2014 — 2018 |
Kay, Jeremy N |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Control of Neuronal Position During Retinal Development
DESCRIPTION (provided by applicant): Retinal neurons are evenly spaced across the retina, a pattern known as a mosaic. Even spacing arises during development through contact-mediated repulsion that occurs specifically between neurons of the same type. The molecular mechanisms that allow homotypic neurons to recognize each other, and consequently to avoid each other, are not known. The objective here is to learn how homotypic recognition signals are initiated, received, and translated into signals that adjust cell position. The central hypothesis s that the transmembrane proteins MEGF10 and MEGF11 constitute a receptor-ligand system that: 1) confers homotypic recognition through binding upon cell-cell contact; and 2) triggers intracellular signaling pathways that produce mutual cell-cell repulsion, thereby creating mosaic spacing. The rationale for this work is that it will provide the first mechanistic explanation of mosaic formation, by revealing how the first identified set of recognition molecules (i.e. MEGF10/11) positions neurons. The mechanisms thus revealed are expected to provide general insight into how retinal neurons recognize and avoid each other, opening the way to understanding both mosaics as well as other neuronal patterning events that influence visual function. To this end, the following Specific Aims are proposed: 1) Determine the intercellular molecular interactions that initiate recognition signals. Preliminary data suggest that MEGF10 and 11 mediate these interactions by binding to themselves and acting as both receptors and ligands. To test this hypothesis the binding specificity of each molecule will be determined biochemically, and their receptor/ligand function will be confirmed in vivo using Megf10 and Megf11 mutant mice. 2) Determine how recognition signals are reported in the cell. Preliminary data show that MEGF10 is required to transduce recognition signals. Using biochemical and in vivo genetic experiments, this Aim will test the hypothesis that ITAM phosphotyrosine motifs in the MEGF10 intracellular domain mediate these recognition signals. 3) Determine how recognition signals alter cellular behavior to produce mosaic spacing. This aim will test the hypothesis that recognition alters the behavior of dendrites. Specifically, it is proposed that recognition causes homotypic dendritic repulsion, through which neurons stake out unique territories that allow them to avoid their neighbors. Recognition will be abrogated genetically in Megf10; Megf11 double mutant mice and dendritic repulsion will be assessed by live imaging of retinal explants. Together, the experiments proposed in these three Aims are expected to reveal for the first time 1) the cell-surface molecules that bind to each other when cells of the same type touch; and 2) how these molecules trigger repulsion in order to specify neuronal position. The approach is innovative because it deploys novel tools and methods to enable the first molecular studies of homotypic recognition in mosaic patterning. The contribution will be significant because molecular events that determine the precise locations of neurons are important for circuit function, both in the retina and throughout the nervous system.
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2016 — 2020 |
Kay, Jeremy N |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Morphology and Image Processing Core
Morphology and Image Processing Module Abstract The objective of the Morphology and Image Processing Module is to enhance the capabilities of individual investigators to conduct cutting edge research in the vision sciences. Our Aims are: 1) to provide resources, support and training required for conducting morphological studies and image analysis at the level exceeding the capabilities of any individual laboratory; 2) to promote collegiality across the community of vision scientists through sharing resources, techniques and expertise; and 3) to engage colleagues into conducting vision research, including support of the next generation of basic and clinician scientists. To achieve these Aims, this Module will support sophisticated facilities equipped with state-of-the-art microscopes and other imaging instruments; microtomes and histology tools; and custom-built software and data processing resources for image analysis. The Module will be supervised and operated by highly experienced personnel, with expertise in conducting a broad array of tissue preparation, image acquisition and automated data processing methodologies. These shared resources will open new research possibilities for both experienced and novice users, and will serve as a platform for fostering interactions among a broad swath of our research community.
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2020 — 2021 |
Kay, Jeremy N |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms of Naturally-Occurring Astrocyte Death During Development
ABSTRACT Naturally-occurring developmental cell death is a fundamental pattern formation mechanism in the nervous system. Whether and how cell death sculpts the astrocyte population is not known. The objective here is to gain insight into astrocyte patterning by learning the mechanisms underlying naturally-occurring astrocyte death in the mouse retina. The central hypothesis is that microglia kill and engulf retinal astrocytes in response to astrocyte-derived ?eat-me? signals, thereby regulating astrocyte numbers and patterning. The rationale for this work is that retinal astrocytes dictate the pattern of developing vasculature. Knowledge of astrocyte death mechanisms will make it possible to study novel factors that shape the ultimate pattern of the astrocyte and vascular networks ? in both normal and pathological developmental contexts. To this end, the following Specif- ic Aims are proposed: 1) Determine cellular mechanisms for developmental cell death of retinal astro- cytes. Preliminary studies show that retinal astrocytes are initially overproduced and then culled between postnatal days 5 and 14. These studies further suggest the working hypothesis that microglia are responsible for killing and eliminating astrocytes during this period. This will be tested in vivo using complementary anatom- ical and chemogenetic approaches. 2) Identify molecular mechanisms responsible for astrocyte elimina- tion during development. Preliminary data show that apoptosis cannot account for developmental loss of ret- inal astrocytes. Instead, a tripartite trans-cellular molecular complex ? comprising phosphatidylserine on the astrocyte surface, the soluble lipid-binding protein MFGE8, and ?v?5 integrins on microglia ? is implicated as a key mediator of astrocyte death. This working hypothesis will be tested using mouse genetic tools in vivo. 3) Determine contribution of developmental death to astrocyte patterning in a disease model. In both mice and humans, neonatal hypoxia exposure can perturb formation of retinal vasculature. Because astrocytes serve as a patterning template for developing vessels, astrocyte patterning defects might contribute to hypoxia- induced vascular pathology. A novel mouse model was developed to study this issue. Preliminary data from this model led to the working hypothesis that microglia-mediated astrocyte death is impaired by hypoxia, caus- ing astrocyte and vessel patterning defects. This will be tested by comparing two mouse strains: a hypoxia- sensitive strain, and a resilient strain that recovers from initial hypoxia-induced pathology. Completion of these aims is expected to: 1) provide the first mechanistic understanding of developmental astrocyte death; and 2) begin to reveal the function of death in patterning the retinal astrocyte population. This contribution will be sig- nificant because it is expected to illuminate how specific pattern formation mechanisms enable astrocyte func- tions, in the retina and throughout the nervous system. The project is innovative because it has strong potential to unveil an entirely new microglia-mediated mechanism for naturally-occurring cell death; this new mechanism may impact development of many cell types and tissues in addition to astrocytes.
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2021 |
Kay, Jeremy N |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Morphology & Image Processing Module
Morphology and Image Processing Module Abstract The objective of the Morphology and Image Processing Module is to enhance the capabilities of individual investigators to conduct cutting edge research in the vision sciences. Our Aims are: 1) to provide resources, support and training required for conducting morphological studies and image analysis at the level exceeding the capabilities of any individual laboratory; 2) to promote collegiality across the community of vision scientists through sharing resources, techniques and expertise; and 3) to engage colleagues into conducting vision research, including support of the next generation of basic and clinician scientists. To achieve these Aims, this Module will support sophisticated facilities equipped with state-of-the-art microscopes and other imaging instruments; microtomes and histology tools; and custom-built software and data processing resources for image and data analysis. The Module will be supervised and operated by highly experienced personnel, with expertise in conducting a broad array of tissue preparation, image acquisition, automated data processing and artificial intelligence methodologies. These shared resources will open new research possibilities for both experienced and novice users, and will serve as a platform for fostering interactions among a broad swath of our research community.
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1 |
2021 |
Kay, Jeremy N |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Precise Assembly of Retinal Circuitry Through Rejection of Inappropriate Synaptic Partners
During development, retinal neurons make exquisitely precise connections with specific synaptic partners. These synaptic choices impact the computational capacity of retinal circuits, and thereby influence visual per- ception. Cell-surface recognition molecules mediate synaptic choices by encoding two kinds of trans-cellular signals: 1) attractive signals that connect neurons with their circuit partners; 2) repulsive signals that shun non- target cells. Both types of cues are needed for precise retinal wiring, but the molecular mechanisms underlying rejection of inappropriate synaptic partners are unknown. The objective here is to identify recognition mecha- nisms that prevent connections between inappropriate synaptic partners. Our central hypothesis is that FLRT and UNC5 families of cell-surface molecules mediate repulsive receptor-ligand interactions that prevent cross- circuit synapse formation. The rationale for this work is that it will reveal a new class of synaptic choice recog- nition molecules that act through repulsive mechanisms. Understanding how the wrong synapses are avoided is a necessary step towards ultimately deciphering the molecular logic underlying synaptic partner choice. To this end, the following Specific Aims are proposed: 1) Identify ligands that prevent retinal neurons from se- lecting inappropriate synaptic partners. Retinal circuits occupy parallel sublayers within the inner plexiform layer (IPL) neuropil. This arrangement facilitates synapse specificity by bringing together arbors of circuit part- ners in a defined location where they are segregated from non-target cells. In preliminary studies using the mouse direction-selective (DS) circuit as a model, we obtained preliminary evidence that the UNC5C cell sur- face protein is a repulsive ligand that confines DS circuit arbors to their appropriate sublayers. This hypothesis will be tested using Unc5c mutant mice and Unc5c misexpression in vivo. 2) Identity receptor-mediated mo- lecular mechanisms that enforce synaptic specificity. Preliminary studies led us to hypothesize that the cell surface protein FLRT2, which is expressed by DS circuit neurons, serves as an UNC5C receptor that con- fines DS circuit arbors to their appropriate sublayers. This hypothesis will be tested using biochemical and in vivo genetic approaches. 3) Determine cellular mechanisms by which retinal neurons shun inappropriate synaptic partners. During dendrite growth, many exploratory branches are eliminated. Our preliminary data suggest that elimination of mistargeted arbors is impaired in Flrt2 and Unc5c mutants. We therefore hypothe- size that UNC5C-FLRT2 repulsion eliminates errant branches to prevent neurons from accessing inappropriate synaptic partners. This idea will be tested by time-lapse imaging of nascent DS circuit dendrites and synapses in Flrt2 and Unc5c mutants. Completion of these Aims is expected to define cellular and molecular mecha- nisms by which neurons avoid incorrect synaptic choices. This contribution will be significant because, once repulsive mechanisms for synapse specificity are known, it will become possible to comprehend how repulsion and attraction work together to produce the overarching molecular logic of synaptic partner choice.
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