1985 — 1995 |
Wyman, Robert |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Neurogenetics of Identified Cells |
0.915 |
1985 |
Wyman, Robert J |
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. |
The Specification of Neural Circuits
Normal functioning of te nervous system depends upon the formation of vast numbers of specific connections between neurons. The mechanisms involved in producing this specificity are unknown, and their discovery presents the outstanding quest in developmental nuerobiology. One profitable approach is the use of genetics: the molecular basis of this specificity may be arrived at through the analysis of mutations which disrupt proper connectivity. Such mutations may alter genetic factors directly involved in neural development. For instance, if surface-bound molecules are mediating the specificity of neural connections, then genes coding for either the molecules themselves or enzymes involved in their synthesis might be identified. However, before genetics can be used rationally as a tool to probe for answers on the molecular level, the effects of mutations on neuronal connectivity must be investigated on the cellular level. We hae isolated several mutations in Drosophila melanogaster which disrupt normal connectivity in the nervous system. The most exciting of thse alters a gene which we believe is directly involved in establishing proper connectivity between two identified neurons of the giant fiber system. This mutation, bendless, deletes the specific branch of the giant fiber which normally makes synaptic contact with the motorneuron to the jump muscle. Our goal in the next several years is to elucidate the role of this gene during development in establishing the proper connectivity between the giant fiber and jump motoneuron. 1. By using normarski optics and lucifer yellow fills we will determine at what time and in what manner mutant giant fibers deviate from the normal developmental sequence. 2. We will isolate new alleles of the bendless gene (especially extreme and temperature sensitive alleles). 3. By the use of mosaics we will determine what cells must have mutant genotype in order for the bendless phenotype to be expressed.
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1986 — 1990 |
Wyman, Robert J |
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. |
Neurogetic Analysis of Synaptic Connectivity
Normal functioning of the nervous system depends upon the formation of vast numbers of specific connections between neurons. The mechanisms involved in producing this specificity are unknown, and their discovery presents the outstanding quest in developmental neurobiology. One key goal is to find the molecular cues which guide nerve cells during axonal growth and final target recognition. Our strategy for identifying such molecules is to first identify the genes which code for them, and then to use the methods of molecular genetics to identify the gene products. We work with 8 identified neurons in Drosophila melanogaster which are responsible for the jump and flight of the escape response. We already know much of the anatomy, physiology and connectivity of these neurons. In particular, we know the role of each individual neuron in the generation of the starting jump and the precisely repeated cycle of activation of the flight mononeurons. We are identifying the genes which specify the properties and interconnections of these cells. We have developed behavioral and physiological screens to recover mutants in which these cells are abnormally connected. We analyze the effect or the mutations by dye fills, light and electron microscopy, electrophysiology, genetic analysis (including mosaic analysis) and molecular cloning. Some of the mutants already recovered affect single branches of individual cells. For these mutations, the goal is to elucidate the role of these genes in establishing proper connectivity by visualizing the growth path of the mutant and normal neurons during development, by using mosaics to determine which cells need to express the gene, and by determining what other effects mutations in these genes have. We are doing the mapping and genetic analyses that are necessary in order to clone these genes (being done in collaborations). We will continue to screen to identify further mutations which cause these neurons to be abnormally connected in order to understand the number, specificity, mode of action and diversity of genes controlling the specificity of connectivity.
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1987 |
Wyman, Robert J |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Genetics of Development and Aging
The participating laboratories share a common interest in understanding Drosophila development and its relevance to human development. Individual research areas include the role of homeotic genes in developmental programming, hormonal regulation of gene expression and cell proliferation, and the development and function of various parts of the nervous and chemosensory systems. The goals are to elucidate the genetic and molecular bases of these complex processes. Since the degenerative phenomena associated with aging are evident in Drosophila, and appear to involve changes of cell differentiation and function, we propose to extend our research, individually and collaboratively, to include a developmental approach to aging in Drosophila. All of the proposed experiments are based on the use of mutants to dissect complex developmental processes, so that the relevant genes can be identified and cloned and their roles in development and aging studied in molecular detail. Since many basic mechanisms of development have been conserved to a significant extent during evolution, information obtained about Drosophila, which offers so many advantages for molecular genetics, is likely to be relevant to related phenomena in other organisms including humans.
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1988 — 1990 |
Wyman, Robert J |
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. |
Transplantation of Myoblasts
The experiments which we are proposing will test the ability of muscle stem cells to survive transplantation, to colonize or augment host muscle to reverse the effects of genetically-induced alterations in the integrity and life span of muscle, and to become immortal. Classic experiments by Hadorn and others in Drosophila melanogaste have shown that the epithelial (cuticle- forming) cells associated with the imaginal disks of the presumptive adult appendages can be immortalized by culturing them in the abdomens of adult hosts. This isolates them from the hormonal cues that induce metamorphic cell differentiation. These cultured imaginal disks can then be induced to differentiate normally, months or even years later, by transplanting them into late-larval hosts and allowing them to undergo metamorphosis. The recent development of techniques for marking Drosophila cells with biochemical tags will enable us to determine whether the life span of the adepithelial cells (adult muscle precursors) within the disks can be similarly prolonged by transplantation. The first aim of this project will be to follow the fates of these muscle stem cells as they are transplanted into successive hosts and to determine whether the differentiation of normal adult muscle stem-cell populations can be retarded or accelerated. Our second aim will be to test the ability of the transplanted myoblasts to reverse the effects of genetic mutations which delete a single specific muscle or cause it to degenerate prematurely. We will screen hosts for behavioral and physiological recovery of function, and individually mark and evaluate the activity of their motorneurons. The out come of these experiments should also help to answer our third aim: to determine whether it is defects in the muscles or in the neurons innervating the muscles that are responsible for the muscle deletions.
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1991 — 1993 |
Wyman, Robert J |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Cellular and Behavioral Neuroscience |
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1993 — 1998 |
Wyman, Robert J |
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. |
Neurogenetic Analysis of Synaptic Connectivity
One of the key problems in developmental neurobiology is to find the molecular cues by which neurons recognize their proper synaptic partners. Our strategy is to use behavioral screens and electrophysiological testing to identify genes which disrupt the specificity of neural connectivity. We study a small set of eight neurons which mediate the visually driven startle/escape response in Drosophila melanogaster. We have done extensive physiological and anatomical characterization of this circuit. We have individually studied each of the large cells of this circuit by stimulating and recording from them, filling them with dyes and examining their synapses in the EM. The Giant Fiber, a large axon which descends from the brain to the thorax, is the command neuron for this response. Mutation of the Passover gene disrupts specific connections between the GF and the motorneuron and interneuron which are its thoracic synaptic targets. We have cloned the gene and it codes for a transmembrane protein from a new gene family. The gene is expressed in the adult brain in a pair of large cells which are probably the GFs and in thorax in cells that are probably the synaptic targets of the GF. It is expressed throughout adult life, so is probably involved in maintenance of the synapses as well as their development. We propose to attempt to rescue the phenotype by P-element mediated transformation. In an attempt to determine the mechanism of action of the Pas gene we will study the temporal and tissue specific expression of the gene's transcripts and proteins in wild-type and mutant strains. Since it seems that only a few cells express this gene strongly, we will use electrophysiological and molecular double labelling to determine the identity of the cells. Electron microscopy will be used to look at specifically affected synapses. We will develop tissue and organ culture techniques to study the cells behavior in vitro. We will begin to study how the genome controls Pas expression in such a small number of cells. Using lowstringency hybridization, we will examine other species, including humans, to see if Pas is a member of a gene family which codes for a set of homologous molecules. We expect that the molecules used for specifying neural connections in Drosophila, once they are identified, will prove to be of general significance in the development of nervous systems in widely different animal groups. There may be homologous genes that are used in the development of the human brain; if so, this research may improve our understanding of genetically based human mental disabilities. If similar molecules are used by human descending motor control neurons, this research may provide a basis for therapeutic measures after spinal cord trauma.
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1998 — 2001 |
Wyman, Robert J |
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. |
Genes Coding For Invertebrate Gap Junctions
We have defined a new gene family, the Passover family, whose members apparently code for the invertebrate gap junction channel. Thirty members sharing high sequence homology are known. Mutants are available for five members: mutations in three family members are viable and alter a few specific gap junctional connections; two genes are expressed widely and are mutable to lethality. The founding member of the family, Passover (Pas), has been expressed in Xenopus oocytes where it forms intercellular channels. This proposal seeks to start defining the properties of the junctions made by pas family genes. The genes will be expressed in specific cells in Drosophila by using the GALA-UAS system and in heterologous systems such as Xenopus oocytes and tissue culture cells by injection of RNA and by transfection. We will determine how different the channels made by different genes are and whether the different channel molecules form homotypic or heterotypic channels. In the animals, PAS is expressed in the neurons of the Giant Fiber system (GFS) which are connected by gap junctions. Pas mutations disconnect these cells. We will determine whether different transcripts rescue different synapses. Chimeric channel genes have been made and will be used to determine whether various aspects of function can be mapped to different molecular domains. A genetic screen will be undertaken to find molecules that interact with pas. Health significance: The pas family proteins have no known homologs in vertebrates. Drugs that target them specifically should be safe for human and vertebrate animals. This could lead to drugs that kill nematodes and other parasitic worms of plants and animals, These drugs should also be effective against insects, like the malaria mosquito, the tsetse fly, the deer tick, etc. for humans, as well as a host of ectoparasitic insects ( and probably mites) of veterinary importance. Of course, a prime target would be insects and other invertebrates that infest and east food crops. Identification of the molecules which form invertebrate gap junctions allows the methods of experimental crops. Identification of the molecules which form invertebrate gap junctions allows the methods of experimental manipulation available in flies and worms to be immediately useable for studies of gap junction communication. This should greatly facilitate studies of developmental mechanisms, channel function and neural systems.
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2005 — 2006 |
Wyman, Robert J |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Genetic Methods For Detecting Gap Junction Communication
DESCRIPTION (provided by applicant): There is strong evidence that gap junctional communication (GJC) is a regulator of cell proliferation and that interruption of this is one of the steps in the malignant transformations of cancer. Gap junctions occur in all animal species and in most tissues from extremely early in development: the eight cell stage in mice, gastrulation in Drosophila and the two-cell stage in nematodes. Yet gap junctions are the cell structures about which the least is known; their role in cell biology and development is still barely explored. Gap junctions are difficult to detect. The standard way to determine whether cells are GJ coupled is to inject dye into one cell and see if it spreads to neighboring cells. In vivo this requires microinjection, which limits the technique to large and unusually accessible cells. We propose to develop a molecular biological method for the in vivo detection of both enduring and transient GJC without the need for intracellular injection. In the simplest version of the technology, transgenic animals will be made with tissue-specific expression of b-galactosidase (b-gal). The intact animal will be injected with a b-gal substrate (e.g., X-gal) which is taken up by cells and is hydrolyzed to a small colored reporter molecule. b-Gal is too large to pass through gap junctions, but the reporter molecule can. Cells expressing b-gal can be detected with antibodies; any cell not expressing b-gal, but filled with the reporter color must have received its color via GJC. The technology will be validated for uniform cells of a single tissue type, different cell types in a complex tissue, in gap junctions made from a variety of GJ proteins, and in heterotypic junctions made from two different GJ proteins. Quantitative measures will be taken of in-animal and across-animal statistical reliability, extent of spread, and spatial and temporal resolution of the method. Aside from b-gal and X-gal as an enzyme-substrate pair, the method will be validated using other b-gal substrates. Another similar method will be tested using tissue specific expression of transporters to load the presynaptic cells and a detection method for trans-junctional passage. The method will be applied to tumors to assess GJ coupling between tumor cells, between tumor cells and normal cells before and after tumor induction. GJ proteins will be expressed in tumors and the method used to assess GJ coupling after expression and to determine whether tumor growth has been suppressed.
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2005 |
Wyman, Robert J |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Using Transgenics to Identify Functional Gap Junctions
[unreadable] DESCRIPTION (provided by applicant): Gap junctions (GJs) occur in all animals from very early stages of development. Yet, despite their ubiquity, gap junctions are the cell structures about which the least is known; their role in cell biology and development is still barely explored. GJs are difficult to detect. In vivo, microinjection is required which limits the technique to large and/or unusually accessible cells. We propose to develop a molecular biological method for the in-vivo detection of both enduring and transient GJs without the need for intracellular injection. [unreadable] Transgenic animals will be made with tissue specific expression of B-galactosidase. The animal will be injected with a B-gal substrate (C12FDG) which is taken up by cells and hydrolysed to a small fluorescent reporter molecule. B-gal is too large to pass through gap junctions, but the reporter molecule can. Cells expressing B-gal can be detected with antibodies; any cell not expressing 6-gal, but exhibiting the reporter color must have received its color via GJs. Another similar method will be tested which makes use of tissue specific expression of transporters to load the presynaptic cells with a small tracer which can pass through GJs and be detected in post-junctional cells. Controls include the demonstration that GJ mutants block the transmission. [unreadable] As a first use, we wish to apply the method, to the following problem. During the last 30 years, many authors have demonstrated temporary gap junctions (GJs) occurring in nervous system development just prior to the period when chemical synapses are formed. GJ communication has been hypothesized to play a direct causal role in the establishment of functional chemical synapses. However, it has not been possible to block the GJs and thus prove a causal link. [unreadable] In the adult eye, there are no retina-to-lamina gap junctions. Yet, we showed that mutations in two gap junction genes disrupt chemical synaptic transmission there. In mutants, transmission can be restored by GJ transgenes, but only if they are expressed during the period of eye development; expression of the genes in the adult does not rescue. We hypothesize that lamina gap junctions during development are essential for the later development of chemical synaptic transmission. We wish to use the method described herein to directly demonstrate such transient GJs [unreadable] [unreadable]
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