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, we identify an extensive reservoir of blood cells (hemocytes) 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, we find no sign of adult hemocyte expansion. 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. Our work identifies a multi-signal relay of organismal humoral immunity, establishing adult Drosophila as model for inter-organ immunity.

Regulation of Drosophila hematopoietic sites by Activin-ß 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. Here we report that Activin-ß, a TGF-ß 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-ß 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.

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.

A systems-level interrogation identifies regulators of Drosophila blood cell number and survival.

In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.

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.

Mutant Rab24 GTPase is targeted to nuclear inclusions.

BACKGROUND: Members of the Rab GTPase family regulate intracellular protein trafficking, but the specific function of Rab24 remains unknown. Several attributes distinguish this protein from other members of the Rab family, including a low intrinsic GTPase activity. RESULTS: The functions of other Rab proteins have been defined through the use of dominant-negative mutants with amino acid substitutions in the conserved N(T)KxD nucleotide binding motif. Surprisingly, when such Rab24 constructs were expressed in cultured cells, they accumulated in nuclear inclusions which disrupted the integrity of the nuclear envelope. The inclusions reacted positively with antibodies against ubiquitin and Hsp70, similar to protein aggregates observed in polyglutamine disorders. They also appeared to sequester importin-beta and GFP-coupled glucocorticoid receptor. Other Rab GTPases with similar mutations in the N(T)KxD motif were never found in inclusions, suggesting that the unusual localization of Rab24 is not related solely to misfolding of its nucleotide-free form. Studies with Rab24/Rab1B chimeras indicated that targeting of the mutant protein to inclusions requires the unique C-terminal domain of Rab24. CONCLUSION: These studies demonstrate that mutations in Rab24 can trigger a cytopathic cellular response involving accumulation of nuclear inclusions. If the N(T)KxD mutants of Rab24 function as dominant suppressors, these studies may point to a unique role for Rab24 in degradation of misfolded cellular proteins or trafficking of proteins to the nuclear envelope. However, we cannot yet eliminate the possibility that these phenomena are related to unusual non-physiological protein interactions with the mutant form of Rab24.