David D. Ginty - US grants

protein structure function; developmental neurobiology; neurotrophic factors; transcription factor; gene induction /repression; apoptosis; protein kinase; regulatory gene; developmental genetics; protooncogene; genetic regulatory element; neurons; neurogenesis; complementary DNA; oligonucleotides; PC12 cells; molecular cloning; transfection; polymerase chain reaction;

DESCRIPTION (Adapted from applicant's abstract): In the nervous system, excitatory neurotransmission markedly influences the expression of genes that are likely to be critical for long-term neuronal adaptive responses, including the maintenance phases of LTP and LTD. Thus, neurotransmitter-sensitive genes may be important substrates of complex neurobiological phenomena underlying learning and memory. The transcription factor CREB has been implicated in regulation of neurotransmitter-sensitive genes and the control of long-term neuronal adaptive responses in both invertebrates and vertebrates. Excitatory neurotransmitters stimulate the phosphorylation of CREB on a single transcriptional regulatory site, serine 133 (Ser133), and this phosphorylation event is critical for CREB-mediated transcription. However, a mammalian model system useful for determining the role of CREB in neurotransmitter signaling, or for identification of genes whose expression is CREB-dependent does not exist. A mouse containing a targeted mutation within the CREB gene exists, but this mouse expresses high levels of a functional CREB protein in brain and other tissues, and the neurons from these mice display neurotransmitter-sensitive, CREB-mediated transcription. The overall objective of the present proposal is to generate two transgenic mouse lines that are either deficient in CREB DNA binding activity or that express a mutant CREB protein that cannot be activated by phosphorylation. Since CREB-dependent transcription is likely to be important for aspects of nervous system development, we will employ new procedures to express our previously characterized dominant negative CREB transgenes exclusively in adult neurons under conditional fashion. These mice will form the basis for future experiments that will address the role of CREB and phosphorylation of CREB Ser133 in neurotransmitter regulation of gene expression, synaptic plasticity and, ultimately, complex neurobiological phenomena such as learning and memory. Moreover, mice deficient in CREB activity exclusively in adult neurons will provide a model system useful for identification of gene whose expression is dependent upon CREB.

DESCRIPTION (Verbatim from the Applicant's Abstract): The longterm objective of our research is to elucidate the mechanism of action of neuronal growth factors that control development of the nervous system and survival of adult neurons. The development of several classes of neurons, and the maintenance of their differentiated state, are regulated by a family of neurotrophic growth factors, the neurotrophins. Nerve growth factor (NGF) is the prototypical target-derived neurotrophic growth factor. In addition to its prominent role during neurodevelopment, NGF can promote survival of populations of adult neurons, including septal cholinergic neurons that normally die in patients with Alzheimer's disease. Our work focuses on the mechanisms by which neurotrophins regulate expression of genes that contribute to growth, differentiation and survival of neurons. Neurotrophins activate the transcription factor CREB (cAMP-response element binding protein) by inducing phosphorylation of CREB on a transcriptional regulatory site, Ser-133. This phosphorylation event is catalyzed by RSK2, a growth factor-sensitive protein kinase. In addition, phosphorylation of CREB Ser-133 is regulated by a retrogradely propagated neurotrophin signal in neonatal sympathetic neurons. Lastly, preliminary results indicate that CREB, or a closely related CREB family member, is critical for NGF induction of transcription of c-fos. Since many, if not most, NGF-sensitive genes contain CREB binding sites within their upstream regulatory regions, it is likely that CREB and CREB family members are critical mediators of the general nuclear response to target-derived NGF. As part of an overall goal to understand NGF regulation of expression of genes that contribute to neuronal differentiation, plasticity and survival, the specific aims of the present proposal are: 1) To characterize the mechanisms of retrograde NGF signaling to transcription factor CREB and other nuclear targets in developing sympathetic neurons; 2) To determine the functional consequences of retrograde NGF signaling to CREB and other nuclear targets, and 3) To establish the requirement of CREB and CREB family members in NGF signal transaction. Together, the proposed research will provide insight into the mechanism of NGF signal transduction, the molecular basis of neurodevelopment, and the control of survival of adult neurons, which are susceptible to death in debilitating neurodegenerative diseases.

DESCRIPTION (Verbatim from the Applicant's Abstract): The longterm objective of our research is to elucidate the mechanism of action of neuronal growth factors that control development of the nervous system and survival of adult neurons. The development of several classes of neurons, and the maintenance of their differentiated state, are regulated by a family of neurotrophic growth factors, the neurotrophins. Nerve growth factor (NGF) is the prototypical target-derived neurotrophic growth factor. In addition to its prominent role during neurodevelopment, NGF can promote survival of populations of adult neurons, including septal cholinergic neurons that normally die in patients with Alzheimer's disease. Our work focuses on the mechanisms by which neurotrophins regulate expression of genes that contribute to growth, differentiation and survival of neurons. Neurotrophins activate the transcription factor CREB (cAMP-response element binding protein) by inducing phosphorylation of CREB on a transcriptional regulatory site, Ser-133. This phosphorylation event is catalyzed by RSK2, a growth factor-sensitive protein kinase. In addition, phosphorylation of CREB Ser-133 is regulated by a retrogradely propagated neurotrophin signal in neonatal sympathetic neurons. Lastly, preliminary results indicate that CREB, or a closely related CREB family member, is critical for NGF induction of transcription of c-fos. Since many, if not most, NGF-sensitive genes contain CREB binding sites within their upstream regulatory regions, it is likely that CREB and CREB family members are critical mediators of the general nuclear response to target-derived NGF. As part of an overall goal to understand NGF regulation of expression of genes that contribute to neuronal differentiation, plasticity and survival, the specific aims of the present proposal are: 1) To characterize the mechanisms of retrograde NGF signaling to transcription factor CREB and other nuclear targets in developing sympathetic neurons; 2) To determine the functional consequences of retrograde NGF signaling to CREB and other nuclear targets, and 3) To establish the requirement of CREB and CREB family members in NGF signal transaction. Together, the proposed research will provide insight into the mechanism of NGF signal transduction, the molecular basis of neurodevelopment, and the control of survival of adult neurons, which are susceptible to death in debilitating neurodegenerative diseases.

DESCRIPTION (Verbatim from the Applicant's Abstract): This K02 application is written for support of my salary and career development. I have been a member of the faculty of the Department of Neuroscience, John Hopkins University School of Medicine for the past four and one-half years. The long-term objective of my research is to elucidate the mechanism of action of neuronal growth factors that control development of the nervous system and survival of adult neurons. Nerve growth factor (NGF) is the prototypical target-derived neurotrophic growth factor. In addition to its prominent role during neurodevelopment, NGF can promote survival of populations of adult neurons, including septal cholinergic neurons that normally die in patients with Alzheimer's disease. Thus, our work should provide insight into neurodevelopment as well as maintenance of neurons that are critical for mental health. Neurotrophins activate the transcription factor CREB (cAMP-response element binding protein) by inducing phosphorylation of CREB on a transcriptional regulatory site, Ser-133. In addition, phosphorylation of CREB Ser-133 is regulated by a retrogradely propagated neurotrophin signal in neonatal sympathetic neurons. Lastly, preliminary results indicate that CREB, or a closely related CREB family member, is critical for NGF induction of transcription of c-fos. Since many, if not most, NGF-sensitive genes contain CREB binding sites within their upstream regulatory regions, it is likely that CREB and CREB family members are critical mediators of the general nuclear response to target-derived NGF. As part of our overall goal to understand NGF regulation of expression of genes that contribute to neuronal differentiation, plasticity and survival, the specific aims of the proposed research are: 1) To characterize the mechanisms of retrograde NGF signaling to transcription factor CREB and other nuclear targets in developing sympathetic neurons; 2) To determine the functional consequences of retrograde NGF signaling to CREB and other nuclear targets, and 3) To establish the requirement of CREB and CREB family members in NGF signal transduction. Together, the proposed research will provide insight into the mechanism of NGF signal transduction, the molecular basis of neurodevelopment, and the control of survival of adult neurons, which are susceptible to death in debilitating neurodegenerative diseases that have profound influence on mental health.

DESCRIPTION (provided by applicant): The long-term objective of our research is to elucidate the mechanism of action of neuronal growth factors that control development of the nervous system and survival of adult neurons. A family of neurotrophic growth factors, the neurotrophins, regulates the development of several classes of neurons and the maintenance of their differentiated state. Nerve growth factor (NGF) is the prototypical target derived neurotrophic growth factor. In addition to its prominent role during neurodevelopment, NGF can promote growth and survival of populations of adult neurons, including septal cholinergic neurons that normally degenerate in patients with Alzheimer's disease. Our work focuses on the mechanisms by which neurotrophin signaling is propagated from distal axons to neuronal nuclei to control the expression of genes that support growth and survival of neurons. We have shown that a retrogradely propagated NGF/TrkA signal in sympathetic neurons supports CREB activity, gene expression and neuronal survival. We also discovered that CREB family transcription factors are critical for growth and survival of neurons. Our more recent findings have shown that CREB activity is controlled not only by phosphorylation on its critical regulatory site, Ser133, but also by a novel neurotrophin-dependent signaling mechanism that governs CREB's DNA binding activity. Lastly, our recent findings indicate that the ubiquitous transcription factor, SRF (Serum Response Factor), appears to cooperate with CREB family members to support neurotrophin dependent gene expression. Thus, as part of our long-term goal of understanding how neurotrophins regulate the expression of genes that contribute to growth and survival of neurons, we propose: To establish how neurotrophin signals are differentially propagated retrogradely in developing neurons;To determine the function(s) and novel modes of regulation of CREB family transcription factors during neurotrophin signaling, and;To establish the role(s) of SRF-mediated gene expression during neurotrophin signaling to the nucleus. Results of the proposed experiments should provide critical insights into how neurotrophic growth factors support growth and survival of neurons during development and in adulthood.

DESCRIPTION (provided by applicant): The Neuroscience Graduate Program, which was begun in 1983, has its headquarters in the Department of Neuroscience at The Johns Hopkins University School of Medicine. Consisting of 64 faculties drawn from various departments across the University, it serves as the hub of a broad spectrum of efforts for the training of graduate students, encompassing molecular, cellular, developmental, systems, cognitive and computational neuroscience as well as neurobiology of disease. Each year, from a pool of-200 applicants, we typically matriculate 10-12 Ph.D. candidates as well as 1-4 candidates for combined M.D./Ph.D. degrees (who are admitted through a separate process). Students enter the program with diverse undergraduate backgrounds ranging from computer science to biochemistry. In the first year they are required to take a year-long integrative lecture course with lab entitled "Neuroscience and Cognition" as well as a seminar on "Science, Ethics and Society". Research opportunities are presented to students through a Departmental Retreat, Lab Lunches (which feature work-in-progress) and a Mini-symposium series by Program Faculty specifically designed to help first-year students choose their research rotations. This information is used to help pick three 12-week lab rotations which are typically completed by the end of the first academic year, following which, a thesis lab is selected. By the end of the second year, students complete 6 additional elective courses, many of which are chosen from a list of 12 small seminar-style courses in Neuroscience specialties. Following completion of a Comprehensive Exam at the end of Year 2, students write and defend a Thesis Proposal which is written in the form of a Pre-doctoral NRSA. Each student is advised by two Prethesis Advisors in Years 1 -2 (at 3 month intervals) and an individualized Thesis Advisory Committee thereafter (at 6 month intervals). Thesis Advisory Committees make reports to the Graduate Program Steering Committee which carefully tracks the progress of each student in the program as well as setting overall program policy. At present, 85 students are enrolled in the Neuroscience Graduate program. The average time to complete the Ph.D. has been 5.3 years. Of the students who have graduated from our program greater than 93% have remained in academic biomedical research. Here, we request stipend support for five students during their first two years in the program. RELEVANCE: The mission of the Program is to train the next generation of neuroscientists to teach and perform research in both basic and clinical neurosciences. The Training Program also serves as a focal point for the faculty and for fostering interactions among students and other investigators doing research in neuroscience.

DESCRIPTION (provided by applicant): The formation of neural circuits relies on axonal and dendritic growth, precise guidance events during development, recognition of appropriate target cells, and the subsequent formation and refinement of synaptic connections. Characterization of each of these stages is critical for understanding the assembly of neuron circuits that mediate all behavior. Deficits in these developmental processes underlie cognitive impairments associated with disease and neurologic disorders. Indeed, aberrant dendritic morphologies and synapses are associated with a range of neuropsychiatric disorders, underscoring the need for understanding the cellular and molecular basis of these events during circuit formation. The objective of this work is to understand how extracellular cues present within the postnatal brain control the morphological development of projection neurons whose cell bodies are located within layer V of the cortex. We will elucidate the functions and mechanisms of action of secreted members of the semaphorin protein family of guidance cues and their neuropilin and plexin receptors on the morphological development of these neurons. Our preliminary work shows that semaphorin 3F (Sema3F) and its neuropilin-2 (Npn-2) receptor function in vivo to govern cortical pyramidal neuron apical dendritic spine morphology and synaptogenesis, whereas semaphorin 3A (Sema3A) promotes the elaboration of basal dendritic arbors. Thus, structurally related cues instruct distinct steps in the development of layer V pyramidal neurons, the location, morphology, and number of synapses that form upon them, and hence the genesis of normal functioning cortical circuits. Mechanistically, we found that localization of the Npn-2 receptor is restricted to primary apical dendritic processes, while Npn-1 is located on both basal and apical dendrites. In addition, Npn-2 is enriched at sites of synapse formation-the PSD. These findings lead to the hypothesis that semaphorin receptor localization underlies Sema3A and Sema3F specificity of action. We propose here to investigate the regulation of neuropilin and plexin receptor distribution, secreted semaphorin receptor signaling mechanisms, and the source and mode of action of these semaphorin ligands during postnatal cortical neuron development. Since our findings will shed light on the mechanisms underlying spatially restricted regulation of dendritic morphology and synapses, the proposed Aims will begin to address how complex cortical connectivity patterns are generated and maintained. Finally, while the focus of the proposed work is on the morphologic and synaptic development of the primary projection neuron of the cortex, the layer V pyramidal neuron, the discoveries made here will have important implications for defining the molecular and cellular basis of neural circuit assembly throughout the brain.

DESCRIPTION (provided by applicant): The goal of this research is to elucidate principles and mechanisms that govern the assembly of neural circuits. The work focuses on the role of the target field, and in particular target-derived growth factors, in the control of neuronal survival and the establishment of synaptic connections between postganglionic sympathetic neurons and their presynaptic partners, and the differentiation of distinct classes of cutaneous sensory neurons. Proposed experiments address mechanisms and functions of the prototypical target-derived neurotrophic growth factor, nerve growth factor (NGF), and its receptor TrkA. NGF, expressed in targets of sympathetic and cutaneous sensory neurons, promotes target field innervation, survival, and synapse formation in the sympathetic nervous system through retrograde NFG/TrkA signaling. Retrograde NGF/TrkA signaling also controls target innervation, survival, and maturation of cutaneous sensory neurons. A main hypothesis to be tested is whether differential sorting and trafficking of TrkA-containing signaling endosomes account for the unique ability of NGF to support retrograde survival;the NGF family member NT3, an intermediate target- derived growth factor, cannot support retrograde survival. This is especially intriguing since both NGF and NT3 promote TrkA activation and TrkA-dependent axonal extension of sympathetic neurons. The work will assess the contribution of the actin cytoskeleton, and its modulation, during TrkA endosome formation, sorting, maturation, and trafficking. Proposed experiments will also address the exciting hypothesis that TrkA endosomes move retrogradely into cell bodies and then throughout the entire dendritic arbor where they instruct the formation of nascent postsynaptic specializations on dendrites. A third aim of the proposed work is to test the hypothesis that target-derived NGF signals retrogradely to promote expression of the transcription factor CBF2, which combines with the sensory neuron-subtype specific transcription factor Runx1 to instruct differentiation of non-peptidergic nociceptors. Thus, proposed work will provide insight into how the target field, and target-derived NGF, controls establishment and maintenance of PNS circuits. Since deficits of axonal transport and signaling and synaptic loss underlie forms of neurodegeneration, findings from the proposed work will be insightful not only for understanding normal development but also for maintenance of the nervous system under normal and disease states. PUBLIC HEALTH RELEVANCE: The proposed studies will provide an understanding of how neuronal differentiation and morphology develop and how cell type specificity, survival, and connectivity are achieved. The particular focus is on understanding how a growth factor, called nerve growth factor, promotes survival and directs the differentiation and connectivity of sympathetic and somatosensory neurons. Importantly, since aberrant neuronal signaling is associated with a range of disorders, including neurodevelopmental disorders and degenerative diseases, this work will inform strategies for treatment of these disorders.

DESCRIPTION (provided by applicant): Inadequate treatment of pain imposes an enormous burden on society, and is due, in part, to a limited understanding of biological events that underlie the transition from acute to chronic pain. Barriers to a better understanding of this transition have included an inability to selectively and efficiently visualize or manipulate specifc sensory neuron populations and the very low throughput of available methods to monitor nociceptor function at the cellular level. In this collaborative proposal, we have teamed together a developmental neurobiologist and several pain biologists to synergistically overcome both of these barriers. Using newly- developed molecular-genetic strategies, we will selectively label each of the four major subpopulations of low- threshold mechanoreceptive (LTMR) neurons, as well as peptidergic and MrgprD-expressing nonpeptidergic nociceptors, in mice. This technology will be combined with selective expression of the genetically encoded calcium indicator, GCAMP3, in mouse peripheral sensory neurons to permit direct visualization of neuronal activity in the skin and DRG. In our first two aims, these tools will allow us to efficiently and with high spatiotemporal resolution monitor changes in the anatomy and function of unambiguously defined nociceptor and LTMR populations during the development of neuropathic pain. These changes will be correlated with corresponding behavioral changes, monitored using classical and newly developed assays of thermal and mechanical sensitivity. In Aim 3, we will selectively ablate two LTMR populations that are candidate participants in pain and define their respective contributions to the establishment, maintenance, and manifestation of neuropathic mechanical hypersensitivity. Together, these studies will provide us with an unprecedented view of the dynamic processes associated with the transition to chronic pain and define cellular targets for the development of improved analgesic therapies. PUBLIC HEALTH RELEVANCE: Chronic pain affects millions of people and is often difficult to treat. This is due, in part, to our poor understanding of why some people, but not others, transition to a state of chronic pain after nerve injury. In this proposal, we will take advantage f new technology to directly visualize specific classes of nerves in the skin to understand how changes in their structure and function after nerve injury enhances the perception of pain. This will allow the rational development of improved therapies to treat chronic pain.

The first vertebrate semaphorin protein characterized, collapsin-1/semaphorin 3A (Sema3A), has potent neuronal growth cone collapsing activity, and the independent discovery by us and others that neuropilin-1 (Npn-1) is a Sema3A receptor revealed the first step by which semaphorin signals are propagated. Npns are obligate ligand binding subunits, and members of the Plexin family are signal transducing subunits of the Class 3 Semaphorin receptors. Sema3F signals predominantly through a Npn-2/PlexinA3 holoreceptor complex, and our work shows that Sema3F-Npn-2/PlexinA3 signaling also controls dendrific spine morphogenesis and the development and function of excitatory synaptic connectivity in the murine hippocampus and cortex. We recently found that Sema3F-Npn-2 signaling contributes to plasficity in an in vitro model system. Cultured Npn-2 null cortical neurons fail to undergo homeostatic scaling of cell surface AMPA receptors in response to a blockade of excitatory synaptic transmission, supporting that Sema3F- Npn-2/PlexinA3 signaling controls both the development and plasticity of adult cortical circuits. We propose addressing in vivo funcfions of Sema3F, Npn-2. and PlexinA3 in an adult model of visual cortex plasticity and homeostafic synapfic scaling. We will record from pyramidal neurons in layer 2/3 of adult primary visual cortex in mouse strains following condifional ablafion of Sema3F, Npn-2, or PlexinA3. We will also use Cre recombinase driver lines to identify the ligand and receptor-expressing cells that mediate this plasticity. We will use our new HA-epitope tagged Npn-2 knock-in mouse line to immunopurify Npn-2/PlexinA3 complexes from forebrain neurons to identify key intracellular signaling components that mediate AMPA receptor trafficking and plasticity of excitatory synapses. These studies will uncover new functions served by developmentally important neuronal guidance molecules during adult neuronal plasticity and will establish the mechanisms by which semaphorin signaling controls AMPA receptor expression and excitatory neurotransmission. RELEVANCE (See instructions): This project will test the hypothesis that Semaphorin signaling pathways, which are essenfial for nervous system development, also govern the strength of excitatory synapses in the mature, adult brain. We will ask how this developmental pathway controls synapse strength. This work will illuminate mechanisms of information processing and storage in the brain, and whether dysfunction of Semaphorin pathways could contribute to psvchiatric disease

Abstract The sense of touch allows us to perceive and respond to the physical world ?we recognize objects held in our hands, discriminate between different textures and shapes, and sensory-motor feedback circuits coordinate our body movements. The sense of touch also underlies forms of social exchange and is thus an essential component of the human experience. The first step leading to touch perception is activation of a group of cutaneous sensory neurons called low- threshold mechanoreceptors (LTMRs). There are several LTMR types, and each has a unique sensitivity, morphology, physiological property and, presumably, function. Understanding the unique functions of each LTMR type, and how ensembles of LTMR activities are integrated and processed in the CNS to form touch percepts are outstanding questions in the field. Therefore, the overall goals of my laboratory, and thus this R35 proposal, are: 1) to elucidate the sensitivities, mechanisms of excitation, and unique functions of the major classes of mammalian cutaneous LTMRs; 2) to define the logic of LTMR circuit organization in the spinal cord and brainstem, and the nature of ascending pathways to the brain that underlie discriminative and affective touch perception; 3) to establish how the peripheral somatosensory system assembles during development; and 4) to determine whether and how dysfunction of touch circuits and their development underlies tactile deficits in autism spectrum disorders and during neuropathic pain. We are achieving these goals using an array of powerful mouse molecular genetic tools, combined with sophisticated electrophysiological, anatomical, behavioral and developmental assays. Successful completion of this R35's goals will thus reveal mechanisms of somatosensory nervous system development and function, and spinal cord touch information processing underlying perception, under normal and disease conditions.

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.

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.