronc in developing epithelial tissues. At least one consequence of ROS production is the activation of hemocytes at undead epithelial disc tissue. Furthermore, the work implies that extracellular ROS and hemocytes are part of the feedback amplification loop between Hid, Dronc, and JNK that occurs during stress-induced apoptosis. Finally, hemocytes release the TNF ligand Eiger, which promotes JNK activation in epithelial disc cells (Fogarty, 2016).

    This work helps to understand why JNK activation occurs mostly in apoptotic/undead cells but occasionally also in neighboring surviving cells. Because the data indicate that hemocytes trigger JNK activation in epithelial cells, the location of hemocytes on the imaginal discs determines which epithelial cells receive the signal for JNK activation. Nevertheless, the possibility is not excluded that there is also an autonomous manner of Dronc-induced JNK activation in undead/apoptotic cells (Fogarty, 2016).

    In the context of apoptosis, hemocytes engulf and degrade dying cells. However, there is no evidence that hemocytes have this role in the undead AiP model. No Caspase-3 (CC3) material is observed in hemocytes attached to undead tissue. Therefore, the role of hemocytes in driving proliferation is less clear and likely context dependent. In Drosophila embryos, hemocytes are required for epidermal wound healing, but this is a nonproliferative process. With respect to tumor models in Drosophila, much of the research to date has focused on the tumor-suppressing role of hemocytes and the innate immune response. However, a few reports have implicated hemocytes as tumor promoters in a neoplastic tumor model. Consistently, in the undead model of AiP, this study found that hemocytes have an overgrowth- and tumor-promoting role. Therefore, the state of the damaged tissue and the signals produced by the epithelium may have differential effects on hemocyte response (Fogarty, 2016).

    In a recent study, ROSs were found to be required for tissue repair of wing imaginal discs in a regenerative (p35-independent) model of AiP, consistent with the current work. Although a role of hemocytes was not investigated in this study, it should be noted that p35-independent AiP models do not cause overgrowth, whereas undead ones such as the ey>hid-p35 AiP model do. It is therefore possible that ROSs in p35-independent AiP models are necessary for tissue repair independent of hemocytes, whereas ROSs in conjunction with ROS-activated hemocytes in undead models mediate the overgrowth of the affected tissue. Future work will clarify the overgrowth-promoting function of hemocytes. These considerations are reminiscent of mammalian systems, where many solid tumors are known to host alternatively activated (M2) tumor-associated macrophages, which promote tumor growth and are associated with a poor prognosis (Fogarty, 2016).

    Because tumors are considered 'wounds that do not heal', the undead model of AiP is seen as a tool to probe the dynamic interactions and intercellular signaling events that occur in the chronic wound microenvironment. Future studies will investigate the specific mechanisms of hemocyte-induced growth and the tumor-promoting role of inflammation in Drosophila as well as roles of additional tissue types, such as the fat body, on modulating tumorous growth (Fogarty, 2016).

    Genetic screen in Drosophila larvae links ird1 function to Toll signaling in the fat body and hemocyte motility

    To understand how Toll signaling controls the activation of a cellular immune response in Drosophila blood cells (hemocytes), a genetic modifier screen was carried out, looking for deletions that suppress or enhance the mobilization of sessile hemocytes by the gain-of-function mutation Toll10b (Tl10b). This study describes the results from chromosome arm 3R, where five regions strongly suppressed this phenotype. The specific genes immune response deficient 1 (ird1), headcase (hdc) and possibly Rab23 were identified as suppressors, and the role of ird1 was studied in more detail. An ird1 null mutant and a mutant that truncates the N-terminal kinase domain of the encoded Ird1 protein affected the Tl10b phenotype, unlike mutations that affect the C-terminal part of the protein. The ird1 null mutant suppressed mobilization of sessile hemocytes, but enhanced other Tl10b hemocyte phenotypes, like the formation of melanotic nodules and the increased number of circulating hemocytes. ird1 mutants also had blood cell phenotypes on their own. They lacked crystal cells and showed aberrant formation of lamellocytes. ird1 mutant plasmatocytes had a reduced ability to spread on an artificial substrate by forming protrusions, which may explain why they did not go into circulation in response to Toll signaling. The effect of the ird1 mutation depended mainly on ird1 expression in hemocytes, but ird1-dependent effects in other tissues may contribute. Specifically, the Toll receptor was translocated from the cell membrane to intracellular vesicles in the fat body of the ird1 mutant, and Toll signaling was activated in that tissue, partially explaining the Tl10b-like phenotype. As ird1 is otherwise known to control vesicular transport, it is concluded that the vesicular transport system may be of particular importance during an immune response (Schmid, 2016).

    The role of variant histone H2AV in D. melanogaster larval hematopoiesis

    Replication-independent histone variants can replace the canonical replication-dependent histones. Vertebrates have multiple H2A variant histones, including H2AZ and H2AX that are present in most eukaryotes. H2AZ regulates transcriptional activation as well as maintenance of gene silencing, while H2AX is important in DNA damage repair. The fruit fly Drosophila melanogaster has only one histone H2A variant (H2AV), which is a chimera of H2AZ and H2AX. This study found that lack of H2AV led to the formation of black melanotic masses in the third instar larvae of Drosophila. The formation of these masses was found in conjunction with a loss of a majority of the primary lymph gland lobes. Interestingly, the cells of the posterior signaling center were preserved in these mutants. Reduction of H2AV levels by RNAi knockdown caused a milder phenotype that preserved the lymph gland structure, but that included precocious differentiation of the prohemocytes located within the medullary zone and secondary lobes of the lymph gland. Mutant rescue experiments suggest that the H2AZ-like rather than the H2AX-like function of H2AV is primarily required for normal hematopoiesis (Grigorian, 2017).

    Absence of the variant histone protein H2AV results in the formation of larval melanotic masses containing plasmatocytes and crystal cells. Previous studies have proposed that the formation of melanotic masses can be due either to the response of a normal immune system to abnormal tissue formed during development, or to a developmental defect in the hemocytes of the lymph gland. The current data showing the loss of a majority of the primary lymph gland lobes in the His2Av810 null mutant, as well as the early differentiation of the medullary zone and secondary lobe prohemocytes when H2AV levels were reduced via RNAi, are consistent with the latter model. The results demonstrate an important role for H2AV during normal hemocyte differentiation and dispersal. Interestingly, studies using a human histiocytic lymphoma cell line or normal macrophages differentiated with macrophage colony stimulating factor (M-CSF; CSF1) have shown an upregulation of the His2Av-related human H2A.Z (H2AFZ) gene during macrophage differentiation. These results imply an evolutionarily conserved role for the closely related H2AV and H2AZ histone variants in blood cell differentiation (Grigorian, 2017).

    The presence of black melanotic masses in Drosophila larvae is not restricted to His2Av mutants. This phenotype has previously been observed in mutants of two different ATP-dependent chromatin-remodeling complexes. Dom, which is a catalytic subunit of the dTip60 complex, plays a role in H2A variant exchange in nucleosomes, as well as in DNA damage repair. dom loss-of-function mutants display black melanotic masses that are composed of melanized lymph glands. Mutants have shown that the vertebrate homolog of dom is required for both embryonic and adult hematopoiesis in the laboratory mouse. Loss of a subunit of another ATP-dependent chromatin-remodeling complex, NURF, also causes melanotic masses. In addition, melanotic masses have been observed in mutations that affect various signaling pathways. For example, constitutive activation of the JAK-STAT pathway via the dominant gain-of-function HopTUM mutation results in the formation of melanotic masses. Constitutive activation of the Toll pathway via the dominant gain-of-function Tl10b mutation also causes melanotic masses. These observations raise the question of whether the closely related variant histones H2AV and H2AZ might be required to repress these evolutionarily conserved signaling pathways in hematopoietic cells (Grigorian, 2017).

    Although the majority of the cells in the primary lymph gland lobes in His2Av mutants are lost, the Antp-positive cells comprising the PSC are spared and can be seen adjacent to the cardioblasts of the dorsal vessel. In addition, these cells express the Hh ligand that normally prevents premature differentiation of hemocyte precursors. The presence of Antp-positive cells can also be observed in posterior lymph gland lobes, where Antp is not normally expressed. In this regard, previous studies have shown that His2Av can function as a Polycomb Group (PcG) gene, and PcG proteins are known to be important for repressing the transcription of homeotic genes such as Antp. In particular, it has been reported that Antp expression is expanded in the central nervous system of larvae that are mutant for His2Av. Reduction of H2AV levels via RNAi in the prohemocytes of the primary lobes, as well as in the secondary lobes, led to increased differentiation of plasmatocytes and crystal cells. This suggests that H2AV also acts downstream of the signals that originate from the PSC and that maintain the prohemocytes of the medullary zone in an undifferentiated state (Grigorian, 2017).

    Reduction of H2AV levels via RNAi causes a less severe phenotype than that of His2Av810 null mutants, in that the primary lobes of the lymph gland are preserved. However, there is a loss of the undifferentiated prohemocytes found within the medullary zone, as these cells differentiate into mature hemocytes. Previous studies in the testis of Drosophila have shown an important role for H2AV in the maintenance of both the germline and cyst stem cells. Together, these results suggest a possible role for H2AV in the transcriptional control of genes important for stem cell maintenance in general. In this regard, the closely related H2AZ protein of mammals has been reported to be important for the differentiation of embryonic stem cells in culturen (Grigorian, 2017).

    H2AV might be exerting its effects on the lymph gland through various signaling pathways that have been shown to orchestrate prohemocyte differentiation. Two pathways that might be affected are the Hh and Wg signaling pathways. Hh has been implicated in maintaining prohemocytes in an undifferentiated state. However, this study observed Hh expression in the PSC of both heterozygous and homozygous mutant His2Av810 larval lymph glands. Wg has been reported to not only maintain the prohemocyte population in an undifferentiated state, but also to dictate PSC cell number. No significant differences were detected in the staining of prohemocytes and PSC cells with anti-Wg antibodies in homozygous versus heterozygous mutant His2Av810 larval lymph glands. These results suggest that loss of H2AV might alter the intracellular responses to these ligands rather than their expression (Grigorian, 2017).

    Drosophila H2AV is a chimeric protein that plays the roles of two widely conserved variant histones, H2AX and H2AZ. H2AX is important for the DNA damage repair response, while H2AZ is important for both transcriptional activation and gene silencing. Previous studies have shown that H2AVCT, which lacks H2AX function, is able to rescue the lethal phenotype seen in His2Av810 null mutants, allowing the organisms to progress to pupation and adulthood. This study found that H2AVCT was able to partially rescue the His2Av810 null larval hematopoietic phenotype, arguing that an H2AZ-like function rather than an H2AX-like function of H2AV is required for hematopoiesis. Nevertheless, differentiation within the lymph gland still appeared disrupted and partial loss of the primary lymph gland lobes could be seen. In addition, the expression of Antp was at times seen to expand into the posterior lobes of the lymph gland. This lack of full rescue could be due to a decreased stability of the H2AVCT protein. However, the presence of H2AVCT in an otherwise His2Av wild-type background was sufficient to cause abnormalities of the lymph gland lobes. Furthermore, overexpression of wild-type or of phosphorylation mutants of H2AV also caused hematopoietic abnormalities. Together, these results imply that a precise dosage of H2AV protein is essential for normal hematopoiesis in Drosophila. Similar alterations in differentiation might also occur in other organs and tissues. In this regard, care should be taken when using His2Av-GFP and His2Av-RFP transgenes, which are popular markers in live imaging (Grigorian, 2017).

    The formation of black melanotic masses in the His2Av810 null mutant establishes larval hemocytes as a useful tool for further studies of H2AV function. Furthermore, given the role that H2AV plays not only in undifferentiated prohemocytes, but also in the germline and cyst stem cells found in the testis, it will be interesting to test whether H2AV also regulates stem cells found in other tissues (Grigorian, 2017).

    Regulation of Drosophila hematopoietic sites by Activin-β from active sensory neurons

    An outstanding question in animal development, tissue homeostasis and disease is how cell populations adapt to sensory inputs. During Drosophila larval development, hematopoietic sites are in direct contact with sensory neuron clusters of the peripheral nervous system (PNS), and blood cells (hemocytes) require the PNS for their survival and recruitment to these microenvironments, known as Hematopoietic Pockets. This study reports that Activin-β, a TGF-β family ligand, is expressed by sensory neurons of the PNS and regulates the proliferation and adhesion of hemocytes. These hemocyte responses depend on PNS activity, as shown by agonist treatment and transient silencing of sensory neurons. Activin-β has a key role in this regulation, which is apparent from reporter expression and mutant analyses. This mechanism of local sensory neurons controlling blood cell adaptation invites evolutionary parallels with vertebrate hematopoietic progenitors and the independent myeloid system of tissue macrophages, whose regulation by local microenvironments remain undefined (Makhijani, 2017).

    This research identified Actβ as one of the elusive genes that govern hemocyte proliferation in the hematopoietic sites (HPs) of the Drosophila larva, as was predicted by previous functional studies. Actβ RNA expression is linked to the level of PNS neuronal activity. This model implies that increased expression of Actβ would give rise to higher levels of active Actβ protein, although the formal demonstration awaits development of a suitable tool for the detection of Actβ protein. In the future, it will be interesting to study specific sensory stimuli that trigger hemocyte responses. Sensory neurons of the PNS have a prime function in detecting innocuous and noxious sensory stimuli such as mechanical strain, temperature, chemicals and light, many of which signal potentially harmful conditions that may cause tissue damage. Thus, linking the detection of challenging conditions with the adaptive expansion of the blood cell pool may be an efficient system to elevate the levels of macrophages, to remove and repair damaged tissues, enhancing the overall fitness of the animal. Because Drosophila larval hemocytes persist into the adult stage, the mechanism of sensory neuron-induced blood cell responses may allow adaptation of the animal beyond the larval stage (Makhijani, 2017).

    In Drosophila self-renewing hemocytes, Actβ/dSmad2 signalling has diverse effects on proliferation, apoptosis and adhesion. The current ex vivo data indicate that hemocyte proliferation is likely a direct effect, which is consistent with similar roles of babo/dSmad2 in other tissues such as Drosophila imaginal discs and brain and TGf-β family dependent proliferation in vertebrate systems. Echoing the findings of babo-CA driven hemocyte apoptosis, TGF-β family mediated direct or indirect effects on apoptosis have been described in invertebrate and vertebrate systems. Overall, TGF-β family signalling is known for its multifaceted biological roles, depending on the cellular contexts and levels of ligand stimulation, which often translate into qualitatively distinct transcriptional and other cellular responses, that are mediated by both Smad and non-Smad signalling mechanisms. While Drosophila Actβ and possibly related TGF-β family ligands are known to signal through the induction of ecdysone receptor (EcR) in some but not all Drosophila tissues, this study found no indication for a link with EcR expression in hemocytes, suggesting other signalling mechanisms in the regulation of larval blood cell responses. In the studied Drosophila system, it further remains to be seen whether Actβ/dSmad2 signalling has direct or indirect effects on hemocyte adhesion, and which other rate-limiting step/s may contribute to this process. Since hemocyte-autonomous loss of dSmad2 signalling causes a more severe phenotype than Actβ lof, it is speculated that other Act family ligands such as daw and myo, which are expressed in various tissues including surface glia, muscle, fat body, gut, and imaginal discs may partially substitute for Actβ in its absence. Overall, Actβ is likely to be only one player in a more complex regulatory network. Future research will identify other inducible signals from neurons that regulate neuron-blood cell communications. This is predicted from Actβ mutants that only partially block carbachol-induced blood cell responses. Actβ/dSmad2 lof and pathway silencing in hemocytes also reveal an underlying ability of the cells to compensate for the lack of this signalling pathway and the associated impairment in proliferation. Time course experiments with various RNAi lines suggest that the amplitude and temporal occurrence of the compensatory response may be proportional to the severity of the block in dSmad2 signalling. Future investigation will address whether the related BMP/Mad pathway might play a part in this, as silencing of Mad in hemocytes appeared to dampen elevated hemocyte numbers seen in dSmad2 null mutants. Similar observations of dSmad2 lof causing Mad overactivation have been reported in the Drosophila wing disc and neuromuscular junction previously (Makhijani, 2017).

    Larval development may comprise distinct sensitive phases for the regulation of hemocyte responses. This is supported by carbachol promoting hemocyte proliferation preferentially in the early-mid 2nd instar larva, that is, at a stage when hemocytes are still tightly localized to the Hematopoietic Pockets (HPs). Likewise, the effects of Actβ lof and pathway silencing in hemocytes are more pronounced in younger larvae, suggesting a possible stronger dependence on the pathway, in addition to the emergence of compensatory mechanisms under lof conditions over time. Moreover, it will be interesting to investigate whether Actβ signalling may not only vary temporally, but also by the ability of cell types to produce active Actβ ligand, thereby influencing signalling outcomes, consistent with the cell type specific processing known for Activins and other ligands of the TGF-β family in both invertebrates and vertebrates (Makhijani, 2017).

    Drosophila Actβ has previously been studied for its role in the formation and function of neuromuscular junctions in the Drosophila larva, where Actβ expressing motor neurons project axons from the CNS, reaching from the center of the larva to the muscle layers of the body wall. However, resident hemocytes are shielded from these areas through the muscle layers of the body wall, which also form the base of the HPs, thereby creating an anatomical space between the muscle layers and epidermis where resident hemocytes and Actβ expressing sensory neurons colocalize (i.e., the Hematopoietic Pocket). The model that sensory neurons signal to adjacent hemocytes in the HPs is further supported by the fact that Actβ silencing in motor neurons did not affect resident hemocyte localization and had, by t-test, no significant effect on hemocyte numbers. However, involvement of alternative or additional scenarios cannot be ruled out, for example, that experimental manipulations of PNS activity, which also feed back to the CNS, would in turn trigger a signal to motor neurons that may respond by secreting Actβ and/or another factor/s, thereby influencing hemocytes and/or the PNS itself. Likewise, although the direct effect of Actβ on hemocytes was confirmed ex vivo, and no signs were found of altered sensory neuron morphology under Actβ lof/silencing, it cannot be ruled out that in the larva, Actβ may contribute to molecular changes in the PNS that in turn might contribute to the observed hemocyte effects (Makhijani, 2017).

    Sensory neurons of the HPs project axons to the CNS, and the current work shows that hemocytes are closely adjacent to and/or form direct contacts with sensory neurons, likely along the neuron cell bodies and dendrites, suggesting the communication involves non-canonical mechanisms. In Drosophila, as in vertebrates, signal transfer along all neuronal membrane surfaces, including dendritic synapses and dendrodendritic connections, have been described, which may also form the interface in neuron-blood cell communication. The transcriptional induction of Actβ in response to sensory stimuli recalls previous reports of the transcriptional upregulation of Actβ in the formation of long-term memory in both flies and vertebrates. This suggests parallels between the neuronal regulation within the CNS, and PNS-blood cell circuits, which will be an interesting subject for future study. Based on these findings and another recent report demonstrating that transcriptional regulation of the related BMP Decapentaplegic (Dpp) in the Drosophila wing epithelium depends on the K+ channel Irk2, it is proposed that cellular electrochemical potential may be a more general theme in the expression of TGF-β family ligands (Makhijani, 2017).

    These findings in the Drosophila model pioneer a new concept that has not been shown in any vertebrate system to date -- the neuronal induction of self-renewing, tissue-resident blood cells. These cells correspond to the broadly distributed system of self-renewing myeloid cells that are present in most vertebrate organs, which by lineage are completely independent from blood cell formation fueled by hematopoietic stem cells. In vertebrates, TGF-β family ligands such as Activin A and TGF-β regulate the activity and immune functions of macrophages, and cellular and humoral immune responses, in multiple ways through autocrine and paracrine signalling. While the autonomic neuronal and glial regulation of hematopoietic stem and progenitor cells in the bone marrow has been recognized, the role of sensory innervation in bone marrow hematopoiesis remains unknown. Even more so, nothing is known about the role of the nervous system in the regulation of the independent, self-renewing myeloid system of tissue macrophages. However, local neurons and sensory innervation of many organs including skin, lung, heart and pancreas and inducible changes in the self-renewal rates of tissue macrophages, suggest that principles of neuronal regulation are likely also at work in vertebrates, providing a link between neuronal sensing and adaptive responses of local blood cell populations (Makhijani, 2017).

    Control of RUNX-induced repression of Notch signaling by MLF and its partner DnaJ-1 during Drosophila hematopoiesis
    In Drosophila, Myeloid Leukemia Factor (MLF) has been shown to control blood cell development by stabilizing the RUNX transcription factor Lozenge (Lz). This study further characterized MLF's mode of action in Drosophila blood cells using proteomic, transcriptomic and genetic approaches. The results show that MLF and the Hsp40 co-chaperone family member DnaJ-1 interact through conserved domains and demonstrate that both proteins bind and stabilize Lz in cell culture, suggesting that MLF and DnaJ-1 form a chaperone complex that directly regulates Lz activity. Importantly, dnaj-1 loss causes an increase in Lz+ blood cell number and size similarly as in mlf mutant larvae. Moreover dnaj-1 was found to genetically interact with mlf to control Lz level and Lz+ blood cell development in vivo. In addition, mlf and dnaj-1 loss was shown to alter Lz+ cell differentiation, and the increase in Lz+ blood cell number and size observed in these mutants is caused by an overactivation of the Notch signaling pathway. Finally, high levels of Lz were shown to be required to repress Notch transcription and signaling. These data indicate that the MLF/DnaJ-1-dependent increase in Lz level allows the repression of Notch expression and signaling to prevent aberrant blood cell development. Thus these findings establish a functional link between MLF and the co-chaperone DnaJ-1 to control RUNX transcription factor activity and Notch signaling during blood cell development in vivo (Miller, 2017).

    Proper blood cell development requires the finely tuned regulation of transcription factors and signaling pathways activity. Consequently mutations affecting key regulators of hematopoiesis such as members of the RUNX transcription factor family or components of the Notch signaling pathway are associated with several blood cell disorders including leukemia. Also, leukemic cells often present recurrent chromosomal rearrangements that participate in malignant transformation by altering the function of these factors. The functional characterization of these genes is thus of importance not only to uncover the molecular basis of leukemogenesis but also to decipher the regulatory mechanisms controlling normal blood cell development. Myeloid Leukemia Factor 1 (MLF1) was identified as a target of the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) more than 20 years ago. Further findings suggested that MLF1 could act as an oncogene or a tumor suppressor depending on the cell context and it was shown that MLF1 overexpression either impairs cell cycle exit and differentiation, promotes apoptosis, or inhibits proliferation in different cultured cell lines. Yet, its function and mechanism of action remain largely unknown (Miller, 2017).

    MLF1 is the founding member of a small evolutionarily conserved family of nucleo-cytoplasmic proteins present in all metazoans but lacking recognizable domains that could help define their biochemical activity . Whereas vertebrates have two closely related MLF paralogs, Drosophila has a single mlf gene encoding a protein that displays around 50% identity with human MLF in the central conserved domain. In the fly, MLF was identified as a partner of the transcription factor DREF (DNA replication-related element-binding factor), for which it acts a co-activator to stimulate the JNK pathway and cell death in the wing disc. MLF has been shown to bind chromatin, as does its mouse homolog, and it can either activate or repress gene expression by a still unknown mechanism. MLF also interacts with Suppressor of Fused, a negative regulator of the Hedgehog signaling pathway, and, like its mammalian counterpart, with Csn3, a component of the COP9 signalosome, but the functional consequences of these interactions remain elusive. Interestingly the overexpression of Drosophila MLF or that of its mammalian counterparts can suppress polyglutamine-induced cytotoxicity in fly and in cellular models of neurodegenerative diseases. Moreover phenotypic defects associated with MLF loss in Drosophila can be rescued by human MLF1. Thus MLF function seems conserved during evolution and Drosophila appears to be a genuine model organism to characterize MLF proteins (Miller, 2017).

    Along this line, the role of MLF during Drosophila hematopoiesis has been studied. Indeed, a number of proteins regulating blood cell development in human, such as RUNX and Notch, also control Drosophila blood cell development. In Drosophila, the RUNX factor Lozenge (Lz) is specifically expressed in crystal cells and it is absolutely required for the development of this blood cell lineage. Crystal cells account for ±4% of the circulating larval blood cells; they are implicated in melanization, a defense response related to clotting, and they release their enzymatic content in the hemolymph by bursting. The Notch pathway also controls the development of this lineage: it is required for the induction of Lz expression and it contributes to Lz+ cell differentiation as well as to their survival by preventing their rupture. Interestingly, the previous analysis revealed a functional and conserved link between MLF and RUNX factors. In particular, MLF was shown to control Lz activity and prevent its degradation in cell culture, and the regulation of Lz level by MLF is critical to control crystal cell number in vivo. Intriguingly, although Lz is required for crystal cell development, mlf mutation causes a decrease in Lz expression but an increase in crystal cell number. In human, the deregulation of RUNX protein level is associated with several pathologies. For instance haploinsufficient mutations in RUNX1 are linked to MDS/AML in the case of somatic mutations, and to familial platelet disorders associated with myeloid malignancy for germline mutations. In the opposite, RUNX1 overexpression can promote lymphoid leukemia. Understanding how the level of RUNX protein is regulated and how this affects specific developmental processes is thus of particular importance (Miller, 2017).

    To better characterize the function and mode of action of MLF in Drosophila blood cells, this study used proteomic, transcriptomic and genetic approaches. In line with recent findings, MLF was found to bind DnaJ-1, a HSP40 co-chaperone, as well as the HSP70 chaperone Hsc70-4, and that both of these proteins are required to stabilize Lz. It was further shown that MLF and DnaJ-1 interact together but also with Lz via conserved domains and that they regulate Lz-induced transactivation in a Hsc70-dependent manner in cell culture. In addition, using a null allele of dnaj-1, it was shown to control Lz+ blood cell number and differentiation as well as Lz activity in vivo in conjunction with mlf. Notably, w mlf or dnaj-1 loss leads to an increase in Lz+ cell number and size due to the over-activation of the Notch signaling pathway. Interestingly, these results indicate that high levels of Lz are required to repress Notch expression and signaling. A model is proposed whereby MLF and DnaJ-1 control Lz+ blood cell growth and number by promoting Lz accumulation, which ultimately turndowns Notch signaling. These findings thus establish a functional link between the MLF/Dna-J1 chaperone complex and the regulation of a RUNX-Notch axis required for blood cell homeostasis in vivo (Miller, 2017).

    Members of the RUNX and MLF families have been implicated in the control of blood cell development in mammals and Drosophila and deregulation of their expression is associated with human hemopathies including leukemia. The current results establish the first link between the MLF/DnaJ-1 complex and the regulation of a RUNX transcription factor in vivo. In addition, these data show that the stabilization of Lz by the MLF/DnaJ-1 complex is critical to control Notch expression and signaling and thereby blood cell growth and survival. These findings pinpoint the specific function of the Hsp40 chaperone DnaJ-1 in hematopoiesis, reveal a potentially conserved mechanism of regulation of RUNX activity and highlight a new layer of control of Notch signaling at the transcriptional level (Miller, 2017).

    MLF binds DnaJ-1 and Hsc70-4, and these two proteins, like MLF, are required for Lz stable expression in Kc167 cells. In addition, these data show that MLF and DnaJ-1 bind to each other via evolutionarily conserved domains and also interact with Lz, suggesting that Lz is a direct target of a chaperone complex formed by MLF, DnaJ-1 and Hsc70-4. Of note, a systematic characterization of Hsp70 chaperone complexes in human cells identified MLF1 and MLF2 as potential partners of DnaJ-1 homologs, DNAJB1, B4 and B6, a finding corroborated by Dyer (2017). Therefore, the MLF/DnaJ-1/Hsc70 complex could play a conserved role in mammals, notably in the regulation of the stability of RUNX transcription factors. How MLF acts within this chaperone complex remains to be determined. In vivo, this study demonstrated that dnaj-1 mutations lead to defects in crystal cell development strikingly similar to those observed in mlf mutant larvae, and these two genes were shown to act together to control Lz+ cells development by impinging on Lz activity. The data suggest that in the absence of DnaJ-1, high levels of MLF lead to the accumulation of defective Lz protein whereas lower levels of MLF allow its degradation. Thus it is proposed that MLF stabilizes Lz and, together with DnaJ-1, promotes its proper folding/conformation. In humans, DnaJB4 stabilizes wild-type E-cadherin but induces the degradation of mutant E-cadherin variants associated with hereditary diffuse gastric cancer. Thus the fate of DnaJ client proteins is controlled at different levels and MLF might be an important regulator in this process (Miller, 2017).

    This work presents the first null mutant for a gene of the DnaJB family in metazoans and the results demonstrate that a DnaJ protein is required in vivo to control hematopoiesis. There are 16 DnaJB and in total 49 DnaJ encoding genes in mammals and the expansion of this family has likely played an important role in the diversification of their functions. DnaJB9 overexpression was found to increase hematopoietic stem cell repopulation capacity and Hsp70 inhibitors have anti-leukemic activity, but the participation of other DnaJ proteins in hematopoiesis or leukemia has not been explored. Actually DnaJ's molecular mechanism of action has been fairly well studied but there are only limited insights as to their role in vivo. Interestingly though, both DnaJ-1 and MLF suppress polyglutamine protein aggregation and cytotoxicity in Drosophila models of neurodegenerative diseases, and this function is conserved in mammals. It is tempting to speculate that MLF and DnaJB proteins act together in this process as well as in leukemogenesis. Thus a better characterization of their mechanism of action may help develop new therapeutic approaches for these diseases (Miller, 2017).

    As shown in this study, mlf or dnaj-1 mutant larvae harbor more crystal cells than wild-type larvae. This rise in Lz+ cell number is not due to an increased induction of crystal cell fate as we could rescue this defect by re-expressing DnaJ-1 or Lz with the lz-GAL4 driver, which turns on after crystal cell induction, and it was also observed in lz hypomorph mutants, which again suggests a post-lz / cell fate choice process. Moreover mlf or dnaj-1 mutant larvae display a higher fraction of the largest lz>GFP+ cell population, which could correspond to the more mature crystal cells. It is thus tempting to speculate that mlf or dnaj-1 loss promotes the survival of fully differentiated crystal cells. RNAseq data demonstrate that mlf is critical for expression of crystal cell associated genes, but both up-regulation and down-regulation of crystal cell differentiation markers were observed in mlf or dnaj-1 mutant Lz+ cells. Also these changes did not appear to correlate with crystal cell maturation status since alterations were found in gene expression in the mutants both in small and large Lz+ cells. In addition the transcriptome did not reveal a particular bias toward decreased expression for 'plasmatocyte' markers in Lz+ cells from mlf- mutant larvae. Thus, it appears that MLF and DnaJ-1 loss leads to the accumulation of mis-differentiated crystal cells (Miller, 2017).

    The data support a model whereby MLF and DnaJ-1 act together to promote Lz accumulation, which in turn represses Notch transcription and signaling pathway to control crystal cell size and number. Indeed, an abnormal maintenance of Notch expression was observed in the larger Lz+ cells as well as an over-activation of the Notch pathway in the crystal cell lineage of mlf and dnaj-1 mutants or when Lz activity was interfered with. Moreover the data as well as previously published experiments show that Notch activation promotes crystal cell growth and survival. Importantly too the increase in Lz+ cell number and size observed in mlf or dnaJ-1 mutant is suppressed when Notch dosage is decreased. Yet, some of the mis-differentiation phenotypes in the mlf or dnaj-1 mutants might be independent of Notch since changes in crystal cell markers expression seem to appear before alterations in Notch are apparent. At the molecular level, the results suggest that Lz directly represses Notch transcription as this study identified a Lz-responsive Notch cis-regulatory element that contains conserved RUNX binding sites. The activation of the Notch pathway in circulating Lz+ cells is ligand-independent and mediated through stabilization of the Notch receptor in endocytic vesicles. Hence a tight control of Notch expression is of particular importance to keep in check the Notch pathway and prevent the abnormal development of the Lz+ blood cell lineage. Notably, Notch transcription was shown to be directly activated by Notch signaling. Such an auto-activation loop might rapidly go awry in a context in which Notch pathway activation is independent of ligand binding. By promoting the accumulation of Lz during crystal cell maturation, MLF and DnaJ-1 thus provide an effective cell-autonomous mechanism to inhibit Notch signaling. Further experiments will now be required to establish how Lz represses Notch transcription. RUNX factors can act as transcriptional repressors by recruiting co-repressor such as members of the Groucho family. Whether MLF and DnaJ-1 directly contribute to Lz-induced-repression in addition to regulating its stability is an open question. MLF and DnaJ-1 were recently found to bind and regulate a common set of genes in cell culture. They may thus provide a favorable chromatin environment for Lz binding or be recruited with Lz and/or favor a conformation change in Lz that allows its interaction with co-repressors. The scarcity of lz>GFP+ cells precludes a biochemical characterization of Lz, MLF and DnaJ-1 mode of action notably at the chromatin level but further genetic studies should help decipher their mode of action. While the post-translational control of Notch has been extensively studied, its transcriptional regulation seems largely overlooked. The current findings indicate that this is nonetheless an alternative entry point to control the activity of this pathway. Given the importance of RUNX transcription factor and Notch signaling in hematopoiesis and blood cell malignancies, it will be of particular interest to further study whether RUNX factors can regulate Notch expression and signaling during these processes in mammals (Miller, 2017).

    References

    Ayyaz, A., Li, H. and Jasper, H. (2015). Haemocytes control stem cell activity in the Drosophila intestine. Nat Cell Biol 17: 736-748. PubMed ID: 26005834

    Casas-Tinto, S., Lolo, F. N. and Moreno, E. (2015). Active JNK-dependent secretion of Drosophila Tyrosyl-tRNA synthetase by loser cells recruits haemocytes during cell competition. Nat Commun 6: 10022. PubMed ID: 26658841

    Crozatier M., et al. (2004). Cellular immune response to parasitization in Drosophila requires the EBF orthologue collier. PLoS Biol. 2: E196. PubMed ID: 15314643

    de Velasco, B., Mandal, L., Mkrtchyan, M. and Hartenstein, V. (2006). Subdivision and developmental fate of the head mesoderm in Drosophila melanogaster. Dev. Genes Evol. 216(1): 39-51. PubMed ID: 16249873

    Dragojlovic-Munther, M. and Martinez-Agosto, J. A. (2013). Extracellular matrix-modulated Heartless signaling in Drosophila blood progenitors regulates their differentiation via a Ras/ETS/FOG pathway and target of rapamycin function. Dev Biol 384: 313-330. PubMed ID: 23603494

    Drechsler, M., Schmidt, A. C., Meyer, H. and Paululat, A. (2013). The conserved ADAMTS-like protein lonely heart mediates matrix formation and cardiac tissue integrity. PLoS Genet 9: e1003616. PubMed ID: 23874219

    Dyer, J. O., Dutta, A., Gogol, M., Weake, V. M., Dialynas, G., Wu, X., Seidel, C., Zhang, Y., Florens, L., Washburn, M. P., Abmayr, S. M. and Workman, J. L. (2017). Myeloid Leukemia Factor acts in a chaperone complex to regulate transcription factor stability and gene expression. J Mol Biol 429(13): 2093-2107. PubMed ID: 27984043

    Fogarty, C. E., Diwanji, N., Lindblad, J. L., Tare, M., Amcheslavsky, A., Makhijani, K., Bruckner, K., Fan, Y. and Bergmann, A. (2016). Extracellular reactive oxygen species drive apoptosis-induced proliferation via Drosophila macrophages. Curr Biol 26(5):575-84. PubMed ID: 26898463

    Ghosh, S., Singh, A., Mandal, S. and Mandal, L. (2015). Active hematopoietic hubs in Drosophila adults generate hemocytes and contribute to immune response. Dev Cell 33(4):478-88. PubMed ID: 25959225

    Gonzalez, E. A., Garg, A., Tang, J., Nazario-Toole, A. E. and Wu, L. P. (2013). A glutamate-dependent redox system in blood cells is integral for phagocytosis in Drosophila melanogaster. Curr Biol 23: 2319-2324. PubMed ID: 24210616

    Grigorian, M., DeBruhl, H. and Lipsick, J. S. (2017). The role of variant histone H2AV in D. melanogaster larval hematopoiesis. Development 144(8):1441-1449. PubMed ID: 28242611

    Holz, A., et al. (2003). The two origins of hemocytes in Drosophila. Development 130: 4955-4962. PubMed ID: 12930778

    Jung, S. H., Evans, C. J., Uemura, C. and Banerjee, U. (2005). The Drosophila lymph gland as a developmental model of hematopoiesis. Development 132(11): 2521-33. PubMed ID: 15857916

    Kalamarz, M. E., Paddibhatla, I., Nadar, C. and Govind, S. (2012). Sumoylation is tumor-suppressive and confers proliferative quiescence to hematopoietic progenitors in Drosophila melanogaster larvae. Biol Open 1: 161-172. PubMed ID: 23213407

    Krzemien J., et al. (2007). Control of blood cell homeostasis in Drosophila larvae by the posterior signalling centre. Nature 446: 325-328. PubMed ID: 17361184

    Krzemien, J., Oyallon, J., Crozatier, M. and Vincent, A. (2010). Hematopoietic progenitors and hemocyte lineages in the Drosophila lymph gland. Dev. Biol. 346(2): 310-9. PubMed ID: 20707995

    Makhijani, K., et al. (2011). The peripheral nervous system supports blood cell homing and survival in the Drosophila larva. Development 138(24): 5379-91. PubMed ID: 22071105

    Makhijani, K., Alexander, B., Rao, D., Petraki, S., Herboso, L., Kukar, K., Batool, I., Wachner, S., Gold, K. S., Wong, C., O'Connor, M. B. and Bruckner, K. (2017). Regulation of Drosophila hematopoietic sites by Activin-β from active sensory neurons. Nat Commun 8: 15990. PubMed ID: 28748922

    Mandal, L., et al. (2007). A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors. Nature 446: 320-324. PubMed ID: 17361183

    Miller, M., Chen, A., Gobert, V., Auge, B., Beau, M., Burlet-Schiltz, O., Haenlin, M. and Waltzer, L. (2017). Control of RUNX-induced repression of Notch signaling by MLF and its partner DnaJ-1 during Drosophila hematopoiesis. PLoS Genet 13(7): e1006932. PubMed ID: 28742844

    Milton, C. C., Grusche, F. A., Degoutin, J. L., Yu, E., Dai, Q., Lai, E. C. and Harvey, K. F. (2014). The Hippo pathway regulates hematopoiesis in Drosophila melanogaster. Curr Biol 24: 2673-2680. PubMed ID: 25454587

    Mondal, B. C., Shim, J., Evans, C. J., Banerjee, U. (2014) Pvr expression regulators in equilibrium signal control and maintenance of Drosophila blood progenitors. Elife: e03626. PubMed ID: 25201876

    Owusu-Ansah, E. and Banerjee, U. (2009). Reactive oxygen species prime Drosophila haematopoietic progenitors for differentiation. Nature 461(7263): 537-41. PubMed ID: 19727075

    Paddibhatla, I., Lee, M. J., Kalamarz, M. E., Ferrarese, R. and Govind, S. (2010). Role for sumoylation in systemic inflammation and immune homeostasis in Drosophila larvae. PLoS Pathog 6: e1001234. PubMed ID: 21203476

    Regan, J. C., Brandao, A. S., Leitao, A. B., Mantas Dias, A. R., Sucena, E., Jacinto, A. and Zaidman-Remy, A. (2013). Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila. PLoS Pathog 9: e1003720. PubMed ID: 24204269

    Reimels, T. A. and Pfleger, C. M. (2015). Drosophila Rabex-5 restricts Notch activity in hematopoietic cells and maintains hematopoietic homeostasis. J Cell Sci [Epub ahead of print]. PubMed ID: 26567216

    Schmid, M. R., Anderl, I., Vo, H. T., Valanne, S., Yang, H., Kronhamn, J., Ramet, M., Rusten, T. E. and Hultmark, D. (2016). Genetic screen in Drosophila larvae links ird1 function to Toll signaling in the fat body and hemocyte motility. PLoS One 11: e0159473. PubMed ID: 27467079

    Sinenko S. A., et al. (2009). Dual role of Wingless signaling in stem-like hematopoietic precursor maintenance in Drosophila. Dev. Cell 16: 756-763. PubMed ID: 19460351

    Sinenko, S. A., Shim, J. and Banerjee, U. (2011). Oxidative stress in the haematopoietic niche regulates the cellular immune response in Drosophila. EMBO Rep. 13(1): 83-9. PubMed ID: 22134547

    Small, C., Ramroop, J., Otazo, M., Huang, L. H., Saleque, S. and Govind, S. (2013). An unexpected link between Notch signaling and ROS in restricting the differentiation of hematopoietic progenitors in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 24318532

    Tokusumi, Y., Tokusumi, T., Stoller-Conrad, J. and Schulz, R. A. (2010). Serpent, suppressor of hairless and U-shaped are crucial regulators of hedgehog niche expression and prohemocyte maintenance during Drosophila larval hematopoiesis. Development 137(21): 3561-8. PubMed ID: 20876645

    Tokusumi, T., Tokusumi, Y., Brahier, M. S., Lam, V., Stoller-Conrad, J. R., Kroeger, P. T. and Schulz, R. A. (2016). Screening and analysis of Janelia FlyLight project enhancer-Gal4 strains identifies multiple gene enhancers active during hematopoiesis in normal and wasp-challenged Drosophila larvae. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27913635

    Wood, W. and Jacinto, A. (2007). Drosophila melanogaster embryonic haemocytes: masters of multitasking. Nat Rev Mol Cell Biol. 2007 Jul;8(7):542-51. PubMed ID: 17565363


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