Gerald Maurice Edelman, MD, PhD - US grants

During embryonic development, cells are brought together by mechanochemical means resulting in embryonic induction. This milieu-dependent differentiation gives rise in turn to tissues and organs. The long term goal of this proposal is to analyze how the cellular interactions of embryonic induction lead to morphogenesis in normal development and how derangements of these interactions give rise to developmental defects. Because of the central role of cell contact in these events, our approach has been to identify and chemically define the structure and activities of cell adhesion molecules (CAMs). Several CAMS have now been identified and characterized in detail and cDNA probes for these molecules have been defined. Immunohistochemical studies show that CAMs are expressed at sites of embryonic induction as well as at salient stages of tissue and organ formation. Early in development, this expression follows a definite set of rules which are general to a great variety of tissues. These observations suggest that locale-specific regulation of defined molecules mediates the pivotal process of cell adhesion in morphogenesis and histogenesis. Two main complementary approaches will be used to extend and deepen this conclusion. First, detailed localization of CAMs in normally developing tissues will be carried out by immunological and gene probe techniques and correlated with known developmental interactions and processes. Second, the function of CAMs will be perturbed at different stages of development and the biochemical and histological changes caused by the perturbations will be analyzed in light of the localization studies. Specifically we will: (1) make a detailed analysis of CAM expression in liver, kidney, limb, and lung and in optic and otic placodes (2) use antibodies to perturb CAM functions in organ and cell cultures of these tissues and in the intact embryo at stages of development when inductive and morphogenetic events are known to occur (3) continue studies to detect, isolate, and characterize new CAMs (4) determine the role of CAMs in the formation of neuronal connections in the cerebellum and the retinotectal system and (5) utilize cultures of skin cells producing appendages such as feathers as well as nervous tissue explants to define factors that are involved in the control of CAM expression.

Several mechanochemical processes coordinated at the cell surface are essential for normal tissue formation and integrity. A guiding theme for our work continues to be the idea that the primary processes of cell migration, cell proliferation and cell adhesion regulate and are regulated by the cross-linkage of cell surface glycoproteins and their interactions with the cytoskeleton (global modulation), and are influenced by changes in the amount, distribution and properties of specific cell surface receptors (local modulation). To corroborate this hypothesis and to examine the role of proteins involved in tissue formation in the control of primary processes, we have isolated and characterized three cell adhesion molecules (N-CAM, L-CAM, and Ng-CAM) and have recently discovered a novel extracellular matrix (ECM) protein (cytotacin) that is involved in both cell-matrix adhesion and cell migration. The distributions of each of these molecules show dynamic alterations during development that are consistent with their identified functions. Furthermore, expression of the CAMs and cytotactin is altered in transformed cells, raising the possibility that alterations in the expression of these molecules is a crucial correlate of the transformed phenotype. We now propose to extend and deepen our study of the dynamics of cell surface modulation. Specifically, we will: 1) determine the structural features of cytotactin at both the protein and nucleic acid levels, with particular regard to known features of CAMs and extracellular matrix proteins; 2) carry out functional assays to describe the role of cytotactin in cell movement and search for its cellular receptors; 3) use monoclonal antibodies to a carbohydrate antigen (NC-1/HNK-1) present on cytotactin, N-CAM, and Ng-CAM to identify other cell surface and ECM proteins that might be involved in adhesion; 4) relate local cell surface modulation to global modulation in nerve-muscle and hepatocyte interactions and determine whether ECM proteins can induce global modulation; and 5) determine whether the effects of viral transformation consistently correlate with modulation of molecules that mediate cell-cell and cell-substrate interactions in neural and epithelial cells. The results of these studies should provide valuable clues to the regulation of fundamental cellular processes, and may be of significant value to our understanding of a variety of disease states including birth defects, tissue disorders, cancer and metastasis.

This is a proposal to establish a Center of Excellence combining the strengths of three large laboratories, each of which is currently approaching major problems of developmental neurobiology at a different level. The primary aim of the project is to develop a concerted, synergistic attack on several major developmental problems: neural cell interactions and regeneration, neuronal viability based on synaptic interactions, and mapping of sensory functions. Each of the laboratories has already developed methodological salients for attacking these problems: molecular cellular adhesion assays, phosphoprotein characterization, tissue culture assays, and electrophysiology and neuroanatomic mapping. By combining these intellectual and operational resources, it is expected that major progress can be made using a multilevel attack and defined systems shared among all three research groups. At the molecular level, we will analyze the expression of cell adhesion molecules in regeneration of neural and neuromuscular systems and in associated cell migration events and the order of gene expression of the phosphoproteins synapsin I, DARPP-32 and G-substrate as a function of synaptogenesis, at the cellular level, we will investigate the laminar, columnar and connectional organization in neural maps instructing such as the striate cortex, using identification of cell specific surface markers and in vitro tissue slice techniques. At the highest level, the contribution of these processes to physiological function will be assessed using electrophysiological measurements. The successful merger of these approaches will require the combined efforts and cooperation of these three laboratories which are in close physical proximity. A Center of Excellence would strongly enhance their intellectual cooperation and the results of this merger are expected to have direct pertinence to the study of degenerative disease and neural regeneration.

This award will support collaborative studies between Dr. Gerald Edelman and his research group at the Neurosciences Institute, Rockefeller University and Dr. Francois Rieger, Unite de Recherche de Biologie et Pathologie Neuromusculaires, Centre National de la Recherche Scientifique, Paris, France. Dr. Edelman, a Nobel Prize winner and eminent researcher in the proposed topic, has collaborated successfully with Dr. Rieger in the past. The neuromuscular system is a complex supracellular structure composed of a number of different cell types: sensory and motor neurons, Schwann cells, muscle cells, fibroblasts, and various support cells. The development, maintenance, and physiological functions of the neuromuscular system depend upon specialized structures, including the neuromuscular junction, the myotendinous junction, and the nodes of Ranvier. In previous research Drs. Edelman and Rieger showed that three glycoproteins involved in cell-cell adhesion are localized to these privileged sites and vary in amount and distribution during development and regeneration of neuromuscular contacts: the neural cell adhesion molecule, N-CAM, the neuron-glia cell adhesion molecule, Ng-CAM, and cytotactin, an extracellular matrix protein. The localization of these molecules was altered in mutant animals with various cellular defects and antibodies to N-CAM blocked the appropriate reinnervation patterns after injury, inhibiting reformation of synapses at old synaptic sites and allowing for extrasynaptic nerve-muscle contacts. The patterns of expression of these molecules correlate with those of other neuromuscular proteins, including acetylcholinesterase, and voltage-dependent calcium channels. During the next phase of collaboration, the investigators will examine the detailed basis for the localized pattern of expression of adhesion molecules and neuromuscular proteins, focusing particularly on cytoskeletal interactions. They will also use cDNA clones specific for muscle forms of N-CAM to transfect cells and produce transgenic animals. These joint experiments would be difficult to accomplish in either laboratory alone. The expertise of Dr. Rieger and his colleagues in electron microscopy, neurophysiology and mutant strains of mice will complement the expertise of Dr. Edelman and his group in the isolation and characterization of cell adhesion molecules, molecular biological techniques, fluorescence microscopy and the production and screening of monoclonal and polyclonal antibodies. These studies should provide new insights into the formation and maintenance of structures crucial to muscle function, as well as to their aberrations in muscle disease and dysfunction.

Cell adhesion molecules (CAMs) play important roles in embryonic tissues and in maintaining the structure and function of adult tissues. Changes in CAM expression accompany a number of disease states, and it has been shown that they are involved in both regenerative and degenerative processes including nerve and muscle. We propose to study the function of CAMs in vivo by microinjecting into mouse embryos the gene for the chicken liver cell adhesion molecule L-CAM under the control of promoters that will cause it to be expressed in transgenic mice in locations where L-CAM is not normally seen. In addition we are making constructs to locations where L- CAM is not normally seen. In addition we are making constructs to truncate, mutate, and delete the endogenous gene for the neural cell adhesion molecule, N-CAM, by transfection of embryonicstem (ES) cells and transplantation into mouse blastocysts to produce transgenic mice. DNA constructs that include the chicken L-CAM gene coupled to the insulin promoter, the neurofilament promoter, and the crystallin promoter have been used in initial experiments in this laboratory to make transgenic mice. Those with the insulin promoter express chicken L-CAM in the beta-cells of the pancreas with some apparent perturbation of cell function. Cultures of ES cells have been established and mouse N-CAM genomic clones have been isolated. Production of mice ectopically expressing L-CAM under control of tissue specific promoters should provide opportunities to locally perturb function by L-CAM expression in the beta-cells of the pancreas, in neurons and in lens cells. These tissues do not express L-CAM normally but express other CAMs, can be assessed. Our preliminary studies show that some of these animals survive and can be used to look for the influence of L-CAM on regenerative processes (eg. nerve-muscle interactions) and degenerative processes that accompany aging. Alteration or ablation of the endogenous N-CAM gene should provide a detailed analysis of the role of each form of the molecule in development, regeneration, and degeneration in vivo. These studies should have extensive implications for a variety of birth defects and genetic defects, and provide important data for assessing the role of CAMs in events associated with aging.

DESCRIPTION (Adapted from applicant's abstract): A number of neural CAMs have been identified, and work in the applicant's laboratory and others has demonstrated the importance of these molecules in the formation and function of the nervous system. It is clear from these studies that when and where a given CAM is expressed during development are critical for its function. The applicant's recent studies have focused on determining the transcription factors that regulate the expression of N-CAM and another neural CAM, L1. He has shown that the genes for these molecules are targets of the homeodomain and paired domain (Pax) proteins that regulate overall neural patterning. He proposes to expand these studies by examining how growth factors of the bone morphogenetic protein (BMP) family and synaptic activity modulate the expression of the N-CAM and L1 genes. The specific aims of the application are: 1) To examine the regulation of N-CAM and L1 gene expression by BMP2 and BMP4 in neural cells; 2) To determine in transgenic mice the contributions of the homeodomain and paired domain binding elements in the N-CAM and L1 genes to their developmental expression patterns and BMP responses; 3) To identify the regulatory elements and transcription factor families involved in the induction of the L1 and N-CAM genes by neural activity. Transfected cells and hippocampal slice cultures from normal and transgenic mice will be used in conjunction with gel shift and supershift assays in these experiments. Overall, these studies will elucidate the role of BMP signaling and of specific homeodomain and Pax gene products in the regulation of the expression of two neural CAMs. It will also provide one of the first demonstrations of the links between transcriptional regulation of CAMs and synaptic activity. These results will be particularly significant in view of the central role of CAMs in brain development, neurological disease and synaptic plasticity.

DESCRIPTION (provided by applicant): Since our discovery of the neural cell adhesion molecule, N-CAM, over 20 years ago, it has become clear that binding by cell adhesion molecules (CAMs) not only mediates cell-cell interactions, but also induces signals that affect embryonic development and adult physiology. In these proposed studies, we focus on the role of N-CAM in regulating critical cellular functions in the nervous system. Our first aim is designed to elucidate the events at and under the cell membrane by which N-CAM binding to the cell surface induces intracellular signals. The ability of N-CAM to induce the transcription factor NF-KB serves as the paradigm, but the studies will be extended to other N-CAM-mediated events. Recombinant proteins corresponding to various extracellular portions of N-CAM will be used to examine binding events, and site-directed mutagenesis of the N-CAM cytoplasmic region will be used to analyze intracellular events. In the second aim, the ability of N-CAM to induce the differentiation of neural stem cells into neurons by binding to heterophilic (non-N-CAM) receptors will be examined in terms of the mechanism of stimulation, the receptors involved, and the types of neurons induced by N-CAM binding. Multivalent recombinant proteins will be used to induce the response and be applied in conjunction with cells from our N-CAM knock-out mice to isolate heterophilic receptors. In the third aim, we will examine the role of N-CAM in inducing long term potentiation (LTP) of synapses, a key process underlying memory and learning. For these studies, synaptic dynamics and morphology will be compared in two strains of N-CAM knock-out mice, one with hippocampal LTP and one without LTP. A new method we have developed for identifying populations of active synapses will be used. The mechanisms defined by these studies should serve as exemplars for the activities of other CAMs. They should also yield new insights into how CAMs regulate gene expression, provide novel reagents for differentiating neural stem cells for potential clinical use, and expand our knowledge of the fundamental mechanisms that underlie memory and learning.