Katja Brueckner - US grants

One of the outstanding questions in animal development is how sensory inputs, through local innervation by the peripheral nervous system (PNS), regulate organ microenvironments and stem cell niches. The proposed research addresses this question using a simple invertebrate model, i.e. the Hematopoietic Pockets (HPs) in the body wall of the optically transparent Drosophila melanogaster larva. In this system, blood cells develop in direct physical contact with segmentally repeated sensory PNS clusters, and functionally rely on the PNS for their attraction, proliferation and trophic survival. The objective of the proposed research is to identify (1) which responses of the hematopoietic cells are regulated by PNS activity, and (2) what are the cellular and molecular mechanisms in the relay of PNS activity to blood cell signaling, using a combination of inducible genetic systems, live imaging and ex vivo analyses. The long-term goal of this research is to understand the signals through which development of the hematopoietic system is modulated by the peripheral nervous system and its sensory inputs. This work is significant because it is expected to reveal general principles how the development of animal tissues is regulated by the PNS and its afferent inputs, allowing adaptation of the animal.
The proposed research will benefit global and local research communities and integrate the education of underprivileged students. Scientists on the project will contribute to the scientific community and the education of students and teachers through traditional approaches and by developing a Drosophila Genetics App for smartphones to map out genetic strategies that will be made publicly available.

DESCRIPTION (provided by applicant): In an emerging paradigm, the peripheral nervous system (PNS) has been identified as a functional component of hematopoietic microenvironments and other stem cell niches. However, it remains largely unknown how the sensory nervous system and its inputs direct hematopoiesis or regulate blood cell responses, To address these questions at the molecular and cellular level, our team developed a simple model for PNS support and regulation of hematopoiesis, using the optically transparent larva of the genetically tractable model organism Drosophila melanogaster (Makhijani et al. Development 2011). The model shows parallels with vertebrate hematopoiesis in the bone marrow niche, and self-renewing tissue macrophages including the microglia of the brain (Makhijani and Brueckner, Fly 2012). Previously, we have shown that, during Drosophila larval development, blood cells (hemocytes) colonize 'hematopoietic pockets', where they accumulate in direct physical contact with segmentally repeated sensory PNS clusters, functionally rely on the PNS for their attraction and trophic survival, and are induced to proliferate in these microenvironments (Makhijani et al. Development 2011). At the molecular level, we identified PNS neuron-produced Activin as a key regulator of hemocyte adhesion and localization and identified N-cadherin (Ncad) as a potential mediator of hemocyte adhesion downstream of dSmad2. Further, we obtained evidence for an interface where environmental sensory stimuli, through activation of PNS neurons, are coupled with blood cell adhesion and localization. We hypothesize that in the Drosophila larval hematopoietic pockets, blood cell adhesion and other responses are controlled by signals emanating from the PNS microenvironment, which are constitutively produced and/or induced by neuronal activity and afferent inputs. The objective of this research is (1) to elucidate the role of Ncad or other adhesion molecules downstream of Activin/dSmad2 signaling in PNS- induced hemocyte adhesion, and (2) to dissect afferent stimulus PNS-hemocyte response circuits in larval hematopoiesis. To address these aims, we will use Drosophila genetics, in vivo and ex vivo analyses, live imaging, and a range of versatile neurobiology genetics tools. This research is innovative, because we established a simple Drosophila model for PNS support and regulation of hematopoiesis, and we will now utilize the system to understand the cellular and molecular principles of PNS-hematopoietic communication in the context of environmental sensory stimuli. This research is significant, because it is expected to provide precedence for specific afferent stimulus- PNS-blood cell response circuits, and identify new concepts of cellular, molecular and electrochemical communication between the PNS and cells of the hematopoietic system that govern blood cell development and homeostasis.

DESCRIPTION (provided by applicant): Cell proliferation, survival and differentiation are commonly known to be regulated by stereotyped developmental programs and physiological feedback mechanisms. However, far less is understood how extrinsic sensory stimuli, through local innervation by the peripheral nervous system, modulate the signaling and responses of cells and tissues in the developmental adaptation and homeostasis of animal tissues. We address this question using a simple Drosophila melanogaster model of niche support by the PNS, focusing on the hematopoietic pockets (HPs) in the body wall of the optically transparent larva. In this system, blood cells (hemocytes) reside in direct physical contact with segmentally repeated sensory PNS clusters, functionally rely on the PNS for their localization and trophic survival, and are induced to proliferate in these microenvironments. We identified PNS neuron-produced Activin as a key regulator of hemocyte adhesion, localization and number, demonstrating that factors from the PNS determine hemocyte signaling and biological responses (Makhijani et al. in prep.). Examining the role of neuron excitation in the HPs, we find that transient silencing of PNS neuronal activity through inducible genetic systems or acetylcholine antagonists results in the rapid scattering or dispersal of resident hemocytes; conversely, PNS stimulants such as the irritant chemicals AITC (wasabi), menthol (mint), or blue light induce recruitment of blood cells to HPs and cause a rise in blood cell numbers over time. Exposure of intact living larvae to these noxious stimuli causes a rapid increase in intracellular calcium (Ca2+), both in PNS neurons and, subsequently, in hemocytes, consistent with an activation of Trp (Transient receptor potential) channels. We hypothesize that activation of the PNS by noxious stimuli regulates blood cell responses through the release or presentation of molecular factors; PNS signals promote hemocyte localization to HPs and facilitate exposure to inductive signals from the microenvironment, resulting in an adaptation of the animal's blood cell pool. The objective of the proposed research is to (1) Determine which aspects of blood cell development are regulated by PNS activity; (2) Identify an inducible mechanism how blood cells are activated by the PNS, focusing on Act and acetylcholine as candidate inducible signals; (3) Dissect the mechanistic sequence by which the irritant AITC (wasabi) regulates blood cell responses. This research is innovative, because we established this simple Drosophila model to study the role of the PNS in the support of a target tissue during development, and now propose to utilize the system to understand the signaling events and biological responses that are triggered in target cells following induction by extrinsic sensory stimuli. This work is significan because it is expected to reveal general principles of how the PNS and its afferent inputs regulate signaling in the homeostasis and developmental adaptation of animal tissues. Interruptions in this communication, such as in peripheral neuropathies, may be the cause of developmental defects and organ degeneration.

PROJECT SUMMARY/ABSTRACT Innate immunity plays important roles as first line defense and primer for adaptive immunity to protect against infection, and its excessive prolonged activation promotes chronic inflammatory diseases. While the main molecular players and signaling pathways involved in innate immunity have been identified, more research is needed to understand how signaling among multiple tissues triggers innate immune responses at the organismal level. Since studying multi-tissue innate immune responses remains challenging in vertebrate systems, we address this question in a simple invertebrate model. Drosophila melanogaster has been key in the discovery of innate immunity, and it is likewise expected to be an excellent model to understand molecular mechanisms that drive more complex, multi-tissue innate immune responses. Specifically, we propose to investigate a new model of an innate immune response in adult Drosophila, which involves the combination of a reservoir of immune cells (hemocytes), respiratory epithelium, and domains of the anatomically colocalizing immune tissue of the fat body. In this model, we focus on the expression of Drosocin as a readout, which promotes survival after bacterial infection. We find that hemocytes, and specifically their signaling by the NFkB- related Imd pathway, are required for the induction of Drosocin expression in the respiratory epithelium and locally restricted domains of the fat body. However, while Imd signaling in hemocytes is required, it is not sufficient to trigger the Drosocin response. We hypothesize that immune cells act as sentinels of bacterial infection that relay a (so far unidentified) signal to the respiratory epithelium and fat body, which in response upregulate Drosocin. Drosocin has, at endogenous expression levels, anti-bacterial function and promotes animal survival after bacterial infection. We propose to (1) identify hemocyte signal/s that trigger the Drosocin response in other tissues, and (2) identify the signaling pathways within tissues that relay the Drosocin response. This work is significant because insights from this Drosophila model are expected to increase our understanding of the molecular mechanisms that drive multi-tissue innate immune responses in a variety of organisms across phyla, thereby extending the concept of `inter-organ/-tissue communication' to innate immunity. New mechanistic principles identified in this model are expected to inform vertebrate research and inspire therapeutic approaches that curb or enhance multi-tissue innate immune responses, which could be tailored toward a variety of medical needs and conditions.