Drosophila tissue and organ development: Haemocytes and the Lymph Gland

Kapoor, A., Padmavathi, A., Madhwal, S. and Mukherjee, T. (2022). Dual control of dopamine in Drosophila myeloid-like progenitor cell proliferation and regulation of lymph gland growth. EMBO Rep 23(6): e52951. PubMed ID: 35476897

Dual control of dopamine in Drosophila myeloid-like progenitor cell proliferation and regulation of lymph gland growth>

In Drosophila, definitive haematopoiesis takes place in a specialized organ termed 'lymph gland'. It harbours multi-potent stem-like blood progenitor cells whose development controls overall growth of this haematopoietic tissue and formation of mature blood cells. With respect to its development, neurotransmitters have emerged as potent regulators of blood-progenitor cell development and function. This study extended the understanding of neurotransmitters and showed that progenitors are self-sufficient with regard to synthesizing dopamine, a well-established neurotransmitter. These cells also have modules for dopamine sensing through the receptor and transporter. Modulating expression of these components in progenitor cells affected lymph gland growth, which suggested growth-promoting function of dopamine in blood-progenitor cells. Cell-cycle analysis of developing lymph glands revealed an unexpected requirement for intracellular dopamine in moderating the progression of early progenitor cells from S to G2 phase of the cell cycle, while activation of dopamine receptor signalling later in development regulated their progression from G2 and entry into mitosis. The dual capacity in which dopamine operated, first intracellularly to coordinate S/G2 transition and later extracellularly in G2/M transition, was critical for the growth of the lymph gland. Overall, the data presented highlight a novel non-canonical use of dopamine in the myeloid system that reveals an uncharacterized function of intracellular dopamine in cell-cycle phasing with outcomes on haematopoietic growth and immunity as well (Kapoor, 2022).

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 N^ts1 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 N^ts1 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 (d_g4), thereby maturing into prohemocytes. The prohemocyte population (S⁺O⁺H⁺Pv⁺d_g4⁺) 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⁺d_g4^lowM⁺). Eventually, dome-gal4 expression is lost entirely in these cells (becoming S⁺O⁺H⁺Pv⁺d_g4^-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

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

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 hh^ts2 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 hh^ts2 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 TCF^DN 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).

Single-cell transcriptomics identifies new blood cell populations in Drosophila released at the onset of metamorphosis

Drosophila blood cells called hemocytes form an efficient barrier against infections and tissue damage. During metamorphosis, hemocytes undergo tremendous changes in their shape and behavior, preparing them for tissue clearance. Yet, the diversity and functional plasticity of pupal blood cells have not been explored. This study combine single-cell transcriptomics and high-resolution microscopy to dissect the heterogeneity and plasticity of pupal hemocytes. This study identified undifferentiated and specified hemocytes with different molecular signatures associated with distinct functions such as antimicrobial, antifungal immune defense, cell adhesion or secretion. Strikingly, a highly migratory and immune-responsive pupal cell population was identified expressing typical markers of the posterior signaling center (PSC), which is known to be an important niche in the larval lymph gland. PSC-like cells become restricted to the abdominal segments and are morphologically very distinct from typical Hemolectin (Hml)-positive plasmatocytes. G-TRACE lineage experiments further suggest that PSC-like cells can transdifferentiate to lamellocytes triggered by parasitoid wasp infestation. In summary, this study presents the first molecular description of pupal Drosophila blood cells, providing insights into blood cell functional diversification and plasticity during pupal metamorphosis (Hirschhauser, 2023).

A single-cell survey of Drosophila blood

Drosophila blood cells, called hemocytes, are classified into plasmatocytes, crystal cells, and lamellocytes based on the expression of a few marker genes and cell morphologies, which are inadequate to classify the complete hemocyte repertoire. This study used single-cell RNA sequencing (scRNA-seq) to map hemocytes across different inflammatory conditions in larvae. Plasmatocytes were resolved into different states based on the expression of genes involved in cell cycle, antimicrobial response, and metabolism, together with the identification of intermediate states. Further, rare subsets within crystal cells and lamellocytes were discovered that express fibroblast growth factor (FGF) ligand branchless and receptor breathless, respectively. These FGF components were identified as required for mediating effective immune responses against parasitoid wasp eggs, highlighting a novel role for FGF signaling in inter-hemocyte crosstalk. This scRNA-seq analysis reveals the diversity of hemocytes and provides a rich resource of gene expression profiles for a systems-level understanding of their functions (Tattikota, 2020).

Previous studies have identified three major Drosophila blood cell types essential for combating infections in this species. This study used scRNA-seq of larval fly blood to gain deeper insights into the different cell types and their transition states in circulation during normal and inflammatory conditions. Comprehensive scRNA-seq data provide information on subpopulations of plasmatocytes and their immune-activated states. Importantly, scRNA-seq could precisely distinguish mature crystal cells and lamellocytes from their respective intermediate states, which are less well understood and for which marker genes were not previously available. Thus, new marker genes identified in this study should facilitate further study of these states. Moreover, it was possible to identify the gene signature of self-renewing plasmatocytes and suggest their role as extra-lymph gland oligopotent precursors. In addition to the identification of various states of mature cell types, this study also suggests novel roles for a number of genes and pathways in blood cell biology. In particular, a putative new Mtk-like anti-microbial peptide (AMP) was identified, and a role was proposed for the FGF signaling pathway in mediating key events leading to the melanization of wasp eggs. Finally, a user-friendly searchable online data mining resource was developed that allows users to query, visualize, and compare genes within the diverse hemocyte populations across conditions (Tattikota, 2020).

Blood cell types are dynamic in nature and several transient intermediate states exist in a continuum during the course of their maturation in several species. This scRNA-seq analysis provides a framework to distinguish cell types from their various states including oligopotent, transient intermediate and activated states (Tattikota, 2020).

Oligopotent state: ScRNA-seq analysis identified PM2 as the oligopotent state of plasmatocytes based on the enrichment of several cell cycle genes including polo and stg. This signature suggests that PM2 corresponds to self-renewing plasmatocytes located in the circulatory and sessile compartments of the Drosophila hematopoietic system where plasmatocytes are the only dividing cells identified. Further, previous studies suggested that lamellocytes derived from embryonic-lineage hemocytes are readily detectable in circulation prior to their release from the lymph gland, and that terminally differentiated crystal cells can also derive from preexisting plasmatocytes in the sessile hub. Hence, it is proposed that PM2 corresponds to the oligopotent state that not only drives expansion of plasmatocytes, but importantly can also give rise to crystal cells and lamellocytes. Monocle3 analysis indicates that cell cycle genes decrease over pseudotime and there is ample evidence in support of the notion that cell cycle arrest may be required for terminal differentiation of various cell types in flies and vertebrates. Our in vivo data also indicates that cell cycle arrest can lead to the generation of terminally differentiated lamellocytes. Interestingly, recent evidence in hemocytes suggests that perturbing cell cycle by knocking down jumu, which is upstream of polo, can also lead to the generation of lamellocytes by activating Toll. In contrast, forced expression of certain oncogenes such as activated Ras and Hopscotch/JAK in hemocytes can also lead to overproduction of plasmatocytes and lamellocytes. It is, however, speculated that the proliferation and differentiation of hemocytes in these contexts may be linked to cell cycle. Thus, it is important to address this paradoxical role of cell cycle in the maintenance of oligopotency and transdifferentiation of plasmatocytes. Studies using lineage tracing methods such as G-TRACE or CRISPR-based in vivo cellular barcoding techniques may help further characterize the contribution of proliferating oligopotent plasmatocytes to blood cell lineages (Tattikota, 2020).

Immune-activated states: PM5 from the scRNA-seq data is enriched in several genes that encode glutathione S-transferase family of metabolic enzymes, which are known to catalyze the conjugation of reduced glutathione (GSH) to xenobiotics for their ultimate degradation. It has been demonstrated that a subset of hemocytes accumulate high GSH levels in Drosophila, in support of these data. Further, the two AMP clusters PM6-7 (PM^AMP) were identified as part of the immune-activated states of plasmatocytes. A recent study has demonstrated that AMPs are highly specific and act in synergy against various pathogens. The scRNA-seq analysis reveals the remarkable difference in the expression of a set of AMPs in the two clusters. Future studies with PM^AMP-specific perturbation of various AMPs identified within plasmatocytes should clarify their contribution in killing specific pathogens. Moreover, the role of Mtkl against pathogens needs further characterization. Pseudotime analysis showed that PM^AMP ends in the same lineage as lamellocytes suggesting a common mode of activation for these cell types and states. Interestingly, induction in hemocytes of Toll, which is upstream of Drs, can lead to the production of lamellocytes, suggesting that LM^int cells may act as the common branch point between immune-activated states and lamellocytes (Tattikota, 2020).

Transient intermediate states: In addition to the oligopotent and immune-activated states, plasmatocytes showed several subpopulations, which most likely are transient intermediate states. Although it remains to be seen whether they exist throughout the larval development, it is possible that these transient states exist along the continuum of cell maturation process. On the other hand, the transcriptomic composition of CC1 and LM1 clusters suggested the presence of intermediates for crystal cells and lamellocytes, respectively. Further analysis by Monocle3, which placed these clusters prior to their terminally differentiated cell types, confirmed the hypothesis that CC1 and LM1 correspond to CC^int and LM^int states, respectively. In the context of the CC lineage within the lymph gland, ultrastructural studies have revealed the presence of immature crystal cells, called procrystal cells, alongside mature crystal cells. This study furthered this observation by demonstrating in vivo that crystal cells exist in a continuum (PPO1^low to PPO1^high), validating the Monocle3 and scRNA-seq data. Moreover, clear gene signatures between the CC^int and LM^int states and their mature counterparts revealed that these intermediates most likely emerge from preexisting Hml⁺ plasmatocytes. With regards to the LM lineage, several groups have speculated that intermediates, called podocytes, or also lamelloblasts, may exist based on cell morphology and size. The scRNA-seq and Monocle3-based data clearly demarcate mature lamellocytes from LM^int at the transcriptomic level. In addition, sub-clustering analysis revealed that LM^int possessed a PM signature demonstrating that these intermediates are presumably derived from PM2 (Tattikota, 2020).

A novel role for the FGF signaling pathway in hemocyte crosstalk: In addition to the known hemocyte - tissue crosstalk, Drosophila hemocytes must act in a coordinated fashion to combat harmful pathogens and foreign entities such as wasp eggs. However, the signaling pathways that mediate the interactions among hemocytes and wound sites or wasp eggs have been unclear. The scRNA-seq uncovered a novel role for the FGF signaling pathway in controlling hemocyte differentiation and subsequent effects on the melanization of wasp eggs. The FGF ligand bnl and its receptor btl were among the genes identified in rare subsets of crystal cells and lamellocytes, respectively, highlighting the power of scRNA-seq in capturing and detecting these small populations of cells. Based on the in vivo data, it is proposed that Bnl⁺crystal cells interact with Btl⁺ lamellocytes to coordinate lamellocyte differentiation and possible migration towards parasitoid wasp eggs. Furthermore, because lamellocytes are also enriched in additional core components of the FGF signaling pathway, future studies involving a comprehensive analysis of this pathway will advance understanding of blood cell communication, differentiation, and migration in the context of immune response (Tattikota, 2020).

In summary, these scRNA-seq data provides a resource for a comprehensive systems-level understanding of Drosophila hemocytes across various inflammatory conditions (Tattikota, 2020).

Differential activation of JAK-STAT signaling reveals functional compartmentalization in Drosophila blood progenitors

Blood cells arise from diverse pools of stem and progenitor cells. Understanding progenitor heterogeneity is a major challenge. The Drosophila larval lymph gland is a well-studied model to understand blood progenitor maintenance and recapitulates several aspects of vertebrate hematopoiesis. However in-depth analysis has focused on the anterior lobe progenitors (AP), ignoring the posterior progenitors (PP) from the posterior lobes. Using in situ expression mapping and developmental and transcriptome analysis, this study revealed PP heterogeneity and identified molecular-genetic tools to study this abundant progenitor population. Functional analysis shows that PP resist differentiation upon immune challenge, in a JAK-STAT-dependent manner. Upon wasp parasitism, AP downregulate JAK-STAT signaling and form lamellocytes. In contrast, this study shows that PP activate STAT92E and remain undifferentiated, promoting survival. Stat92E knockdown or genetically reducing JAK-STAT signaling permits PP lamellocyte differentiation. How heterogeneity and compartmentalization allow functional segregation in response to systemic cues and could be widely applicable is discussed (Rodrigues, 2021).

Rab5 and Rab11 maintain hematopoietic homeostasis by restricting multiple signaling pathways in Drosophila

The hematopoietic system of Drosophila is a powerful genetic model for studying hematopoiesis, and vesicle trafficking is important for signal transduction during various developmental processes; however, its interaction with hematopoiesis is currently largely unknown. Three endosome markers, Rab5, Rab7, and Rab11, were selected for study that play a key role in membrane trafficking, and it was determined whether they participate in hematopoiesis. Inhibiting Rab5 or Rab11 in hemocytes or the cortical zone (CZ) significantly induced cell overproliferation and lamellocyte formation in circulating hemocytes and lymph glands and disrupted blood cell progenitor maintenance. Lamellocyte formation involves the JNK, Toll, and Ras/EGFR signaling pathways. Notably, lamellocyte formation was also associated with JNK-dependent autophagy. In conclusion, Rab5 and Rab11 were identified as novel regulators of hematopoiesis, and the results advance the understanding of the mechanisms underlying the maintenance of hematopoietic homeostasis as well as the pathology of blood disorders such as leukemia (Yu, 2021).

Toward a Consensus in the Repertoire of Hemocytes Identified in Drosophila

The catalog of the Drosophila immune cells was until recently limited to three major cell types, based on morphology, function and few molecular markers. Three recent single cell studies highlight the presence of several subgroups, revealing a large diversity in the molecular signature of the larval immune cells. Since these studies rely on somewhat different experimental and analytical approaches, this study compared the datasets and identify eight common, robust subgroups associated to distinct functions such as proliferation, immune response, phagocytosis or secretion. Similar comparative analyses with datasets from different stages and tissues disclose the presence of larval immune cells resembling embryonic hemocyte progenitors and the expression of specific properties in larval immune cells associated with peripheral tissues (Cattenoz, 2021).

A gap-junction-mediated, calcium-signaling network controls blood progenitor fate decisions in hematopoiesis

Stem cell homeostasis requires coordinated fate decisions among stem cells that are often widely distributed within a tissue at varying distances from their stem cell niche. This requires a mechanism to ensure robust fate decisions within a population of stem cells. This study shows that, in the Drosophila hematopoietic organ, the lymph gland (LG), gap junctions (see Drosophila Ogre) form a network that coordinates fate decisions between blood progenitors. Using live imaging of calcium signaling in intact LGs, it was found that blood progenitors are connected through a signaling network. Blocking gap junction function disrupts this network, alters the pattern of encoded calcium signals, and leads to loss of progenitors and precocious blood cell differentiation. Ectopic and uniform activation of the calcium-signaling mediator CaMKII restores progenitor homeostasis when gap junctions are disrupted. Overall, these data show that gap junctions equilibrate cell signals between blood progenitors to coordinate fate decisions and maintain hematopoietic homeostasis (Ho, 2021).

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

Immune challenges demand the gearing up of basal hematopoiesis to combat infection. Little is known about how during development, this switch is achieved to take care of the insult. This study shows that the hematopoietic niche of the larval lymph gland of Drosophila senses immune challenge and reacts to it quickly through the nuclear factor-κB (NF-κB), Relish, a component of the immune deficiency (Imd) pathway. During development, Relish is triggered by ecdysone signaling in the hematopoietic niche to maintain the blood progenitors. Loss of Relish causes an alteration in the cytoskeletal architecture of the niche cells in a Jun Kinase dependent manner, resulting in the trapping of Hh implicated in progenitor maintenance. Notably, during infection, downregulation of Relish in the niche tilts the maintenance program towards precocious differentiation, thereby bolstering the cellular arm of the immune response (Ramesh, 2021).

A Conserved Role for Asrij/OCIAD1 in Progenitor Differentiation and Lineage Specification Through Functional Interaction With the Regulators of Mitochondrial Dynamics

The echanism by which Mitochondria morphology and dynamics regulate cell differentiation and lineage choice remains incompletely understood. Asrij/OCIAD1 is a conserved protein that governs mitochondrial morphology, energy metabolism and human embryonic stem cell (hESC) differentiation. This study compared hESC phenotypes with those of Drosophila hematopoiesis, where Asrij is shown to regulate blood progenitor maintenance by conserved mechanisms. In concordance with hESC studies, this study found that Drosophila Asrij also localizes to mitochondria of larval blood cells and its depletion from progenitors results in elongated mitochondria. Live imaging of asrij knockdown hemocytes and of OCIAD1 knockout hESCs showed reduced mitochondrial dynamics. It was hypothesized that mitochondrial fission and fusion may control progenitor maintenance or differentiation in an Asrij-dependent manner. Knockdown of the fission regulator Drp1 in Drosophila lymph gland progenitors specifically suppressed crystal cell differentiation whereas depletion of the fusion regulator Marf (Drosophila Mitofusin) increased the same with concomitant upregulation of Notch signaling. These phenotypes were stronger in anterior progenitors and were exacerbated by Asrij depletion. Asrij is known to suppress Notch signaling and crystal cell differentiation. This study demonstrates a conserved role for Asrij/OCIAD1 in linking mitochondrial dynamics and progenitor differentiation (Ray, 2021).

Chk2-p53 and JNK in irradiation-induced cell death of hematopoietic progenitors and differentiated cells in Drosophila larval lymph gland

Ionizing radiation (IR) induces DNA double-strand breaks that activate the DNA damage response (DDR), which leads to cell cycle arrest, senescence, or apoptotic cell death. Understanding the DDR of stem cells is critical to tissue homeostasis and the survival of the organism. Drosophila hematopoiesis serves as a model system for sensing stress and environmental changes; however, their response to DNA damage remains largely unexplored. The Drosophila lymph gland is the larval hematopoietic organ, where stem-like progenitors proliferate and differentiate into mature blood cells called hemocytes. It was found that apoptotic cell death was induced in progenitors and hemocytes after 40 Gy irradiation, with progenitors showing more resistance to IR-induced cell death compared to hemocytes at a lower dose. Furthermore, it was found that Drosophila ATM (tefu), Chk2 (lok), p53, and reaper were necessary for IR-induced cell death in the progenitors. Notably, IR-induced cell death in mature hemocytes required tefu, Drosophila JNK (bsk), and reaper, but not lok or p53. In summary, this study found that DNA damage induces apoptotic cell death in the late third instar larval lymph gland and identified lok/p53-dependent and -independent cell death pathways in progenitors and mature hemocytes, respectively (Nguyen, 2021).

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

Mechanistic studies of Drosophila lymph gland hematopoiesis are limited by the availability of cell-type specific markers. Using a combination of bulk RNA-Seq of FACS-sorted cells, single cell RNA-Seq, and genetic dissection, this study identified new blood cell subpopulations along a developmental trajectory with multiple paths to mature cell types. This provides functional insights into key developmental processes and signaling pathways. Metabolism is highlighted as a driver of development, graded Pointed expression is shown to allow distinct roles in successive developmental steps, and mature crystal cells are shown to specifically express an alternate isoform of Hypoxia-inducible factor (Hif/Sima). Mechanistically, the Musashi-regulated protein Numb facilitates Sima-dependent non-canonical, and inhibits canonical, Notch signaling. Broadly, it was found that prior to making a fate choice, a progenitor selects between alternative, biologically relevant, transitory states allowing smooth transitions reflective of combinatorial expressions rather than stepwise binary decisions. Increasingly, this view is gaining support in mammalian hematopoiesis (Girard, 2021).

The Drosophila lymph gland is the major hematopoietic organ that develops during the larval stages for the purpose of providing blood cells during later pupal/adult periods. Hematopoietic function for the larva itself is largely provided by a separate set of sessile or circulating blood cells outside of the lymph gland. The only time the lymph gland provides blood cells to the circulating larval hemolymph is if the larva faces a stress or immune challenge. This study entirely concentrates on the primary/anterior lobes of the lymph gland, which display the highest hematopoietic activity during normal larval development (Girard, 2021).

Past work has identified specific functional zones. The PSC (Posterior Signaling Center) is marked by expression of Antp and knot/collier (kn/col). The PSC signals progenitors that belong to the medullary zone (MZ) and are marked by domeMESO (mesodermal enhancer of domeless) and Tep4. Differentiating cells form the cortical zone (CZ), expressing Hemolectin (Hml), Peroxidasin (Pxn), lozenge (lz), and other differentiating cell markers. A narrow band of cells that are double positive for domeMESO and HmlΔ occupy the edge abutting these two zones in the early third instar, and is referred to as the intermediate zone (IZ), which contains intermediate progenitors (IPs) (Girard, 2021).

Invertebrates predate the evolution of the lymphoid system for adaptive immunity. Accordingly, Drosophila blood cells are all similar in function to cells of the vertebrate myeloid lineage. The most predominant class of blood cells, the plasmatocytes (PLs; 95% of all hemocytes), share a monophyletic relationship with vertebrate macrophages. PLs function in the engulfment of microbes and apoptotic cells, and they produce extracellular matrix proteins. A minor (2-5%), but important class is represented by crystal cells (CCs) named for their crystalline inclusions of the pro-phenoloxidase enzymes, PPO1 and PPO2. CCs are necessary for melanization, blood clot formation, immunity against bacterial infections, and to help mitigate hypoxic stress. The transcription factor Lozenge (Lz) cooperates with Notch signaling to express a number of target genes (such as hindsight/pebbled) to specify CCs, whereas the Sima (vertebrate HIF-1α) protein is required for their maintenance. The orthologue of Lz in mammals is RUNX1, with broad hematopoietic function at many developmental stages, and RUNX1 is often dysregulated in acute myeloid leukemias. The third class of blood cells, lamellocytes (<1%), is usually present only during parasitization by wasps (Girard, 2021).

In early genetic studies, the MZ appeared to consist of a fairly homogeneous group of cells, although a small number of cells clustered near the heart (dorsal vessel) are identified as pre-progenitors. More recent reports have noted considerable heterogeneity and complexity within the progenitor population. Particularly noteworthy, in this context, is the functional distinction into a Hh-sensitive and a Hh-resistant group of progenitors within the MZ (Girard, 2021).

Hematopoiesis requires complex collaborations between direct cell to cell signals (e.g., Serrate/Notch), interzonal communication (e.g., Hedgehog), signals from the neighboring cardiac tube, and systemic signals (e.g., olfactory and nutritional). An important type of interzonal signaling mechanism relevant to this paper involves multiple cell types across the zones. In brief, progenitors are maintained not only through PSC-derived signals but also through a signaling relay mediated by the differentiating cells. This backward signal from the differentiating cells to the precursors is named the Equilibrium Signal. In this process, Pvf1 (PDGF- and VEGF-related factor 1) produced by the PSC, trans-cytoses through the MZ to bind its receptor Pvr (PDGF/VEGF receptor), which is expressed at high levels in the CZ. This initiates a STAT-dependent but JAK-independent signaling cascade that ultimately leads to the secretion of the extracellular enzyme ADGF-A (adenosine deaminase-related growth factor A). This enzyme breaks down adenosine, preventing its mitogenic signal and proliferation of MZ progenitors. Acting together the niche and the backward signal maintain a balance between progenitor and differentiated cell types. The genetic studies broadly implicated the CZ cells as originators of this backward signal. Finer analysis, afforded by cell-separated bulk and single-cell RNA-Seq in this study, allows this role to be attributed to a smaller and more specific subset of cells (Girard, 2021).

RNA-Seq has been used recently as a technique to study Drosophila blood cells. Four of the cited studies analyze circulating blood cells that have a completely different developmental profile than the lymph gland. Cho (2020) utilized the lymph gland and validated its zonal structure at the level of gene expression. Additionally, new markers and sub-zones were identified. The broader picture revealed in the current work is largely consistent with Cho (2020), but several important details and interpretations vary. The results and conclusions of the two independent studies are compared and contrasted in this paper. Importantly, the primary motivation of this current study is to use the combined strategies of several RNA-Seq analyses as a tool to provide data that can be combined seamlessly with the powerful genetics available in Drosophila. This functional validation of the two approaches is an advancement over the use of transcriptomics to distinguish cell types by their expressed markers. This is a level of in vivo mechanistic analysis that is not yet available for many mammalian systems, but for which Drosophila could serve as a model. While this work also describes subzones and their characteristic markers, the primary emphasis that makes it distinct is the use of a complex strategy that allows this study to extend beyond cell type identification and to dissect mechanisms that define alternate paths and pathways that were not solvable by earlier genetic methods alone (Girard, 2021).

The novel conclusions from this analysis include a clear characterization of the IZ cells (IPs), and a demonstration of the IPs as a distinct cell type; identification of two separate transitional populations that define distinct paths between progenitors and differentiated cells fates; the role of metabolism in a zone-specific developmental program; previously uncharacterized functional aspects of transcriptional regulation by the JNK and RTK pathways; the unique mechanism of CC maturation by a novel and specific isoform of Sima identified in the RNA-Seq analysis and a previously uncharacterized interaction of this Sima isoform with Notch, Numb, and Musashi, which provides a full mechanism for CC formation and maintenance (Girard, 2021).

This combination of molecular genetics and whole genome approaches makes it clear that hematopoietic cells are far more heterogeneous and diverse than previously realized by genetics alone, and helps shift the view of hematopoiesis from being a series of discrete steps to a more continuous journey of cells with similar, but not identical transcriptomic profiles along multiple paths. The multiplicity in layers of decision points creates new routes, which can each lead to a distinct differentiated endpoint, or, alternatively, follow their parallel trajectories to a single final outcome (Girard, 2021).

The cells of the small, hematopoietic lymph gland tissue are far more complex at the genome-wide expression level than could have been anticipated by earlier marker and genetic analyses. This is now confirmed by this work, and by the earlier results of (Cho, 2020). The first step in this analysis was to separate cells by FACS based on the canonical markers that classically define each zone within the lymph gland. When probed for the presence of known 'hallmark genes,' the separated cells expressing them match up with their corresponding zones, providing early validation of the methods used. This process also allows identification of zone enriched gene expression for less well-characterized cell types, including the IZ cells (IPs), as well as immature and mature CC types (iCC and mCC). This bulk RNA-Seq approach was further extended using scRNA-Seq and genetics to identify possible combinations of markers that identify each cell type. However, the primary goal of this work is not to identify more tissue-specific hallmark genes (although several were found), but to utilize RNA-Seq as a tool with other genetic strategies to understand cell-fate specification, the multiple developmental paths available to a cell, and the mechanistic links between expression trends and developmental function. Many individual examples, and two complete case studies are presented that solve long-standing questions in Drosophila hematopoiesis (Girard, 2021).

The transcriptomic data are most useful in determining trends in the collective behavior of a set of related genes. At the core of this assertion is the fact that most developmentally relevant genes function in a context-dependent manner, and their individual expression is therefore not exclusively limited to a single cell type, but certain combinations of expressed genes could approximate their identities. Obvious exceptions are genes marking functions of terminal states such as lz or NimC1, but even in such cases, RNA expression begins in multipotent precursors and continues in the terminal cell types. The case studies presented in this work demonstrate this concept, showing that a graded expression pattern of a transcription factor allows the identification of specific phenotypes for each developmental step. Similarly, expression of an alternate isoform for the protein Sima and the RNA-binding protein Msi explains why Numb inhibits canonical Ser/Notch function but not non-canonical Sima/Notch function in the same cell type. Thus the motivation for this study is to provide multiple examples that take advantage of the ready access to genetic tools that make Drosophila a particularly attractive system in which to establish detailed mechanistic aspects of complex pathways. Based on the long history of conservation of basic principles, it is not unreasonable to expect that parallels to such mechanisms will be found in mammalian hematopoiesis (Girard, 2021).

Employing fairly conservative criteria for cluster separation in scRNA-Seq, this study identified eight primary clusters. The CCs were subclustered to yield iCC and mCC giving rise to the following nine groups of cells: a single cluster each for PSC, X (a mitosis and replication stress-related cluster), PL, and CC (subclustered into iCC and mCC). Two clusters each were identified for MZ (MZ1 and MZ2), and one for the two transitional populations (IZ and proPL). The compact arrangement of the majority of clusters implies smooth developmental transitions between them even as, from a gene-enrichment point of view, they represent different cell types. However, from a developmental biology point of view, it is the functional differences between clusters that must be used to define them as distinct cell types. It is virtually impossible to find any transcript that is 100% cell-specific, and therefore this analysis focused on trends and enrichments in transcriptional patterns. Sometimes, as in the case of pnt, the changes in expression along each developmental step can be very small, but the trend defines its multiple functions and only functional data from mutant analysis provides validation for the gene expression patterns (Girard, 2021).

RNA-Seq is by now a commonly used technique in many fields, although its first use in lymph gland hematopoiesis was relatively recent (Cho, 2020). That study identified new markers and validated the expression of a representative number of the expressed genes. A detailed comparison of the transcriptional map comparing the clusters and subclusters of Cho, with those generated in the current single-cell RNA-Seq is presented. By comparing the sizes of the clusters/subclusters, the overlapping gene lists, and the expression patterns and genetic profiles, this study found that MZ1 is similar to the PH1 and PH2 subclusters in Cho; MZ2 is similar to PH3 and PH4; IZ to PH5 and PH6; proPL to PM1; PL to PM2, PM 3, and PM4; PSC to PSC; iCC to CC1; mCC to CC2; and X is most similar to the 'GST-rich' cluster of Cho. The differences in where boundaries are drawn could arise from many sources, such as the experimental technique (drop Seq by Cho vs. 10x), genetic background (Oregon R vs. w1118), and perhaps most importantly, the computational strategy (manual curation and aggregation of the clusters based on known gene expression by Cho. vs. unsupervised graph-based clustering in this study). Both studies provide useful data. The strength of the current study is that FACS was used to sort populations defined as MZ, CZ, IZ, CC, and so on, and therefore, it is certain that the two clusters MZ1 and MZ2, for example, belong to the traditionally defined 'MZ' and the same is true for the others. The second strength is that the current strategy requires the use of multiple backgrounds and biological replicates, and the results are very consistent. Finally, given that most expression patterns represent trends rather than specific cells, and often different from the proteins they encode (such as for numb), the strongest validation of expression data, is thought to be when it is in agreement with genetic strategies based on loss of function in a subset of cells (such as with pnt or Mmp1) (Girard, 2021).

The results of this study are presented as a model of lymph gland development (see Summary of markers, case studies, and a model for the developmental progression of lymph gland cells). This analysis is based on a single time point in development but the occupancy states in pseudotime allow maturation states to be used as a form of developmental clock. The model is largely based on adjacencies, genetic compositions, and validation by mutant analysis. Transition from pre-progenitors to progenitors, then through transitional IZ or proPL populations, finally on to PLs or CCs is a continuous process traversing gradually through a permissive landscape. It does not appear to be a set of pre-programmed, quantal decisions that a cell makes based on the expression of a single fate-specifying gene. This idea is gaining increased traction in the newer reports on mammalian hematopoiesis (Girard, 2021).

The developmental trajectory for Drosophila hematopoiesis is branched, and the subdivision of 9 expression-based clusters into 22 subpopulations is based on both cell type and the trajectory state in which they reside. It is important to point out that in this context, the cluster name (e.g., MZ1 or MZ2) represents cell types distinguishable by their gene-enrichment profile, whereas the 'states' (such as MZ2-1, MZ2-2, and MZ2-3) represent the same cell type (MZ2), but appearing at different pseudo-times (1, 2, or 3). Although the analysis is a snapshot of a particular real-time point in development, many developmental steps of a single cell type are represented as progress in pseudotime. For example, the MZ2-3 state is composed of the most mature cells of the MZ2 cell type. The next transitions to either of the two separate transitional cell types, IZ or proPL, that define alternate developmental paths. The cell states MZ2-3, IZ-5, and PL-7a/b are nodes of bifurcation based on this model. Some details of the model require further functional confirmation in vivo that is beyond the scope of the current manuscript. It is anticipated that such details of cell identity will change with future refinements. However, the model provides a blueprint and a rich opportunity to study changes in signaling, cell cycle, or possible modes of cell divisions that promote alternate cell fates (Girard, 2021).

An important finding of this study is the demonstration of alternate paths that initiate with the same progenitor types and terminate in the same differentiated fate, but they traverse through distinct transitory cell types. The distinction between transitional states such as IZ and proPL would be less remarkable, if they did not also have additional unique characteristics and functions. For example, together the genetic and RNA-Seq data suggest that proPL is likely a major source of the equilibrium signal, whereas IZ largely contributes to the JNK signal. The two cell types are largely non-overlapping and virtually non-adjacent in a 3D t-SNE representation of the clusters. These alternate routes are reminiscent of the concept of progression through alternate epigenetic landscapes proposed by Waddington at the very dawn of Developmental Biology. Finally, in T cell development, there is evidence to suggest that intermediate cells bridge the major singly and doubly marked populations, but even less is known about their possible developmental roles (Girard, 2021).

Minor paths not involving either of the two major transitional states (IZ or proPL) are consistent with, but not fully established yet by the data. For instance, the earliest PL clusters (PL-3) are sandwiched between MZ2 and PL-7 with no intervening proPL or IZ cells, suggesting a direct MZ to PL path, or perhaps one that involves X as an intermediary. As another example of a minor path, a small number of iCC cells follow the path PL-7/iCC-7/iCC-6/mCC-6. The iCC-7 to iCC-6 transition is a reversal in pseudotime. Although unexpected, this supports the concepts of transdifferentiation and dedifferentiation proposed in Drosophila hematopoiesis. It will be interesting to determine in future studies if paths that are minor during homeostasis become more prominent under stress or immune challenge when a rapid and amplified response is prioritized over orderly development (Girard, 2021).

Contrary to a commonly held viewpoint, metabolic pathways are regulated in a cell-specific manner and their participation is not limited to 'housekeeping' roles during development. Indeed, data on both cancer and developmental metabolism show that selective use of such pathways can drive certain critical developmental decisions instead of the other way around (Girard, 2021).

The analysis presented in this paper demonstrates that in Drosophila hematopoiesis, cells within individual zones are not only defined by their position within the organ and the markers that they express, but also by their metabolic status that is foreshadowed by the content of their transcriptome. The PSC cells, as a group, for example, are well represented by most upper glycolysis genes that are then used, not for bioenergetic purposes, but to increase the PPP flux of glucose metabolism that aids in maintaining an NADPH/GSH-dependent low ROS status for these cells. This is important as high ROS in the PSC is a trigger for a specific immune response that must be repressed during homeostasis. Interestingly, the immediately adjacent MZ cells are lower in NADPH-forming enzymes, and their genes controlling oxidative phosphorylation are higher than in the PSC. This would lead to higher ROS even during homeostasis. Indeed, the MZ ROS levels are high and this physiological amount is essential for progenitor differentiation. A very interesting example of metabolic control is in the IZ cluster. Surprisingly, this narrow band of cells is enriched for genes required for both synthesis and clearance of free ceramide from a cell. This is important given the known role of ceramide in the activation of the JNK pathway, and genetic and immunohistochemical evidence is provided of transient activation of JNK and MMP1 in this group of cells (Girard, 2021).

Unlike cancer metabolism, developmental metabolism is at a surprisingly early phase of research, and Drosophila hematopoiesis could be a very attractive system to study this phenomenon during homeostasis. More broadly, the results point to the continued relevance of the use of Drosophila as the singular invertebrate hematopoietic model, which provides a logical framework within which to establish less-studied concepts such as the characterization of parallel transitory populations, the roles of developmental metabolism, mechanisms of unusual signaling paradigms, and genetic dissection of pleiotropy (Girard, 2021).

Broad Ultrastructural and Transcriptomic Changes Underlie the Multinucleated Giant Hemocyte Mediated Innate Immune Response against Parasitoids

Multinucleated giant hemocytes (MGHs) represent a novel type of blood cell in insects that participate in a highly efficient immune response against parasitoid wasps involving isolation and killing of the parasite. Previously, this study showed that circulating MGHs have high motility and the interaction with the parasitoid rapidly triggers encapsulation. However, structural and molecular mechanisms behind these processes remained elusive. This study used detailed ultrastructural analysis and live cell imaging of MGHs to study encapsulation in Drosophila ananassae after parasitoid wasp infection. Dynamic structural changes were found, mainly driven by the formation of diverse vesicular systems and newly developed complex intracytoplasmic membrane structures, and abundant generation of giant cell exosomes in MGHs. In addition, RNA sequencing was used to study the transcriptomic profile of MGHs and activated plasmatocytes 72 h after infection, as well as the uninduced blood cells. This revealed that differentiation of MGHs was accompanied by broad changes in gene expression. Consistent with the observed structural changes, transcripts related to vesicular function, cytoskeletal organization, and adhesion were enriched in MGHs. In addition, several orphan genes encoding for hemolysin-like proteins, pore-forming toxins of prokaryotic origin, were expressed at high level, which may be important for parasitoid elimination. These results reveal coordinated molecular and structural changes in the course of MGH differentiation and parasitoid encapsulation, providing a mechanistic model for a powerful innate immune response (Cinege, 2021).

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

Genetic and genomic analysis in Drosophila suggests that hematopoietic progenitors likely transition into terminal fates via intermediate progenitors (IPs) with some characteristics of either, but perhaps maintaining IP-specific markers. In the past, IPs have not been directly visualized and investigated owing to lack of appropriate genetic tools. This study reports a Split GAL4 construct, CHIZ-GAL4, that identifies IPs as cells physically juxtaposed between true progenitors and differentiating hemocytes. IPs are a distinct cell type with a unique cell-cycle profile and they remain multipotent for all blood cell fates. In addition, through their dynamic control of the Notch ligand Serrate, IPs specify the fate of direct neighbors. The Ras pathway controls the number of IP cells and promotes their transition into differentiating cells. This study suggests that it would be useful to characterize such intermediate populations of cells in mammalian hematopoietic systems (Spratford, 2021).

Progenitor and differentiated cell types have been well described in Drosophila hematopoiesis. Genetic evidence suggested that certain cells have an intermediate characteristic in that they express some progenitor as well as mature cell markers. Although these cells could be identified during Drosophila hematopoiesis owing to their overlapping expression patterns, the absence of tools to directly detect such populations has thus far prevented a detailed analysis of these transitional cells. These cells have been designated IPs and they bridge medullary zone (MZ) progenitors with the cortical zone (CZ) hemocytes. This study used a Split GAL4 strategy to generate CHIZ-GAL4 that allows identification and genetical manipulation of IPs. The IPs of the intermediate zone (IZ) represent a unique cell type that have some characteristics that are distinct from and others that are similar to the cells of the MZ and CZ. For example, IPs express dome, but not E-cad, both of which are MZ markers. Similarly, IPs express Hemolectin (Hml), but not the maturity markers P1 (plasmatocytes) and Hnt (crystal cells). Interestingly, the IP cells share the property of multipotency with cells of the MZ in that both can contribute to all three populations of mature hemocytes. Importantly, it is believed IPs are a unique cell type, as their numbers can be expanded or reduced upon genetic manipulation as shown, for example, with modulation of the Ras pathway. In addition, bulk and single cell RNA-seq data obtained recently in the laboratory identifies several genes that are highly enriched within IPs when compared with their expression in all other cell types in the LG. In future studies, these will serve well as specific IZ markers and provide further functional relevance for this population (Spratford, 2021).

The MZ cells are fairly quiescent; they are largely held in G2, and will undergo mitosis in a limited number of cells. In contrast, IP cells are found in G1, S and G2 but with a very limited extent of mitosis. It is proposed that before entering the IP state, a dome+ progenitor is released from G2 and it undergoes mitosis. Subsequently, Hml is initiated and continues to be expressed as IPs progress through G1, S and G2. At this point dome expression ceases, thus ending the CHIZ-state. The dome-negative post-CHIZ cell likely undergoes a round of mitosis before it progresses to a differentiated state. The IPs are multipotent and contribute to all of the three mature hemocyte populations. It should be noted that the data presented in this study do not preclude the possibility that a few of the hemocytes might form by a parallel mechanism that does not involve the IPs (Spratford, 2021).

As in many developmental systems, entry into a proliferative state and fate determination are intimately intertwined and this applies as well to the transition from the IZ to the CZ. It is presumed that a mitotic event must closely follow exit from the IP state and is linked to differentiation into a hemocyte. It is also known that the Ras/Raf pathway is required for exit out of the IP state. In other systems, Ras/Raf activity has largely been associated with proliferation, but in Drosophila, this pathway often governs cell fate determination, as seen, for example, during the development of the eye imaginal disc. Thus, it remains uncertain at the present moment whether Ras/Raf initiates the mitotic process and this allows differentiation signals to be sensed to turn on markers, or whether another mechanism controls the entry into mitosis and Ras is responsible for turning off a marker such as dome. In a manner similar to that seen in other well-defined developmental situations in Drosophila, the Ras/Raf and Notch pathways play dueling roles in the post-CHIZ stage of defining cell fate. The IPs express Ser in a dynamic pattern and induce neighbors to take on a crystal cell fate. The expression of Ser is downregulated after the mid-third instar, and its restricted spatial and temporal pattern of expression limits crystal cell number. Crystal cells do not have active Ras signaling as established by their expression of the Yan protein. The Ras/Raf signal promotes plasmatocyte fate, whereas crystal cells are dependent on Notch signaling. Upstream events that activate Ras in the IPs are currently unknown and will be of great interest for future investigation. It is possible that a canonical ligand-dependent RTK may be involved; however, other autonomous molecular mechanisms such as changes in metabolism could feed into Ras (Spratford, 2021).

IPs may provide an opportunity to synchronize the assignment of cell fate during normal development, and maintain plasmatocytes and crystal cells in a stereotypical ratio. It is also likely that IPs have unique signaling functions as inferred from their regulation of Ser expression to induce direct neighbors to take on a crystal-cell fate. It is interesting to note that this transitional population acts autonomously as multipotent progenitors while they also non-autonomously induce one of the specific blood cell fates. Investigation into the expression of receptors and ligands in IPs will expand current understanding of the role these cells play in regulating the balance between progenitors and the various determined blood cell types during homeostasis. If all progenitors in the MZ were to directly differentiate into mature hemocytes without going through the buffer zone provided by the IPs, then a relatively steady pool of progenitors will be difficult to preserve, and the spatio-temporal order of hemocyte specification will not be maintained. Under stress conditions or immune challenge this buffer could be altered in favor of faster production of hemocytes at the cost of progenitor number (Spratford, 2021).

The experimental strategy used to develop CHIZ-GAL4 has been successfully adapted for identifying cell types based on the co-expression of other genes in Drosophila, particularly in the nervous system. There is nothing about this strategy that is Drosophila-specific and one hopes that its most useful application might be to uncover cryptic cell types in the context of the significantly more complex transitions described in mammalian hematopoietic development (Spratford, 2021).

The vascular niche controls Drosophila hematopoiesis via fibroblast growth factor signaling

In adult mammals, hematopoiesis, the production of blood cells from hematopoietic stem and progenitor cells (HSPCs), is tightly regulated by extrinsic signals from the microenvironment called 'niche'. Bone marrow HSPCs are heterogeneous and controlled by both endosteal and vascular niches. The Drosophila hematopoietic lymph gland is located along the cardiac tube which corresponds to the vascular system. In the lymph gland, the niche called Posterior Signaling Center controls only a subset of the heterogeneous hematopoietic progenitor population indicating that additional signals are necessary. This study reports that the vascular system acts as a second niche to control lymph gland homeostasis. The FGF ligand Branchless produced by vascular cells activates the FGF pathway in hematopoietic progenitors. By regulating intracellular calcium levels, FGF signaling maintains progenitor pools and prevents blood cell differentiation. This study reveals that two niches contribute to the control of Drosophila blood cell homeostasis through their differential regulation of progenitors (Destalminil-Letourneau, 2021).

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 (bsk^DN) 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 Egfr^TS/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 (Egfr^DN) 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 spi^RNAi 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 Hml^Δ-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 Egfr^DN is not expressed in these cells when Hml^Δ-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 (Egfr^Act) 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).

Inflammatory production of reactive oxygen species by Drosophila hemocytes activates cellular immune defenses

The production of reactive oxygen species (ROS) is a prominent response to infection among innate immune cells such as macrophages and neutrophils. To better understand the relationship between antimicrobial and regulatory functions of blood cell ROS, this study has characterized the ROS response to infection in Drosophila hemocytes. Using fluorescent probes, a biphasic hemocyte ROS response was found to bacterial infection. In the first hour, virtually all hemocytes generate a transient ROS signal, with nonphagocytic cells including prohemocytes and crystal cells displaying exceptionally strong responses. A distinct, and more delayed ROS response starting at 90min is primarily within cells that have engulfed bacteria, and is sustained for several hours. The early response has a clear regulatory function, as dampening or intensifying the intracellular ROS level has profound effects on plasmatocyte activation. In addition, ROS are necessary and sufficient to activate JNK signalling in crystal cells, and to promote JNK-dependent crystal cell rupture. These findings indicate that Drosophila will be a promising model in which to dissect the mechanisms of ROS stimulation of immune activation (Myers, 2018).

Proto-pyroptosis: An ancestral origin for mammalian inflammatory cell death mechanism in Drosophila melanogaster

Pyroptosis has been described in mammalian systems to be a form of programmed cell death that is important in immune function through the subsequent release of cytokines and immune effectors upon cell bursting. This form of cell death has been increasingly well-characterized in mammals and can occur using alternative routes however, across phyla, there has been little evidence for the existence of pyroptosis. This study provide evidence for an ancient origin of pyroptosis in an in vivo immune scenario in Drosophila melanogaster. Crystal cells, a type of insect blood cell, were recruited to wounds and ruptured subsequently releasing their cytosolic content in a caspase-dependent manner. This inflammatory-based programmed cell death mechanism fits the features of pyroptosis, never before described in an in vivo immune scenario in insects and relies on ancient apoptotic machinery to induce proto-pyroptosis. Further, this study unveiled key players upstream in the activation of cell death in these cells including the apoptosome which may play an alternative role akin to the inflammasome in proto-pyroptosis. Thus, Drosophila may be a suitable model for studying the functional significance of pyroptosis in the innate immune system (Dziedziech, 2021).

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 p27^KIP1 or p21^CIP1/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; Phago^less 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).

Identification of Bipotential Blood Cell/Nephrocyte Progenitors in Drosophila: Another Route for Generating Blood Progenitors

The Drosophila lymph gland is the larval hematopoietic organ and is aligned along the anterior part of the cardiovascular system, composed of cardiac cells, that form the cardiac tube and its associated pericardial cells or nephrocytes. By the end of embryogenesis the lymph gland is composed of a single pair of lobes. Two additional pairs of posterior lobes develop during larval development to contribute to the mature lymph gland. This study describes the ontogeny of lymph gland posterior lobes during larval development and identifies the genetic basis of the process. By lineage tracing it was shown that each posterior lobe originates from three embryonic pericardial cells, thus establishing a bivalent blood cell/nephrocyte potential for a subset of embryonic pericardial cells. The posterior lobes of L3 larvae posterior lobes are composed of heterogeneous blood progenitors and their diversity is progressively built during larval development. It was further established that in larvae, homeotic genes and the transcription factor Klf15 regulate the choice between blood cell and nephrocyte fates. These data underline the sequential production of blood cell progenitors during larval development (Morin-Poulard, 2022).

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 Hop^TUM mutation results in the formation of melanotic masses. Constitutive activation of the Toll pathway via the dominant gain-of-function Tl^10b 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 H2AV^CT, 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 H2AV^CT 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 H2AV^CT protein. However, the presence of H2AV^CT 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).

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

Drosophila hemocytes are akin to mammalian myeloid blood cells that function in stress and innate immune-related responses. A multi-potent progenitor population responds to local signals and to systemic stress by expanding the number of functional blood cells. This study shows mechanisms that demonstrate an integration of environmental carbon dioxide (CO2) and oxygen (O2) inputs that initiate a cascade of signaling events, involving multiple organs, as a stress response when the levels of these two important respiratory gases fall below a threshold. The CO2 and hypoxia-sensing neurons interact at the synaptic level in the brain sending a systemic signal via the fat body to modulate differentiation of a specific class of immune cells. These findings establish a link between environmental gas sensation and myeloid cell development in Drosophila. A similar relationship exists in humans, but the underlying mechanisms remain to be established (Cho, 2018).

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

Drosophila larval hematopoiesis occurs in a specialized multi-lobed organ called the lymph gland. Extensive characterization of the organ has provided mechanistic insights into events related to developmental hematopoiesis. Spanning from the thoracic to the abdominal segment of the larvae, this organ comprises a pair of primary, secondary, and tertiary lobes. Much understanding arises from the studies on the primary lobe, while the secondary and tertiary lobes have remained mostly unexplored. Previous studies have inferred that these lobes are composed of progenitors that differentiate during pupation; however, the mechanistic basis of this extended progenitor state remains unclear. This study shows that posterior lobe progenitors are maintained by a local signaling center defined by Ubx and Collier in the tertiary lobe. This Ubx zone in the tertiary lobe shares several markers with the niche of the primary lobe. Ubx domain regulates the homeostasis of the posterior lobe progenitors in normal development and an immune-challenged scenario. This study establishes the lymph gland as a model to tease out how the progenitors interface with the dual niches within an organ during development and disorders (Kanwal, 2021).

Myogenic contraction of a somatic muscle powers rhythmic flow of hemolymph through Drosophila antennae and generates brain pulsations

Hemolymph is driven through the antennae of Drosophila melanogaster by the rhythmic contraction of muscle 16 (m16), which runs through the brain. Contraction of m16 results in the expansion of an elastic ampulla, opening ostia and filling the ampulla. Relaxation of the ampullary membrane forces hemolymph through vessels into the antennae. This study shows that m16 is an auto-active rhythmic somatic muscle. The activity of m16 leads to the rapid perfusion of the antenna by hemolymph. In addition, it leads to the rhythmic agitation of the brain, which could be important for clearing the interstitial space (Kay, 2021).

Characterization of the Drosophila Adult Hematopoietic System Reveals a Rare Cell Population With Differentiation and Proliferation Potential

While many studies have described Drosophila embryonic and larval blood cells, the hematopoietic system of the imago remains poorly characterized and conflicting data have been published concerning adult hematopoiesis. Using a combination of blood cell markers, This study shows that the adult hematopoietic system is essentially composed of a few distinct mature blood cell types. In addition, the transcriptomics results indicate that adult and larval blood cells have both common and specific features and it appears that adult hemocytes reactivate many genes expressed in embryonic blood cells. Interestingly, this study identify a small set of blood cells that does not express differentiation markers but rather maintains the expression of the progenitor marker dome^Meso. Yet, this study shows that these cells are derived from the posterior signaling center, a specialized population of cells present in the larval lymph gland, rather than from larval blood cell progenitors, and that their maintenance depends on the EBF transcription factor Collier. Furthermore, while these cells are normally quiescent, this study found that some of them can differentiate and proliferate in response to bacterial infection. In sum, the results indicate that adult flies harbor a small population of specialized cells with limited hematopoietic potential and further support the idea that no substantial hematopoiesis takes place during adulthood (Boulet, 2021).

Pvr and distinct downstream signaling factors are required for hemocyte spreading and epidermal wound closure at Drosophila larval wound sites

Tissue injury is typically accompanied by inflammation. In Drosophila melanogaster, wound-induced inflammation involves adhesive capture of hemocytes at the wound surface followed by hemocyte spreading to assume a flat, lamellar morphology. The factors that mediate this cell spreading at the wound site are not known. This study discoverd a role for the Platelet-derived growth factor (PDGF)/Vascular endothelial growth factor (VEGF)-related receptor (Pvr) and its ligand, Pvf1, in blood cell spreading at the wound site. Pvr and Pvf1 are required for spreading in vivo and in an in vitro spreading assay where spreading can be directly induced by Pvf1 application or by constitutive Pvr activation. In an effort to identify factors that act downstream of Pvr, a genetic screen was performed in which select candidates were tested to determine if they could suppress the lethality of Pvr overexpression in the larval epidermis. Some of the suppressors identified are required for epidermal wound closure, another Pvr-mediated wound response, some are required for hemocyte spreading in vitro, and some are required for both. One of the downstream factors, Mask, is also required for efficient wound-induced hemocyte spreading in vivo. These data reveals that Pvr signaling is required for wound responses in hemocytes (cell spreading) and defines distinct downstream signaling factors that are required for either epidermal wound closure or hemocyte spreading (Tsai, 2021).

Proteasome α6 Subunit Negatively Regulates the JAK/STAT Pathway and Blood Cell Activation in Drosophila melanogaster

JAK/STAT signaling regulates central biological functions such as development, cell differentiation and immune responses. In Drosophila, misregulated JAK/STAT signaling in blood cells (hemocytes) induces their aberrant activation. This study identified several components of the proteasome complex as negative regulators of JAK/STAT signaling in Drosophila. A selected proteasome component, Prosα6, was studied further. In S2 cells, Prosα6 silencing decreased the amount of the known negative regulator of the pathway, ET, leading to enhanced expression of a JAK/STAT pathway reporter gene. Silencing of Prosα6 in vivo resulted in activation of the JAK/STAT pathway, leading to the formation of lamellocytes, a specific hemocyte type indicative of hemocyte activation. This hemocyte phenotype could be partially rescued by simultaneous knockdown of either the Drosophila STAT transcription factor, or MAPKK in the JNK-pathway. These results suggest a role for the proteasome complex components in the JAK/STAT pathway in Drosophila blood cells both in vitro and in vivo (Jarvela-Stolting, 2021).

Jumu is required for the activation of JAK/STAT in Drosophila lymph gland development and epidermal wounds

The regulatory mechanism of hematopoiesis and innate immunity in Drosophila is highly similar to that in mammals, and Drosophila has become a suitable model to understand vertebrate hematopoiesis and the immune response. JAK-STAT signaling pathway components are widely conserved during evolution, and contribute to hematopoiesis and multiple tissue damage and immune responses. This study demonstrates that Stat92E is widely expressed in the lymph gland, and the loss of jumu inhibits the maintenance of the JAK/STAT pathway in the CZ and MZ but not in the PSC of the lymph gland. Furthermore, this study found that clean puncture wounding of the larval epidermis can lead to the activation of JAK/STAT signaling and the generation of lamellocytes, and Jumu is required for the activation of JAK/STAT in response to epidermal wounds (Hao, 2021).

Fos regulates macrophage infiltration against surrounding tissue resistance by a cortical actin-based mechanism in Drosophila

The infiltration of immune cells into tissues underlies the establishment of tissue-resident macrophages and responses to infections and tumors. Yet the mechanisms immune cells utilize to negotiate tissue barriers in living organisms are not well understood, and a role for cortical actin has not been examined. This study found that the tissue invasion of Drosophila macrophages, also known as plasmatocytes or hemocytes, utilizes enhanced cortical F-actin levels stimulated by the Drosophila member of the fos proto oncogene transcription factor family (Dfos, Kayak). RNA sequencing analysis and live imaging show that Dfos enhances F-actin levels around the entire macrophage surface by increasing mRNA levels of the membrane spanning molecular scaffold tetraspanin TM4SF, and the actin cross-linking filamin Cheerio, which are themselves required for invasion. Both the filamin and the tetraspanin enhance the cortical activity of Rho1 and the formin Diaphanous and thus the assembly of cortical actin, which is a critical function since expressing a dominant active form of Diaphanous can rescue the Dfos macrophage invasion defect. In vivo imaging shows that Dfos enhances the efficiency of the initial phases of macrophage tissue entry. Genetic evidence argues that this Dfos-induced program in macrophages counteracts the constraint produced by the tension of surrounding tissues and buffers the properties of the macrophage nucleus from affecting tissue entry. This study thus identifies strengthening the cortical actin cytoskeleton through Dfos as a key process allowing efficient forward movement of an immune cell into surrounding tissues (Belyaeva, 2022).

A Novel Drosophila Model to Investigate Adipose tissue Macrophage Infiltration (ATM) and Obesity highlights the Therapeutic Potential of Attenuating Eiger/TNFα Signaling to Ameliorate Insulin Resistance and ATM

This study presents a novel Drosophila model to investigate the mechanisms underlying adipose tissue macrophage(ATM) infiltration. This study demonstrated the therapeutic potential of attenuating Eiger/TNFα signaling to ameliorate insulin resistance and ATM. To study ATM infiltration and its consequences, a novel Drosophila model (OBL) was developed that mimics key aspects of human adipose tissue. Genetic manipulation was used to reduce ecdysone levels to prolong the larval stage. These animals are hyperphagic, and exhibit features resembling obesity in mammals, including increased lipid storage, adipocyte hypertrophy, and high levels of circulating glucose. Moreover, a significant infiltration of immune cells (hemocytes) in the fat bodies was observed, accompanied by insulin resistance and systemic metabolic dysregulation. Furthermore, it was found that attenuation of Eiger/TNFα signaling and using metformin and anti-oxidant bio-products like anthocyanins led to a reduction in ATM infiltration and improved insulin sensitivity. These data suggest that the key mechanisms that trigger immune cell infiltration into adipose tissue are evolutionarily conserved and may provide the opportunity to develop Drosophila models to better understand pathways critical for immune cell recruitment into adipose tissue, in relation to the development of insulin resistance in metabolic diseases such as obesity and type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD). This OBL model can also be a valuable tool and provide a platform either to perform genetic screens or to test the efficacy and safety of novel therapeutic interventions for these diseases (Mirzoyan, 2023).

Blood progenitor redox homeostasis through olfaction-derived systemic GABA in hematopoietic growth control in Drosophila

The role of reactive oxygen species (ROS) in myeloid development is well established. However, its aberrant generation alters hematopoiesis. Thus, a comprehensive understanding of events controlling ROS homeostasis forms the central focus of this study. It was shown that, in homeostasis, myeloid-like blood progenitor cells of the Drosophila larvae, which reside in a specialized hematopoietic organ termed the lymph gland, use TCA (the Krebs cycle) to generate ROS. However, excessive ROS production leads to lymph gland growth retardation. Therefore, to moderate blood progenitor ROS, Drosophila larvae rely on olfaction and its downstream systemic GABA. GABA internalization and its breakdown into succinate by progenitor cells activates pyruvate dehydrogenase kinase (PDK), which controls inhibitory phosphorylation of pyruvate dehydrogenase (PDH). PDH is the rate-limiting enzyme that connects pyruvate to the TCA cycle and to oxidative phosphorylation. Thus, GABA metabolism via PDK activation maintains TCA activity and blood progenitor ROS homeostasis, and supports normal lymph gland growth. Consequently, animals that fail to smell also fail to sustain TCA activity and ROS homeostasis, which leads to lymph gland growth retardation. Overall, this study describes the requirement of animal odor-sensing and GABA in myeloid ROS regulation and hematopoietic growth control (Goyal, 2022).

Single Cell Analysis of the Fate of Injected Oncogenic RasV12 Cells in Adult Wild Type Drosophila

=Dish-cultured oncogenic RasV12 cells into adult male flies and single cell transcriptomics was used to examine their destiny within the host after 11 days. The preinjection samples were identified in the 11-day postinjection samples in all 16 clusters of cells, of which 5 disappeared during the experiment in the host. The other cell clusters expanded and expressed genes involved in the regulation of cell cycle, metabolism, and development. In addition, three clusters expressed genes related to inflammation and defense. Predominant among these were genes coding for phagocytosis and/or characteristic for a plasmatocytes (the fly equivalent of macrophages). A pilot experiment indicated that the injection into flies of oncogenic cells, in which two of most strongly expressed genes had been previously silenced by RNA interference, into flies resulted in a dramatic reduction of their proliferation in the host flies as compared to controls. As has been shown earlier, the proliferation of the injected oncogenic cells in the adult flies is a hallmark of the disease and induces a wave of transcriptions in the experimental flies. It is hypothesized that this results from a bitter dialogue between the injected cells and the host, while the experiments presented in this study should contribute to deciphering this dialogue (Chen, 2023).

Lipid content evaluation of Drosophila tumour associated haemocytes through Third Harmonic Generation measurements

Non-linear microscopy is a powerful imaging tool to examine structural properties and subcellular processes of various biological samples. The competence of Third Harmonic Generation (THG) includes the label free imaging with diffraction-limited resolution and three-dimensional visualization with negligible phototoxicity effects. In this study, THG records and quantifies the lipid content of Drosophila haemocytes, upon encountering normal or tumorigenic neural cells, in correlation with their shape or their state. The lipid accumulations of adult haemocytes were shown to be similar before and after encountering normal cells. In contrast, adult haemocytes prior to their interaction with cancer cells have a low lipid index, which increases while they are actively engaged in phagocytosis only to decrease again when haemocytes become exhausted. This dynamic change in the lipid accrual of haemocytes upon encountering tumour cells could potentially be a useful tool to assess the phagocytic capacity or activation state of tumour-associated haemocytes (Mari, 2023). vv

Ectopic expression of matrix metalloproteinases and filopodia extension via JNK activation are involved in the invasion of blood tumor cells in Drosophila mxc mutant

Drosophila mxc(mbn1) mutant exhibits severe hyperplasia in larval hematopoietic tissue called the lymph glands (LGs). However, the malignant nature of these cells remains unknown. This study aimed to identify if mxc(mbn1) LG cells behave as malignant tumor cells and uncover the mechanism(s) underlying the malignancy of the mutant hemocytes. When mutant LG cells were allografted into normal adult abdomens, they continued to proliferate; however, normal LG cells did not proliferate. Mutant circulating hemocytes also attached to the larval central nervous system (CNS), where the basement membrane was disrupted. The mutant hemocytes displayed higher expression of matrix metalloproteinase (MMP) 1 and MMP2 and higher activation of the c-Jun N-terminal kinase (JNK) pathway than normal hemocytes. Depletion of MMPs or JNK mRNAs in LGs resulted in reduced numbers of hemocytes attached to the CNS, suggesting that the invasive phenotype involved elevated expression of MMPs via hyperactivation of the JNK pathway. Moreover, hemocytes with elongated filopodia and extra lamellipodia were frequently observed in the mutant hemolymph, which also depended on JNK signaling. Thus, the MMP upregulation and overextension of actin-based cell protrusions were also involved in hemocyte invasion in mxc(mbn1) larvae. These findings contribute to the understanding of molecular mechanisms underlying mammalian leukemic invasion (Takarada, 2023).

Pan-neuronal expression of human mutant huntingtin protein in Drosophila impairs immune response of hemocytes

Huntington's disease (HD) is a late-onset; progressive, dominantly inherited neurological disorder marked by an abnormal expansion of polyglutamine (poly Q) repeats in Huntingtin (HTT) protein. The pathological effects of mutant Huntingtin (mHTT) are not restricted to the nervous system but systemic abnormalities including immune dysregulation have been evidenced in clinical and experimental settings of HD. Indeed, mHTT is ubiquitously expressed and could induce cellular toxicity by directly acting on immune cells. However, it is still unclear if selective expression of mHTT exon1 in neurons could induce immune responses and hemocytes' function. This study intended to monitor perturbations in the hemocytes' population and their physiological functions in Drosophila, caused by pan-neuronal expression of mHTT protein. A measure of hemocyte count and their physiological activities caused by pan-neuronal expression of mHTT protein highlighted the extent of immune dysregulation occurring with disease progression. It was found that pan-neuronal expression of mHTT significantly alters crystal cells and plasmatocyte count in larvae and adults with disease progression. Interestingly, plasmatocytes isolated from diseased conditions exhibit a gradual decline in phagocytic activity ex vivo at progressive stages of the disease as compared to age-matched control groups. In addition, diseased flies displayed elevated reactive oxygen species (ROS) in circulating plasmatocytes at the larval stage and in sessile plasmatocytes of hematopoietic pockets at terminal stages of disease. These findings strongly implicate that neuronal expression of mHTT alone is sufficient to induce non-cell-autonomous immune dysregulation in vivo (Dhankhar, 2022).

Two Isoforms of serpent Containing Either One or Two GATA Zinc Fingers Provide Functional Diversity During Drosophila Development

GATA transcription factors play crucial roles in various developmental processes in organisms ranging from flies to humans. In mammals, GATA factors are characterized by the presence of two highly conserved domains, the N-terminal (N-ZnF) and the C-terminal (C-ZnF) zinc fingers. The Drosophila GATA factor Serpent (Srp) is produced in different isoforms that contains either both N-ZnF and C-ZnF (SrpNC) or only the C-ZnF (SrpC). This study investigated the functional roles ensured by each of these isoforms during Drosophila development. Using the CRISPR/Cas9 technique, new mutant fly lines were generated deleted for one (ΔsrpNC) or the other (ΔsrpC) encoded isoform, and a third one with a single point mutation in the N-ZnF that alters its interaction with its cofactor, the Drosophila FOG homolog U-shaped (Ush). Analysis of these mutants revealed that the Srp zinc fingers are differentially required for Srp to fulfill its functions. While SrpC is essential for embryo to adult viability, SrpNC, which is the closest conserved isoform to that of vertebrates, is not. However, to ensure its specific functions in larval hematopoiesis and fertility, Srp requires the presence of both N- and C-ZnF (SrpNC) and interaction with its cofactor Ush. These results also reveal that in vivo the presence of N-ZnF restricts rather than extends the ability of GATA factors to regulate the repertoire of C-ZnF bound target genes (Mousalems, 2021).

The conserved Pelado/ZSWIM8 protein regulates actin dynamics by promoting linear actin filament polymerization

Actin filament polymerization can be branched or linear, which depends on the associated regulatory proteins. Competition for actin monomers occurs between proteins that induce branched or linear actin polymerization. Cell specialization requires the regulation of actin filaments to allow the formation of cell type-specific structures, like cuticular hairs in Drosophila, formed by linear actin filaments. This study reports the functional analysis of CG34401/pelado, a gene encoding a SWIM domain-containing protein, conserved throughout the animal kingdom, called ZSWIM8 in mammals. Mutant pelado epithelial cells display actin hair elongation defects. This phenotype is reversed by increasing actin monomer levels or by either pushing linear actin polymerization or reducing branched actin polymerization. Similarly, in hemocytes, Pelado is essential to induce filopodia, a linear actin-based structure. This study further showed that this function of Pelado/ZSWIM8 is conserved in human cells, where Pelado inhibits branched actin polymerization in a cell migration context. In summary, these data indicate that the function of Pelado/ZSWIM8 in regulating actin cytoskeletal dynamics is conserved, favoring linear actin polymerization at the expense of branched filaments (Molina-Pelayo, 2022).

Cells extend membrane protrusions like lamellipodia and filopodia from the leading edge to sense, to move and to form new contacts. The Arp2/3 complex sustains lamellipodia formation, and in conjunction with the actomyosin contractile system, provides mechanical strength to the cell. Drosophila p53-related protein kinase (Prpk), a Tsc5p ortholog, has been described as essential for cell growth and proliferation. In addition, Prpk interacts with proteins associated to actin filament dynamics such as α-spectrin and the Arp2/3 complex subunit Arpc4. This study investigated the role of Prpk in cell shape changes, specifically regarding actin filament dynamics and membrane protrusion formation. Reductions in Prpk alter cell shape and the structure of lamellipodia, mimicking the phenotypes evoked by Arp2/3 complex deficiencies. Prpk co-localize and co-immunoprecipitates with the Arp2/3 complex subunit Arpc1 and with the small GTPase Rab35. Importantly, expression of Rab35, known by its ability to recruit upstream regulators of the Arp2/3 complex, could rescue the Prpk knockdown phenotypes. Finally, the requirement of Prpk was evaluated in different developmental contexts, where it was shown to be essential for correct Arp2/3 complex distribution and actin dynamics required for hemocytes migration, recruitment, and phagocytosis during immune response (Molina, 2022).

Actin remodeling mediates ROS production and JNK activation to drive apoptosis-induced proliferation

Stress-induced cell death, mainly apoptosis, and its subsequent tissue repair is interlinked although knowledge of this connection is still very limited. An intriguing finding is apoptosis-induced proliferation (AiP), an evolutionary conserved mechanism employed by apoptotic cells to trigger compensatory proliferation of their neighboring cells. Studies using Drosophila as a model organism have revealed that apoptotic caspases and c-Jun N-terminal kinase (JNK) signaling play critical roles to activate AiP. For example, the initiator caspase Dronc, the caspase-9 ortholog in Drosophila, promotes activation of JNK leading to release of mitogenic signals and AiP. Recent studies further revealed that Dronc relocates to the cell cortex via Myo1D, an unconventional myosin, and stimulates production of reactive oxygen species (ROS) to trigger AiP. During this process, ROS can attract hemocytes, the Drosophila macrophages, which further amplify JNK signaling cell non-autonomously. However, the intrinsic components connecting Dronc, ROS and JNK within the stressed signal-producing cells remain elusive. This study identified LIM domain kinase 1 (LIMK1), a kinase promoting cellular F-actin polymerization, as a novel regulator of AiP. F-actin accumulates in a Dronc-dependent manner in response to apoptotic stress. Suppression of F-actin polymerization in stressed cells by knocking down LIMK1 or expressing Cofilin, an inhibitor of F-actin elongation, blocks ROS production and JNK activation, hence AiP. Furthermore, Dronc and LIMK1 genetically interact. Co-expression of Dronc and LIMK1 drives F-actin accumulation, ROS production and JNK activation. Interestingly, these synergistic effects between Dronc and LIMK1 depend on Myo1D. Therefore, F-actin remodeling plays an important role mediating caspase-driven ROS production and JNK activation in the process of AiP (Farrell, 2022).

ROS-directed activation of Toll/NF-κB in the hematopoietic niche triggers benzene-induced emergency hematopoiesis

Hematopoietic stem cells/progenitor cells (HSC/HPCs) orchestrate the hematopoietic process, effectively regulated by the hematopoietic niche under normal and stressed conditions. The hematopoietic niche provides various soluble factors which influence the differentiation and self-renewal of HSC/HSPs. Unceasing differentiation/proliferation/high metabolic activity of HSC/HPCs makes them susceptible to damage by environmental toxicants like benzene. Oxidative stress, epigenetic modifications, and DNA damage in the HSC/HPCs are the key factors of benzene-induced hematopoietic injury. However, the role of the hematopoietic niche in benzene-induced hematopoietic injury/response is still void. Therefore, the current study aims to unravel the role of the hematopoietic niche in benzene-induced hematotoxicity using a genetically tractable model, Drosophila melanogaster. The lymph gland is a dedicated hematopoietic organ in Drosophila larvae. A group of 30-45 cells called the posterior signaling center (PSC) in the lymph gland acts as a niche that regulates Drosophila HSC/HPCs maintenance. Benzene exposure to Drosophila larvae (48 h) resulted in aberrant hemocyte production, especially hyper-differentiation of lamellocytes followed by premature lymph gland dispersal and reduced adult emergence upon developmental exposure. Subsequent genetic experiments revealed that benzene-induced lamellocyte production and premature lymph gland dispersal were PSC mediated. The genetic experiments further showed that benzene generates Dual oxidase (Duox)-dependent Reactive Oxygen Species (ROS) in the PSC, activating Toll/NF-κB signaling, which is essential for the aberrant hemocyte production, lymph gland dispersal, and larval survival. Together, this study establishes a functional perspective of the hematopoietic niche in a benzene-induced hematopoietic emergency in a genetic model, Drosophila, which might be relevant to higher organisms (D'Souza, 2022)

The Drosophila CD36 Homologue croquemort Is Required to Maintain Immune and Gut Homeostasis during Development and Aging

Phagocytosis is an ancient mechanism central to both tissue homeostasis and immune defense. Both the identity of the receptors that mediate bacterial phagocytosis and the nature of the interactions between phagocytosis and other defense mechanisms remain elusive. This study reports that Croquemort (Crq), a Drosophila member of the CD36 family of scavenger receptors, is required for microbial phagocytosis and efficient bacterial clearance. Flies mutant for crq are susceptible to environmental microbes during development and succumb to a variety of microbial infections as adults. Crq acts parallel to the Toll and Imd pathways to eliminate bacteria via phagocytosis. crq mutant flies exhibit enhanced and prolonged immune and cytokine induction accompanied by premature gut dysplasia and decreased lifespan. The chronic state of immune activation in crq mutant flies is further regulated by negative regulators of the Imd pathway. Altogether, these data demonstrate that Crq plays a key role in maintaining immune and organismal homeostasis (Guillou, 2016).

This study shows that Crq is required for the engulfment of microbes by plasmatocytes and their clearance. The mild immune deficiency due to crq mutation is associated with increased susceptibility to infection, defects in immune homeostasis, gut hyperplasia, and decreased lifespan. This study also re-confirmed a role for crq in apoptotic cell clearance, although the phagocytosis defect of crq^ko plasmatocytes is less severe than what had been previously observed with two lethal crq deficiency mutants, Df(2L)al and Df(2L)XW88. A possible explanation is that these deficiencies may have deleted at least one other gene required for apoptotic cell clearance. Additionally, morphological defects associated with secondary mutations could have exacerbated the crq phagocytosis defect by preventing efficient plasmatocyte migration to apoptotic cells. These same deficiency mutants had been assessed qualitatively for phagocytosis of bacteria by injecting embryos with E. coli or S. aureus; their plasmatocytes had no obvious defect in their ability to engulf these bacteria. However, a role for crq in phagocytosis of S. aureus, but not that of E. coli, was subsequently proposed based on S2 cell phagocytosis assays following knock-down of crq by RNAi. The current study shows that crq is required in vivo for uptake and phagosome maturation of both S. aureus and E. coli. A simple explanation of this discrepancy with E. coli could be that knocking down crq by RNAi is not sufficient to affect its role in E. coli phagocytosis (but sufficient to affect its role in S. aureus phagocytosis) and that completely abrogating crq expression by in vivo knock-out leads to a stronger phenotype with both bacteria. The in vivo data in crq^ko flies further demonstrate that crq is required to resist multiple microbial infections, such as Ecc15, E. faecalis, B. bassiana, and C. albicans. These data therefore argue that crq plays a more general role in microbial phagocytosis than was previously anticipated. Previous experiments to test whether crq is required for bacterial phagocytosis in embryos were qualitative rather than quantitative and did not allow identification of a role for crq at that stage. In contrast, the current experiments in adult crq^ko flies are quantitative and allowed identification of a delay in phagocytosis, followed by a defect in bacterial clearance in crq^ko hemocytes. A possible explanation for this discrepancy would be that hemocytes may differ in their expression profile, behavior, and phagocytic ability at various developmental stages due to differences in their microenvironment and/or sensitivity to stimuli. Accordingly, it has recently been shown that the phagocytic activity of embryonic hemocytes acts as a priming mechanism, increasing the ability of primed cells to phagocytose bacteria at later stages. It is therefore possible that embryonic, larval and adult hemocytes display very different levels of priming and bacterial phagocytic activity, and that crq is required mostly in larval/adult bacterial phagocytosis. Alternatively, a potential defect in phagocytosis of bacteria by embryonic hemocytes of the crq deficiencies may have been suppressed by the deletion of (an)other gene(s) in that genomic region (Guillou, 2016).

Because the immune competence of hemocytes varies during development, the potential role for crq in innate immunity was examined by knocking it out. This study shows that Crq is a major plasmatocyte marker at all developmental stages of the fly. crq^ko flies are homozygous viable, but short-lived, and can hardly be maintained as a homozygous stock in a non-sterile environment; crq^ko pupae become susceptible to environmental bacteria and their microbiota during pupariation. In a recent study, Arefin (2015) induced the pro-apoptotic genes hid or Grim in plamatocytes and crystal cells using the Hml-Gal4 driver (Hml-apo). A similar pupal lethality was observed but also associated with an induction of lamellocyte differentiation and the apparition of melanotic tumors of hemocyte origin. It was therefore concluded that the death of hemocytes triggered lamellocyte accumulation and melanotic tumor phenotypes. In contrast, no obvious melanotic tumors were observed in crq^ko flies, despite observing a loss of hemocytes in aging crq^ko flies and crq^ko flies subjected to Ecc15 infection. One possible explanation is that hemocytes do not die of apoptosis in crq^ko flies, but of a distinct mechanism. Alternatively, crq mutation could affect more hemocytes than Hml-apo flies, as crq is expressed in all plasmatocytes while Hml is only expressed in 72.4% of all plasmatocytes expressing crq. Thus the 27.6% of non-Hml plasmatocytes (thus non induced for apoptosis, which is Hml-Gal4 dependent) may respond to the death of the other plasmatocytes by inducing a signal that triggers the induction of lamellocytes and the subsequent formation of melanotic tumors. Considering the role of crq in apoptotic cell clearance, this signal may require a functional crq, which could explain why crq^ko flies do not develop melanotic tumors. Strikingly, in the Arefin study, as well as in previous studies, targeted ablation of plasmatocytes also made resulting 'hemoless' pupae more susceptible to environmental microbes. Extensive tissue remodeling takes place at pupariation, and plasmatocytes are essential to remove dying cells, debris, and bacteria. Thus, it was argued that this increased susceptibility was likely due to environmental bacteria invading the body cavity after disruption of the gut. In addition, it was found that the gut microbiome of Hml-apo flies could influence pupal lethality, as the eclosure rate of Hml-apo flies varied depending on the quality of the food they were reared on. Accordingly, the rescue of the crq^ko pupal lethality with antibiotics demonstrates that their premature aging and death are indeed due to infection by normally innocuous environmental bacteria. Altogether, these data suggest that phagocytes and crq are important actors regulating the interaction between a host and its microbiome (Guillou, 2016).

Hosts use both resistance and tolerance mechanisms to withstand infection and survive a specific dose of microbes. crq^ko flies exhibit a shorter lifespan when compared to control flies, but they are equally tolerant to aseptic wounds and infections. The crq^ko flies are less resistant to infection, as crq is required to promote efficient microbial phagocytosis. crq^ko plasmatocytes can still engulf bacteria, albeit at a lower efficiency than their controls. The data also demonstrate that crq plays a major role in phagosome maturation during bacterial clearance. This is in agreement with a recent study showing that crq promotes phagosome maturation during the clearance of neuronal debris by epithelial cells (Han, 2014). Thus, crq is required at several stages of phagocytosis. Similar observations have been made for the C. elegans Ced-1 receptor and for Drpr, as both promote engulfment of apoptotic corpses and their degradation in mature phagosomes (Guillou, 2016).

'Hemoless', Hml-apo and crq^ko flies are all more susceptible to environmental microbes and their microbiota. While it is not known whether mutants of eater, which encodes a phagocytic receptor for bacteria but does not play a role in phagosome maturation, are more susceptible to environmental microbes during pupariation, both eater mutants and 'hemoless' flies showed either decreased or unaffected systemic responses. Hml-apo larvae however, showed an upregulation in Toll-dependent constitutive Drs mRNA levels whereas Dpt expression was suppresse. In contrast, crq^ko flies showed no significant difference in constitutive or infection induced expression of Drs, but showed an increased expression of Dpt with age, and infection induced an increased and chronic expression of Dpt. Altogether the results argue that phagosome maturation defects in crq^ko flies lead to persistence of bacteria and thus to an increased and persistent systemic immune response via the Imd pathway. Such defects in phagosome maturation are not present in hemocyte ablation experiments, which could explain different outcomes for the host immunity and survival (Guillou, 2016).

This study have found that Crq acts in parallel to the Toll and Imd pathways. In the mealworm Tenebrio molitor, hemocytes and cytotoxic enzymatic cascades eliminate most bacteria early during infection, and AMPs are required to eliminate persisting bacteria. These data suggest that AMPs act in parallel with hemocytes to fight infections. It was also found that crq^ko flies are more susceptible to infection with S. aureus than wild-type and Toll pathway-deficient flies. These results are consistent with S. aureus infection being mainly resolved via phagocytosis and Crq having a major role in this process. Surprisingly, the opposite was observed for infection with other Gram-negative or positive bacteria and fungi. Drosophila mutants for AMP production were more susceptible to infection than crq^ko flies, arguing that AMPs are critical to eliminate the bulk of pathogens. Indeed, crq (thus phagocytosis) is not essential for Ecc15 elimination, but accelerates bacterial clearance. The results also suggest that the defects in phagosome maturation may allow some bacteria to persist and grow within hemocytes, where they are hidden from systemic AMPs. Thus, depending on the microbe, humoral and cellular immune responses can act at distinct stages of infection. In this context, phagocytosis acts as a main defense mechanism against pathogens that may escape AMPs or modulate their production (Guillou, 2016).

Chronic activation of immune pathways can be detrimental to organismal health. In Drosophila, multiple negative regulators of the Imd pathway, including PGRP-LB, act in concert to maintain immune homeostasis. This study has observed that crq^ko flies sustain high production levels of the AMP Dpt and the cytokine Upd3, demonstrating that defects in phagocyte function can lead to chronic immune activation. Notably, the level of Dpt expression induced by activation of the Imd pathway in unchallenged conditions is stronger in crq^ko flies than was previously observed in mutants of three negative regulators of the Imd pathway, namely pirk^EY, PGRP-SC^Δ, and PGRP-^LBΔ, and over 1,000-fold higher in PGRP-LB^LBΔ, crq^ko double mutants. This is despite the persistence of only a few hundred bacteria in these mutants. This phenotype may be due solely to the accumulation of these persistent bacteria, or Crq may also function in plasmatocytes to remove immunostimulatory molecules from the hemolymph. Nonetheless, this study shows that plasmatocytes, Crq, and phagocytosis are all key factors in the immune response, and that losing crq induces a state of chronic immune induction (Guillou, 2016).

The ability of a host to control microbes decreases with age, a phenomenon called immune senescence. The causes of immune senescence remain elusive, but the loss of immune cells with age and a decline in their ability to phagocytose have been suggested. Recent studies have argued that microbial dysbiosis and disruption in gut homeostasis contribute to early aging. In addition, persistent activation of the JAK-STAT pathway in the gut has been linked to age-related decline in gut structure and function. Aging crq^ko flies lose a greater number of hemocytes than wild-type flies after infection, which may be the result of accumulating bacteria in these hemocytes in which phagosomes fail to mature. The premature death of crq^ko flies could be partially rescued by the presence of antibiotics. This demonstrates that phagocytosis, and phagosome maturation in particular, plays a crucial role in managing the response to environmental microbes and potentially, the gut microbiota directly to promote normal aging. This study also found that chronic upd3 expression in crq^ko flies triggers premature midgut hyperplasia, which is known to alter host physiology and promote premature aging. It has recently been proposed that plasmatocytes can influence gut homeostasis by secreting dpp ligands and modulating stem cell activity. These results reinforce the possibility of an interaction between plasmatocyte function and gut homeostasis, and suggests that cytokines derived from hemocytes can trigger cell responses in the gut. These results are also in agreement with a recent publication showing that Upd3 from hemocytes can trigger intestinal stem cell proliferation. Altogether, these results demonstrate that the interaction between hemocytes and the gut tissue are central to host health, and the data demonstrate that phagocytic defects can be associated with chronic gut inflammation and aberrant intestinal stem cell turn-over. As gut aging and barrier integrity are in turn important to maintain bodily immune homeostasis, the following model is proposed: in crq^ko flies, plasmatocyte-derived cytokines accelerate gut aging promoting loss of gut homeostasis and microbial dysbiosis, with immune and plasmatocyte activation acting in a positive feedback loop (Guillou, 2016).

Collectively, these data show that Crq is essential in development and aging to protect against environmental microbes. Interestingly, the impact of mutating crq on host physiology is strikingly different from previously reported phagocytic receptor mutations. It is speculated that this could be due to its dual role in uptake and phagosome maturation during phagocytosis. Crq is required for microbial elimination in parallel to the Toll and Imd pathways and acts to maintain immune homeostasis. This situation is surprisingly reminiscent of inflammatory disorders, such as Crohn's disease, that result from primary defects in bacterial elimination and trigger chronic immune activation and disruption of gut homeostasis. Further characterization of the crq mutation in Drosophila will provide an interesting conceptual framework to understand auto-inflammatory diseases and their repercussions on immune homeostasis and host health (Guillou, 2016).

Hemocytes are critical for Drosophila melanogaster post-embryonic development, independent of control of the microbiota

Proven roles for hemocytes (blood cells) have expanded beyond the control of infections in Drosophila. Despite this, the critical role of hemocytes in post-embryonic development has long thought to be limited to control of microorganisms during metamorphosis. This has previously been shown by rescue of adult development in hemocyte-ablation models under germ-free conditions. This study shows that hemocytes have a critical role in post-embryonic development beyond their ability to control the microbiota. Using a newly generated, strong hemocyte-specific driver line for the GAL4/UAS system, it was shown that specific ablation of hemocytes is early pupal lethal, even under axenic conditions. Genetic rescue experiments prove that this is a hemocyte-specific phenomena. RNA-seq data suggests that dysregulation of the midgut is a prominent consequence of hemocyte ablation in larval stages, resulting in reduced gut lengths. Dissection suggests that multiple processes may be affected during metamorphosis. It is believed that this novel role for hemocytes during metamorphosis is a major finding for the field (Stephenson, 2022).

Drosophila hemocytes recognize lymph gland tumors of mxc mutants and activate the innate immune pathway in a reactive oxygen species-dependent manner

Mechanisms of cancer cell recognition and elimination by the innate immune system remains unclear. The immune signaling pathways are activated in the fat body to suppress the tumor growth in mxcmbn1 hematopoietic tumor mutants in Drosophila by inducing antimicrobial peptides (AMP). This study investigated the regulatory mechanism underlying the activation in the mutant. Firstly, it was found that reactive oxygen species (ROS) accumulated in the hemocytes due to induction of dual oxidase and one of its activators. This was required for the AMP induction and the tumor growth suppression. Next, more hemocytes transplanted from normal larvae were associated with the mutant tumor than normal lymph glands (LGs). Matrix metalloproteinase 1 and 2 (MMP2) were highly expressed in the tumors. The basement membrane components in the tumors were reduced and ultimately lost inside. Depletion of the MMP2 rather than MMP1 resulted in a significantly reduced AMP expression in the mutant larvae. The hemocytes may recognize the disassembly of basement membrane in the tumors and activate the ROS production. These findings highlight the mechanism via which macrophage-like hemocytes recognize tumor cells and subsequently convey the information to induce AMPs in the fat body. They contribute to uncover the role of innate immune system against cancer (Kinoshita, 2022).

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

Stem cell compartments in metazoa get regulated by systemic factors as well as local stem cell niche-derived factors. However, the mechanisms by which systemic signals integrate with local factors in maintaining tissue homeostasis remain unclear. Employing the Drosophila lymph gland, which harbors differentiated blood cells, and stem-like progenitor cells and their niche, this study demonstrates how a systemic signal interacts and harmonizes with local factor/s to achieve cell type-specific tissue homeostasis. Genetic analyses uncovered a novel function of Lar, a receptor protein tyrosine phosphatase. Niche-specific loss of Lar leads to upregulated insulin signaling, causing increased niche cell proliferation and ectopic progenitor differentiation. Insulin signaling assayed by PI3K activation is downregulated after the second instar larval stage, a time point that coincides with the appearance of Lar in the hematopoietic niche. It was further demonstrated that Lar physically associates with InR and serves as a negative regulator for insulin signaling in the Drosophila larval hematopoietic niche. Whether Lar serves as a localized invariable negative regulator of systemic signals such as insulin in other stem cell niches remains to be explored (Kaur, 2019).

An effort to understand the maintenance of the hematopoietic niche led to the discovery of the role of Lar in regulating insulin signaling in the niche, which is crucial for lymph gland homeostasis. Lar in the hematopoietic niche acts as a rheostat, restricting excessive insulin signaling to limit proliferation in later developmental stages. A physiological consequence of insulinemia in the niche is upregulated ROS. As a result, the ROS/Spitz/EGFR/ERK circuit that is evoked during an immune response gets activated during normal development. Lar abrogation from the niche also activates JNK to bolster niche cell proliferation. In addition to Dpp, insulin signaling is also known to stimulate cell proliferation via Myc in the hematopoietic niche. It is, therefore, also possible that the InR/Pi3K activation and immense proliferation observed upon Lar loss from the hematopoietic niche might also involve Myc activation (Kaur, 2019).

Lar is a transmembrane type IIA receptor protein tyrosine phosphatase, which has two intracellular phosphatase domains (D1 and D2) and extracellular immunoglobulin (Ig) and fibronectin type III (FNIII) domains. The different domains of Lar provide this single molecule the ability to carry out diverse functions. A major interactor of Lar in Drosophila is the actin cytoskeleton. This interaction is often encountered in the developing nervous system, in which it plays a significant role in axonal migration and synapse morphogenesis (Kaur, 2019).

In addition, in oocytes, Lar is implicated in follicle cell development and patterning through actin organization. A study in Drosophila germline stem cells (GSCs) has demonstrated that Lar can act as a cell adhesion molecule by localizing E-cadherin at the GSC-hub interface, thereby maintaining the attachment between male GSCs and hub cells. Moreover, evidence of the physical interaction of Lar with N-cadherin in the Drosophila embryo further endorses the interaction of Lar with cell adhesion molecules (Kaur, 2019).

Many in-vitro studies in the mammalian system have revealed that Lar interacts with various tyrosine kinases, thereby modulating different signaling pathways. In vitro studies using mammalian cell lines demonstrated that InR physically associates with Lar. SPR has shown that the most preferred substrate for Drosophila Lar is InR, but the evidence for physical interaction remains to be demonstrated. The current study provides the first in vivo physical association of Lar with InR in Drosophila (Kaur, 2019).

Besides the evidence of physical interaction, genetic data demonstrate that loss of Lar in the hematopoietic niche results in hyperactivated insulin signaling. Upregulated PI3K expression suggests that Lar directly acts on InR and not on any other regulators of InR signaling such as Pten or Tsc1/2 in the hematopoietic niche. No alteration in InR expression upon Lar loss from the niche further confirms that Lar-InR interaction impinges on PI3K-Akt-insulin signaling and not on InR expression. Furthermore, the results show that the catalytic function of Lar protein that resides in the PTPD1 domain is crucial for Lar-InR interaction (Kaur, 2019).

LAR can modulate multiple tyrosine kinases; it appears that the spatial distribution of LAR gives it specificity for its cell-type tyrosine kinase receptor. Equivalent evidence comes from the current in vivo study, in which it was successfully demonstrated that, in hematopoietic tissue, wherever there is a high membranous tGPH (reporting insulin signaling), Lar expression is low, and vice versa. This reciprocal expression, coupled with the genetic analyses, projects a mechanism underpinning the activation of receptor tyrosine kinases by RPTPs in a cell-type-specific manner (Kaur, 2019).

Insulin signaling helps to coordinate nutritional status with systemic growth control both in invertebrates and in the mammalian system. Through its receptor, insulin is now known to have a much broader pleiotropic role, controlling several physiological processes. Therefore, inappropriate activation of insulin signaling has been linked with various aberrant scenarios such as infection, cancer and diabetes (Kaur, 2019).

The hematopoietic niche cells can sense the systemic insulin level, which is essential for their proliferation. Overactivation of InR increases the niche cell number, whereas downregulation causes it to decline. The systemic insulin level is also directly sensed by the hemocyte progenitors. Knockdown of insulin signaling in the progenitors results in their precocious differentiation, demonstrating that physiological levels of insulin signaling are essential for their maintenance. The expression data and genetic analyses unravel a differential requirement of insulin signaling in the niche compared with progenitor cells (Kaur, 2019).

This study further illustrated how the pleiotropic effect of excessive insulin signaling in the niche affects the homeostasis of the organ. The hyperactivated insulin signaling in the niche generates excessive ROS. This high level of ROS in the niche has a two-prong effect in two different cell types of the developing lymph gland. First, within the niche, it evokes JNK to provide a thrust to the ongoing proliferation of the niche cells. Previous literature has demonstrated that oxidative stress leads to the activation of JNK, which is known to have both pro- and anti-proliferative functions. Thus, by the stimuli, strength and duration of the JNK activation, diverse responses ranging from apoptosis, survival and altered proliferation can be evoked. The current study demonstrates that the ectopic activation of JNK due to the gradual accumulation of ROS collaborates with hyperactivated insulin signaling to boost cell proliferation. The second effect of the elevated ROS in the niche is the activation of ERK in the hemocyte progenitors. Activation of the Spitz/EGFR pathway by ectopic ROS in the niche is known to activate ERK in the progenitors. The activated ERK causes ectopic differentiation and lamellocyte generation (Kaur, 2019).

The cumulative effect of deregulated signals disturbs the homeostasis of the organ Interestingly, hyperactivation or hypoactivation of insulin signaling is also associated with deregulated hematopoiesis in the vertebrate system. For example, altered insulin signaling in diabetic mice affects the composite microenvironment of the bone marrow leading to compromised function of the hematopoietic niche. This work provides a strong genetic link between Lar and Insulin signaling, which should be tested in vertebrates. Although LAR is expressed in T-cell lineages in vertebrates, it remains to be seen whether Lar is also present in vertebrate hematopoietic niche/s and functions similarly. It is also intriguing to observe that, similar to the vertebrate system, a low level of ROS is present in the Drosophila hematopoietic niche. It will be fascinating to see whether the hyperactivation of insulin signaling also generates ROS in the vertebrate niche and affects cell fate specification via the same mechanism that is elucidated in this study (Kaur, 2019).

This study unravels a check on insulin signaling by Lar that authorizes the hematopoietic niche to act as the 'interlocutor', evaluating the physiological state of an organism and thereby relaying it to the hemocyte progenitors for their homeostasis (Kaur, 2019).

Temporal specificity and heterogeneity of Drosophila immune cells

Immune cells provide defense against non-self and have recently been shown to also play key roles in diverse processes such as development, metabolism, and tumor progression. The heterogeneity of Drosophila immune cells (hemocytes) remains an open question. Using bulk RNA sequencing, this study found that the hemocytes display distinct features in the embryo, a closed and rapidly developing system, compared to the larva, which is exposed to environmental and metabolic challenges. Through single-cell RNA sequencing, fourteen hemocyte clusters were identified present in unchallenged larvae and associated with distinct processes, e.g., proliferation, phagocytosis, metabolic homeostasis, and humoral response. Finally, this study characterizes the changes occurring in the hemocyte clusters upon wasp infestation, which triggers the differentiation of a novel hemocyte type, the lamellocyte. This first molecular atlas of hemocytes provides insights and paves the way to study the biology of the Drosophila immune cells in physiological and pathological conditions (Cattenoz, 2020).

The innate immune response has made the object of intense investigation in Drosophila melanogaster, as this model shows mechanisms that are conserved throughout evolution, from pattern recognition molecules to immune molecular cascades. Given the importance of innate immunity in a variety of physiological and pathological processes including tumour progression, the current challenge is to characterize immune cell heterogeneity and identify specific hemocyte populations. This is the aim of the present work (Cattenoz, 2020).

Three classes of hemocytes have so far been identified: plasmatocytes (PL), crystal cells (CC) and lamellocytes (LM). PL are the most abundant cell type and are responsible for the main functions of the hemocytes: phagocytosis, secretion of extracellular matrix proteins (ECM), signalling molecules and antimicrobial peptides (AMPs). The CC account for less than 5% of the total hemocyte population with distinctive crystals inside them that are composed of prophenoloxidases (PPO). These enzymes are released in large quantity upon wounding and are key component for the melanization process. The LM are flat and large cells that only appear upon challenge. They are considered activated immune cells that arise through PL trans-differentiation or from a mitotic dedicated precursor (Anderl, 2016; Cattenoz, 2020 and references therein).

In the embryo, the hemocytes contribute to the clearance of apoptotic cells and the deposition of ECM-related molecules including Peroxidasin (Pxn) and Viking (Vkg). By the larval stage, the organism interacts with the external environment and responds to metabolic and oxidative stress as well as to infection or injury related stimuli. The hemocytes must therefore adapt to these new, highly demanding, settings. In addition, while during embryogenesis the hemocytes are highly motile and patrol the whole organism, during the larval life a large fraction of them, called resident hemocytes, colonize segmentally repeated epidermal-muscular pockets in which cell proliferation is enhanced. Upon wounding, septic infection or infestation by parasitic wasps, the resident hemocytes are mobilized and enter in circulation to reach the site of the immune challenge. Thus, hemocyte localization adapts to homeostatic and challenged conditions (Cattenoz, 2020).

This study has characterize the transcriptional changes occurring during development and the different types of hemocytes present in the larva. Comparing the bulk RNA sequencing data allows definition of stage-specific features: hemocytes contribute to the shaping of the tissues and are glycolytic whereas larval hemocytes show a strong phagocytic potential and a metabolic switch toward internalization of glucose and lipid and beta-oxidation. The single cell RNA sequencing (sc RNA seq) assays allows identification of fourteen clusters of larval PL and to assign specific molecular and cellular features, including nutrient storage, proliferative potential, antimicrobial peptide production and phagocytosis (Cattenoz, 2020).

Finally, as a first characterization of the immune response at the single cell level, this study assesses the transcriptional changes induced by infestation by the parasitic wasp Leptopilina boulardi, one of the most studied cellular immune pathways. The wasp lays eggs in the Drosophila larva and triggers hemocyte proliferation as well as LM differentiation, with subsequent encapsulation of the wasp egg and its death through the increase of the levels of reactive oxygen species (ROS). The sc RNA seq sequencing assay identifies two LM populations, a mature one with a strong glycolytic signature, and a population that expresses both LM and PL features, likely originating through trans-differentiation (Anderl, 2016; Cattenoz, 2020 and references therein).

The response to wasp infestation involves the embryonic hemocytes that differentiate from the procephalic mesoderm (1st wave of hematopoiesis), as well as the hemocytes that originate from the lymph gland, the site of the 2nd hematopoietic wave. While in not infested (NI) conditions, the lymph gland histolyses and releases hemocytes in circulation during the pupal life, upon wasp infestation (WI), it undergoes precocious histolysis so that both lymph gland and embryo derived hemocytes populate the larva. Single cell RNA sequencing assay identifies the same number of PL clusters as that observed in normal conditions, showing that the PL from the first and from the second hematopoietic wave share the same features (Cattenoz, 2020).

In sum, this work characterizes the transcriptional changes occurring during hemocyte development and the hemocyte populations present in the Drosophila larva. It also provides the molecular signature and the initial characterization of the larval hemocyte repertoire as well numerous novel markers in NI and in WI conditions. These first bulk and single cell RNA seq data pave the way to understand the role of the immune system in development and physiology (Cattenoz, 2020).

This work provides the first atlas of the Drosophila hemocytes, by specifically focusing on those that originate from the first hematopoietic wave. These hemocytes undergo a molecular and metabolic shift during development. This study shows the existence of distinct hemocyte populations and identifies a large panel of novel markers specific to the different populations. Monitoring the larval response against the wasp L. boulardi reveals the hemocyte behavior upon challenge and defines intermediate and mature LM populations. Finally, multiple bioinformatics tools are used to predict a temporal progression among the different hemocyte clusters in control and in challenged conditions (Cattenoz, 2020).

Immune cells are considered as static components of the defense system, however these cells constantly interact with the ever-changing environment. In addition, the cells that are born in the early embryo experience the extensive rearrangements that occur during development, including tissue and organ formation. We here show that the Drosophila hemocytes undergo a significant transcriptional shift that fully complies with the requirements of the embryonic and larval stages (Cattenoz, 2020).

The highly migratory hemocytes present in the differentiated embryo display a strong developmental role: they allow tissue reshaping by secreting several constituents of the extracellular matrix and by engulfing dead cells through specific scavenger receptors such as NimC4. They also display high levels of gluconeogenesis and TAG synthesis, processes that provide adequate levels of glucose and fatty acid for tissue/organ development. The larval hemocytes, on the other hand, express high levels of transcripts that are linked to the immune response, in accordance with the exposure to pathogens, and are highly phagocytic. Moreover, they express fewer molecules associated with the extracellular matrix as compared to those observed in the embryo. Finally, they strongly express the molecular pathways that release stored energy (beta oxidation, TCA cycle), most likely in preparation for the metamorphosis and to help building the adult tissues (Cattenoz, 2020).

The single cell analysis on the NI animals reveals the presence of 14 different hemocyte clusters, in addition to the classical distinction between PL and CC. This provides a battery of novel specific markers that will make it possible to investigate the role of the different hemocyte populations and to generate more targeted genetic tools. Excitingly, it is already possible to define distinct features and functions of the different clusters, based on the profile of gene expression, on the enrichment in specific GO terms and regulons as well as on the in vivo validation. Indeed a number of clusters are identified by a single regulon or by a specific combination of regulons in the case of related clusters (Cattenoz, 2020).

The PL-Rel cluster (12%) likely provides a cellular reservoir for a specific immune response, the closely related PL-AMP hemocytes (0.5%) seem more specifically dedicated to the humoral response, whereas PL-vir1 hemocytes (4%) seem dedicated to the anti-viral response. These three clusters share GO terms and regulons associated with immune functions, suggesting that they respond to a variety of challenges. The PL-Lsp hemocytes (>3%) represent the nutrient reservoir that stores amino acids and, in agreement with a role in homeostasis, they are mostly located in circulation. The PL-Lsp and PL-AMP hemocytes are associated with the major roles of the fat body, the metabolic homeostasis and the humoral immune response, suggesting that they contribute to the fat body -- hemocyte axis acting in physiological and pathological conditions. This axis is bidirectional. For example: (1) the small secreted peptide Edin produced in the fat body controls the number of plasmatocytes in circulation upon wasp infestation; (2) the hemocyte expression of the Spaetzle ligand controls the activity of Toll signaling in the fat body and affects the response to infection as well as tumor growth; (3) the metabolically induced production of the NimB5 protein from the fat body adjusts the number of hemocytes to the physiological state of the larva (Ramond, 2020; Cattenoz, 2020 and references therein).

The PL-robo2 hemocytes (6% of the total population) are associated with phagocytosis but are also enriched for the lipid scavenger receptor Crq involved in the inflammatory response upon high fat diet. This cluster shares features with the large PL-0 and PL-2 clusters that are mildly enriched for the regulon related to the phagocytic abilities (srp), in agreement with the finding that the vast majority of the larval hemocytes is phagocytic. The PL-0, PL-2, PL-1 and PL-3 large clusters, which do not display strong specific molecular features, may indeed represent cells that can serve different purposes, perhaps less efficiently than the more specialized hemocytes. They may also constitute a reservoir of cells that have a basal activity but express enhanced potentials in response to challenges, in line with the trajectories identified by the bioinformatic analyses (Cattenoz, 2020).

The data on the small CC cluster validate the role of these cells in melanization and reveal a distinct metabolism compared to PL. The CC seem to use glucose as energy source and the PL mostly lipids. The genes associated with mitosis are enriched in a single cluster that preferentially localizes to the resident compartment. This indicates that PL-prolif, which represents less than 1% of the total population, provides the pool of mitotic precursors in the larva. In agreement with these data, the bioinformatics analyses reveal a sequential progression leading from PL-prolif to most of the hemocyte clusters identified in the larva. PL-Inos, which is the cluster the most closely related to PL-prolif, is also associated with the resident compartment and likely represents immature progenitors (Cattenoz, 2020).

According to the bioinformatics analyses, the PL-Impl2 hemocytes seem not to originate from PL-prolif hemocyte. These may represent hemocytes that are set aside in the embryo and indeed the comparison of all the clusters reveals that the PL-Impl2 hemocytes are significantly enriched for transcripts that are specific to the E16 hemocytes (Cattenoz, 2020).

The identification of different populations of specialized plasmatocytes in the Drosophila larva prompts drawing of parallels with the mammalian immune cells. The closest relative to plasmatocytes are the monocytes and the macrophages. Monocytes are equipped with Toll-like receptors, scavenger receptors and their main function is to patrol as well as remove microorganisms, lipids and dying cells via phagocytosis. Upon inflammation, they infiltrate specific tissues and differentiate into macrophages. The macrophages keep phagocytosing, induce an inflammatory response by releasing cytokines and participate to the repair of the tissue. The sc RNA seq assay reveals that hemocytes express markers such as Integrin alphaPS2 (If), EcR/Hr96, Lamp1, Rgh, Tfc/lectin-46Cb/CG34033 and Lz, which are the Drosophila orthologues of CD11b, PPARγ, CD68, Dectin, CD207 and RUNX, respectively. In mammals these proteins are responsible for migration, adhesion, phagocytosis, differentiation from monocytes to macrophages and pathogen recognition (Cattenoz, 2020).

The single cell analysis upon WI reveals the reduced representation of some clusters such as PL-Rel and the expanded representation of 'early' clusters (e.g., PL-prolif, PL-Inos). Thus, specific hemocyte clusters may preferentially survive/proliferate upon challenge. The majority of the clusters, however, remain equally represented in the two conditions and the correlation between the average transcriptomes in NI and WI conditions reveals strong similarity between most of the identified clusters. This implies that the hemocytes produced by the 1st and the 2nd hematopoietic waves share major features. Two new populations of cells appear, LM1 and LM2, the second one representing an intermediate state characterized by the co-expression of LM and PL genes. Interestingly, LM2 also expresses a specific identity that is linked to energy supply (e.g. respiratory chain, NADH activity), whereas LM1 cells are mostly devoted to encapsulation (Cattenoz, 2020).

According to the bioinformatics predictions (RNA Velocity and Monocle) on the WI dataset, the PL-prolif cluster seems to rapidly branch out: one arm gives rise to the LM clusters, partly associated with the PL-vir1 cluster, whereas the other gives rise to the other plasmatocyte clusters. This early separation could support the hypothesis of a dedicated precursor, the lamelloblast, producing a second population of LM that is not generated through plasmatocyte trans-differentiation (Anderl, 2016). In this model, the 1st hematopoietic wave would produce LM through trans-differentiation (the expression of LM2 markers only increases in the first 24 h after infestation), whereas the 2nd wave would do it (also) through the mitotically active lamelloblast. At the level of resolution provided by the sc RNA seq assay, the lamelloblast cluster may have been lost (Cattenoz, 2020).

In sum, the different clusters identified by the sc RNA seq assay exhibit distinct features, which can be now tested functionally using the newly identified markers and the associated genetic tools that are publically available (Gal4 drivers, RNAi and overexpressing transgenes, mutations). Future technological refinements may enhance the depth of the analyses, as the current sc RNA seq assays only allows for the identification of a subset of genes for each cluster, the most expressed ones. As an example, the larval hemocytes do not all phagocytose with the same efficiency, but we cannot allocate the different potentials to specific clusters. Nevertheless, the data on the bulk and single cell transcriptomes of the Drosophila hemocytes provide a powerful framework to understand the role of immune cells in physiological and pathological conditions (Cattenoz, 2020).

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

Cell-intrinsic and extrinsic signals regulate the state and fate of stem and progenitor cells. Recent advances in metabolomics illustrate that various metabolic pathways are also important in regulating stem cell fate. However, understanding of the metabolic control of the state and fate of progenitor cells is in its infancy. Using Drosophila hematopoietic organ: lymph gland, this study demonstrated that Fatty Acid Oxidation (FAO) is essential for the differentiation of blood cell progenitors. In the absence of FAO, the progenitors are unable to differentiate and exhibit altered histone acetylation. Interestingly, acetate supplementation rescues both histone acetylation and the differentiation defects. It was further shown that the CPT1/whd (withered), the rate-limiting enzyme of FAO, is transcriptionally regulated by Jun-Kinase (JNK), which has been previously implicated in progenitor differentiation. This study thus reveals how the cellular signaling machinery integrates with the metabolic cue to facilitate the differentiation program (Tiwari, 2020).

Single-cell RNA sequencing identifies novel cell types in Drosophila blood

Drosophila has been extensively used to model the human blood-immune system, as both systems share many developmental and immune response mechanisms. However, while many human blood cell types have been identified, only three were found in flies: plasmatocytes, crystal cells and lamellocytes. To better understand the complexity of fly blood system, single-cell RNA sequencing technology was used to generate comprehensive gene expression profiles for Drosophila circulating blood cells. In addition to the known cell types, two new Drosophila blood cell types were identified: thanacytes and primocytes. Thanacytes, which express many stimulus response genes, are involved in distinct responses to different types of bacteria. Primocytes, which express cell fate commitment and signaling genes, appear to be involved in keeping stem cells in the circulating blood. Furthermore, the data revealed four novel plasmatocyte subtypes (Ppn⁺, CAH7⁺, Lsp⁺ and reservoir plasmatocytes), each with unique molecular identities and distinct predicted functions. Cross-species markers from Drosophila hemocytes to human blood cells were identified. This analysis unveiled a more complex Drosophila blood system and broadened the scope of using Drosophila to model human blood system in development and disease (Fu, 2020).

Drosophila has been used extensively to study the human blood-vascular system. At first glance both systems might seem to have little in common; however, at the molecular level, they are highly conserved. For example, they share specific transcription factors and signaling pathways during development. And, in both systems the terminally differentiated blood cell lineages are derived from common progenitor cells. Moreover, at the functional level, Drosophila blood cells demonstrate phagocytosis, innate immunity, wound healing, engulfing of large particles (e.g., wasp infestation), and sensing of environmental gasses like oxygen levels, similar to the human myeloid blood cell system. (Fu, 2020).

Decades of research have revealed a complex and numerous cell system in human blood, comprising erythrocytes, leukocytes (neutrophils, T lymphocytes and B lymphocytes), natural killer (NK) cells, macrophages and thrombocytes. In contrast, to date, only three terminally differentiated blood cell types have been described in Drosophila: plasmatocytes, crystal cells and lamellocytes (Fu, 2020).

Plasmatocytes are the most numerous cell type (~90-95% of hemocytes). During embryogenesis, plasmatocytes constitute a major source of extracellular matrix proteins which are essential for embryonic renal tubule morphogenesis, further they engulf apoptotic cells by endocytosis. Within the immune system, they provide phagocytic and antimicrobial functions to remove any invading particles, similar to human macrophages. Plasmatocytes are known to express the free radical scavenging enzyme Peroxidasin (Pxn), and several cell surface molecules involved in phagocytosis, including Nimrod C1 (NimC1; P1 antigen) and Eater receptors (Fu, 2020).

Crystal cells make up a much smaller portion of Drosophila hemocytes (~2-5%). They are named for their distinct crystalline structures, and these inclusions contain Prohenoloxidase (ProPO) enzymes that mediate melanization in response to injury. Further, crystal cells facilitate innate immunity and the hypoxic response, not unlike human platelet and granulocyte functions. Early markers include lozenge (lz) and pebbled (peb), while later markers include the ProPO enzyme coding genes PPO1 and PPO2 (Fu, 2020).

Lamellocytes are generally very rare in healthy flies; however, their numbers increase rapidly in response to wasp infection. They arise from trans-differentiation of plasmatocytes. Lamellocyte morphology stands out as they form large flat disc-shaped cells, ideal for enveloping intrusions. They often contain more lysosomes and phagocytic vacuoles than plasmatocytes but lamellocytes do not exhibit phagocytic activity. Their human equivalents are the multinucleated giant cells that arise when monocytes or macrophages fuse together during an infection. The expressed markers include PPO3, atilla, Integrin alphaPS4 subunit (ItgaPS4), misshapen (msn), L6 or L2 antigens, and myospheroid (mys; encodes β subunit of the integrin dimer). Mys is also expressed by hemocyte progenitors and plasmatocytes (Fu, 2020).

Cells, including those from the Drosophila blood system, have traditionally been classified based on their morphology and the expression of a limited number of defined protein markers. However, single-cell RNA sequencing (scRNA-seq) technology can assay gene expression of an individual cell at a genome-wide scale, for thousands of cells in a single experiment, and has provided many new insights into the richness of cell type variety that makes up different tissues and organs (Fu, 2020).

This study applied scRNA-seq technology to study the cellular heterogeneity of the total circulating blood cells in Drosophila wandering third instar larvae. This analysis revealed four previously unknown plasmatocytes subtypes: Ppn+ plasmatocytes, CAH7+ plasmatocytes, Lsp+ plasmatocytes and reservoir plasmatocytes. And new markers that uniquely distinguish crystal cells and lamellocytes among hemocytes. These findings also uncovered two new Drosophila blood cell types: thanacytes and primocytes, which display distinct gene expression profiles with unique markers. By silencing Tep4, a thanacyte-specific gene, this study showed that thanacytes are responsible for the distinct response to different type of bacterial infection. The expression profiles of these newly identified cells were further compared to those of the human blood system, and cross-species markers were identified linking fly and human blood cell types. This analysis unveiled a more complex Drosophila blood system and broader scope for using Drosophila to model human blood system (Fu, 2020).

This study identified four plasmatocyte subtypes (Ppn+, CAH7+, Lsp+, and reservoir PM), each with a unique expression profile and pathways that indicate specialized functions. Interestingly, these subtypes appear to fulfill defined roles in the immune response system. For example, pathway analysis indicates phagocytic activity is specific to the Ppn+ PM and CAH7+ PM, suggesting they are at the front line of eliminating pathogens and cell debris (Fu, 2020).

Reservoir PM subtype makes up the majority of plasmatocytes (60%). They show a very broad response to immune and non-immune stimuli and unique molecular cell markers to define this group seem to be missing, suggesting they might represent a plastic plasmatocyte cell state. For example, similar to the human naïve T cells, which are mature T cells but have not yet been exposed to an antigen upon which their molecular profile changes drastically. This is further supported by pseudotemporal trajectory tracing, which places the reservoir PM at the root with the other PM subtypes branching off based on differing transcriptional profiles. Of note, lamellocyte numbers are known to rapidly increase in response to wasp infection, arising from trans-differentiating plasmatocytes. These findings suggest that reservoir PM might be the source for these immune-induced lamellocytes. Further studies are needed to tease out the functional responsibilities and contributions of the individual blood cell types and subtypes, and how these might change in response to an immune challenge (Fu, 2020).

The scRNA-seq data provided a wealth of information at the single-cell level, and revealed five distinct cell types, including the known plasmatocytes, crystal cells and lamellocytes. The two additional cell types expressed the pan-hemocyte marker srp, but expression of established blood cell type-specific markers did not rise above the detection threshold. Based on their unique gene expression profiles, they were identified as novel Drosophila blood cell types. The first is a small, quite distinct cell cluster as it only selectively expresses early pan-hemocyte genes. Further analysis shows pathways of cell fate commitment and regulation of differentiation are particularly active, indicating a hitherto undescribed circulating progenitor cell type in Drosophila blood, which were named primocytes. Primocytes have high level of expression of transcription factors that regulate cell fate commitment, including Antp, kn, Mad, and ham. Interestingly, Antp and Kn are also highly expressed in the Posterior Signaling Center (PSC) of lymph gland primary lobe. This role of the PSC is reminiscent of the 'niche', the micro-environment of hematopoietic stem cells in vertebrates. Therefore, it is speculated that the primocytes might be involved in keeping stem cell fate in the Drosophila blood. The PSC cells in lymph gland only consist of a small number of cells, consistent with the small percentage of primocytes in the circulating hemocytes. Thanacytes, on the other hand, express common hemocyte markers, but show no expression of pan-plasmatocyte markers above threshold. Tep4 is highly expressed in thanacytes, but nearly absent in all other hemocyte cell types. These Tep4+ cells show a striking contrast with plasmatocytes in that they do not express NimC1 (P1, marker of plasmatocytes) at detectable levels, which further supports the scRNA-seq findings that these cells represent a novel blood cell type. Tep4 is known to play a role in the Drosophila cellular immune response to certain Gram-negative bacteria. Tep4 expression was used as a proxy for thanacytes and an immune response assay was carried out. The thanacyte-specific Tep4 expression, if silenced, led to distinct responses to certain types of bacteria. This finding suggests that thanacytes have distinct responses to different types of bacteria. It was also found that thanacytes express several proteases that might aid to remove infected cells, similar to those found in cytoplasmic granules of cytotoxic T cells and NK cells. Further studies are warranted to deduce the exact nature and limitations of the thanacyte response (Fu, 2020).

Drosophila models have long been used to study the human blood-vascular system. The prevailing view has been that the mammalian hematopoietic cells differentiate into lymphoid and myeloid lineages. Hemocytes in Drosophila, and other invertebrates, are considered to be restricted to the myeloid lineage, even though they show strong conservation of genetic homology with human immune cells. In line with these thoughts, this study found that Drosophila Aldh is uniquely expressed in Ppn+ PM, while its human homolog ALDH2 is specific to CD14+ monocytes and dendritic cells of the myeloid lineage. CD14+ monocytes make up 2%-10% of all leukocytes, while Ppn+ PM make up ~12% of all plasmatocytes. Both CD14+ monocytes and dendritic cells are part of the mammalian innate immune system, and they have three main functions phagocytosis, antigen presentation, and cytokine production. Interestingly, Ppn+ PM in fly is one of two blood cell subtypes in Drosophila, in which phagocytic activity was denoted (Fu, 2020).

Further, data was uncovered that might blur the invertebrate-mammalian division and expand the notion that the mammalian myeloid and lymphoid blood cells at times diverge from their traditional lineages, especially under challenging conditions. For example, CAH7, expression of which is specific to the CAH7+ PM, encodes a carbonic anhydrase. An enzyme essential for homeostasis of oxygen-hemoglobin binding, which was first discovered in red blood cells. Drosophila has no known red blood cell equivalent. Furthermore, the human homologs to two prominent thanacyte-specific molecular markers, GZMB (Drosophila CG30088) and GZMH (Drosophila CG30090), both encode members of the granzyme family. These serine proteases are released by cytotoxic T cells and NK cells via cytoplasmic granules, and induce targeted lysis of infected cells through endocytosis (Trapani, 2001). Thanacytes express several proteases and show enriched endocytosis pathways, suggesting they might operate in a similar fashion (Fu, 2020).

The transcriptome-wide molecular profiles obtained by scRNA-seq uncovered new cell types and subtypes, and identified new markers to aid in future studies of their functions. Also this analysis unveiled a much more complex Drosophila blood system than described to date. Moreover, it provides hints of potential non-myeloid activity in Drosophila blood cells that might blur the lines of conventional lineage restriction. Studies directly comparing the molecular profiles of human and Drosophila blood cell types, as well as their dynamics in response to immune challenges will be fundamental. The data presented in this study contribute knowledge and identified resources towards an increased understanding of how both systems relate, thereby widening the scope of the Drosophila model to study the human blood system in development, health and disease (Fu, 2020).

Single-cell transcriptome maps of myeloid blood cell lineages in Drosophila

The Drosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and mature hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells, yet molecular underpinnings of the lymph gland hemocytes have been less investigated. This study used single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discover previously undescribed hemocyte types including adipohemocytes, stem-like prohemocytes, and intermediate prohemocytes. Additionally, this study identified the developmental trajectory of hemocytes during normal development as well as the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. Finally, similarities and differences were established between embryonically derived- and larval lymph gland hemocytes. Altogether, this study provides detailed insights into the hemocyte development and cellular immune responses at single-cell resolution (Cho, 2020).

Blood cells are highly specialized cells that play crucial roles in the elimination of foreign threats during immune responses and in various forms of stress responses and development1. Blood cells in Drosophila, collectively called hemocytes, are reminiscent of myeloid-lineage blood cells in vertebrates, and are represented by at least three morphologically distinct hemocyte populations: plasmatocytes (PM), crystal cells (CC), and lamellocytes (LM). Plasmatocytes, which comprise ~95% of the hemocytes, play a role in phagocytosis, tissue remodeling, and cellular immune responses-much like macrophages, their vertebrate counterpart. Crystal cells account for ~5% of the blood population and are characterized by crystalline inclusions made up of prophenoloxidase (ProPO), an enzyme responsible for activating the melanization cascade. Finally, lamellocytes, which are seldom seen in healthy animals grown at normal conditions, mostly differentiate upon parasitic wasp infestation or environmental challenges (Cho, 2020).

Blood development in vertebrates involves the primitive and definitive waves of hematopoiesis. Reminiscent of vertebrate hematopoiesis, two hematopoietic waves have been described during Drosophila development, embryonic and larval lymph gland hematopoiesis. Hematopoiesis in the lymph gland is initiated from hemangioblast-like cells in the embryonic cardiogenic mesoderm, which give rise to the primary lobe of the larval lymph gland. Medially located prohemocytes, which sustain the developmental potential to generate all three mature hemocyte types, constitute the medullary zone (MZ) and continue to proliferate until the early third instar. Mature hemocytes emerge at the distal edge of the lymph gland from mid-second instar and comprise the cortical zone (CZ). Located between the undifferentiated medullary zone and the differentiated cortical zone, is the intermediate zone (IZ) that contains a group of differentiating cells expressing markers for both the medullary zone and the cortical zone. The posterior signaling center (PSC), a small group of cells that secrete various ligands, is located at the medio-posterior side of the lymph gland and regulates proper growth of the rest of the lymph gland. Lymph glands from healthy larvae reared under normal lab conditions generally follow fixed developmental states until late third instar. Remarkably, following the onset of pupariation, the lymph gland disintegrates, allowing hemocytes to disperse into circulation (Cho, 2020).

Female wasps, including those of the genus Leptopilina, attack second-instar larvae via a sharp needle-like ovipositor that efficiently delivers their eggs. Wasp eggs trigger cellular immune responses that accompany lamellocyte differentiation from both embryonically-derived and lymph gland-derived hemocytes. Lamellocytes are seen in circulation by 24h post-infestation; yet, lamellocytes generated in the lymph gland remain in their original location. Within 48h after infestation, a massive differentiation of lamellocytes takes place followed by disruption of the lymph gland. Hemocytes in the lymph gland eventually dissociate into circulation, and mature lamellocytes derived from the lymph gland and hematopoietic pockets encapsulate and neutralize wasp eggs (Cho, 2020).

The Drosophila lymph gland has been largely characterized based on genetic markers and cellular morphology. However, the molecular underpinnings of hematopoietic cells such as different states and the gene regulatory network of each cell type have been less investigated. In addition, questions as to how prohemocytes and mature hemocytes differentiate into lamellocytes upon active immunity, and to what extent hemocytes derived from the embryonic and the lymph gland hematopoiesis differ have been unanswered (Cho, 2020).

This study built a census of myeloid-like Drosophila hemocytes by taking advantage of single-cell RNA sequencing (scRNA-seq) technology and establish a detailed map for larval hemocytes in the developing lymph gland. Classes of hemocytes and their differentiation trajectories were detected and molecular and cellular changes of myeloid-like hemocytes upon immune challenges are described. Furthermore, both distinct and common characteristics of hemocytes originating from embryonic and larval lymph gland lineages were identified. Altogether, This work will stimulate future studies on the development and diverse functions of the myeloid-like blood cell lineage (Cho, 2020).

This study used Drop-seq to build libraries from dissociated cells of the lymph gland. Given that hemocytes are highly susceptible to multiple stresses, it was suspected that cell dissociation process might stress the lymph gland hemocytes. This is apparent in the unexpectedly high number of lamellocytes detected in the scRNA-seq. Generally, wild-type lymph glands rarely produce lamellocytes. These lamellocytes plausibly differentiated following dissociation-induced stress response. Alternatively, the number under normal condition may be undervalued due to the lack of markers to identify early lamellocytes. Another possibility is that lamellocytes, that are usually larger than other hemocytes, are better captured than other hemocytes in the Drop-seq. Nonetheless, to reduce the stress-induced bias, additional measures wetr introduced in this analyses, including the comparison of the single-cell transcriptome with bulk RNA-seq. As a result, scRNA-seq datasets faithfully display single-cell transcriptomes of all known cell types, as well as two other previously undescribed cell types. GST-rich cells, enriched with ROS-responsive and DNA damage genes, emerge during prohemocyte development. Considering that genes enriched in GST-rich cells are also evident in the lymph gland bulk RNA-seq and GST-rich-specific marker genes are detected in wild-type lymph glands, this population cannot be considered a consequence of stressed hemocytes. Rather, this subtype may represent a state that prohemocytes experience during development, or may play an active role in ROS-mediated or GABA-mediated stress responses. Adipohemocytes, on the other hand, share hallmarks of both mature plasmatocytes and lipid metabolism, appearing only at 120h AEL of the lymph gland. Macrophages in vertebrates readily take up lipids and lipoproteins, and accumulation of lipid-containing macrophages, called foam cells, is highlighted in various pathological conditions. In Drosophila, the presence of lipid-containing hemocytes has not been reported. Given these results, and that adipohemocytes are frequently observed in insects, including Aedes aegypti, it is possible that flies also conserve metabolism-oriented hemocytes to coordinate immunity and metabolism (Cho, 2020).

Prohemocytes have been widely considered to represent a uniform cell population based on the expression of marker genes, domeless or Tep4. However, recent studies have suggested their heterogeneity based on uneven expressions of cell cycle markers or bifurcated col expressions. In support of these studies, unbiased subclustering of primary clusters identified different statuses of prohemocytes. First, prohemocytes differ in the expression of cell cycle regulators, implying an asynchrony of prohemocyte development and their states. This observation also accounts for the stochastic cell cycle patterns visualized with the UAS-FUCCI system. Second, dynamic expression patterns were observed of development-related or DNA replication-related and proliferation-related genes in early or late prohemocytes, respectively. In addition, the 120h AEL-specific PH6 (an auxiliary trajectory) denotes unique pathways including steroid biosynthesis-related genes. Lastly, the presence of prohemocytes with more differentiated states is also indicative of their dynamics. Although the presence of the intermediate zone has been recognized, the biological significance of various intermediary states and the previously undescribed functions of endogenous genes including Nplp2 in these subclusters require further investigations (Cho, 2020).

As the most naïve subcluster identified in this study, PH1 (Prohemocyte1), demarcates a group of cells that has not been annotated by previous markers such as Tep4, Antp or col. Discovery of the hidden cell population-PH1, will shed light on understanding the hierarchy of prohemocyte differentiation and enhance the relevance of the lymph gland as a hematopoietic model. Roles for Notch, Stat92E, or scalloped in the earliest state of prohemocytes have been previously suggested by recent studies. Moreover, clonal analyses have shown that cells adjacent to the PSC generate the largest population in the lymph gland. These studies are consistent with the hypothesis that Notch/Delta and JAK/STAT+ cells nearby the PSC sustain latent capacities to produce the entire lymph gland hemocytes. Since this analyses focus on the second to the third instar lymph gland, it will be important to further delineate an ancestor of PH1 and understand its developmental association with Notch+ cells described in the first-instar lymph gland (Cho, 2020).

Comparative analyses on wild-type and wasp-infested lymph glands revealed that wasp infestation exerts a biased differentiation of hemocytes to the lamellocyte lineage. By 24h PI, lymph glands physically remain in place; yet, cells within lymph glands undergo a dynamic differentiation towards early lamellocytes. Interestingly, iPH1 and iPH2 significantly reduce their numbers at 24h PI while amplifying iGST-rich, iPH4, iLM1, and iLM2. The depletion of early prohemocytes and augmentation of the following subclusters could be tightly associated with the PSC considering its role in the PH1 maintenance. An expansion of iPH4 provides a designated pool for the iLMs and could be critical to sufficiently meeting the high demand for immune cells upon wasp parasitism similar to the circulation. In addition to iPH4 committed to lamellocyte differentiation, NimC1+ PM1 transdifferentiates into lamellocytes, revealing two independent routes for lamellocyte differentiation. Unlike other hemocytes, the iPSC remains stationary in its number and transcriptome profile. Hence, PSC may likely function through a post-transcriptional modification in controlling the immune response (Cho, 2020).

Drosophila metamorphosis involves hemocyte mediated macroendocytosis and efferocytosis

Drosophila hemocytes are majorly associated with immune responses, but they also undertake several non-immune functions that are crucial during various stages of development. The activity and behaviour of hemocytes are least documented during the metamorphic phase of fly development. This study describes the activity, form and behaviour of the most abundant type of hemocyte in Drosophila melanogaster, the "plasmatocyte," throughout pupal development. This study reveals different forms of plasmatocytes laden with varying degrees of histolyzing debris (muscle and fat) which extend beyond the size of the cell itself, highlighting the phagocytic capacity of these plasmatocytes. Interestingly, the engulfment of apoptotic debris by plasmatocytes is an actin-dependent process, and by the end of metamorphosis, clearance is achieved. The uptake of apoptotic debris consisting of muscles and lipids by the plasmatocytes provides a model that can be employed to dissect out the relevant components of macroendocytosis and lipid-loaded phagocytosis. This understanding, by itself, is crucial for addressing the emerging role of phagocytes in physiology and pathophysiology (Ghosh, 2020).

Adult Drosophila lack hematopoiesis but rely on a blood cell reservoir at the respiratory epithelia to relay infection signals to surrounding tissues

The use of adult Drosophila melanogaster as a model for hematopoiesis or organismal immunity has been debated. Addressing this question, an extensive reservoir of blood cells (hemocytes) was identified at the respiratory epithelia (tracheal air sacs) of the thorax and head. Lineage tracing and functional analyses demonstrate that the majority of adult hemocytes are phagocytic macrophages (plasmatocytes) from the embryonic lineage that parallels vertebrate tissue macrophages. Surprisingly, no sign of adult hemocyte expansion was observed. Instead, hemocytes play a role in relaying an innate immune response to the blood cell reservoir: through Imd signaling and the Jak/Stat pathway ligand Upd3, hemocytes act as sentinels of bacterial infection, inducing expression of the antimicrobial peptide Drosocin in respiratory epithelia and colocalizing fat body domains. Drosocin expression in turn promotes animal survival after infection. This work identifies a multi-signal relay of organismal humoral immunity, establishing adult Drosophila as model for inter-organ immunity (Sanchez Bosch, 2019).

Drosophila melanogaster has greatly promoted understanding of innate immunity and blood cell development, but the capacity of the adult animal as a model remains a matter of debate. Most studies reported lack of new blood cell production and increasing immunosenescence, while one publication claimed continued hematopoietic activity in adult Drosophila (Sanchez Bosch, 2019).

Drosophila blood cells, or hemocytes, emerge from two lineages that persist into the adult, showing parallels with the two myeloid systems in vertebrates. First, hemocytes originating in the embryo parallel vertebrate tissue macrophages, as they quickly differentiate into plasmatocytes (macrophage-like cells), and subsequently proliferate extensively, mainly in the hematopoietic pockets (HPs) of the larva (Gold, 2014; Gold, 2015; Makhijani, 2011; Makhijani, 2012). At least some of these plasmatocytes can further differentiate into other blood cell types such as crystal cells and, under immune challenge, lamellocytes. Second, hemocytes originating in the lymph gland (LG) also give rise to plasmatocytes, crystal cells, and lamellocytes, yet in the lymph gland they are predominantly generated from blood cell progenitors (prohemocytes). At the beginning of metamorphosis, hemocytes from both the hematopoietic pockets and the lymph gland enter the open circulatory system and intermix. The subsequent fate and capacity of adult blood cells has remained largely unclear. Accordingly, this study comprehensively investigated the hematopoietic capacity of the blood cell system in adult Drosophila. A second part of this study focused on the role of adult blood cells in the humoral immune response, identifying a system of organismal innate immunity that centers on the respiratory epithelia in Drosophila (Sanchez Bosch, 2019).

Historically, Drosophila has been instrumental in the discovery of innate immunity and Toll like receptor (TLR) signaling. Toll and the related immune deficiency (Imd) signaling are evolutionary conserved NFκB family pathways, studied in detail regarding their upstream activation by pathogens and other inputs, and downstream signal transduction components and mechanisms. Targets include antimicrobial peptides (AMPs), which have been investigated for their transcriptional gene regulation and functional properties. TLR signaling has been well established also in vertebrate systems for its roles in infection and inflammation. However, it has been far less understood how multiple tissues or organs communicate with each other to elicit local innate immune responses (Sanchez Bosch, 2019).

Addressing these questions, this study clarifies basic principles of the blood cell system in adult Drosophila and its role in multi-tissue organismal immunity. An extensive blood cell reservoir was identified at the respiratory epithelia and fat body, its dynamics were investigated and probed for various signs of hematopoiesis. A key role of adult blood cells is demonstrated as sentinels of bacterial infection that trigger a humoral response in their reservoir, i.e., the respiratory epithelia and colocalizing domains of the fat body. This response culminates in the expression of the AMP gene Drosocin, which is shown to be significant for animal survival after bacterial infection. This work identifies Imd signaling and Upd3 expression in hemocytes as required steps in this relay of organismal immunity, laying the foundation for the use of adult Drosophila to dissect additional mechanisms of multi-tissue innate immunity in the future (Sanchez Bosch, 2019).

This study discovered a central role for an extensive blood cell reservoir at the respiratory epithelia and fat body of adult Drosophila. The reservoir serves as major receptacle of blood cells and foreign particles, and in addition executes a local humoral immune response of Drosocin expression that promotes animal survival after bacterial infection. Both functions are tied together by hemocytes acting as sentinels of infection, that signal through the Imd pathway and Upd3 to induce Drosocin expression in the tissues of their surrounding reservoir, i.e., the respiratory epithelia and colocalizing domains of the fat body (Sanchez Bosch, 2019).

Historic literature on Drosophila and other insects focused on the adult heart as the site of hemocyte accumulation. It described clusters of hemocytes at the ostia of the heart as 'immune organ', locations where hemocytes and bacteria accumulate. More recently, adult blood cell production at the heart was proposed (Ghosh, 2015). Some studies described functions of hemocytes in other locations, such as at the ovaries or along the gut of adult flies. Taking a more global cryosectioning approach afforded the identification of the largest reservoir of hemocytes in adult Drosophila, which surrounds the respiratory epithelia and is lined by fat body of the thorax and head. It is concluded that hemocytes and particles are delivered to these areas by the streaming hemolymph, even though the detailed anatomy of the open circulatory system remains to be mapped in more detail. Hemocytes may be physically caught in these locations, or in addition may engage in active adhesion. The intimate relationship of hemocytes with the respiratory epithelia, hemolymph, and adjacent fat body may serve interconnected roles, (1) guarding the respiratory epithelia as a barrier to the environment through functions of hemocytes in both phagocytosis and the induction of humoral immunity, and (2) facilitating gas exchange of hemocytes and nearby immune tissues, which in turn may again benefit defense functions. The former may be particularly advantageous in the defense against fungal pathogens that invade Drosophila via the tracheal system as primary route of infection, such as the entomopathogenic fungus B. bassiana. Regarding the latter, a study in caterpillars described the association of hemocytes with trachea, proposing a function for the respiratory system to supply hemocytes with oxygen (Sanchez Bosch, 2019).

Drosophila adult blood cells derive from two lineages: one that originates in the embryo and resembles vertebrate tissue macrophages, and another that produces blood cells in the lymph gland through a progenitor-based mechanism. It is estimated that more than 60% of adult hemocytes derive from the embryonic lineage is surprising, considering past views that the majority of adult hemocytes would derive from the lymph gland. It places more importance on the Drosophila embryonic lineage of hemocytes and suggests additional parallels with tissue macrophages in vertebrates, which persist into adulthood and form a separate myeloid system independent of the progenitor-derived monocyte lineage. Future research will show whether the relative contribution of the two hemocyte lineages to the adult blood cell pool will be the same or different under conditions of stress and immune challenges (Sanchez Bosch, 2019).

Given that embryonic-lineage plasmatocytes are highly proliferative in the hematopoietic pockets of the larva, and lymph gland hemocyte progenitors and some lymph gland plasmatocytes proliferate during larval development, the absence of hemocyte proliferation in the adult may be surprising. Nevertheless, combining the broad evidence supporting lack of significant hematopoietic activity in adult Drosophila, and evidence that Srp in adult Drosophila is not a progenitor marker, the findings robustly contradict an adult hematopoiesis model. The findings further reveal important differences to embryonic development, where Srp is required for the specification of undifferentiated prohemocytes. This study shows that during maturation of the adult animal, hemocytes relocate to the respiratory epithelia and the heart, upon completion of cytolysis of larval fat body cells, thereby refuting claims of new blood cell production at the heart. Similarly, seemingly increased numbers of fluorescently labeled hemocytes following bacterial infection are likely based on infection-induced upregulation of hemocyte-specific genes and their respective enhancers including the reporter HmlΔ-GAL4, UAS-GFP. Enhanced hemocyte expression of Hemolectin (Hml) and other hemocyte-specific markers post-infection has been described previously (Sanchez Bosch, 2019).

Taken together, this broad evidence speaks to a lack of significant hematopoietic capacity of the blood cell system in adult Drosophila. The findings are in agreement with other studies that have reported a lack of hemocyte proliferation in adult Drosophila and functional immunosenescence in aging flies. Despite the scope of conditions tested, the possibility cannot be excluded that some other specific immune challenge or stress might exist that would be potent enough to trigger proliferation- or differentiation-based blood cell production in adult Drosophila. Likewise, it cannot be excluded that adult Drosophila may possess small numbers of proliferation- and/or differentiation-competent progenitors that may have persisted e.g. from the lymph gland posterior lobes; such cells might give rise to new differentiated hemocytes, although according to the current data they would remain insignificant in number (Sanchez Bosch, 2019).

Taking into account the short reproductive phase and relatively short life span of Drosophila, the adult fly may be sufficiently equipped with the pool of hemocytes that is produced in the embryo and larva. In fact, hemocytes do not seem essential for the immediate survival of adult flies: Drosophila ablated of hemocytes, and mutants devoid of hemocytes, survive to adulthood although they are more prone to, and succumb more rapidly to infection. A model is proposed that places emphasis on larval development as the sensitive phase for the expansion and regulation of the adult blood cell pool. In the larva, hemocytes of both the embryonic and lymph gland lineage integrate signals from a variety of internal and external stimuli to adapt to existing life conditions (Sanchez Bosch, 2019).

This work reveals a role for hemocytes in a local humoral immune response of the fat body and respiratory epithelia. Previous studies on hemocyte-ablated flies have reported increases in Defensin and IM1 expression. In contrast, this study finds a positive role for hemocytes in the induction of Drosocin in tissues that form the hemocyte reservoir, i.e., the respiratory epithelia and fat body domains of the head and thorax. The concept of hemocytes promoting AMP expression in other tissues is well established. A role for AMP expression in surface epithelia that interface with the environment was reported in a previous study, and Drosocin expression was described in embryonic and larval trachea and the abdominal tracheal trunks of adult Drosophila, albeit not in the respiratory epithelia (Sanchez Bosch, 2019).

In adult Drosophila, hemocytes tightly localize between the respiratory epithelia and fat body tissue that occupies the space toward to the cuticle exoskeleton. It is proposed that this close anatomical relationship facilitates rapid local signaling. Consistent with previous knowledge that Drosocin expression is lost in imd mutant backgrounds, this study found that hemocyte-autonomous Imd signaling is required, albeit not sufficient, to trigger the infection-induced Drosocin response. Likewise, the Imd pathway upstream receptor PGRP-LC is required in hemocytes, suggesting that DAP-type peptidoglycan recognition and initiation of Imd signaling are a critical step in triggering the Drosocin response. Transcriptional induction of upd3 by Imd signaling is supported by putative Rel binding sites identified in the upd3 genomic region, two of which are even fully conserved across seven Drosophilids including Drosophila melanogaster. The data suggest roles for hemocyte-expressed upd3, and corresponding Jak/Stat signaling in cells of the fat body and respiratory system, all of which are required albeit not sufficient. Overactivation of the pathway paradoxically suppresses Drosocin expression, and even temporally restricted expression of activated hopTumL in trachea was largely lethal, possibly indicating leaky expression of the transgene. Overall, it can only be speculated that the unexpected effects of Jak/Stat overactivation might be due to activation of some negative feedback loop or other complex signaling changes that remain a matter of future investigation (Sanchez Bosch, 2019).

Several reports provide precedent for a role of hemocyte-expressed Upd3 in the induction of immune responses in other target tissues. Following septic injury, upregulation of upd3 in hemocytes triggers induction of stress peptide genes of the turandot family including totA in fat body. Similarly, in response to injury, hemocyte-produced Upd3 induces Jak/Stat signaling in the fat body and gut. Under lipid-rich diet, upd3 is induced in hemocytes, causing impaired glucose homeostasis and reduced lifespan in adult Drosophila. In the larva, hemocyte-derived Upd2 and -3 activate Jak/Stat signaling in muscle, which are required for the immune response against parasitic wasps. However, in the Drosocin response around the reservoir of hemocytes, the data predict that additional signal/s and/or signaling pathway/s are needed to initiate Drosocin expression and potentially restrict its expression to defined fat body domains of the head and thorax. Additional events may include signaling through Toll or other signaling pathways in hemocytes and/or other tissues including the respiratory epithelia and fat body. Likewise, other types of signals may be required, such as reactive oxygen species (ROS) or nitric oxide (NO), which play roles in the relay of innate immune responses to infection and stress, or non-peptide hormones including ecdysone, which confers competence in the embryonic tracheal Drosocin response to bacterial infection and enhances humoral immunity under conditions of dehydration. Lastly, there could be requirement for additional processing to make bacterial ligands accessible for receptors in other tissues, as has been reported for Psidin, a lysosomal protein required in blood cells for degradation of engulfed bacteria and expression of Defensin in the fat body, although this mechanism may not be universal in all systems (Sanchez Bosch, 2019).

This work reveals an active role of endogenous Drosocin expression in survival after bacterial infection. Since the cloning of Drosocin and its classification as inducible antibacterial peptide, Drosocin has been studied for its transcriptional regulation, illustrating its induction under a variety of bacterial and other immune challenges. Drosocin structure and antimicrobial function have been studied in vitro and by overexpression from transgenes in Drosophila and in heterologous vertebrate systems. Consistent with the current findings, a recent study on CRISPR-based Drosocin null mutants reached similar conclusions regarding the requirement of endogenous Drosocin expression for animal survival following E. cloacae infection (Hanson, 2019). Expanding from these findings, this study reveals the anatomical features of Drosocin expression and its unique path of induction. In addition to Drosocin's role in animal survival after bacterial infection, the data suggest contribution of Drosocin to animal survival after injury through PBS injection. Injury has emerged as a factor that affects survival, a phenomenon for which the molecular mechanisms still remain to be determined. Alternatively, considering that the fly surface and living conditions are not sterile and survival experiments are performed over extended periods of time, it cannot be ruled out that PBS injections may have led to inadvertent infection with some low level contaminating microbes. A role for endogenous Drosocin levels in the antimicrobial response is strongly supported by independent data in the literature. Specifically, the minimum inhibitory concentration (MIC) of Drosocin against E. coli and E. cloacae was determined to be well within the range or below the endogenous concentration of Drosocin in the Drosophila hemolymph (MIC is 1 or 2 μM for the glycosylated forms, and 8 or 10 μM for the unglycosylated form, respectively, compared to 40 μM Drosocin in the Drosophila hemolymph (Sanchez Bosch, 2019).

In conclusion, this study revokes the use of adult Drosophila as effective model to study hematopoiesis, and establishes it as promising system for organismal immunity centering on the immune signaling relay at the reservoir of blood cells. At the evolutionary level, this model shows parallels with vertebrate immune cells of the lung and innate immune responses to bacterial infection. The Drosophila model opens countless avenues for exciting future research, e.g., to investigate additional molecular and cellular mechanisms in the immune signaling relay, the role and regulation of the system in the defense against pathogens that invade the trachea as natural route of infection, the use of the same axis by gram-positive or non-bacterial pathogens, and the induction of other AMPs and immune effector genes in the same axis of regulation (Sanchez Bosch, 2019).

Subpopulation of Macrophage-Like Plasmatocytes Attenuates Systemic Growth via JAK/STAT in the Drosophila Fat Body

Drosophila hemocytes, like those of mammals, are given rise from two distinctive phases during both the embryonic and larval hematopoiesis. Embryonically derived hemocytes, mostly composed of macrophage-like plasmatocytes, are largely identified by genetic markers. However, the cellular diversity and distinct functions of possible subpopulations within plasmatocytes have not been explored in Drosophila larvae. This study shows that larval plasmatocytes exhibit differential expressions of Hemolectin (Hml) and Peroxidasin (Pxn) during development. Moreover, removal of plasmatocytes by overexpressing pro-apoptotic genes, hid and reaper in Hml-positive plasmatocytes, feeding high sucrose diet, or wasp infestation results in increased circulating hemocytes that are Hml-negative. Interestingly these Hml-negative plasmatocytes retain Pxn expression, and animals expressing Hml-negative and Pxn-positive subtype largely attenuate growth and abrogate metabolism. Furthermore, elevated levels of a cytokine, Unpaired 3, are detected when Hml-positive hemocytes are ablated, which in turn activates JAK/STAT activity in several tissues including the fat body. Finally, it was observed that insulin signaling is inhibited in this background, which can be recovered by concurrent loss of upd3. Overall, this study highlights heterogeneity in Drosophila plasmatocytes and a functional plasticity of each subtype, which reaffirms extension of their role beyond immunity into metabolic regulation for cooperatively maintaining internal homeostatic balance (Shin, 2020).

Macrophages Use Distinct Actin Regulators to Switch Engulfment Strategies and Ensure Phagocytic Plasticity In Vivo

Macrophages must not only be responsive to an array of different stimuli, such as infection and cellular damage, but also perform phagocytosis within the diverse and complex tissue environments found in vivo. This requires a high degree of morphological and therefore cytoskeletal plasticity. This study uses the exceptional genetics and in vivo imaging of Drosophila embryos to study macrophage phagocytic versatility during apoptotic corpse clearance. Macrophage phagocytosis is highly robust, arising from their possession of two distinct modes of engulfment that utilize exclusive suites of actin-regulatory proteins. "Lamellipodial phagocytosis" is Arp2/3-complex-dependent and allows cells to migrate toward and envelop apoptotic corpses. Alternatively, Diaphanous and Ena drive filopodial phagocytosis to reach out and draw in debris. Macrophages switch to "filopodial phagocytosis" to overcome spatial constraint, providing the robust plasticity necessary to ensure that whatever obstacle they encounter in vivo, they fulfil their critical clearance function (Davidson, 2020).

Parasitoid wasp venom vesicles (venosomes) enter Drosophila melanogaster lamellocytes through a flotillin/lipid raft-dependent endocytic pathway

Venosomes are extracellular vesicles found in the venom of Leptopilina endoparasitoids wasps, which transport and target virulence factors to impair the parasitoid egg encapsulation by the lamellocytes of their Drosophila melanogaster host larva. Using the co-immunolocalization of fluorescent L. boulardi venosomes and one of the putative-transported virulence factors, LbGAP, with known markers of cellular endocytosis, this study showed that venosomes endocytosis by lamellocytes is not a process dependent on clathrin or macropinocytosis and internalization seems to bypass the early endosomal compartment Rab5. After internalization, LbGAP colocalizes strongly with flotillin-1 and the GPI-anchored protein Atilla/L1 (a lamellocyte surface marker) suggesting that entry occurs via a flotillin/lipid raft-dependent pathway. Once internalized, venosomes reach all intracellular compartments, including late and recycling endosomes, lysosomes, and the endoplasmic reticulum network. Venosomes therefore enter their target cells by a specific mechanism and the virulence factors are widely distributed in the lamellocytes' compartments to impair their functions (Wan, 2020).

Intramacrophage ROS Primes the Innate Immune System via JAK/STAT and Toll Activation

Tissue injury is one of the most severe environmental perturbations for a living organism. When damage occurs in adult Drosophila, there is a local response of the injured tissue and a coordinated action across different tissues to help the organism overcome the deleterious effect of an injury. This study shows a change in the transcriptome of hemocytes at the site of tissue injury, with pronounced activation of the Toll signaling pathway. Induction of the cytokine upd-3 and Toll receptor activation occur in response to injury alone, in the absence of a pathogen. Intracellular accumulation of hydrogen peroxide in hemocytes is essential for upd-3 induction and is facilitated by the diffusion of hydrogen peroxide through a channel protein Prip. Importantly, hemocyte activation and production of reactive oxygen species (ROS) at the site of a sterile injury provide protection to flies on subsequent infection, demonstrating training of the innate immune system (Chakrabarti, 2020).

bfc, a novel serpent co-factor for the expression of croquemort, regulates efferocytosis in Drosophila melanogaster

Efferocytosis is the process by which phagocytes recognize, engulf, and digest (or clear) apoptotic cells during development. Impaired efferocytosis is associated with developmental defects and autoimmune diseases. In Drosophila melanogaster, recognition of apoptotic cells requires phagocyte surface receptors, including the scavenger receptor CD36-related protein, Croquemort (Crq, encoded by crq). In fact, Crq expression is upregulated in the presence of apoptotic cells, as well as in response to excessive apoptosis. This study identified a novel gene bfc (booster for croquemort), which plays a role in efferocytosis, specifically the regulation of the crq expression. Bfc protein interacts with the zinc finger domain of the GATA transcription factor Serpent (Srp), to enhance its direct binding to the crq promoter; thus, they function together in regulating crq expression and efferocytosis. Overall, this study shows that Bfc serves as a Srp co-factor to upregulate the transcription of the crq encoded receptor, and consequently boosts macrophage efferocytosis in response to excessive apoptosis. Therefore, this study clarifies how phagocytes integrate apoptotic cell signals to mediate efferocytosis (Zheng, 2021).

Apoptosis is a developmentally programmed cell death process in multicellular organisms essential for the removal of excessive or harmful cells; whereby apoptotic cells (ACs) are swiftly removed by phagocytes to prevent the release of toxins and induction of inflammation, a process crucial for organ formation, tissue development, homeostasis, and normal immunoregulation. In fact, defects in AC clearance (efferocytosis) can lead to the development of various inflammatory and autoimmune diseases. During efferocytosis, the effective clearance of ACs is accomplished through the recognition and binding of engulfment receptors or bridging molecules on the surface of phagocytes to 'eat me' signals exposed on the surface of ACs. After receptor activation, downstream signals trigger actin cytoskeleton rearrangement and membrane extension around the ACs to form phagosomes. Finally, mature phagosomes fuse with lysosomes to form phagolysosomes, where the internalized ACs are ultimately digested and cleared (Zheng, 2021).

Since efferocytosis is conserved throughout evolution, it has been studied not only in mammals but also in Drosophila melanogaster. Of note, in D. melanogaster, ACs are removed by non-professional phagocytes, such as epithelial cells and professional phagocytes, such as macrophages and glial cells. Importantly, Drosophila macrophages perform similar functions to those of mammalian macrophages; they participate in both the phagocytosis of ACs and pathogens. Several engulfment receptors have been identified as key players in the recognition and removal of ACs in Drosophila. Franc and colleagues first characterized Croquemort (Crq), a Drosophila CD36-related receptor required by macrophages to engulf ACs. Additionally, Draper (Drpr, a homolog of CED-1/MEGF10) also mediates AC clearance in both glia and macrophages; JNK signaling plays a role in priming macrophages to rapidly respond to injury or microbial infections. Of note, Drpr and its adapter Dmel\Ced-6 (GULP homolog) also seemed important for axon pruning and the engulfment of degenerating neurons by glial cells. The Src tyrosine kinase Src42A (Frk homolog) promotes Drpr phosphorylation and its association with another soluble tyrosine kinase, Shark (ZAP70 homolog), which in turn activates the Drpr pathway. In addition to Drpr, Six-Microns-Under (SIMU) [10] and integrin αPS3 [21] contribute to efferocytosis. SIMU, a Nimrod family cell surface receptor, functions upstream of Drpr to mediate the recognition and clearance of ACs as well as of non-apoptotic cells at wound sites through the recognition of phosphatidylserine (PS). Importantly, the transcriptional factor Serpent (Srp), a GATA factor homolog, was recently found to be required for the efficient phagocytosis of ACs in the context of Drosophila embryonic macrophages and acted via the regulation of SIMU, Drpr, and Crq (Zheng, 2021).

Searching for other genes required for efferocytosis, this study performed transcriptomic analysis (RNA-seq) and RNAi screening, and discovered 12 genes required for AC clearance in Drosophila S2 cells. In particular, a novel gene, bfc (booster for croquemort), involved in efferocytosis, encodes a specific regulatory factor for the crq transcriptional expression, both in vitro and in vivo. Importantly, this study demonstrated that the GATA factor Srp directly binds to the crq promoter, while Bfc strengthens this binding by interacting with the Srp zinc finger domain. Therefore, a model is proposed in which Bfc cooperates with Srp to enhance crq expression and subsequently induce efferocytosis in D. melanogaster (Zheng, 2021).

In mammals, ACs are recognized by CD36, one of the several phagocyte cell surface receptors, with the AC surface molecules serving as cognate 'eat-me' signals/ligands. ACs also secrete molecules that attract distant phagocytes and modulate the immune response or phagocytic receptor activity. However, the mechanisms underlying this effect remain unclear. Crq is a CD36-related scavenger receptor in Drosophila and is expressed immediately after the onset of apoptosis in embryonic macrophages. The expression of Crq is regulated by the extent of apoptosis, although the regulatory mechanisms by which ACs control the expression of Crq and subsequently induce phagocytosis in embryonic macrophages have not been described (Zheng, 2021).

This study has revealed a novel protein, Bfc (Booster for Crq), that plays a key role in efferocytosis via specifically regulating the expression of crq in a manner dependent on the extent of apoptosis. Bfc interacts with the zinc finger domain of the transcription factor Srp as a cofactor to enhance the binding of Srp to the crq promoter, leading to the upregulation of crq expression and the consequent induction of efferocytosis in Drosophila melanogaster. Importantly, the data reveal the molecular mechanisms by which ACs affect Crq expression, as well as how the phagocytic ability of embryonic macrophages is boosted in the presence of excessive apoptosis (Zheng, 2021).

This study found that in S2 cells, the ACs induced the transcriptional upregulation of crq. In vivo, the macrophages developed as early as the first wave of developmentally programmed apoptosis began at embryogenic stage 11, when the expression of crq was activated and subsequently became widespread throughout the embryo. Importantly, these results are similar to the regulatory mechanisms associated with the expression of other phagocytic receptors, such as Drpr and integrin. For instance, studies showed that AC engulfment rapidly triggers an intracellular calcium burst followed by increased levels of drpr transcripts in Drosophila macrophages; similarly, Draper and integrins become apically enriched soon after the engulfment of apoptotic debris in epithelial follicle cells (Zheng, 2021).

That the expression of crq was elevated early after the co-culture of ACs and S2 cells, but gradually decreased to the basal levels as efferocytosis continued, suggesting that the regulation of AC clearance and crq expression follow a similar pattern. It was demonstrated that most AC samples added to live S2 cells were composed of apoptotic cells rather than necrotic cells. However, the upregulated expression of genes in response to the presence of a few necrotic cells cannot eliminated. Indeed, based on transcriptome analysis, 12 genes were identified that are required for AC clearance, which was confirmed by subsequent efferocytosis assays using their individual knockdown in S2 cells. Interestingly, among the 12 genes, two were related to innate immunity. CecA1, regulated at the transcriptional level encodes an antibacterial peptide, as well as a secreted protein that mediates the activation of the Toll pathway during bacterial infection. This result may contradict the discreet nature of the apoptotic process. However, ATPs released by bacteria are known to mediate inflammation, and the toll-like receptor 4 (TLR4) is activated by ACs to promote dendritic cell maturation and innate immunity in human monocyte-derived dendritic cells. These results indicate that innate immune pathways are activated in the presence of ACs, and may contribute to their recognition or clearance in Drosophila (Zheng, 2021).

Among these 12 genes, CG9129 (bfc) and CG30172 regulated the expression of crq and hence, efferocytosis. Further studies must be performed to elucidate the role of CG30172 in efferocytosis. On the other hand, The role played by bfc in efferocytosis as well as the underlying mechanism was clearly dissected. Using several different experimental approaches, this study demonstrated that bfc regulates crq expression in response to excessive apoptosis. First, bfc RNAi treatment decreased the crq expression levels in S2 cells exposed to ACs, but not in the absence of ACs. Second, the increase in crq transcription was proportional to the extent of apoptosis in embryos, which was blocked by the loss of bfc. Notably, other phagocytic receptors have been reported to be activated by dying cells. The integrin heterodimer αPS3/βPS can be enriched in epithelial follicle cells after the engulfment of dying germline cells. In addition, Drpr expression increases in follicle and glial cells, which activates the downstream JNK signaling during the clearance of apoptotic germline cells and neurons, respectively. Collectively, the available scientific literature suggests that the expression of phagocytic receptors can be stimulated by the presence of excessive ACs to improve the phagocytic activity of macrophages or epithelial cells in different tissues (Zheng, 2021).

Bioinformatics analysis of the conserved domains and gene structure indicated that Bfc does not likely function directly as a transcription factor. This study identified Srp as a Bfc interaction partner using yeast two-hybrid and Co-IP analyses. Shlyakhover (2018) reported that Srp is required for the expression of SIMU, Drpr, and Crq receptors in embryonic macrophages; however, the current results demonstrated that bfc only affects the expression of crq expression through interaction with Srp, with no impact on the expression of several other genes. A plausible hypothesis for this phenotype is that Bfc assistance for Srp binding to the promoters of simu and drpr, may have limited effects. Thus, the results suggest that Bfc may regulate the Crq expression levels in the first wave of AC recognition via binding to Srp, whereas other regulatory factors participate in the Srp-mediated regulation of Drpr and SIMU (Zheng, 2021).

Srp directly binds to the DNA consensus sequence GATA of the crq promoter via its highly conserved Cys-X2-Cys-X17-Cys-X2-Cys zinc finger binding domain (C4 motif). Meanwhile, Srp also interacts with Bfc through its zinc finger domain; curiously, while the mutation of the C4 motif did not affect the latter interaction, it completely blocked the former. Importantly, it was also shown that mutation in the GATA site abolished the expression of the crq in Drosophila embryo macrophages. As a potential Srp cofactor, Bfc increased the ability of Srp to bind to the crq promoter, while bfc knockdown inhibited the crq transcriptional activity. Ush (homolog of FOG-2 in Drosophila), a cofactor of GATA transcriptional factors, can bind Srp and limit crystal cell production during Drosophila blood cell development. Interestingly, genetic studies have demonstrated that Ush acts with Srp to maintain the pluripotency of hemocyte progenitors and suppresses their differentiation. Ush was reported to repress crq expression by interacting with the isoform of Srp, SrpNC (with two GATA zinc finger) while the other isoform of Srp, SrpC (with one GATA zinc finger) induced crq expression, which may indicate Bfc and Ush act on different isoforms of Srp to regulate crq expression by opposite mechanisms (Zheng, 2021).

Although the results elucidate several factors that contribute to efferocytosis in Drosophila embryos, some mechanistic details remain unresolved; for instance, how ACs induce Bfc-mediated regulation of crq expression in macrophages remains unclear. Bfc regulates Crq expression and efferocytosis, but not macrophage development. Moreover, this study found that Bfc-mediated activation of crq transcription and Crq accumulation leads to positive feedback to promote increased Bfc expression, which is required for engulfment. As expected, the upregulation of Bfc expression occurred earlier than that of crq in S2 cells after incubation with ACs. Therefore, further studies are required to elucidate the upstream signals in the context of the crq-mediated regulation of bfc expression. As previous studies have shown that Crq is required for phagosome maturation during the clearance of neuronal debris by epithelial cells and bacterial clearance, further studies should be conducted to determine whether Bfc is involved in the clearance of neuronal debris (Zheng, 2021).

This study is not without limitations. For instance, other potential regulators of efferocytosis, whose expression is not affected by ACs could not be detected in this study. In mammals, CD36 is involved in the clearance of ACs and regulates the host inflammatory response. As a CD36 family homolog, Crq promotes the clearance of ACs and bacterial uptake via efferocytosis. Researchers have reported that the GATA factor Srp is required for Crq expression; this study confirmed this finding and showed that Srp directly binds to the crq promoter via its GATA binding site, which is enhanced by Bfc. However, no apparent Bfc homologs exist in vertebrates, and whether GATA factors regulate the CD36 family in a mechanism similar to that in flies remains unclear. Nevertheless, it is predicted that one or more functional homologs of Bfc may exist in mammals and are likely involved in apoptotic cell clearance. Unraveling them as well as determining whether and how bfc participates in eliminating pathogens and innate immunity is essential (Zheng, 2021).

In summary, this study has shown that the expression of the engulfment receptor Crq is transcriptionally regulated by the presence of ACs, via Srp, and its newly identified cofactor, Bfc. Altogether, these findings imply that macrophages adopt a precise mechanism to increase the expression of engulfment receptors to boost their phagocytic activity, in the presence of excessive ACs. A similar role and mechanism is anticipated in the context of mammalian engulfment receptors in response to excessive ACs. Therefore, the findings of this study have significant implications for a wide range of human diseases, including those associated with aberrant apoptotic cell death and efferocytosis, such as tumor progression, neurodegenerative disorders, and other severe inflammatory conditions (Zheng, 2021).

Cell division in tissues enables macrophage infiltration

Cells migrate through crowded microenvironments within tissues during normal development, immune response, and cancer metastasis. Although migration through pores and tracks in the extracellular matrix (ECM) has been well studied, little is known about cellular traversal into confining cell-dense tissues. This study found that embryonic tissue invasion by Drosophila macrophages requires division of an epithelial ectodermal cell at the site of entry. Dividing ectodermal cells disassemble ECM attachment formed by integrin-mediated focal adhesions next to mesodermal cells, allowing macrophages to move their nuclei ahead and invade between two immediately adjacent tissues. Invasion efficiency depends on division frequency, but reduction of adhesion strength allows macrophage entry independently of division. This work demonstrates that tissue dynamics can regulate cellular infiltration (Akhmanova, 2022).

Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure

Changes in cell morphology require the dynamic remodeling of the actin cytoskeleton. Calcium fluxes have been suggested as an important signal to rapidly relay information to the actin cytoskeleton, but the underlying mechanisms remain poorly understood. This study identified the EF-hand domain containing protein EFhD2/Swip-1 as a conserved lamellipodial protein strongly upregulated in Drosophila macrophages at the onset of metamorphosis when macrophage behavior shifts from quiescent to migratory state. Loss- and gain-of-function analysis confirm a critical function of EFhD2/Swip-1 in lamellipodial cell migration in fly and mouse melanoma cells. Contrary to previous assumptions, TIRF-analyses unambiguously demonstrate that EFhD2/Swip-1 proteins efficiently cross-link actin filaments in a calcium-dependent manner. Using a single-cell wounding model, this study showed that EFhD2/Swip-1 promotes wound closure in a calcium-dependent manner. Mechanistically, these data suggest that transient calcium bursts reduce EFhD2/Swip-1 cross-linking activity and thereby promote rapid reorganization of existing actin networks to drive epithelial wound closure (Lehne, 2022).

MicroRNA miR-274-5p supresses Found-in-Neurons associated with Melanotic mass Formation and Developmental Growth in Drosophila

The hematopoietic system plays a crucial role in immune defense response and normal development, and it is regulated by various factors from other tissues. The dysregulation of hematopoiesis is associated with melanotic mass formation; however, the molecular mechanisms underlying this process are poorly understood. This study observed that the overexpression of miR-274 in the fat body resulted in the formation of melanotic masses. Moreover, abnormal activation of the and JAK/STAT signaling pathways was linked to these consequences. In addition to this defect, miR-274 overexpression in the larval fat body decreased the total tissue size, leading to a reduction in body weight. miR-274-5p was found to directly supress the expression of found-in-neurons (fne), which encodes an RNA-binding protein. Similar to the effects of miR-274 overexpression, fne depletion led to melanotic mass formation and growth reduction. Collectively, miR-274 plays a regulatory role in the fne-JNK signaling axis in melanotic mass formation and growth control (Kim, 2023).

Gbb Regulates Blood Cell Proliferation and Differentiation through JNK and EGFR Signaling Pathways in the Drosophila Lymph Gland

The Drosophila lymph gland is an ideal model for studying hematopoiesis, and unraveling the mechanisms of Drosophila hematopoiesis can improve understanding of the pathogenesis of human hematopoietic malignancies. Bone morphogenetic protein (BMP) signaling is involved in a variety of biological processes and is highly conserved between Drosophila and mammals. Decapentaplegic (Dpp)/BMP signaling is known to limit posterior signaling center (PSC) cell proliferation by repressing the protooncogene dmyc. However, the role of two other TGF-β family ligands, Glass bottom boat (Gbb) and Screw (Scw), in Drosophila hematopoiesis is currently largely unknown. This study showed that the loss of Gbb in the cortical zone (CZ) induced lamellocyte differentiation by overactivation of the EGFR and JNK pathways and caused excessive differentiation of plasmatocytes, mainly by the hyperactivation of EGFR. Furthermore, it was found that Gbb was also required for preventing the hyperproliferation of the lymph glands by inhibiting the overactivation of the Epidermal Growth Factor Receptor (EGFR) and c-Jun N-terminal Kinase (JNK) pathways. These results further advance understanding of the roles of Gbb protein and the BMP signaling in Drosophila hematopoiesis and the regulatory relationship between the BMP, EGFR, and JNK pathways in the proliferation and differentiation of lymph gland hemocytes (Zhang, 2023).

Transcriptome profile in Drosophila Kc and S2 embryonic cell lines. G3 (Bethesda)

Drosophila melanogaster cell lines are an important resource for a range of studies spanning genomics, molecular genetics and cell biology. Amongst these valuable lines are Kc167 and S2 cells, which were originally isolated in the late 1960s from embryonic sources and have been used extensively to investigate a broad spectrum of biological activities including cell-cell signaling and immune system function. Whole-genome tiling microarray analysis of total RNA from these two cell types was performed as part of the modENCODE project over a decade ago and revealed that they share a number of gene expression features. This study expands on these earlier studies by using deep coverage RNA sequencing approaches to investigate the transcriptional profile in Kc and S2 cells in detail. Comparison of the transcriptomes reveals that approximately 75% of the 13,919 annotated genes are expressed at a detectable level in at least one of the cell lines, with the majority of these genes expressed at high levels in both cell lines. Despite the overall similarity of the transcriptional landscape in the two cell types, 2588 differentially expressed genes are identified. Many of the genes with the largest fold change are known only by their "CG" designations, indicating that the molecular control of Kc and S2 cell identity may be regulated in part by a cohort of relatively uncharacterized genes. These data also indicate that both cell lines have distinct hemocyte-like identities, but share active signaling pathways and express a number of genes in the network responsible for dorsal-ventral patterning of the early embryo (Klonaros, 2023).

Lipopolysaccharide -mediated resistance to host antimicrobial peptides and hemocyte-derived reactive-oxygen species are the major Providencia alcalifaciens virulence factors in Drosophila melanogaster

Bacteria from the genus Providencia are ubiquitous Gram-negative opportunistic pathogens, causing "travelers' diarrhea", urinary tract, and other nosocomial infections in humans. Some Providencia strains have also been isolated as natural pathogens of Drosophila melanogaster. This study investigated the virulence factors of a representative Providencia species-P. alcalifaciens. A P. alcalifaciens transposon mutant library was generated, and an unbiased forward genetics screen was performed in vivo for attenuated mutants. The screen uncovered 23 mutants with reduced virulence. The vast majority of them had disrupted genes linked to lipopolysaccharide (LPS) synthesis or modifications. These LPS mutants were sensitive to cationic antimicrobial peptides (AMPs) in vitro and their virulence was restored in Drosophila mutants lacking most AMPs. Thus, LPS-mediated resistance to host AMPs is one of the virulence strategies of P. alcalifaciens. Another subset of P. alcalifaciens attenuated mutants exhibited increased susceptibility to reactive oxygen species (ROS) in vitro and their virulence was rescued by chemical scavenging of ROS in flies prior to infection. Using genetic analysis, it was found that the enzyme Duox specifically in hemocytes is the source of bactericidal ROS targeting P. alcalifaciens. Consistently, the virulence of ROS-sensitive P. alcalifaciens mutants was rescued in flies with Duox knockdown in hemocytes. Therefore, these genes function as virulence factors by helping bacteria to counteract the ROS immune response. This reciprocal analysis of host-pathogen interactions between D. melanogaster and P. alcalifaciens identified that AMPs and hemocyte-derived ROS are the major defense mechanisms against P. alcalifaciens, while the ability of the pathogen to resist these host immune responses is its major virulence mechanism (Shaka, 2022)

The aim of this study was to dissect the host-pathogen interactions between Providencia and D. melanogaster. To achieve this aim, various genetic approaches were used that enabled determination of the contributions of both pathogen and host to the outcome of the infection. First, the responses of the fruit fly to Pa infection was characterized and, using mutant analysis, the Imd pathway and iron sequestration were identified as prominent defense mechanisms against Pa. Second, an unbiased forward genetics screen was performed using a transposon mutant library that were generated for this purpose, and Pa virulence factors necessary to infect the fly were identified. This mutant library has the potential to serve as a valuable resource for exploring the genetic basis for all Pa traits. Third, mutants of the major immune pathways in Drosophila were used, and they were infected with attenuated Pa mutants to identify pathogen virulence factors that allow the bacteria to respond to specific immune defenses and evade immune clearance. Thereby, this study dissected both sides of host-pathogen relationship in a Drosophila-Providencia model and provided the first insights into the molecular mechanisms of Pa virulence (Shaka, 2022)

To identify Pa virulence factors, an in vivo screen was performed which yielded 23 attenuated mutants. The majority of these mutants (15/23) had transposon insertions in genes involved in LPS biosynthesis and LPS modifications, pointing towards a vital role of intact LPS in Pa pathogenesis. This finding is consistent with a well-known role of LPS in host-pathogen interactions. At the mechanistic level, LPS protects Pa from Drosophila Imd pathway-dependent AMPs, particularly Drosocin. Consistent with this, Pa LPS mutants showed increased susceptibility in vitro to the cationic AMP polymyxin B and their virulence was restored in Relish and ΔAMP mutant flies deficient for Imd-dependent AMPs. The finding that Pa LPS mediates resistance to host AMPs complements numerous previous studies in diverse pathogens that reported a similar protective function of LPS against host innate defenses. Several studies that used Drosophila as an infection model also discovered LPS as an essential protective barrier against insect AMPs. For example, another study found that LPS O-antigen-deficient Serratia marcescens mutants were attenuated in wild-type flies but not in an Imd pathway mutant. A similar phenotype was reported for F. novicida mutants with affected LPS. These data demonstrate that a major determinant of virulence in several pathogens is the LPS-mediated ability to resist the systemic immune response. Additionally, LPS was shown to facilitate microbiota-host interactions. For instance, LPS biosynthesis mutants of Acetobacter fabarum, a Drosophila commensal, had a reduced ability to colonize the fruit fly intestine. While the mechanism behind this phenotype has not been investigated yet, increased sensitivity to intestinal AMPs is a likely reason, as shown for the human commensal Bacteroides thetaiotaomicron. Among the LPS mutants, ArnA (pmrA) (PL11H9) was found that encodes an enzyme that catalyzes the formation of modified arabinose UDP-L-4-formamido-arabinose (UDP-L-Ara4FN). The modified arabinose reduces the negative charge of lipid A and the binding of cationic AMPs. This is the most commonly observed LPS modification implicated in cationic AMP resistance. This modification is also crucial for Yersinia pestis resistance to the insect cecropin-like AMP cheopin (Shaka, 2022)

In addition to mutations affecting LPS, several were uncovered that disrupt lipoproteins, like OmpA (PL13H10), NlpI (PL7D10), and YbaY (PL5A4). While YbaY is poorly characterized, OmpA and NlpI were previously implicated in the virulence of different pathogens. Whereas OmpA contributes to virulence in various ways ranging from facilitating adhesion and invasion to conferring resistance to serum, NlpI function in virulence is less clear. The results suggest that all three lipoproteins mutants behave like LPS mutants–they are susceptible to polymyxin B and their virulence is rescued in an AMP mutant, indicating that their reduced virulence is due to an increased susceptibility to host AMPs. The mechanism behind this phenotype requires further investigation, however NlpI was shown to be essential for cell envelop integrity, which might contribute to increased sensitivity to AMPs. The screen uncovered two additional peptidoglycan-associated lipoproteins, TolB (PL2D4) and Pal (PL4B5), that are part of a multiprotein complex, the Tol-Pal system. It bridges between the peptidoglycan and the outer membrane and is important for proper structure and function of the outer membrane. Importantly, TolA and Pal are necessary for correct surface polymerization of O-antigen chains, likely explaining the sensitivity of tol and pal mutants to detergents and several antibiotics. Similar to the Pa tol and pal mutants, F. novicida mutants in these genes were attenuated in Drosophila infection and more sensitive to host AMPs (Shaka, 2022)

The second largest group of mutants with reduced virulence that were identified constitutes ROS-sensitive mutants. Since it was possible rescue the virulence of these mutants by chemical or genetic ROS scavenging, their attenuated virulence is likely due to an inability to resist host ROS produced in response to infection. Among such ROS-sensitive mutants, only the one lacking cytochrome oxidase (PL1A3) was previously shown to be required for virulence in other bacteria by enhancing the tolerance to oxidative stress. Some other genes, like dihydrolipoyl dehydrogenase (PL4F11) and typA (PL6B7), were also linked to virulence but not necessarily via ROS sensitivity. No previous evidence was found of the role of ATPase RavA stimulator ViaA (PL14C2) in virulence, however there seems to be a link to ROS response in E. coli. Therefore, further investigation of the identified genes is required to clarify their role in bacterial virulence and ROS sensitivity. While previous studies identified several sources of ROS in flies, including melanisation, hemocytes, Nox and, Duox, the results showed that Duox specifically in hemocytes is the major producer of ROS in case of Pa infection. Notably, in case of F. novicida melanisation played a prominent role as a source of ROS. An interesting avenue for future studies would be to understand the differences between Duox- and melanisation-derived ROS and their preferential activity against specific pathogens (Shaka, 2022)

The screen also identified several hypothetical proteins. Using ROS and polymyxin B sensitivity assays and rescue in AMP- and ROS-deficient flies, it was shown that PL4E6 and PL11H8 contribute to bacterial resistance to host AMPs, while PL6D10 is necessary to survive ROS exposure. Thus, with this approach a mechanism of virulence could be assigned to hypothetical proteins with unknown function. However, how those protein contribute to ROS or AMP sensitivity remains unknown (Shaka, 2022)

One Pa mutant (Sigma-E factor regulatory protein rseB, PL13C10), was identified that was not sensitive to ROS and polymyxin in vitro. However, the virulence of this mutant was rescued in Relish and AMP-deficient flies. Very likely the rescue phenotype could be due to sensitivity to additional antimicrobial peptides produced by flies. Such increased sensitivity to AMPs is possible given the role of Sigma-E factor in cell envelope integrity (Shaka, 2022)

Among all AMPs tested, Drosocin proved to be particularly important in controlling Pa infection. Consistent with the Pa LPS mutants, F. novicida mutants in LPS were particularly sensitive to Drosocin. Considering that Drosocin is known to bind bacterial LPS, alterations in LPS might promote Drosocin interactions with LPS and bacterial killing or make intracellular targets more accessible. A previous in vivo analysis of AMP specificity has shown that Drosocin plays a critical role in controlling Enterobacter cloacae infection. A recent study confirmed this finding, however additionally reported that the Drosocin gene encodes not one, but two AMPs: Drosocin and IM7 (newly named as Buletin). Buletin but not Drosocin contributes to host defense against Providencia burhodogranariea infection. Since the Drosocin mutant that was used lacked both Drosocin and Buletin and the Drosocin overexpression line similarly produced both peptides, it remains to be tested whether Drosocin or Buletin or both peptides together are involved in the defense against Pa (Shaka, 2022)

While in vivo experiments demonstrate that AMPs are the major Relish-regulated molecules controlling Pa LPS mutants, in vitro assays with synthesized Drosophila AMPs were not conclusive. None of the three Drosophila AMPs that were tested, Cecropin A, Cecropin B, and Diptericin B, showed activity against Pa. Considering the high specificity of some AMP-microbe interactions, it could be that the peptides that were tested have no effect on Pa. Indeed, based on in vivo results, Drosocin, which was not available for an in vitro test, is the primary AMP controlling Pa infection. Additionally, in vitro effects of AMPs can be different than in vivo effects of mutants or knockdowns for the same AMPs, suggesting that physiological context or interaction among peptides is important. Also, there are a number of technical reasons why in vitro assays may not reflect in vivo activities, including AMPs adhering to plastic assay plates, differences in salt concentrations or pH, stress on microbes, interactions among AMPs and between AMPs and other components of the immune system. These potential issues have to be considered when interpreting the results of in vitro antimicrobial tests performed with AMPs (Shaka, 2022)

Contrary to expectations, the screen did not hit any bacterial effectors, like toxins, that might be responsible for damaging the host. Since toxins are likely to be redundant, disruption of an individual toxin gene may not give a phenotype. Similarly, no mutants were identified in secretion systems, suggesting that Pa does not require effector translocation to infect Drosophila. The only toxin that was so far implicated in Pa pathogenesis is cytolethal distending toxin which blocks eukaryotic cell proliferation. Interestingly, Pa LPS was shown to cause epithelial barrier dysfunction by reducing occludin levels in Caco-2 cell monolayers and induced apoptosis in calf pulmonary artery endothelial cells. Thus, LPS might not only mediate resistance to host AMPs but also act as an effector-like molecule (Shaka, 2022)

By discovering the mechanisms of Pa resistance to host AMPs and ROS, this study opens the doors to potential strategies to exploit such Pa mechanisms and sensitize the pathogen to host defenses to improve infection treatment. To illustrate the feasibility of such an approach, polymyxin B treatment was used to disrupt Pa LPS in vivo, and it was found to be sufficient to improve Drosophila survival after infection. Such beneficial effect of polymyxin B required functional Imd pathway signalling and was independent of direct bactericidal activity, suggesting that disruption of the major barrier against AMPs sensitizes the pathogen to host defenses. These results suggest that affecting LPS function might be a useful strategy to treat Providencia infections, particularly those resistant to antibiotics (Shaka, 2022)

Sensitizing Pa to host ROS also appears to be an attractive anti-virulence strategy, considering that resistance to host ROS is one of the key Pa virulence mechanisms that was identified. Some compounds were shown to sensitize the pathogens to oxidative stress and immune clearance but in a species-specific manner. For example, 2-[2-nitro-4-(trifluoromethyl) benzoyl]-1,3-cyclohexanedione (NTBC) treatment inhibits production of pyomelanin pigment and increases sensitivity of pyomelanogenic Pseudomonas aeruginosa strains to oxidative stress. Similarly, BPH-642 –cholesterol biosynthesis inhibitor, blocked biosynthesis of staphyloxanthin antioxidant pigment in S. aureus, resulting in increased immune clearance in a mouse infection model. However, to date there are no known compounds that would predispose Pa or generally any pathogen to ROS without being toxic to the host, thus limiting the development of ROS-potentiating anti-infectives (Shaka, 2022)

In summary, reciprocal analysis of interactions between D. melanogaster and P. alcalifaciens revealed that the host relies on Imd-dependent AMPs and hemocyte-derived ROS as major branches of immunity that are important for fighting infection with P. alcalifaciens. On the pathogen side, it was found that the ability to resist these host immune responses is the major virulence mechanism of P. alcalifaciens. Leveraging this knowledge has great potential to improve P. alcalifaciens infection treatment either by potentiating the host defenses or disrupting pathogen virulence (Shaka, 2022).

Effects of valproate on seizure-like activity in Drosophila melanogaster with a knockdown of Ube3a in different neuronal populations as a model of Angelman Syndrome

Drosophila blood cells called hemocytes form an efficient barrier against infections and tissue damage. During metamorphosis, hemocytes undergo tremendous changes in their shape and behavior, preparing them for tissue clearance. Yet, the diversity and functional plasticity of pupal blood cells have not been explored. This study combine single-cell transcriptomics and high-resolution microscopy to dissect the heterogeneity and plasticity of pupal hemocytes. We identified undifferentiated and specified hemocytes with different molecular signatures associated with distinct functions such as antimicrobial, antifungal immune defense, cell adhesion or secretion. Strikingly, a highly migratory and immune-responsive pupal cell population was identified expressing typical markers of the posterior signaling center (PSC), which is known to be an important niche in the larval lymph gland. PSC-like cells become restricted to the abdominal segments and are morphologically very distinct from typical Hemolectin (Hml)-positive plasmatocytes. G-TRACE lineage experiments further suggest that PSC-like cells can transdifferentiate to lamellocytes triggered by parasitoid wasp infestation. In summary, this study presents the first molecular description of pupal Drosophila blood cells, providing insights into blood cell functional diversification and plasticity during pupal metamorphosis (Moore, 2023).

Ush regulates hemocyte-specific gene expression, fatty acid metabolism and cell cycle progression and cooperates with dNuRD to orchestrate hematopoiesis

U-shaped (Ush), a multi-zinc finger protein, maintains the multipotency of stem cell-like hemocyte progenitors during Drosophila hematopoiesis. Using genomewide approaches this study has revealed that Ush binds to promoters and enhancers and that it controls the expression of three gene classes that encode proteins relevant to stem cell-like functions and differentiation: cell cycle regulators, key metabolic enzymes and proteins conferring specific functions of differentiated hemocytes. Complementary biochemical approaches were employed to characterise the molecular mechanisms of Ush-mediated gene regulation. Distinct Ush isoforms were uncovered, one of which binds the Nucleosome Remodeling and Deacetylation (NuRD) complex (see HDAC1) using an evolutionary conserved peptide motif. Remarkably, the Ush/NuRD complex specifically contributes to the repression of lineage-specific genes but does not impact the expression of cell cycle regulators or metabolic genes. This reveals a mechanism that enables specific and concerted modulation of functionally related portions of a wider gene expression programme. Finally, genetic assays were used to demonstrate that Ush and NuRD regulate enhancer activity during hemocyte differentiation in vivo and that both cooperate to suppress the differentiation of lamellocytes, a highly specialised blood cell type. These findings reveal that Ush coordinates proliferation, metabolism and cell type-specific activities by isoform-specific cooperation with an epigenetic regulator (Lenz, 2021).

Drosophila Hox genes induce melanized pseudo-tumors when misexpressed in hemocytes

Hox genes are early determinants of cell identity along the anterior-posterior body axis across bilaterians. Several late non-homeotic functions of Hox genes have emerged in a variety of processes involved in organogenesis in several organisms, including mammals. Several studies have reported the misexpression of Hox genes in a variety of malignancies including acute myeloid leukemia. The Hox genes Dfd, Ubx, abd-A and Abd-B were overexpressed via the UAS-Gal4 system using Cg-Gal4, Lsp2-Gal4, He-Gal4 and HmlD3-Gal4 as specific drivers. Genetic interaction was tested by bringing overexpression lines in heterozygous mutant backgrounds of Polycomb and trithorax group factors. Larvae were visually scored for melanized bodies. Circulating hemocytes were quantified and tested for differentiation. Pupal lethality was assessed. Expression of Dfd, Ubx and abd-A, but not Abd-B in the hematopoietic compartment of Drosophila led to the appearance of circulating melanized bodies, an increase in cell number, cell-autonomous proliferation, and differentiation of hemocytes. Pupal lethality and melanized pseudo-tumors were suppressed in Psc¹ and esc² backgrounds while polycomb group member mutations Pc¹ and Su(z)12³ and trithorax group member mutation Trl^R85 enhanced the phenotype. Dfd, Ubx and abd-A are leukemogenic. Mutations in Polycomb and trithorax group members modulate the leukemogenic phenotype. This RNAseq of Cg-Gal4 > UAS-abd-A hemocytes may contain genes important to Hox gene induced leukemias (Ponrathnam, 2021).

The Epidermal Growth Factor Ligand Spitz Modulates Macrophage Efferocytosis, Wound Responses and Migration Dynamics During Drosophila Embryogenesis

How multifunctional cells such as macrophages interpret the different cues within their environment and undertake an appropriate response is a key question in developmental biology. Understanding how cues are prioritized is critical to answering this - both the clearance of apoptotic cells (efferocytosis) and the migration toward damaged tissue is dependent on macrophages being able to interpret and prioritize multiple chemoattractants, polarize, and then undertake an appropriate migratory response. This study investigate the role of Spitz, the cardinal Drosophila epidermal growth factor (EGF) ligand, in regulation of macrophage behavior in the developing fly embryo, using activated variants with differential diffusion properties. The results show that misexpression of activated Spitz can impact macrophage polarity and lead to clustering of cells in a variant-specific manner, when expressed either in macrophages or the developing fly heart. Spitz can also alter macrophage distribution and perturb apoptotic cell clearance undertaken by these phagocytic cells without affecting the overall levels of apoptosis within the embryo. Expression of active Spitz, but not a membrane-bound variant, can also increase macrophage migration speeds and impair their inflammatory responses to injury. The fact that the presence of Spitz specifically undermines the recruitment of more distal cells to wound sites suggests that Spitz desensitizes macrophages to wounds or is able to compete for their attention where wound signals are weaker. Taken together these results suggest this molecule regulates macrophage migration and their ability to dispose of apoptotic cells. This work identifies a novel regulator of Drosophila macrophage function and provides insights into signal prioritization and integration in vivo. Given the importance of apoptotic cell clearance and inflammation in human disease, this work may help in understanding the role EGF ligands play in immune cell recruitment during development and at sites of disease pathology (Tardy, 2021).

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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

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Zheng, Q., Gao, N., Sun, Q., Li, X., Wang, Y. and Xiao, H. (2021). bfc, a novel serpent co-factor for the expression of croquemort, regulates efferocytosis in Drosophila melanogaster. PLoS Genet 17(12): e1009947. PubMed ID: 34860835

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Genes involved in tissue development

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