1985 — 1986 |
Johnson, Wayne A |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Dopa Decarboxylase Gene Expression in Drosophila @ Harvard University (Medical School) |
0.957 |
1987 |
Johnson, Wayne A |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Regulation of Dopa Decarboxylase Gene in Drosophila @ Harvard University (Medical School) |
0.957 |
1990 — 1992 |
Johnson, Wayne A |
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. |
Role of Pou-Factors in Neuronal Development
The primary objectives of the proposed project are (i) determination of the role of POU factors such as the Drosophila factor Cf1a in development, (ii) characterization of how the Cf1a gene is regulated and its relationship to other pattern formation genes and (iii) confirmation of the putative relationship between Cf1a and the neuron-specific expression of the dopa decarboxylase (Ddc)gene. The proposed specific aims are to characterize the Cf1a transcription unit by mapping and sequencing of CF1a genomic clones and construction of P-element transformant strains expressing Cf1a/beta-galactosidase fusion genes. Analysis of Cf1a/beta-galactosidase fusion genes with mutations within Cf1a regulatory sequences should identify sequences necessary for wild-type expression. The wild-type expression pattern of the Cf1a gene during development will be characterized both by the development of a Cf1a-antiserum for immunohistological labeling, the expression of Cf1a/-galactosidase fusion genes in P-element transformant strains and by continued application of in situ hybridization using digoxigenin-labeled Cf1a probes (see Preliminary Results). The phenotype of mutations within the Cf1a gene will be analyzed by the generation of Cf1a P-element insertion mutants using hybrid dysgenesis as well as a thorough genetic characterization of overlapping deficiencies which move the Cf1a gene. The phenotypic effects of altering the restricted expression of Cf1a by ubiquitously expressing the Cf1a protein under the control of the hsp70 promoter in heat-shocked P-element transformant strains will determine whether restricted expression both spatially and temporally is required for the correct function of Cf1a. A conclusive demonstration that Cf1a directly regulates Ddc expression in specific neurons would be especially significant since it would like a lineage determinant to regulation of a downstream gene involved in the phenotypic expression of a neuronal cell-type. The ubiquitous expression of Cf1a protein could also potentially demonstrate a direct effect upon Ddc gene expression of abherrant CF1a expression causes Ddc expression in inappropriate neurons. If this effect is dependent upon an interaction with the Cf1 binding site, then it would not be seen in transformant strains containing only a Ddc gene with a clustered point mutation in the Cf1 binding site, rendering it incapable of binding the Cf1a protein. A correlation of results from studies on how Cf1a is regulated with the phenotypic effects of over- and under-expression of the Cf1a gene in mutant strains will suggest possible developmental functions for POU factors such as Cf1a. The remarkable conservation of structure between human and Drosophila POU-domains suggests that results from molecular studies in Drosophila can be readily extrapolated to more clinically relevant applications. The suspected role of the Cf1a gene in the development of dopaminergic neurons should be of interest for clinicians investigating various human pathologies such as Parkinsonism which involve the abherrant development of premature degeneration of specific neurons.
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1994 — 1996 |
Johnson, Wayne A |
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. |
Pou-Factors and Neuronal Development
The primary objectives of the proposed project are (i) determination of the in vivo function of the Drosophila POU factor, Cf1a, during CNS development, (ii) identification of downstream target genes and upstream regulators of the Cf1a gene and (iii) identification and characterization of genes encoding proteins which may interact directly with the Cf1a protein as heterodimers or coactivators. The proposed specific aims are to generate a collection of Cf1a mutant alleles by ems mutagenesis. Hypomorphic, temperature-sensitive or tissue-specific mutations collected in such screens will help to characterize the Cf1a mutant phenotype in detail and deduce specific cellular requirements for Cf1a protein during embryonic development. Mutant strains will be analyzed by immunological labeling for various cellular markers. Hypomorphic or temperature- sensitive Cf1a alleles resulting from mutagenic screens will also be mutagenized to generate enhancer or suppressor mutations in genes encoding proteins which may interact directly with the Cf1a protein. Protein- protein interactions involving the Cf1a protein will also be investigated using a direct binding screen of a lambda-gt11 library using 32P-labeled Cf1a protein fragments as probe to isolate potential coactivators or detect heterodimer formation. In collaboration with three other Drosophila laboratories at Iowa, an enhancer-trap insertion library will be generated and screened for potential Cf1a target genes. Putative candidates will be tested for Cf1a-dependent expression using Cf1a mutant alleles and the ubiquitous expression of the Cf1a protein from an Hsp70-Cf1a insertion strain. Upstream regulators of the Cf1a gene will be identified by a detailed characterization of Cf1a regulatory sequences using previously cloned genomic sequences. DNase footprinting using embryonic nuclear extracts, transgenic expression of modified Cf1a-lacZ fusion genes and a comparison of evolutionarily conserved sequence elements will be combined to identify sequence elements and corresponding DNA-binding factors which are essential for the correct temporal and cell-specific expression of the Cf1a gene. Known genes which may directly regulate Cf1a expression will be examined by analyzing Cf1a protein expression in various mutant backgrounds affecting midline and tracheal development using a previously characterized Cf1a antiserum. POU-domain transcription factors have been shown in other species to be important in the designation of cell lineage identities. By examining POU-factor function in a system highly amenable to genetic and molecular manipulation, such as Drosophila, information gathered concerning the interactions of transcription factors during development can be extrapolated to higher order systems which cannot be as easily examined. A potential relationship between the Cf1a gene and the differentiation of glial and neuronal cells in the developing CNS should be of interest for clinicians investigating various human neurological syndromes which involve degeneration of neurons or myelinating oligodendrocytes.
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1997 — 2000 |
Johnson, Wayne A |
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. |
Pou Factors and Neuronal Development
The primary objectives of the proposed project are (i) determination of the in vivo function of the Drosophila POU factor, Cf1a, during CNS development, (ii) identification of downstream target genes and upstream regulators of the Cf1a gene and (iii) identification and characterization of genes encoding proteins which may interact directly with the Cf1a protein as heterodimers or coactivators. The proposed specific aims are to generate a collection of Cf1a mutant alleles by ems mutagenesis. Hypomorphic, temperature-sensitive or tissue-specific mutations collected in such screens will help to characterize the Cf1a mutant phenotype in detail and deduce specific cellular requirements for Cf1a protein during embryonic development. Mutant strains will be analyzed by immunological labeling for various cellular markers. Hypomorphic or temperature- sensitive Cf1a alleles resulting from mutagenic screens will also be mutagenized to generate enhancer or suppressor mutations in genes encoding proteins which may interact directly with the Cf1a protein. Protein- protein interactions involving the Cf1a protein will also be investigated using a direct binding screen of a lambda-gt11 library using 32P-labeled Cf1a protein fragments as probe to isolate potential coactivators or detect heterodimer formation. In collaboration with three other Drosophila laboratories at Iowa, an enhancer-trap insertion library will be generated and screened for potential Cf1a target genes. Putative candidates will be tested for Cf1a-dependent expression using Cf1a mutant alleles and the ubiquitous expression of the Cf1a protein from an Hsp70-Cf1a insertion strain. Upstream regulators of the Cf1a gene will be identified by a detailed characterization of Cf1a regulatory sequences using previously cloned genomic sequences. DNase footprinting using embryonic nuclear extracts, transgenic expression of modified Cf1a-lacZ fusion genes and a comparison of evolutionarily conserved sequence elements will be combined to identify sequence elements and corresponding DNA-binding factors which are essential for the correct temporal and cell-specific expression of the Cf1a gene. Known genes which may directly regulate Cf1a expression will be examined by analyzing Cf1a protein expression in various mutant backgrounds affecting midline and tracheal development using a previously characterized Cf1a antiserum. POU-domain transcription factors have been shown in other species to be important in the designation of cell lineage identities. By examining POU-factor function in a system highly amenable to genetic and molecular manipulation, such as Drosophila, information gathered concerning the interactions of transcription factors during development can be extrapolated to higher order systems which cannot be as easily examined. A potential relationship between the Cf1a gene and the differentiation of glial and neuronal cells in the developing CNS should be of interest for clinicians investigating various human neurological syndromes which involve degeneration of neurons or myelinating oligodendrocytes.
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2001 — 2004 |
Johnson, Wayne A |
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. |
Synaptic Connectivity in Central Brain
The overall goal of this proposal is to characterize the molecular mechanisms by which synapses between central interneurons are established and modulated. We will work toward that broad goal by (1) further characterizing the function of the Drosophila POU domain transcriptional regulator, Acj6, and its role in establishing correct synaptic connections in the central brain, (2) identifying other components of the pathway(s) regulated by Acj6 during synapse formation and (3) characterizing the role of the amino terminal Acj6 POUIV box in regulating Acj6 function. The specific alms of this proposal are: (1) to identify other components of the pathway(s) regulated by Acj6 through completion of a genetic screen for enhancers and suppressors of the acj6 mutant phenotype, (2) to further characterize the acj6 mutant defects in synaptic connectivity by generating Acj6 neuron-specific expression transposons for use both as cell-specific markers and for misexpression experiments, (3) to characterize the neuron-specific expression of Acj6 isoforms resulting from alternative splicing of the amino terminal POUIV box; and (4) to complete a yeast two-hybrid screen for heterologous proteins interacting with the Acj6 POUIV box. The process of synaptogenesis requires precise initial selection of the correct synaptic partners and subsequent refinement of terminal processes and connections. This process of synaptic selection, refinement, strengthening and/or elimination is thought to be important for learning and memory formation and may lie at the root of a number of neuropathologies. Genetic analysis of synaptic function in the fruit fly, Drosophila melanogaster, has contributed significantly to our knowledge of learning and memory mechanisms. In preliminary studies, we have shown that the Drosophila transcriptional regulator, Acj6, is essential for regulation of terminal axon branching and synaptogenesis in a subset of central interneurons. The significant evolutionary conservation of transcription factor interactions shown over the past several years has demonstrated the value of detailed characterization of developmental regulatory mechanisms in an organism such as Drosophila which can be more readily manipulated experimentally. Information gained from an analysis of the fundamental developmental roles played by the Acj6 protein in Drosophila can be extrapolated to higher organisms to better understand analogous processes involved in synaptic connectivity and certain developmental abnormalities.
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2009 — 2010 |
Johnson, Wayne Arlon |
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. |
Sensory Control of Oxygen-Dependent Taxis Behavior
DESCRIPTION (provided by applicant): The evolution and survival of higher life forms on Earth has relied upon an oxygen-enriched atmosphere to drive efficient energy metabolism. Physiological and behavioral mechanisms have evolved to maintain adequate levels of respiratory oxygen while at the same time limiting toxic reactive oxygen species formation. The ability of all organisms, whether simple or complex, to monitor external and internal oxygen levels is key to survival under normal environmental conditions. In clinical situations where patients may be confronted with pathological conditions such as cardiovascular disease, cancer or respiratory illness, these mechanisms can be disrupted or pushed to their limits. Essential oxygen-sensing mechanisms are diverse and as yet poorly understood. Use of the broad range of experimental tools available in model genetic systems such as C. elegans and Drosophila can contribute significantly to advancing our understanding of mechanisms involved in oxygen-sensing. We have utilized genetic, molecular and physiological approaches to relate a fundamental aerotactic behavior in Drosophila larvae to the activation of a subset of peripheral sensory neurons. Preliminary studies suggest that larvae utilize an oxygen-sensing mechanism to coordinate behaviors necessary for food exit prior to pupariation and ultimate survival. These larval aerotactic preferences require function of a small subset of peripheral sensory neurons specifically expressing the DEG/ENaC ion channel subunit, Pickpocket1 (PPK1). Transgenic hypersensitization of ppk1-expressing neurons causes a dramatic increase in larval sensitivity to increased oxygen levels. Transgenic inactivation of ppk1-expressing neurons using tetanus toxin causes a loss of aversion to increased oxygen levels. We propose a model in which PPK1-expressing neurons may function as sensors of environmental oxygen during larval stages. In arthropods such as Drosophila, oxygen is supplied to essentially every cell and tissue by diffusion of air through a complex tracheal tubule system. During larval foraging stages, when larvae display the highest aversion to increased oxygen levels, the external openings of the larval tracheal system, the posterior spiracles, are the only part of the larva projected above the surface of the food to allow access to the atmosphere. Preliminary results show that a single ppk1-expressing neuron innervates each of the posterior spiracles. The focus of this proposal will be on those posterior spiracle neurons and their potential role in oxygen-sensing and aerotactic behavior. Specific Aim I will utilize Ca2+sensitive G-CaMP imaging techniques to examine direct oxygen-dependent activation of the PPK1-expressing neurons innervating the posterior spiracles. Specific Aim II will seek to correlate the function of the same PPK1-expressing posterior spiracle neurons with larval aerotactic behavior by generating single cell hypersensitized neuronal clones using the MARCM technique. Successful completion of these aims should establish a versatile and productive genetic model for use in future genetic and molecular studies of environmental oxygen sensing mechanisms. PUBLIC HEALTH RELEVANCE: The ability of all organisms, whether simple or complex, to monitor external and internal oxygen levels is key to survival under normal environmental conditions. In clinical situations where patients may be confronted with pathological conditions such as cardiovascular disease, cancer or respiratory illness, these mechanisms can be disrupted or pushed to their limits. Despite the central role of oxygen sensing in the survival of life on this planet, we know relatively little concerning the diverse mechanisms that are utilized to balance oxygen supply with metabolic demand.
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