Hematopoiesis at the onset of metamorphosis: terminal differentiation and dissociation of the <Emphasis Type="Italic">Drosophila</Emphasis> lymph gland

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

JAK/STAT and the GATA factor Pannier control hemocyte maturation and differentiation in Drosophila

The lymph gland is the major site of hematopoiesis in Drosophila. During late larval stages three types of hemocytes are produced, plasmatocytes, crystal cells, and lamellocytes, and their differentiation is tightly controlled by conserved factors and signaling pathways. JAK/STAT is one of these pathways which have essential roles in vertebrate and fly hematopoiesis. We show that Stat has opposing cell-autonomous and non-autonomous functions in hemocyte differentiation. Using a clonal approach we established that loss of Stat in a set of prohemocytes in the cortical zone induces plasmatocyte maturation in adjacent hemocytes. Hemocytes lacking Stat fail to differentiate into plasmatocytes, indicating that Stat positively and cell-autonomously controls plasmatocyte differentiation. We also identified the GATA factor pannier (pnr) as a downstream target of Stat. By analyzing the phenotypes resulting from clonal loss and over-expression of pnr in lymph glands, we find that Pnr is positively regulated by Stat and specifically required for the differentiation of plasmatocytes. Stat and Pnr represent two essential factors controlling blood cell maturation in the developing lymph gland and exert their functions both in a cell-autonomous and non-cell-autonomous manner.     

Asrij maintains the stem cell niche and controls differentiation during Drosophila lymph gland hematopoiesis

Several signaling pathways control blood cell (hemocyte) development in the Drosophila lymph gland. Mechanisms that modulate and integrate these signals are poorly understood. Here we report that mutation in a conserved endocytic protein Asrij affects signal transmission and causes aberrant lymph gland hematopoiesis. Mammalian Asrij (Ociad1) is expressed in stem cells of the blood vascular system and is implicated in several cancers. We found that Drosophila Asrij is a pan-hemocyte marker and localizes to a subset of endocytic vesicles. Loss of asrij causes hyperproliferation of lymph gland lobes coupled with increased hemocyte differentiation, thereby depleting the pool of quiescent hemocyte precursors. This co-relates with fewer Col+ cells in the hematopoietic stem cell niche of asrij mutants. Asrij null mutants also show excess specification of crystal cells that express the RUNX factor Lozenge (Lz), a target of Notch signaling. Asrij mutant lymph glands show increased N in sorting endosomes suggesting aberrant trafficking. In vitro assays also show impaired traffic of fluorescent probes in asrij null hemocytes. Taken together our data suggest a role for Asrij in causing increased Notch signaling thereby affecting hemocyte differentiation. Thus, conserved endocytic functions may control blood cell progenitor quiescence and differentiation.     

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.     

Regulation of larval hematopoiesis in Drosophila melanogaster: a role for the multi sex combs gene

Drosophila larval hematopoietic organs produce circulating hemocytes that ensure the cellular host defense by recognizing and neutralizing non-self or noxious objects through phagocytosis or encapsulation and melanization. Hematopoietic lineage specification as well as blood cell proliferation and differentiation are tightly controlled. Mutations in genes that regulate lymph gland cell proliferation and hemocyte numbers in the body cavity cause hematopoietic organ overgrowth and hemocyte overproliferation. Occasionally, mutant hemocytes invade self-tissues, behaving like neoplastic malignant cells. Two alleles of the Polycomb group (PcG) gene multi sex combs (mxc) were previously isolated as such lethal malignant blood neoplasm mutations. PcG genes regulate Hox gene expression in vertebrates and invertebrates and participate in mammalian hematopoiesis control. Hence we investigated the need for mxc in Drosophila hematopoietic organs and circulating hemocytes. We show that mxc-induced hematopoietic hyperplasia is cell autonomous and that mxc mainly controls plasmatocyte lineage proliferation and differentiation in lymph glands and circulating hemocytes. Loss of the Toll pathway, which plays a similar role in hematopoiesis, counteracted mxc hemocyte proliferation but not mxc hemocyte differentiation. Several PcG genes tested in trans had no effects on mxc hematopoietic phenotypes, whereas the trithorax group gene brahma is important for normal and mutant hematopoiesis control. We propose that mxc provides one of the regulatory inputs in larval hematopoiesis that control normal rates of plasmatocyte and crystal lineage proliferation as well as normal rates and timing of hemocyte differentiation.     

Zfrp8, the Drosophila ortholog of PDCD2, functions in lymph gland development and controls cell proliferation

We have identified a new gene, Zfrp8, as being essential for hematopoiesis in Drosophila. Zfrp8 (Zinc finger protein RP-8) is the Drosophila ortholog of the PDCD2 (programmed cell death 2) protein of unknown function, and is highly conserved in all eukaryotes. Zfrp8 mutants present a developmental delay, lethality during larval and pupal stages and hyperplasia of the hematopoietic organ, the lymph gland. This overgrowth results from an increase in proliferation of undifferentiated hemocytes throughout development and is accompanied by abnormal differentiation of hemocytes. Furthermore, the subcellular distribution of gamma-Tubulin and Cyclin B is affected. Consistent with this, the phenotype of the lymph gland of Zfpr8 heterozygous mutants is dominantly enhanced by the l(1)dd4 gene encoding Dgrip91, which is involved in anchoring gamma-Tubulin to the centrosome. The overgrowth phenotype is also enhanced by a mutation in Cdc27, which encodes a component of the anaphase-promoting complex (APC) that regulates the degradation of cyclins. No evidence for an apoptotic function of Zfrp8 was found. Based on the phenotype, genetic interactions and subcellular localization of Zfrp8, we propose that the protein is involved in the regulation of cell proliferation from embryonic stages onward, through the function of the centrosome, and regulates the level and localization of cell-cycle components. The overproliferation of cells in the lymph gland results in abnormal hemocyte differentiation.     

敲低piwi基因对黑腹果蝇血细胞增殖及分化的影响

[目的]探究敲低piwi基因对黑腹果蝇Drosophila melanogaster血细胞增殖及分化的影响.[方法]利用黑腹果蝇e33C-Gal4和Hml-Gal4-UAS-2×EGFP品系分别与野生型W1118和UAS-piwi RNAi品系杂交,实现在黑腹果蝇游离血细胞或淋巴腺中降低piwi基因的表达;采用免疫荧光...     

Developmental control of blood cell migration by the Drosophila VEGF pathway.

We show that a vascular endothelial growth factor (VEGF) pathway controls embryonic migrations of blood cells (hemocytes) in Drosophila. The VEGF receptor homolog is expressed in hemocytes, and three VEGF homologs are expressed along hemocyte migration routes. A receptor mutation arrests progression of blood cell movement. Mutations in Vegf17E or Vegf27Cb have no effect, but simultaneous inactivation of all three Vegf genes phenocopied the receptor mutant, and ectopic expression of Vegf27Cb redirected migration. Genetic experiments indicate that the VEGF pathway functions independently of pathways governing hemocyte homing on apoptotic cells. The results suggest that the Drosophila VEGF pathway guides developmental migrations of blood cells, and we speculate that the ancestral function of VEGF pathways was to guide blood cell movement.     

Kicking it up a Notch for the best in show: Scalloped leads Yorkie into the haematopoietic arena

Maintenance and differentiation of progenitor cells is essential for proper organ development and adaptation to environmental stress and injury. In Drosophila melanogaster, the haematopietic system serves as an ideal model for interrogating the function of signaling pathways required for progenitor maintenance and cell fate determination. Here we focus on the role of the Hippo pathway effectors Yorkie and Scalloped in mediating and facilitating Notch signaling-mediated lineage specification in the lymph gland, the primary center for haematopoiesis within the developing larva. We discuss the regulatory mechanisms which promote Notch activity during normal haematopoiesis and its modulation during immune challenge conditions. We provide additional evidence establishing the hierarchy of signaling events during crystal cell formation, highlighting the relationship between Yorkie, Scalloped and Lozenge, while expanding on the role of Yorkie in promoting hemocyte survival and the developmental regulation of Notch and its ligand, Serrate, within the lymph gland. Finally, we propose additional areas of exploration that may provide mechanistic insight into the environmental and non-cell autonomous regulation of cell fate in the blood system.     

Kicking it up a Notch for the best in show: Scalloped leads Yorkie into the haematopoietic arena

Maintenance and differentiation of progenitor cells is essential for proper organ development and adaptation to environmental stress and injury. In Drosophila melanogaster, the haematopietic system serves as an ideal model for interrogating the function of signaling pathways required for progenitor maintenance and cell fate determination. Here we focus on the role of the Hippo pathway effectors Yorkie and Scalloped in mediating and facilitating Notch signaling-mediated lineage specification in the lymph gland, the primary center for haematopoiesis within the developing larva. We discuss the regulatory mechanisms which promote Notch activity during normal haematopoiesis and its modulation during immune challenge conditions. We provide additional evidence establishing the hierarchy of signaling events during crystal cell formation, highlighting the relationship between Yorkie, Scalloped and Lozenge, while expanding on the role of Yorkie in promoting hemocyte survival and the developmental regulation of Notch and its ligand, Serrate, within the lymph gland. Finally, we propose additional areas of exploration that may provide mechanistic insight into the environmental and non-cell autonomous regulation of cell fate in the blood system.     

Yantar, a conserved arginine-rich protein is involved in Drosophila hemocyte development

To identify novel factors involved in Drosophila hematopoiesis, we screened a collection of lethal recessive mutations that also affected normal hemocyte composition in larvae. We present the characterization of the gene yantar (ytr) for which we isolated null and hypomorphic mutations that were associated with severe defects in hemocyte differentiation and proliferation; ytr is predominantly expressed in the hematopoietic tissue during larval development and encodes an evolutionary conserved protein which is predominantly localized in the nucleus. The hematopoietic phenotype in ytr mutants is consistent with a defect or block in differentiation of precursor hemocytes: mutant larvae have enlarged lymph glands (LGs) and have an excess of circulating hemocytes. In addition, many cells exhibit both lamellocyte and crystal cell markers. Ytr function has been preserved in evolution as hematopoietic specific expression of the Drosophila or mouse Ytr proteins rescue the differentiation defects in mutant hemocytes.     

Apoptosis in medfly hemocytes is regulated during pupariation through FAK, Src, ERK, PI-3K p85a, and Akt survival signaling

Focal adhesion kinase (FAK) and its downstream signaling targets are implicated in the process of apoptosis induced by external stimuli, in several mammalian systems. In this report, we demonstrate, that medfly (Ceratitis capitata) hemocytes do undergo apoptosis during larval development. In particular, we show using Western blot, ELISA and flow cytometry analysis, that FAK expression silencing in transfected by FAK double-stranded RNA (dsRNA) hemocytes, enhances twofold hemocyte apoptosis, by signaling through Src, MEK/ERK, and PI-3K/Akt signaling pathways. FAK expression silencing, in response to FAK dsRNA treatment, blocks partially the phosphorylation of its downstream targets. Pre-incubation of hemocytes, with specific inhibitors of FAK downstream signaling molecules, demonstrated that all these inhibitors reduced hemocyte viability and enhanced the magnitude of apoptosis about threefold. This data suggest that these pathways contribute to hemocyte survival and/or death during development. The expression and phosphorylation of FAK, Src, PI-3K p85a, Akt, and ERK signaling molecules appear to be dependent upon developmental stages. The expression and phosphorylation of the above signaling molecules, in annexin-positive and annexin-negative hemocytes is also distinct. The maximum expression and phosphorylation of FAK, Src, PI-3K p85a, Akt, and ERK appeared in annexin-positive hemocytes, in both early and late apoptotic hemocytes. The novel aspect of this report is based on the fact that hemocytes attempt to suppress apoptosis, by increasing the expression/phosphorylation of FAK and, hence its downstream targets signaling molecules Src, ERK, PI-3K p85a, and Akt. Evidently, the basic survival pathways among insects and mammals appear to remain unchanged, during evolution.     

Dual role for Insulin/TOR signaling in the control of hematopoietic progenitor maintenance in Drosophila

The interconnected Insulin/IGF signaling (IlS) and Target of Rapamycin (TOR) signaling pathways constitute the main branches of the nutrient-sensing system that couples growth to nutritional conditions in Drosophila. Here, we addressed the influence of these pathways and of diet restriction on the balance between the maintenance of multipotent hematopoietic progenitors and their differentiation in the Drosophila lymph gland. In this larval hematopoietic organ, a pool of stem-like progenitor blood cells (prohemocytes) is kept undifferentiated in response to signaling from a specialized group of cells forming the posterior signaling center (PSC), which serves as a stem cell niche. We show that, reminiscent of the situation in human, loss of the negative regulator of IIS Pten results in lymph gland hyperplasia, aberrant blood cell differentiation and hematopoietic progenitor exhaustion. Using site-directed loss- and gain-of-function analysis, we demonstrate that components of the IIS/TOR pathways control lymph gland homeostasis at two levels. First, they cell-autonomously regulate the size and activity of the hematopoietic niche. Second, they are required within the prohemocytes to control their growth and maintenance. Moreover, we show that diet restriction or genetic alteration mimicking amino acid deprivation triggers progenitor cell differentiation. Hence, our study highlights the role of the IIS/TOR pathways in orchestrating hematopoietic progenitor fate and links blood cell fate to nutritional status.     

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, we specified two separate anlagen 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. Our 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.     

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. Here, we 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. Our results show that the embryonic LG contains primordial hematopoietic cells which actively divide to give rise to a pool of pro-hemocytes. We found no evidence 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. We 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.     

Distinct mechanisms regulate hemocyte chemotaxis during development and wound healing in Drosophila melanogaster

Drosophila melanogaster hemocytes are highly motile macrophage-like cells that undergo a stereotypic pattern of migration to populate the whole embryo by late embryogenesis. We demonstrate that the migratory patterns of hemocytes at the embryonic ventral midline are orchestrated by chemotactic signals from the PDGF/VEGF ligands Pvf2 and -3 and that these directed migrations occur independently of phosphoinositide 3-kinase (PI3K) signaling. In contrast, using both laser ablation and a novel wounding assay that allows localized treatment with inhibitory drugs, we show that PI3K is essential for hemocyte chemotaxis toward wounds and that Pvf signals and PDGF/VEGF receptor expression are not required for this rapid chemotactic response. Our results demonstrate that at least two separate mechanisms operate in D. melanogaster embryos to direct hemocyte migration and show that although PI3K is crucial for hemocytes to sense a chemotactic gradient from a wound, it is not required to sense the growth factor signals that coordinate their developmental migrations along the ventral midline during embryogenesis.     

Cell interactions in the differentiation of a melanotic tumor in Drosophila.

The cellular events in the formation of melanotic tumors in the tu-W mutant larva of Drosophila melanogaster are described. The first step is the differentiation of spherical hemocytes to flattened cells, the lamellocyte variants. Subsequently, the surface of the caudal fat body undergoes changes to which the hemocytes respond by forming cellular capsules. The hemocytes utilize two mechanisms in this process: (1) phagocytosis of small particulate materials escaping from the adipose cells, (2) adhesion to form a multilayered wall of lamellocytes. Differentiating hemocytes in the vicinity of the tumor-forming site extrude membrane-bound vesicles that tend to adhere to the hemocyte surfaces. These vesicles are trapped between the lamellocytes as they pile in layers to form the capsule wall. It is suggested that the vesicles play a role in lamellocyte-to-lamellocyte adhesion during the initial stages of hemocyte aggregation at the tumor-forming site.