Of blood cells and the nervous system

Hematopoiesis is well-conserved between Drosophila and vertebrates. Similar as in vertebrates, the sites of hematopoiesis shift during Drosophila development. Blood cells (hemocytes) originate de novo during hematopoietic waves in the embryo and in the Drosophila lymph gland. In contrast, the hematopoietic wave in the larva is based on the colonization of resident hematopoietic sites by differentiated hemocytes that arise in the embryo, much like in vertebrates the colonization of peripheral tissues by primitive macrophages of the yolk sac, or the seeding of fetal liver, spleen and bone marrow by hematopoietic stem and progenitor cells. At the transition to the larval stage, Drosophila embryonic hemocytes retreat to hematopoietic “niches,” i.e., segmentally repeated hematopoietic pockets of the larval body wall that are jointly shared with sensory neurons and other cells of the peripheral nervous system (PNS). Hemocytes rely on the PNS for their localization and survival, and are induced to proliferate in these microenvironments, expanding to form the larval hematopoietic system. In this process, differentiated hemocytes from the embryo resume proliferation and self-renew, omitting the need for an undifferentiated prohemocyte progenitor. Larval hematopoiesis is the first Drosophila model for blood cell colonization and niche support by the PNS. It suggests an interface where innocuous or noxious sensory inputs regulate blood cell homeostasis or immune responses. The system adds to the growing concept of nervous system dependence of hematopoietic microenvironments and organ stem cell niches, which is being uncovered across phyla.     

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. Here, we introduce 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, we show that these resident hemocytes tightly colocalize with peripheral neurons and we demonstrate 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.     

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage. HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.     

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.     

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.     

Slit-Robo signalling prevents sensory cells from crossing the midline in Drosophila

Maintenance of bilateral symmetry throughout animal development requires that both left and right halves of the body follow nearly identical patterns of cell proliferation, differentiation, death and migration. During formation of the perfectly bilateral Drosophila larval peripheral nervous system (PNS), the sensory precursor cells of the ventral multidendritic neuron vmd1a originating from each hemisegment migrate away from the ventral midline. Our observations indicate that in slit mutant embryos, as well as in robo, robo2 double mutants, sensory precursor cells of the left and right vmd1a neurons aberrantly cluster at the midline and then the pair of vmd1a neurons migrate to their final position on the same side of the embryo. This results in disruption of PNS bilateral symmetry. Expression of slit at the midline rescues the slit mutant vmd1a phenotype, suggesting that midline-secreted Slit activates Robo/Robo2 signalling to control the migration of the vmd1a sensory precursor cells. Our study indicates that midline-secreted Slit prevents vmd1a sensory cells from crossing the midline and thereby maintains PNS bilateral symmetry during development.     

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.     

The insect cellular immune response

The innate immune system of insects is divided into humoral defenses that include the production of soluble effector molecules and cellular defenses like phagocytosis and encapsulation that are mediated by hemocytes. This review summarizes current understand- ing of the cellular immune response. Insects produce several terminally differentiated types of hemocytes that are distinguished by morphology, molecular and antigenic markers, and function. The differentiated hemocytes that circulate in larval or nymphal stage insects arise from two sources： progenitor cells produced during embryogenesis and mesodermally derived hematopoietic organs. Regulation of hematopoiesis and hemocyte differentiation also involves several different signaling pathways. Phagocytosis and encapsulation require that hemocytes first recognize a given target as foreign followed by activation of downstream signaling and effector responses. A number of humoral and cellular receptors have been identified that recognize different microbes and multicellular parasites. In turn, activation of these receptors stimulates a number of signaling pathways that regulate different hemocyte functions. Recent studies also identify hemocytes as important sources Of a number of humoral effector molecules required for killing different foreign invaders.     

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, we define the developmental steps in the maturation of blood cells (hemocytes) from their precursors. 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.     

Multiple roles of mouse Numb in tuning developmental cell fates.

BACKGROUND: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. RESULTS: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest--derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons. CONCLUSIONS: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.     

The Hematopoietic Niche: A Drosophila Model,at Last

The niche provides a specialised microenvironment necessary for maintenance of stem cells in a non differentiated state. While the hematopoietic stem cell (HSC) niche in vertebrates was the first to be recognized, Drosophila niches supporting germline stem cells were characterised first. Recent evidence for the existence of a niche maintaining hematopoietic precursors in Drosophila opens the way to study in vivo the niche/hematopoietic precursors interactions. The availability of a large collection of cell markers, mutants and sophisticated genetic tools makes Drosophila an attractive model for investigating the cellular and molecular mechanisms that are involved in these interactions.     

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.     

Induction of embryonic hematopoietic and endothelial stem/progenitor cells by hedgehog-mediated signals

Blood and vascular endothelial cells form in all vertebrates during gastrulation, a process in which the mesoderm of the embryo is induced and then patterned by molecules whose identity is still largely unknown. Blood islands' of primitive hematopoietic cell clusters surrounded by a layer of endothelial cells form in the yolk sac, external to the developing embryo proper. These lineages arise from a layer of extraembryonic mesoderm that is closely apposed with a layer of primitive (visceral) endoderm. Despite the identification of genes such as Flk1, SCL/tal-1, Cbfa2/Runx1/AML1 and CD34 that are expressed during the induction of primitive hematopoiesis and vasculogenesis, the early molecular and cellular events involved in these processes are not well understood. Recent work has demonstrated that extracellular signals secreted by visceral endoderm surrounding the embryo are essential for the initiation of these events. A member of the Hedgehog family of signaling molecules (Indian hedgehog) is produced by visceral endoderm, can induce formation of blood and endothelial cells in explant cultures and can reprogram prospective neurectoderm along hematopoietic and endothelial cell lineages. Hedgehog proteins also stimulate proliferation of definitive hematopoietic stem/progenitor cells. These findings may have important implications for regulating hematopoiesis and vascular development for therapeutic purposes in humans and for the development of new sources of stem cells for transplantation and gene therapy.     

Neurogenesis and asymmetric cell division

The astonishing cellular diversity in the central nervous system (CNS) arises from neural progenitors which can undergo different modes of symmetric and asymmetric divisions to self-renew as well as produce differentiated neuronal and glial progeny. Drosophila CNS neural progenitor cells, neuroblasts, have been utilised as a model to stimulate the understanding of the processes of asymmetric division, generation of neuronal lineages and, more recently, stem cell biology in vertebrates. Here we review some recent developments involving Drosophila and mammalian neural progenitor cells, highlighting some similarities and differences in the mechanisms that regulate their divisions during neurogenesis.     

In vitro studies of hematopoiesis in the silkworm: cell proliferation in and hemocyte discharge from the hematopoietic organ

The lepidopteran hematopoietic process is poorly understood. We therefore examined the fundamental properties of hematopoiesis in the silkworm Bombyx mori using hematopoietic organ culture. In a medium containing larval plasma taken from the fourth day of the final larval stadium, over 50,000 hemocytes per hematopoietic organ were discharged within 48 h, with the number of cells comprising the hematopoietic organ simultaneously increasing from approximately 20,000 to 40,000. However, in the absence of plasma, cell numbers comprising the hematopoietic organ were unchanged and the number of discharged cells was much less. Hematopoietic organs cultured with plasma showed strong mitotic indices in a BrdU incorporation assay, but did not when cultured without plasma, indicating that plasma contains hematopoietic factor(s). The hematopoietic stimulation ability of larval plasma was observed from the last day of the penultimate larval stadium to the prepupal stage. The response of the hematopoietic organs to larval plasma was highest at the beginning of the final larval stadium and decreased with aging. Most cells discharged from the hematopoietic organ were plasmatocytes and prohemocytes, irrespective of location and developmental stage. Using this in vitro culture method, we tested the effects of 20-hydroxyecdysone (20E) and juvenile hormone-I (JH-I) on B. mori hematopoiesis. 20E showed a weak, but significant, hematopoietic activity, whereas JH-I did not, suggesting that a part of larval hematopoiesis is endocrinally regulated.