Matthew Y. Pecot - US grants

? DESCRIPTION (provided by applicant): The nervous system comprises tremendous cellular complexity yet its function relies on neurons forming precise patterns of synaptic connections. How individual neurons find and form synapses with the correct partners amidst so many inappropriate ones remains poorly understood. Recent evidence indicates that defects in neural connectivity are an underlying cause of neurological disorders. Thus, identifying molecular mechanisms underlying synaptic connectivity is of major importance to biomedical research and human health. Within the visual systems of vertebrates and invertebrates neurons target axons or dendrites to discrete layers wherein they form synaptic connections, thereby providing a structural basis for the parallel processing of different visual information. In the fly optic lobe synaptic layers contain synapses from many neurons, yet specific neurons within a layer synapse with only a subset of these. How synaptic specificity within layers is achieved is unknown. We have discovered that two families of immunoglobulin (Ig) domain-containing proteins known to engage in heterothallic inter-family interactions are expressed complementarily in a cell-type and layer-specific manner within the fly optic lobe. Different afferent cell types express unique combinations of Dprs (21 genes), and target neurons express Dpr interacting proteins or DIPs (11 members). We hypothesize that different heterothallic Dpr-DIP interactions provide a common mechanism by which afferent neurons establish unique patterns of synaptic connections. To test this hypothesis we will investigate Dpr and DIP function in regulating synaptic specificity within a single afferent cell type, L3 lamina monopolar neurons which synapse with multiple partners within their target layer. We will identify cognate Dpr-DIP pairs expressed by L3 neurons and their synaptic partners and investigate their role in synapse formation. We will also perform gain of function experiments to assess if these Dpr-DIP interactions are sufficient to promote synaptic connectivity. The goal of this research is to identify a molecular strategy underlying synaptic specificity. These studies are designed to address a fundamental gap in our knowledge of the molecular mechanisms underlying neural connectivity and establish a platform for the long term investigation of this issue. We anticipate this research will shed light on strategies for rewiring neural circuits in individuals affected by neurological disease and for creating neural circuits with novel functions. To achieve my short term career goal of establishing an independent research program and earning promotion to Associate Professor in the Department of Neurobiology at Harvard Medical School, and my long term career goal of achieving tenure within the Department, I have assembled a team of mentors consisting of tenured faculty at Harvard Medical School who have helped me establish a career development plan. David Ginty, a Professor in the Department of Neurobiology will serve as my primary mentor, and Michael Greenberg, Professor and Chair of the Department of Neurobiology, and David Van Vactor, Professor in the Department of Cell Biology will be co-mentors. Each member will contribute to my growth as an independent investigator in complementary ways based on their scientific expertise and experience. My career development activities will be focused on: (1) Improvement of mentoring, management and lab organization skills (2) Development of my research program (3) Learning how to best fulfill my institutional responsibilities. Based on my strong career development plan, the expertise of my mentor team and the supportive environment within the Department of Neurobiology and Harvard Medical School I believe I have an excellent opportunity to achieve my career goals.

PROJECT SUMMARY/ABSTRACT The ability of neurons to selectively synapse with correct cell types amidst many alternatives (here referred to as synaptic specificity) underlies the structure and function of the nervous system. With respect to progress made in illuminating mechanisms governing the guidance and patterning of axons and dendrites, our knowledge of how synaptic specificity is achieved is severely limited. Addressing this gap in knowledge is essential to understanding how the precision of neural connectivity is established. Our goal is to identify general molecular strategies underlying synaptic specificity. Progress in this area has been limited by the difficulty in studying synapse formation with precise molecular and cellular resolution in complex regions. Therefore, we focus on the Drosophila visual system, wherein cell types and synapses between them are well- characterized, and it is feasible to interrogate gene function in a cell autonomous manner. It is widely believed that neurons identify correct synaptic partners through use of complementary cell surface tags that function like a ?lock and key?. However, evidence supporting this idea is scarce. Previously, we found that members of two subfamilies of the immunoglobulin superfamily (IgSF), dprs (21 members) and dpr-interacting proteins (DIPs) (9 members) which bind heterophilically, are expressed in a matching manner between synaptic partners in the Drosophila visual system. Based on our preliminary findings, we hypothesize that dpr-DIP interactions regulate synaptic specificity by biasing synapse formation towards specific cell types, thereby preventing promiscuous synapse formation with incorrect partners. In this model, dpr-DIP interactions are not necessary for synaptogenesis, but promote synapse formation between specific cell types, potentially by controlling the location of synaptic machinery. We will test this hypothesis in 3 Specific AIMs. In AIMs I and II, we perform focused studies at specific synapses in the lamina to determine if dpr-DIP interactions (I) are necessary to prevent synapse formation with incorrect partners, and (II) have the capacity to promote synapse formation between specific cell types. In AIM III, we will test whether dpr-DIP interactions generally control synaptic connectivity in the visual system through broader studies in a different region of the optic lobe (medulla), which address (1) the function of diverse dpr-DIP interactions at multiple synapses, and (2) whether complementary dpr/DIP expression is generally predictive of synaptic connectivity. In general, our data support the longstanding idea that neurons identify correct synaptic partners through complementary cell surface tags that function like a ?lock and key?. However, we propose that such molecules are not necessary for synaptogenesis, and rather control synaptic specificity by limiting promiscuous synapse formation. This research will advance fundamental knowledge of how neurons selectively form synapses. As dpr-DIP complexes are similar to complexes of mammalian IgSF proteins our findings will be widely transferable. In the long-term, we expect our findings to inform strategies for restoring brain function in the context of disease.

The precise assembly of neural circuits provides the basis for nervous system function and animal behavior. Laminar arrangement of neural connections is a primary strategy for organizing neural circuits in vertebrates and invertebrates. Previous research has illuminated how particular neuron types target to and arborize within specific layers in isolated contexts. However, how the targeting and morphogenesis of different neuron types is coordinated to establish layered networks of connections is unknown. Addressing this gap in knowledge is fundamentally important to understanding how neural circuits are established. The goal of our research is to identify general molecular and cellular principles underlying the construction of layered neural networks. To accomplish this, our strategy is to determine how cells are coordinated to specific layers, and identify commonalities in how different layers assemble to illuminate general mechanisms. This approach requires precise knowledge of the cell types that innervate specific layers and genetic access to these cell types during development. Therefore, we study layer assembly in the Drosophila visual system, wherein well-characterized genetically accessible cell types synapse within specific layers in a stereotyped manner. In the Drosophila medulla, more than 60 uniquely identifiable neuron types synapse within 10 parallel layers. Previous studies indicate that medulla layers are refined during development from broad domains through a precise sequence of interactions between specific cell types. Similar findings in the mouse retina suggests this is a conserved developmental strategy for building synaptic layers. The main thrust of the proposal is to determine the molecular logic governing broad domain organization and the refinement of layers from these regions. We recently showed that Drosophila Fezf (dFezf), a conserved transcription factor, controls the assembly of a specific layer by coordinating the layer-specific innervation of different cell types. Based on preliminary findings, we hypothesize that (1) dFezf acts through a network of transcriptional regulators to control a gene program that regulates early and late stages of layer refinement, and (2) the use of transcriptional modules (like dFezf) to coordinate layer-specific innervation represents a general mechanism for constructing discrete layers. We will test this in 3 Specific AIMs. In AIMs I and II, we use dFezf as a handle to address the molecular underpinnings of (I) broad domain organization within the early medulla, and (II) the stepwise refinement of a specific layer. In AIM III we determine if transcriptional modules analogous to dFezf function generally to orchestrate the assembly of medulla layers. As the Drosophila visual system is analogous to the vertebrate retina in structure and function, and research in the mouse cortex is consistent with Fezf2 regulating the assembly of laminar circuitry, we expect our findings will have broad significance for the development of diverse nervous systems. In the long-term, we expect our findings to inform strategies for re-wiring neural circuits to restore brain function in the context of disease.