Elwood Linney - US grants

This research is an extension of previous work using polyoma mutant viruses and viral sequences with recombinant DNA probes to study the gene expression requirements of teratocarcinoma cells. Teratocarcinoma cells consist of a stem-cell embryonal carcinoma (EC), which can be grown as a cell line under cell culture conditions, and then depending upon the cell line used, can be induced to differentiate to other cell types. We are using the viral and recombinant DNA probes to examine what DNA sequences are necessary for expression of genes in these different cell types and to try to select for cellular sequences which might play a role in cell type-specific gene expression. This work makes use of various gene transfer techniques, manipulation of small restriction fragments containing viral enhancement sequences, oligonucleotide synthesis to reconstruct regulatory sequences, and competition transfection experiments to examine whether regulatory sequences are competing for cellular factors, which play a role in enhancing gene expression in teratocarcinoma cells. (G)

nucleic acid sequence; genetic recombination; gene expression; regulatory gene; developmental genetics; hematopoietic stem cells; genetic manipulation; murine leukemia virus; provirus; embryo /fetus; genome; cell differentiation; mammalian embryology; molecular cloning; teratoma; structural genes; virus genetics; growth /development; virus RNA; radiotracer; electron microscopy;

This study is directed towards examining the onset and control, during development, of glucocortocoid receptor inducible regulatory systems. We plan to examine the developmental generation of this system using early mouse stem cell systems and also the mouse embryo. Our starting point is the mouse embryonal carcinoma cell (or EC cell) since we and others have shown the absence of a glucocortocoid system in these stem cells. We plan to examine the basis(es) for the absence of a glucocortocoid response in mouse embryonal carcinoma cells through examination of glucocortocoid receptor levels, the expression and nature of glucocortocoid receptor mRNA levels, and through introducing vectors for expression of the glucocortocoid receptor. Using stem cell lines and mouse embryos, we plan to examine the developmental timing of the onset of the glucocortocoid response, its possible role in early differentiation and whether we might interfere with this role through gene transfer experiments.

biomedical facility; genetically modified animals; microinjections; laboratory mouse;

DESCRIPTION: The principal investigator proposes five years of work to determine the sensitivity and nature of responses to estrogens and anti-estrogens in different tissues of zebrafish embryos and young fish. Methods will be employed to visualize estrogen-induced signal transduction, examine expression of estrogen receptor mRNA, and translate these results into 3-dimensional imaging of gene expression and mRNA localization in the intact organism. It is asserted that a need exists for a system where one can see estrogen signal transduction in the whole organism as it develops, because estrogens affect several different organ systems, and because it is important to determine the extent to which compounds acting as anti-estrogens in some tissues have estrogen-like activity in other tissues. This approach also can be extended to the examination of synthetic and natural estrogenic/anti-estrogenic compounds. The principal investigator poses several questions. Can estrogenic compounds affect where and when the estrogen receptor mRNA is expressed during development? Do estrogenic antagonists and agonists have different developmental target specificities, activating expression at different developmental times and places? Does "artificial" estrogen exposure in early development affect reproductive capability and sexual phenotype of the resulting fish? These questions will be approached by a combination of molecular techniques including the use of fluorescent reporter genes and transgenic animal derivation, and laser scanning confocal microscopy followed by computer-assisted image analysis. Five specific aims are identified. (1) Examine development in presence of beta-estradiol and other compounds identified as estrogenic antagonists or agonists in mammalian systems. (2) Isolate, characterize and computer archive the location of the zebrafish estrogen receptor during development from 24 hours post-fertilization through 3 weeks post-hatching. (3) Examine estrogen response in zebrafish to different estrogen-responsive elements (ERE) by co-injection of zebrafish estrogen receptor and ERE-thymidine kinase-promoter-green fluorescent protein (GFP) plasmid constructs to determine expression and sensitivity to different estrogens and anti-estrogens. (4) Generate transgenic zebrafish with chosen constructs to examine the estrogen response. (5) Examine regulation of the transgenic response and 3-dimensional expression of estrogen receptor mRNA in response to exposure to different estrogens and anti-estrogens.

The proposal is focused upon using the zebrafish embryo as a model system for examining the nature of retinoid signaling during embryogenesis. This laboratory's past experience in working with mouse embryos has provided us with a picture of retinoid signaling during embryogenesis but the mouse system is much more difficult to examine in real-time because of the absence of in vitro culture systems that would allow for following events directly and continuously throughout development. In this proposal we plan to use fluorescent reporter techniques (the expression of green fluorescent protein and mutants thereof as a reporter gene and fusion proteins and the use of fluorescent whole mount in situ hybridization), transgenic approaches, microinjection of in vitro synthesized fusion mRNAs, confocal laser scanning microscopy, and three-dimensional computer reconstruction of digitized fluorescent data to develop a picture of retinoid signal during development of the embryo. We plan to computer archive the appearance of the zebrafish retinoic acid receptors and retinoid-X receptors during embryonic development; to develop transgenic zebrafish which will allow us to determine where retinoic acid receptor activity occurs; to introduce the expression of fusion proteins which will interfere with this expression. Our experience in the mouse embryo suggests that one does not observe retinoid signaling just because receptors and retinoids co-exist at specific locations in the embryos. We have isolated a putative zebrafish homologue to the human SMRT co-repressor and plan to examine and isolate a zebrafish homologue to the N-Cor co-repressor. These co-repressor will be characterized, their 3-dimensional expression patterns will be determined and computer archived using fluorescent whole mount in situ hybridization, and their possible role in selecting or limiting retinoid signaling in the embryo will be examined.

This research core will facilitate the use of gene transfer and transgenes of fish in the different projects necessitating these technologies. The core will be setup to facilitate advice on transgenic construction, it will provide vectors and reporter genes, it will produce transgenic fish and screen the fish for the transgene. It will also provide instruction on handling and analyzing transgenic fish. Eventually, as new transgenic lines are produced that have firefly luciferase reporter genes, it will provide instructions on assaying the treated embryos and larvae for firefly induction by toxicants. In this context. the specific aims of this core will be: 1) To provide advice, vectors, cloned regulatory elements and reporter genes for the construction of transgenic vectors. For those laboratories not familiar with working with DNA, it will instruct individuals on how to make the transgenic constructs and/or make them for the laboratory. 2) To produce transgenic fish via DNA microinjection or pseudotyped retroviral vector infection. To screen the progeny of the potential transgenic founders to identify transgenic expressing progeny for the development of transgenic lines. To instruct individual, if desired, on the examination, breeding, and transgenic screening of the transgenic lines. 3) To collect and freeze down sperm from the transgenic lines for long- term storage. 4) To setup breedings so that embryos can be provided for treatment studies and to aid in the luciferase analysis of the IRES transgenic fish.

Transgenic fish will be used to study the effect of pesticides upon gene expression of transgenic reporter genes that report changes in the estrogen-responsive, retinoic acid-responsive pathways in developing fish and in the development of the nervous system. Our hypothesis are: 1) Liver reporter transgenic fish embryos and larvae can be used as biosensors to detect whole organism effects of environmental toxicants. 2) There may be unique windows of sensitivity of chemicals which impact upon the estrogen receptor signal transduction pathway that can be detected and evaluated in fish models. 3) Dicistronic reporter genes will allow for both tissue specific expression studies plus quantitative evaluation of toxicants upon whole fish embryos. 4) Transgenic techniques developed for the zebrafish can be used for indigenous species such as the killifish, Fundulus heteroclitus. The specific aims of this proposal are: I. To explore the use of transgenic zebrafish models to screen several superfund chemicals for detectable differences of GFP expression. II. To examine the behavior and incorporation of the GFP positive primordial germ cells into the developing reproductive tract and to see if there are biological effects upon sexual determination of the fish through treatment with pesticides investigated in specific aim I. III. To develop dicistronic vectors for the appropriate transgenic promoters that will allow for ligand inducible expression of both a luciferase gene (for quantitative measurement) and a GFP gene (for anatomical localization of signal). IV. To develop Fundulus models with the most appropriate transgenes based upon the results of specific aims I and II.

DESCRIPTION (provided by applicant):Transgenic mouse, rat and fish models have been constructed which allow the investigation of the effects of chemicals upon producing mutations. In all these organisms the process involves challenge with the chemical followed by isolation of the DNA and screening for mutations in transgenes representing selectable bacterial markers. While this allows for some degree of quantitativeness, the results uncovered are limited by the processing time and the selection of tissue from which DNA is obtained. In this proposal we plan to develop an imaging assay in transgenic embryos and larvae which will allow for a live, whole animal screen for mutations produced by potential cancer therapeutics. This approach will take advantage of the small size of zebrafish, allowing for microscopical observation throughout and beyond embryonic development. By evaluating live individual organs as they develop, the potential toxicity of new cancer therapeutics can be easily evaluated as to dose, and whether biochemical processing by the organism might create a by-product that is localized by the organ that does the processing. This technology will be developed by using transgenic technologies in combination with fluorescent reporter targets which, in unmutated form, will localize fluorescence to specific regions of the cells of the organism and, if mutated, a signal change would occur allowing one to scan the whole embryo for somatic mutations. The specific aims are: 1) to develop fluorescent indicator reporter genes that will, if mutated in a target region, cause fluorescence to re-locate to another region of the cell 2) to construct, identify and characterize germ-line transgenic zebrafish with these indicator genes using DNA microinjection technology and pseudotyped retroviral vector infection technology 3) to evaluate the transgenic lines with a characterized mutagen(ENU) to test the proof of principle

DESCRIPTION (provided by applicant) Despite the tremendous inter-individual variability in the respond to environmental toxins, the investigators simply do not understand why certain people develop disease when challenged with environmental agents and others remain healthy. Yet, there is emerging consensus that many of the complex (and prevalent) diseases that humans develop occur as a result of multiple biologically unique gene-gene and gene-environment interactions. The recent advances in human and molecular genetics has provided an unparalleled opportunity to understand how genes and genetic changes interact with environmental stimuli to either preserve health or cause disease. The theme of this Center is to use gene expression profiling to understand the effect of environmental stresses on human health. This will be accomplished by establishing an interdisciplinary Center that supports the use of complementary biologic systems (humans, mice, zebrafish and worms) to investigate the role of genetic susceptibility in the pathogenic response to specific types of environmental stress (bacteria, malnutrition, and metals). This approach will enable the investigators to develop and investigate environmental models of human disease that represent biologically unique gene-environment- pathophysiological phenotypes. Microarray analyses will be used to comprehensively evaluate the biological response to environmental stress and to identify pathogenic mechanisms that are relevant to innate immunity, neural tube defects, and transition metal toxicity. The end result is a broad based yet highly integrated program that has the potential to make a number of novel, related observations. The overall hypothesis unifying this research program is that gene expression profiling will identify genes and pathogenic processes that are critical to human environmental health and disease. In aggregates the coupled scientific findings from the proposed Program will substantially enhance our understanding of environmental toxicology and genomics.

DESCRIPTION (provided by applicant) Despite the tremendous inter-individual variability in the respond to environmental toxins, the investigators simply do not understand why certain people develop disease when challenged with environmental agents and others remain healthy. Yet, there is emerging consensus that many of the complex (and prevalent) diseases that humans develop occur as a result of multiple biologically unique gene-gene and gene-environment interactions. The recent advances in human and molecular genetics has provided an unparalleled opportunity to understand how genes and genetic changes interact with environmental stimuli to either preserve health or cause disease. The theme of this Center is to use gene expression profiling to understand the effect of environmental stresses on human health. This will be accomplished by establishing an interdisciplinary Center that supports the use of complementary biologic systems (humans, mice, zebrafish and worms) to investigate the role of genetic susceptibility in the pathogenic response to specific types of environmental stress (bacteria, malnutrition, and metals). This approach will enable the investigators to develop and investigate environmental models of human disease that represent biologically unique gene-environment- pathophysiological phenotypes. Microarray analyses will be used to comprehensively evaluate the biological response to environmental stress and to identify pathogenic mechanisms that are relevant to innate immunity, neural tube defects, and transition metal toxicity. The end result is a broad based yet highly integrated program that has the potential to make a number of novel, related observations. The overall hypothesis unifying this research program is that gene expression profiling will identify genes and pathogenic processes that are critical to human environmental health and disease. In aggregates the coupled scientific findings from the proposed Program will substantially enhance our understanding of environmental toxicology and genomics.

Our working hypothesis is that there is a hierarchy of effects of organophosphate embryonic organophosphate exposure but selective targets and developmental windows are responsible for adult effects. We plan to use zebrafish to screen organophosphate pesticides to examine embryonic and larval effects upon exposure and in collaboration with the Levin laboratory, to investigate and compare effects of these exposures upon learning. Our intention is to screen at least 3 additional esterase inhibitors (along with chlorpyrifos: parathion, diazinon from the superfund list, and the non-superfund list but commonly used malathion). The four specific aims are: 1) to perform dosage studies with selected transgenic lines for the first 5 days post-fertilization and to score for a)inhibition of acetylcholine esterase and b)morphological effects?preliminary behavioral differences will be noted for potential analysis of adults by E. Levin 2) to perform 5 day time courses with selected exposures and to perform microarray analyses to identify genes, clusters of genes and time course of gene expression effects 3) through analysis of expression work to a) identify potential unique genes(per chemical) that are markedly up-regulated(as candidate genes for isolating their promoters to create inhibitor-specific transgenic biosensor) and b) to identify potential genes and their time-course that are positively or negatively affected by the exposure to differentiate effects of different inhibitors and potentially identify pathways affected?this could include in situ localization of the genes affected by the exposure 4) from specific aim 3) the generation of organophosphate- specific biosensor transgenic fish based upon unique genes up-regulated by each inhibitor, evaluate transgenics with inhibitor exposures and determine whether battery of transgenic fish can be used to assay mixtures of inhibitors.

This proposal will be testing the hypothesis that exposure of the very early embryo to compounds that can impact upon neuronal signaling will result in adults with learning and or behavioral problems. Its three specific aims are: 1) to examine how ectopic signaling through the acetylcholine pathway results in changes that eventually influence adult learning/behavior;2) to test whether signaling interference in the glycine and GABA pathways impact upon adult learning/behavior and to examine the molecular/developmental changes imposed and 3) to evaluate whole embryo effects of environmental chemicals that might affect neuronal signaling. Its potential value to human health is related to the large number of chemicals that have been and are being introduced into our environment without an adequate amount of pediatric or embryonic neurological examination. We have previously found that very early exposure of the zebrafish embryo to chlorpyrifos results in adults with learning deficiencies. In this proposal we plan to examine the early events affected by this challenge and to challenge other neurotransmitter pathways to test the generality of the hypothesis. The technologies used will include behavioral analyses, the creation and use of a number of fluorescent transgenic reporter lines to fish, microarray analysis, and HPLC analysis of neurotransmitter levels.

DESCRIPTION (provided by applicant): This proposal is based upon the hypothesis that phase 1 enzymes are involved in an embryonic switch and that the default position for the switch at the time of fertilization is OFF. This would occur in a subset of genes that have retinoic acid response elements (RAREs) in their regulatory DNA. The OFF position would be due to a function of retinoic acid receptors that has not received sufficient notice: in the absenc of retinoic acid, corepressor molecules bind to the RAR? on the RAREs, the corepresor prevents the RAR? from interacting with a coactivator and also brings in a histone deacetylase that condenses the chromatin. As development proceeds, retinoic acid is precisely parsed out through aldehyde dehydrogenases, and as it leaves its cellular source the retinoic acid can enter adjacent cells but is limited by boundaries of cells expressing specific cytochrome p450 activity (cyp26a1, cy26b1, cyp26c1) that oxidize retinoic acid to a nonfunctional ligand. All of these components are carefully regulated in the developing embryo and have direct and/or indirect feedback loops that are affected by retinoic acid. The allotment of retinoic acid then derepresses genes and affects the differentiation of the embryonic cells. So what does this mean and why is it important? Genes recognized as functional Phase I enzymes in the adult play a critical in the development of the embryo-drugs; hormones that affect these genes or designed to interact with them could then unknowingly play dramatic or subtle roles in the development of the embryo in general and specifically, the nervous system. We will be testing this hypothesis mechanistically with a toolbox of antisense morpholinos, indicator transgenic embryos, and additional reagents and approaches to identify the genes repressed and to see if experimental manipulation of these components can have transgenerational effects upon the germline. Specifically we will be using transgenic retinoic acid indicator zebrafish embryos (developed in this laboratory) to help us identify what genes might be repressed via this mechanism at the time of zygotic gene expression initiation. We will confirm this association by immunoprecipitating zebrafish smrt corepressor associated chromatin using anti-Smrt antisera. We will compare the identified family of genes that go through this double screen with genes others have found to turn on when embryonic stem cells differentiate and we will interrogate our 400+ Agilent microarray zebrafish developmental database to identify general developmental expression patterns and genes that appear to be coregulated in time. Through this work we hope to not only test the hypothesis but to begin to identify an important subset of genes held-back by this RAR-corepressor mechanism PUBLIC HEALTH RELEVANCE: The developmental expression of Phase I enzymes can play a critical role in developmental decisions in the embryos. In this proposal, the molecular nature and control of these events will be described and evaluated.