David I. Gottlieb - US grants

The major long range goal of this work is to understand the mechanisms that control the expression of important neuronal properties during the development of the mammalian central nervous system. We plan to use monoclonal antibodies (MAbs) which recognize subsets of neurons to determine when cell specific antigens are first expressed. In particular, we wish to find out if cell specific antigen expression occurs in dividing stem cells that are the progenitors of the mature neuron or if the final mitosis must occur before cell specific antigen expression begins. We would also like to find out if the formation of appropriate connections is necessary in order to either initiate or maintain expression of cell specific antigens. One part of the proposal is to investigate these questions using two newly discovered MAbs. RB-8 stains a subset of rat olfactory sensory cells and their axons. We will study the distribution of RB-8 staining in the embryonic and adult olfactory bulb. In the adult we will map the distribution of RB-8 positive fibers over the entire extent of the bulb. We will do this in several adult rats to see if this pattern is invariant or if it varies from animal to animal. The development of RB-8 staining will be carefully followed in embryonic and young animals. Finally, the pattern of RB-8 staining in regenerating olfactory projections will be determined. RB-6 was seleted because, in the retina, it stains only the retinal ganglion cells. In the case of RB-6 we will observe the appearance of antigen positive cells in retinas before, during and after the birth of ganglion cells. The expression of RB-6 antigen in vitro will also be studied to determine if the presence of target cells influences antigen expression. Monoclonal antibodies which recognize cell surface antigens on subsets of neurons will prove important in future research in this area. Part of our efforts will be directed towards selecting new monoclonal antibodies with these attributes. We will use both neural tumors and membranes from the developing brain to immunize mice and produce additional hybridomas. All work will be carried out using rat cells and membranes as the rat now appears to be the most intensively studied vertebrate as far as CNS biology is concerned.

The mammalian nervous system is comprised of a vast repertoire of neuronal types. Its reasonable to assume that this repertoire is generated by a complex network of genetic control. However, to date very little is known about this network. We propose to analyze a very fundamental developmental choice made by neurons: whether to be excitatory or inhibitory. The decision to be inhibitory usually involves the selective expression of the genes for glutamic acid decarboxylase (GAD). We propose to analyze the mechanisms that lead to the expression of the GAD67 gene in the correct neurons of the mammalian brain. This should give insight into the larger problem of how the genome can guide the development of so many separate neuronal types. Preliminary data strongly suggests that there are several transcriptional start sites for the GAD67 gene. Segments of DNA surroundin the transcriptional start sites will be cloned and transfected into cell lines and used to create lines of transgenic mice. In this way we will determine the minimal unit of DNA necessary to give cell type specific expression of the GAD67 gene. Once this is determined, deletion analysis will be performed to locate regulatory sites in and adjacent to the GAD67 gene. Knowledge of the location and sequence of these sites will make it possible to systematically search for trans-acting factors that control the expression of the GAD gene. P19 mouse teratocarcinoma cells can be induced to differentiate into neuronal and glial like cells by treatment with retinoic acid. After induction levels of GAD67 RNA transcripts rise dramatically. However the functional properties of the cells which are expressing GAD are not known. The ability of these cells to make functiona GAD enzyme and GABAergic presynaptic terminals will be investigated. Since there are 2 genes coding for GAD it will be important to know the relative contribution of each one. As a step in that direction we will create mice in which both alleles of the GAD67 gene are interrupted. We will then determine the effect of eliminating the functional GAD67 gene on development. Taken together these studies will provide basic information o mechanisms that lead to the emergence of a major subset of brain neurons. The work will have relevance to human diseases that are caused by or involv disturbances of normal brain development and gene expression. Recently GAD has been shown to be a dominant autoantigen in juvenile diabetes and the stiff man syndrome. This work will provide additional basic data about thi important disease-related set of genes.

The mammalian nervous system is comprised of a vast repertoire of neuronal types. Its reasonable to assume that this repertoire is generated by a complex network of genetic control. However, to date very little is known about this network. We propose to analyze a very fundamental developmental choice made by neurons: whether to be excitatory or inhibitory. The decision to be inhibitory usually involves the selective expression of the genes for glutamic acid decarboxylase (GAD). We propose to analyze the mechanisms that lead to the expression of the GAD67 gene in the correct neurons of the mammalian brain. This should give insight into the larger problem of how the genome can guide the development of so many separate neuronal types. Preliminary data strongly suggests that there are several transcriptional start sites for the GAD67 gene. Segments of DNA surroundin the transcriptional start sites will be cloned and transfected into cell lines and used to create lines of transgenic mice. In this way we will determine the minimal unit of DNA necessary to give cell type specific expression of the GAD67 gene. Once this is determined, deletion analysis will be performed to locate regulatory sites in and adjacent to the GAD67 gene. Knowledge of the location and sequence of these sites will make it possible to systematically search for trans-acting factors that control the expression of the GAD gene. P19 mouse teratocarcinoma cells can be induced to differentiate into neuronal and glial like cells by treatment with retinoic acid. After induction levels of GAD67 RNA transcripts rise dramatically. However the functional properties of the cells which are expressing GAD are not known. The ability of these cells to make functiona GAD enzyme and GABAergic presynaptic terminals will be investigated. Since there are 2 genes coding for GAD it will be important to know the relative contribution of each one. As a step in that direction we will create mice in which both alleles of the GAD67 gene are interrupted. We will then determine the effect of eliminating the functional GAD67 gene on development. Taken together these studies will provide basic information o mechanisms that lead to the emergence of a major subset of brain neurons. The work will have relevance to human diseases that are caused by or involv disturbances of normal brain development and gene expression. Recently GAD has been shown to be a dominant autoantigen in juvenile diabetes and the stiff man syndrome. This work will provide additional basic data about thi important disease-related set of genes.

embryonic stem cell; cell line; biotechnology; cell differentiation; surface antigens; mesoderm; ectoderm; drug resistance; neomycin; hybrid cells; cell fusion; endoderm; monoclonal antibody; gene targeting; hamsters; flow cytometry; genetically modified animals; laboratory mouse; hybridomas;

SUBPROJECT ABSTRACT NOT AVAILABLE

The long-term goal of this research is two-fold. One is to gain insights into the mechanisms that control fate choice in the early developing nervous system. The principal tool is ES cells differentiated in culture to produce a model of normal CNS development. The model is powerful because large numbers of cells are produced and because gone targeting and other genetic manipulations are feasible. Using the advantages of this model we will investigate how early neural precursor cells choose a fate defined by expression of the Olig2 gene. Cells from this pathway eventually differentiate into motor neurons and oligodendrocytes. Retinoic acid and sonic hedgehog interact to specify na'ive cells to follow this fate. We will learn how these 2 pathways interact. Specified cells are capable of cell division. We have discovered how to induce dividing cells to undergo long-term division. Even though they divide continuously the cells keep their identity as members of the Olig2 lineage. Our working hypothesis is that normal early neural stem cells are indeed capable of sustained division. If true, this will provide large numbers of specified neural stem cells for many applications. The final stage of stem cell life is differentiation. We will investigate signals that are responsible for differentiation of Olig2 pathway cells. We have already discovered how to efficiently differentiate these cells into astrocytes and oligodendrocytes and will extend this work to neurons. The second long-term goal is to harness the potential that ES cells have for neural transplantation. While the potential is real, practical application demands major advances in basic understanding. It is theoretically possible to direct ES cells to coordinately differentiate into any single type of neural cell. Achievement of this goal even for a few cell lineages would open up new opportunities in transplantation research. Results of this project will provide proof-of-principle for this idea. A combination of instructive and selective strategies is being applied to the goal of getting pure populations. Cells from each stage of the Olig2 lineage will be available in large numbers and at high purity. Lessons learned from this lineage will be applicable to others. One strong possibility is that genetically engineered cells will be most efficacious in transplantation. The ES cell-based system is ideally suited to providing genetically engineered cells.

DESCRIPTION (provided by applicant): The Mediator Co-activator Complex (Mediator) is a 25-30 protein complex that plays a pivotal role in regulating gene transcription in eukaryotic cells. Mediator is expressed in brain cells but currently its precise functions have not been defined. In particular we lack of understanding of the roles of Mediator subunits that interact wit neural transcription factors. In this pilot project we'll investigate the function of the Mediator1 subunit (Med1) in neural stem cells using conditional Med1 knockout mice. We will determine if neural stem cells survive in absence of Med1. We will also use a combination of in vivo and cell culture analyses to define the basic biological processes that are perturbed by lack of Med1. These experiments will give a first look at the role of Med1 in neural stem cells and provide a framework for future large-scale studies of this and other Mediator subunits. Gene transcription is a fundamental aspect of neural cell life and Mediator is likely to be an integral part of it. A number of human neural genetic disorders arise from mutation of Mediator subunits so this work is highly relevant to clinical and translational neuroscience. PUBLIC HEALTH RELEVANCE: This project investigates a complex of 30 proteins that is essential for transcription of most genes in humans. Three of the proteins are mutated to give serious developmental disorders in humans including severe malformations of the neural tube and brain. Very little is known about the complex in neural stem cells, neurons and glia so the results will likely be relevant to understanding neural diseases and in their diagnosis and treatment.