Longitudinal tracking of hemocyte populations in vivo indicates lineage relationships and supports neural progenitor identity in adult neurogenesis.

Adult neurogenesis, which takes place in both vertebrate and invertebrate species, is the process by which new neurons are born and integrated into existing functional neural circuits, long after embryonic development. Most studies in mammals suggest that self-renewing stem cells are the source of the new neurons, although the extent of self-renewal is a matter of debate. In contrast, research in the crayfish Procambarus clarkii has demonstrated that the neural progenitors producing adult-born neurons are capable of both self-renewing and consuming (non-self-renewing) divisions. However, self-renewing divisions are relatively rare, and therefore the production of adult-born neurons depends heavily on progenitors that are not replenishing themselves. Because the small pool of neural progenitors in the neurogenic niche is never exhausted throughout the long lives of these animals, we hypothesized that there must also be an extrinsic source of these cells. It was subsequently demonstrated that the neural progenitors originate in hemocytes (blood cells) produced by the immune system that travel in the circulation before ultimately integrating into niches where the neural lineage begins. The current study examines the developmental lineage of the three hemocyte types - hyaline (HC), semigranular (SGC) and granular (GC) cells - with the goal of understanding the origins of the progenitor cells that produce adult-born neurons. Longstanding qualitative metrics for hemocyte classification were validated quantitatively. Then, in a longitudinal study, proliferation markers were used to label the hemocytes in vivo, followed by sampling the circulating hemocyte population over the course of two months. Hemolymph samples were taken at intervals to track the frequencies of the different hemocyte types. These data reveal sequential peaks in the relative frequencies of HCs, SGCs and GCs, which were identified using qualitative and quantitative measures. These findings suggest that the three hemocyte types comprise a single cellular lineage that occurs in the circulation, with each type as a sequential progressive stage in hemocyte maturation beginning with HCs and ending with GCs. When combined with previously published data, this timeline provides additional evidence that HCs serve as the primary neural progenitor during adult neurogenesis in P. clarkii.

A MASP-like functions as PRR to regulate the mRNA expressions of inflammatory factors in the Pacific oyster Crassostrea gigas.

Mannose-binding lectin-associated serine protease (MASP) is a type of central serine protease in the complement lectin pathway. In the present study, a MASP-like was identified from the Pacific oyster Crassostrea gigas, defined as CgMASPL-2. The cDNA sequence of CgMASPL-2 was of 3399 bp with an open reading frame of 2757 bp and encoded a polypeptide of 918 amino acids containing three CUB domains, an EGF domain, two IG domains, and a Tryp_SPC domain. In the phylogenetic tree, CgMASPL-2 was firstly clustered with Mytilus californianus McMASP-2-like, and then assigned into the invertebrate branch. CgMASPL-2 shared similar domains with M. californianus McMASP-2-like and Littorina littorea LlMReM1. CgMASPL-2 mRNA was expressed in all the tested tissues with the highest expression in haemolymph. CgMASPL-2 protein was mainly distributed in the cytoplasm of haemocytes. The mRNA expression of CgMASPL-2 increased significantly in haemocytes after Vibrio splendidus stimulation. The recombinant 3 × CUB-EGF domains of CgMASPL-2 displayed binding activities to diverse polysaccharides (lipopolysaccharide, peptidoglycan and mannose) and microbes (Staphylococcus aureus, Micrococcus luteus, Pichia pastoris, Vibrio anguillarum, V. splendidus and Escherichia coli). In anti-CgMASPL-2 treated oysters, the mRNA expressions of CgIL17-1 and CgIL17-2 in haemocytes decreased significantly after V. splendidus stimulation. The results indicated that CgMASPL-2 could directly sense microbes and regulate the mRNA expressions of inflammatory factors.

ROS function as an inducer of autophagy to promote granulocyte proliferation in Pacific oyster Crassostrea gigas.

Hematopoiesis is the biological process to generate new blood cells in the living body and reactive oxygen species (ROS) contribute significantly to the regulation of haematopoietic cell homeostasis. In the present study, the involvement of ROS in the proliferation of haemocytes was examined in Pacific oyster Crassostrea gigas. The ROS content in haemocytes increased significantly after lipopolysaccharide (LPS) treatment, but decreased after the treatment with antioxidant N-Acetyl-L-cysteine (NAC, a scavenger of ROS). The percentage of 5-ethynyl-2'-deoxyuridine labeled (EdU+) granulocytes in total haemocytes significantly increased at 12 h (4.12-fold, p < 0.001) and 24 h (2.36-fold, p < 0.001) after LPS treatment, while decreased at 12 h (0.26-fold, p < 0.001) and 24 h (0.61-fold, p < 0.05) after NAC treatment, respectively. Meanwhile, the percentage of haemocytes with autophagosome positive signals significantly increased at 12 h (1.17-fold, p < 0.01) and 24 h (1.19-fold, p < 0.05) after LPS treatment, but significantly reduced at 12 h (0.41-fold, p < 0.001) and 24 h (0.28-fold, p < 0.001) after the NAC treatment, respectively. After ammonium chloride (NH4Cl) treatment, the percentage of haemocytes with autophagosome and EdU+ granulocytes significantly increased at 12 h, which was 1.27-fold (p < 0.01) and 1.70-fold (p < 0.01) of control group, respectively. These results collectively suggested that ROS produced after LPS treatment could act as an inducer for autophagy and involved in regulating the proliferation of some granulocytes in C. gigas.

Integrin ß2 and ß3: Two plasmatocyte markers deepen our understanding of the development of plasmatocytes in the silkworm Bombyx mori.

Insect hemocytes play important biological roles at developmental stages, metamorphosis, and innate immunity. As one of the most abundant cell types, plasmatocytes can participate in various innate immune responses, especially in encapsulation and node formation. Here, 2 molecular markers of plasmatocytes, consisting of integrin ß2 and ß3, were identified and used to understand the development of plasmatocytes. Plasmatocytes are widely distributed in the hematopoietic system, including circulating hemolymph and hematopoietic organs (HPOs). HPOs constantly release plasmatocytes with high proliferative activity in vitro; removal of HPOs leads to a dramatic reduction in the circulating plasmatocytes, and the remaining plasmatocytes gradually lose their ability to proliferate in vivo. Our results demonstrated that the release of plasmatocytes from HPOs is regulated by insulin-mediated signals and their downstream pathways, including PI3K/Akt and MAPK/Erk signals. The insulin/PI3K/Akt signaling pathway can significantly irritate the hematopoiesis, and its inhibitor LY294002 could inhibit the hemocytes discharged from HPOs. While the insulin/MAPK/Erk signaling pathway plays a negative regulatory role, inhibiting its activity with U0126 can markedly promote the discharge of plasmatocytes from HPOs. Our results indicate that the circulating plasmatocytes are mainly generated and discharged by HPOs. This process is co-regulated by the PI3K/Akt and MAPK/Erk signals in an antagonistic manner to adjust the dynamic balance of the hemocytes. These findings can enhance our understanding of insect hematopoiesis.

Intrinsic and Extrinsic Regulation of Hematopoiesis in Drosophila.

Drosophila melanogaster lymph gland, the primary site of hematopoiesis, contains myeloid-like progenitor cells that differentiate into functional hemocytes in the circulation of pupae and adults. Fly hemocytes are dynamic and plastic, and they play diverse roles in the innate immune response and wound healing. Various hematopoietic regulators in the lymph gland ensure the developmental and functional balance between progenitors and mature blood cells. In addition, systemic factors, such as nutrient availability and sensory inputs, integrate environmental variabilities to synchronize the blood development in the lymph gland with larval growth, physiology, and immunity. This review examines the intrinsic and extrinsic factors determining the progenitor states during hemocyte development in the lymph gland and provides new insights for further studies that may extend the frontier of our collective knowledge on hematopoiesis and innate immunity.

Histone lysine-specific demethylase 1 regulates the proliferation of hemocytes in the oyster Crassostrea gigas.

Background: Lysine-specific demethylase 1 (LSD1) is an essential epigenetic regulator of hematopoietic differentiation, which can specifically mono-methylate H3K4 (H3K4me1) and di-methylate H3K4 (H3K4me2) as a transcriptional corepressor. Previous reports have been suggested that it participated in hematopoiesis and embryonic development process. Here, a conserved LSD1 (CgLSD1) with a SWIRM domain and an amino oxidase (AO) domain was identified from the Pacific oyster Crassostrea gigas. Methods: We conducted a comprehensive analysis by various means to verify the function of CgLSD1 in hematopoietic process, including quantitative real-time PCR (qRT-PCR) analysis, western blot analysis, immunofluorescence assay, RNA interference (RNAi) and flow cytometry. Results: The qRT-PCR analysis revealed that the transcripts of CgLSD1 were widely expressed in oyster tissues with the highest level in the mantle. And the transcripts of CgLSD1 were ubiquitously expressed during larval development with the highest expression level at the early D-veliger larvae stage. In hemocytes after Vibrio splendidus stimulation, the transcripts of CgLSD1 were significantly downregulated at 3, 6, 24, and 48 h with the lowest level at 3 h compared to that in the Seawater group (SW group). Immunocytochemical analysis showed that CgLSD1 was mainly distributed in the nucleus of hemocytes. After the CgLSD1 was knocked down by RNAi, the H3K4me1 and H3K4me2 methylation level significantly increased in hemocyte protein. Besides, the percentage of hemocytes with EdU-positive signals in the total circulating hemocytes significantly increased after V. splendidus stimulation. After RNAi of CgLSD1, the expression of potential granulocyte markers CgSOX11 and CgAATase as well as oyster cytokine-like factor CgAstakine were increased significantly in mRNA level, while the transcripts of potential agranulocyte marker CgCD9 was decreased significantly after V. splendidus stimulation. Conclusion: The above results demonstrated that CgLSD1 was a conserved member of lysine demethylate enzymes that regulate hemocyte proliferation during the hematopoietic process.

PGE2 mediates hemocyte-spreading behavior by activating aquaporin via cAMP and rearranging actin cytoskeleton via Ca2.

Spreading behavior of hemocytes (= insect blood cells) is essential for cellular immune responses against various microbial pathogens. It is activated by prostaglandin E2 (PGE2) via its membrane receptor associated with secondary messenger, cAMP, in insects. This study observed an increase of calcium ion (Ca2+) level after an acute increase of cAMP induced by PGE2 treatment and clarified the intracellular signals underlying the hemocyte-spreading behavior. Inhibition of Ca2+ flux significantly impaired the hemocyte-spreading and subsequent cellular immune response, phagocytosis. The up-regulation of intracellular Ca2+ in response to PGE2 was dependent on cAMP because RNA interference (RNAi) of PGE2 receptor expression or inhibiting adenylate cyclase prevented Ca2+ mobilization. The up-regulation of Ca2+ was induced by inositol triphosphate (IP3) via its specific IP3 receptor. Furthermore, inhibition of ryanodine receptor impaired Ca2+ mobilization, suggesting Ca2+-induced Ca2+ release. However, the effective spreading behavior of hemocytes was dependent on both secondary messengers. Ca2+ signal stimulated by cAMP was required for activating small G proteins because RNAi treatments of small G proteins such as Rac1, RhoA, and Cdc42 failed to stimulate hemocyte-spreading. In contrast, aquaporin was activated by cAMP. Its activity was necessary for changing cell volume during hemocyte-spreading. These results indicate that PGE2 mediates hemocyte-spreading via cAMP signal to activate aquaporin and via Ca2+ signal to activate actin cytoskeletal rearrangement.

Crayfish hemocytes develop along the granular cell lineage.

Despite the central role of hemocytes in crustacean immunity, the process of hemocyte differentiation and maturation remains unclear. In some decapods, it has been proposed that the two main types of hemocytes, granular cells (GCs) and semigranular cells (SGCs), differentiate along separate lineages. However, our current findings challenge this model. By tracking newly produced hemocytes and transplanted cells, we demonstrate that almost all the circulating hemocytes of crayfish belong to the GC lineage. SGCs and GCs may represent hemocytes of different developmental stages rather than two types of fully differentiated cells. Hemocyte precursors produced by progenitor cells differentiate in the hematopoietic tissue (HPT) for 3 ~ 4 days. Immature hemocytes are released from HPT in the form of SGCs and take 1 ~ 3 months to mature in the circulation. GCs represent the terminal stage of development. They can survive for as long as 2 months. The changes in the expression pattern of marker genes during GC differentiation support our conclusions. Further analysis of hemocyte phagocytosis indicates the existence of functionally different subpopulations. These findings may reshape our understanding of crustacean hematopoiesis and may lead to reconsideration of the roles and relationship of circulating hemocytes.

Methotrexate negatively acts on inflammatory responses triggered in Drosophila larva with hyperactive JAK/STAT pathway.

Drosophila is a valuable paradigm for studying tumorigenesis and cancer. Mutations causing hematopoietic aberrations and melanotic-blood-tumors found in Drosophila mutants are vastly studied. Clear understanding about the blood cells, signaling pathways and the tissues affected during hematopoietic tumor formation provide an opportunity to delineate the effects of cancer therapeutics. Using this simple hematopoietic archetype, we elucidated the effects of the anti-cancer drug, Methotrexate (MTX) on immune responses in two scenarios i.e. against wasp infection and in hematopoietic mutant, hopTum-l. Through this in vivo study we show that MTX impedes the immune responses against wasp infection including the encapsulation response. We further observed that MTX reduces the tumor penetrance in gain-of-function mutants of JAK/STAT pathway, hopTum-l. MTX is anti-inflammatory as it hinders not only the immune responses of acute inflammation as observed after wasp infestation, but also chronic inflammatory responses associated with constitutively activated JAK/STAT pathway mutant (hopTum-l) carrying blood tumors.

A fibrinogen-related protein mediates the recognition of various bacteria and haemocyte phagocytosis in oyster Crassostrea gigas.

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like (FBG) domains, which play important roles as pattern recognition receptors (PRRs) in the innate immune responses. In the present study, a fibrinogen-like protein was identified from the oyster Crassostrea gigas (defined as CgFREP1). The open reading frame of CgFREP1 was of 966 bp that encoded a predicted polypeptide of 321 amino acids comprising a signal peptide and a fibrinogen-like domain. The mRNA expression of CgFREP1 was detected in all the examined tissues. The recombinant CgFREP1 (rCgFREP1) displayed binding activities to lipopolysaccharide (LPS), mannose (MAN), as well as Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and Gram-negative bacteria (Vibrio splendidus and Escherichia coli). The rCgFREP1 displayed the agglutinating activity towards M. luteus, V. splendidus and E. coli in the presence of Ca2+. rCgFREP1 was able to enhance the phagocytic activity of haemocytes towards V. splendidus, and exhibited binding activity to the CUB domain of CgMASPL-1. These results suggest that CgFREP1 not only serves as a PRR to recognize and agglutinate different bacteria but also mediates the haemocytes phagocytosis towards V. splendidus.

The larval haematopoietic organs of Manduca sexta (Insecta, Lepidoptera): An insight into plasmatocyte development and larval haematopoiesis.

Haematopoietic organs (HOs) in Lepidoptera are widely recognised as the source for at least two haemocyte types. With new specific markers for oenocytoids and spherule cells and a method to identify prohaemocytes, the haemocytes formed in and released by the HOs of Manduca sexta are characterised. Differentiation of HO-cells to haemocytes other than plasmatocytes and prohaemocytes neither occurs in the organ itself nor in cells released in vitro by the HOs. Differential labelling patterns evidence the existence of plasmatocyte subpopulations and prohaemocytes, which might represent a gradual differentiation of haemocytes within the organs. Prohaemocytes can be identified by PNA-labelling of the cell membrane. These prohaemocytes are found in circulation and in the HOs and are released by the organs. Circulating prohaemocytes possess characteristics for granular cells, plasmatocytes or oenocytoids while HO derived prohaemocytes share characteristics only with plasmatocytes. Ablation of the HOs diminishes the plasmatocyte and prohaemocyte number, indicating a true larval haematopoietic function.

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

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

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

Deletion mutant of PGE2 receptor using CRISPR-Cas9 exhibits larval immunosuppression and adult infertility in a lepidopteran insect, Spodoptera exigua.

Prostaglandins (PGs) mediate various physiological processes in insects and other invertebrates, but there is very little information on PG receptors. This study identified a PGE2 receptor (SePGE2R) in the lepidopteran insect, Spodoptera exigua, and addressed its functional association with cellular immunity, development, and reproduction. SePGE2R is expressed in most developmental stages and tissues. After SePGR2R expression knock down by RNA interference (RNAi), larval nodule formation (clears bacterial infections from circulating hemolymph) was severely suppressed coupled with reduced F-actin growth in hemocytes. Treating female adults with RNAi prevented nurse cell dumping in follicles and interfered with oocyte development. SePGE2R was heterologously expressed in Sf9 cells, in which the endogenous S. frugiperda PGE2R was knocked down by small interfering RNA. This transiently expressed SePGE2R responded to PGE2, but not other PGs, with dose-dependent up-regulation of intracellular cAMP concentrations. Treating S. exigua larvae with PGE2 led to activation of a trimeric Gαs subunit, protein kinase A (PKA), and Rho family small intracellular G proteins in hemocytes. A deletion mutant of SePGE2R was generated using CRISPR/Cas9 which exhibited severely retarded larval development and adult reproduction. We infer that PGE2R mediates insect immune and reproductive processes via a PKA signal pathway.

A novel Adiponectin receptor (AdipoR) involved in regulating cytokines production and apoptosis of haemocytes in oyster Crassostrea gigas.

Adiponectin receptors (AdipoRs) comprise a seven-transmembrane domain-containing protein family, which specifically recognize adiponectin (APN) and play critical roles in the immunological and physiological processes in vertebrates. In the present study, a novel AdipoR is identified from oyster Crassostrea gigas (designated as CgAdipoR). The full-length cDNA of CgAdipoR is of 1209 bp encoding a polypeptide of 343 amino acids. There is an N-terminal domain, a Hly III domain, and a C-terminal domain in CgAdipoR. After the transfection of CgAdipoR, the level of intracellular Ca2+ into HEK293T cells increases significantly (1.36-fold, p < 0.05) after APN incubation. The mRNA transcripts of CgAdipoR are widely distributed in all the tested tissues, with the highest expression level in haemocytes (3.20-fold of that in hepatopancreas, p < 0.05). After lipopolysaccharide (LPS), Vibrio splendidus and polyinosinic-polycytidylic acid (poly (I:C)) stimulations, the mRNA expression of CgAdipoR in haemocytes is significantly up-regulated and reached the highest level at 24 h (15.07-fold, p < 0.01), 6 h (4.39-fold, p < 0.01) and 24 h (5.62-fold, p < 0.01) compared to control group, respectively. After CgAdipoR is interfered by specific CgAdipoR-dsRNA, the expression level of interleukins (CgIL17-1, CgIL17-2, CgIL17-3 and CgIL17-5) in haemocytes decreases significantly (p < 0.01) at 24 h post LPS stimulation, while the expression level of CgTNF-1 increases significantly (1.68-fold, p < 0.01), compared to that in the dsEGFP group. In CgAdipoR dsRNA-injected oysters, the mRNA expressions of anti-apoptotic B-cell lymphoma-2 (Bcl-2) in haemocytes significantly decreases at 24 h after LPS challenge, which is (0.58-fold, p < 0.05) of that in dsEGFP-injected oysters, while the apoptotic rate of haemocytes is significantly up-regulated (1.93-fold of that in dsEGFP group, p < 0.05). These results collectively suggest that CgAdipoR plays an important role in the immune response of oysters by regulating the expressions of inflammatory cytokines and haemocyte apoptosis.

A novel site of haematopoiesis and appearance and dispersal of distinct haemocyte types in the Manduca sexta embryo (Insecta, Lepidoptera).

With a set of haemocyte specific markers novel findings on haematopoiesis in the Manduca sexta embryo are presented. We identify a hitherto unknown paired haematopoietic cluster, the abdominal haemocyte cluster in abdominal segment 7 (A7-HCC). These clusters are localised at distinct positions and are established at around katatrepsis. Later in embryogenesis, the A7-HCCs disintegrate, thereby releasing numerous embryonic plasmatocytes which disperse both anteriorly and posteriorly. These cells follow stereotypic migration routes projecting anteriorly. The thoracic larval haematopoietic organs are established at around midembryogenesis. We identify embryonic oenocytoids in the M. sexta embryo for the first time. They appear in the head region roughly at the same time as the A7-HCCs occur and successively disperse in the body cavity during development. Localisation of the prophenoloxidase (proPO) mRNA and of the proPO protein are identical. Morphological, cytometric and antigenic traits show three independently generated haemocyte types during embryogenesis.

The N-terminal peptide generated after activation of prophenoloxidase affects crayfish hematopoiesis.

The circulating hemocytes of invertebrates are important mediators of immunity, and hemocyte homeostasis is of high importance for survival and health of crustaceans. The prophenoloxidase (proPO)-activating system is one of the most essential immune reactions, which can be activated by pattern recognition proteins from microorganisms. Activation of proPO by the proPO activating enzyme generates an N-terminal peptide, with cleavage site after Arg176, as well as the active enzyme phenoloxidase, which is the key enzyme for melanization. In the present study we demonstrate a role for the N-terminal proPO-peptide in hematopoiesis. Injection of this proPO-peptide increased the number of circulating hemocytes and especially granular hemocytes. We also show that the reactive oxygen species (ROS) production in the anterior proliferative center was enhanced after proPO peptide injection, which is a prerequisite for rapid hemocyte release from the hematopoietic tissue. Moreover, this peptide had an effect on ROS production in in vitro cultured hematopoietic cells and induced spreading of these cells within 72 h. Taken together, our findings show a role of the N-terminal proPO peptide in stimulation of hematopoiesis in crayfish, Pacifastacus leniusculus.

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. Here, we show 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, we 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.

Pericardin, a Drosophila collagen, facilitates accumulation of hemocytes at the heart.

Hematopoietic cell lineages support organismal needs by responding to positional and systemic signals that balance proliferative and differentiation events. Drosophila provides an excellent genetic model to dissect these signals, where the activity of cues in the hemolymph or substrate can be traced to determination and differentiation events of well characterized hemocyte types. Plasmatocytes in third instar larvae increase in number in response to infection and in anticipation of metamorphosis. Here we characterize hemocyte clustering, proliferation and transdifferentiation on the heart or dorsal vessel. Hemocytes accumulate on the inner foldings of the heart basement membrane, where they move with heart contraction, and are in proximity to the heart ostia and pericardial nephrocytes. The numbers of hemocytes vary, but increase transiently before pupariation, and decrease by 4 h before pupa formation. During their accumulation at the heart, plasmatocytes can proliferate and can transdifferentiate into crystal cells. Serrate expressing cells as well as lamellocyte-like, Atilla expressing ensheathing cells are associated with some, but not all hemocyte clusters. Hemocyte aggregation is enhanced by the presence of a heart specific Collagen, Pericardin, but not the associated pericardial cells. The varied and transient number of hemocytes in the pericardial compartment suggests that this is not a hematopoietic hub, but a niche supporting differentiation and rapid dispersal in response to systemic signals.

Identification and functional characterization of lactadherin, an agglutinating glycoprotein from the chordate Styela clava.

Lactadherin is an extracellular matrix glycoprotein with stimulating agglutination ability that plays crucial roles in animal immunology. In the present study, a novel lactadherin, Sc-lactadherin, was identified from the marine invertebrate chordate, Styela clava. Its full-length cDNA consisted of 579 bps, encoding 193 amino acids with a coagulation FA58C domain. Recombinant Sc-lactadherin via a prokaryotic expression system showed strong hemocyte fusion activity. Therefore, we further examined its effects on cell behaviors using human umbilical vein endothelial cells (HUVECs) and human cervical cancer (HeLa) cells. Recombinant Sc-lactadherin significantly increased the proliferation rate of HUVECs and HeLa cells and improved the cell migration rate of HUVECs. These results demonstrated that the lactadherin identified from the marine ascidian displayed the agglutinating activity. Functional characterization of the recombinant protein showed that it promoted cell proliferation and migration, indicating the potential roles of Sc-lactadherin in immunology and organogenesis in marine ascidians.