Barbara Ranscht - US grants

The central theme of this program project is the study of interactions of cell surface proteins in the development of the vertebrate embryo. Two of these proteins are involved in the differentiation of the nervous system and two others will be tested for their role in the morphogenesis of skin and the development of the bony skeleton. These surface proteins are being investigated at three levels of biological organization; the cell, the tissue and the organism. The NILE glycoprotein will be studied for its role in mediating interactions between nerve cells, and a new surface glycoprotein, GP90, will be investigated for its function in the communication of nerve cells with non-neuronal tissues such as muscle. A cell surface-associated heparan sulfate proteoglycan will be studied for its role in the morphogenesis of the embryonic skin, and the membrane glycoprotein alkaline phosphatase will be considered for a possible structural role in the morphogenesis of the skin and the bony skeleton. All projects will use the most up-to-date methods in recombinant DNA technology, immunology, protein chemistry and cell biology to investigate in detail the structure and function of these surface proteins that are highly relevant to vertebrate development. The structure of the proteins will be deduced from the nucleotide sequence of cDNAs that encode them. Their functions will be investigated by identifying the functional domains on the molecules themselves and of the ligands with which they interact. An animal model system for the human inherited disorder hypophosphatasia will be developed as part of one project by introducing mutated forms of the normal allele into the germ line of mice. This transgenic system will serve as a paradigm for later studies on the role of the other cell surface molecules in the development of vertebrate embryos. Two core facilities will be established for this program project. One will support the molecular biological studies and the second one will be established for the quantitative analysis of the behavior of cells as a function of the surface molecules that are being studied. With this facility we will be able to record, store and process images of cells behaving under defined conditions and evaluate changes in their shape and motility as a response to other cells or their extracellular matrix.

The central theme of this program project is the study of interactions of cell surface proteins in the development of the vertebrate embryo. Two of these proteins are involved in the differentiation of the nervous system and two others will be tested for their role in the morphogenesis of skin and the development of the bony skeleton. These surface proteins are being investigated at three levels of biological organization; the cell, the tissue and the organism. The NILE glycoprotein will be studied for its role in mediating interactions between nerve cells, and a new surface glycoprotein, GP90, will be investigated for its function in the communication of nerve cells with non-neuronal tissues such as muscle. A cell surface-associated heparan sulfate proteoglycan will be studied for its role in the morphogenesis of the embryonic skin, and the membrane glycoprotein alkaline phosphatase will be considered for a possible structural role in the morphogenesis of the skin and the bony skeleton. All projects will use the most up-to-date methods in recombinant DNA technology, immunology, protein chemistry and cell biology to investigate in detail the structure and function of these surface proteins that are highly relevant to vertebrate development. The structure of the proteins will be deduced from the nucleotide sequence of cDNAs that encode them. Their functions will be investigated by identifying the functional domains on the molecules themselves and of the ligands with which they interact. An animal model system for the human inherited disorder hypophosphatasia will be developed as part of one project by introducing mutated forms of the normal allele into the germ line of mice. This transgenic system will serve as a paradigm for later studies on the role of the other cell surface molecules in the development of vertebrate embryos. Two core facilities will be established for this program project. One will support the molecular biological studies and the second one will be established for the quantitative analysis of the behavior of cells as a function of the surface molecules that are being studied. With this facility we will be able to record, store and process images of cells behaving under defined conditions and evaluate changes in their shape and motility as a response to other cells or their extracellular matrix.

light microscopy; biomedical facility; digital imaging; image processing; histology; DVD /CD ROM;

charge coupled device camera; tissue /cell preparation; image processing; neurogenesis; biomedical facility;

DESCRIPTION (Adapted from the applicant's abstract): The goal of this application is to understand the molecular signals that underlie the formation of cytoarchitecture in cerebellar development. Over the past few years, a number of studies have addressed cell adhesion/recognition molecules of the immunoglobulin gene superfamily (IgSF) in the nervous system. However, studies on the function of these molecules in neural development in vivo are sparse, and the understanding of their roles in establishing neuronal architecture remains largely elusive. The current application will address the role of the IgSF cell adhesion/recognition molecule contactin in cerebellar development using a combination of in vivo and in vitro approaches. Several lines of evidence indicate that contactin plays a pivotal role in regulating cellular interactions that control cerebellar development. This investigator has generated mice with disrupted contactin gene function which develop a severely ataxic phenotype with an onset of postnatal day 9. The mutation is lethal by postnatal day 18. Preliminary analyses of the mutant cerebellum have identified defects in granule neuron development. This neuron population, which, in the wild type, expresses contactin throughout postnatal cerebellar development, provides an opportune model to study the role of contactin in neural development. This project will focus on two major functions of contactin that are indicated from preliminary analyses of the knockout mice. First, using the knock-out mice, the investigator has obtained evidence for a role of contactin in regulating the number of granule cell precursors in the external germinal layer (EGL). The project will test several hypotheses, which will address if contactin-mediated cellular interactions are required for the proliferation, survival and/or maintenance of the undifferentiated state of granule neuron precursors in the EGL. Second, additional preliminary data have revealed a later role of contactin in the fasciculation of parallel fiber axons in the cerebellar molecular layer. The investigator proposes to investigate the mechanism by which contactin regulates this function, and will determine in vitro the ligands with which contactin interacts in controlling axon-axon interactions. These studies will move forward the understanding of the molecular basis of cerebellar development, and, in particular, provide an opportunity to dissect the function of an IgSF cell adhesion/recognition molecule in a biological setting. Thus, this work may set an example for future studies on a host of similar proteins.

Description All of the proposed projects require conventional and fluorescence microscopy, image analysis, and time lapse cinematography. Thus, to efficiently accomplish the microscopic analysis of our data, we request a microscopy Core Facility. This Facility will be staffed with an experienced senior research technician (to be recruited when the program is funded), and will provide assistance with sample preparation, execution of the experiments, image collection, data analysis and editing of confocal and other microscopic images. The Program PI's concurred that this facility is essential to efficiently achieve the goals of the proposed program. In our current set-up, individual postdocs are trained in the use of the Metamorph Image Analysis system and the confocal microscope by the Manager of the Cell Analysis Facility, Dr. Edward Monosov. This training provides members of the program with skills to operate the systems to address a specific question. By nature, however, this training provides neither continuity within the program, nor depth of knowledge to an individual to independently sept-up and operate the Confocal Microscope and Image Analysis Systems for numerous applications. In order to overcome these problems, the Program PI's want to place these operations in the hands of an experienced microscopist/computer analyst. This person will be trained by and interface with the manager of the Institute's Cell Analysis Facility in order to conduct confocal and image analyses for the Program. Operation The core will be house in a 300 square foot room on the first floor on the Burnham Institute's Fitch Building (Building 4), adjacent to three of the Program Laboratories. This room currently houses the Program's 405M Axiovert and the attached image analysis system, and the Burnham Institute's second Confocal microscope, a Zeiss LSM 410. Plans are to accommodate the staff of Program Project Core B in this room. A core staff person with extensive experience in microscopy and computing will be recruited when this grant is funded. The staff will report to Dr. Ranscht, who will serve as the core director (6% effort). Training of the staff will be provided by Dr. Edward Monosov, the manager of the Institute's Cell Analysis Shared Facility The Burnham Institute charges for training time $40/hour, and for unsupervised time on the confocal microscope $20 per hour to offset costs for service contracts and maintenance of the MRC1024-MP confocal instrument. Equipment 1) Zeiss 405M inverted Microscope and Metamorph Image Analysis System. The Program Project extensively uses an inverted Zeiss 405M Microscope that was acquired for the Core of the existing Program Grant in year 1 (1989). This is an excellent microscope that is equipped with optics for phase contrast, differential interference contrast, darkfield, bright field and fluorescence in the red, green and ultra violet spectra. The instrument has been updated over the years with new filter sets and objectives. Attached peripherals included a heated stage for the cultures and an Eppendorff microinjection system suited for the injection of cultured cell. We have extended the capability of the system to do lapse time cinematography using an Optronics ZVS-47E intensified CCD video camera, a Sony Trinitron Color Video Monitor (analog) and a time lapse laser videodisk recorder (Sony LVR 3000N, analog).

DESCRIPTION (Adapted from the applicant's abstract): The goal of this application is to understand the molecular signals that underlie the formation of cytoarchitecture in cerebellar development. Over the past few years, a number of studies have addressed cell adhesion/recognition molecules of the immunoglobulin gene superfamily (IgSF) in the nervous system. However, studies on the function of these molecules in neural development in vivo are sparse, and the understanding of their roles in establishing neuronal architecture remains largely elusive. The current application will address the role of the IgSF cell adhesion/recognition molecule contactin in cerebellar development using a combination of in vivo and in vitro approaches. Several lines of evidence indicate that contactin plays a pivotal role in regulating cellular interactions that control cerebellar development. This investigator has generated mice with disrupted contactin gene function which develop a severely ataxic phenotype with an onset of postnatal day 9. The mutation is lethal by postnatal day 18. Preliminary analyses of the mutant cerebellum have identified defects in granule neuron development. This neuron population, which, in the wild type, expresses contactin throughout postnatal cerebellar development, provides an opportune model to study the role of contactin in neural development. This project will focus on two major functions of contactin that are indicated from preliminary analyses of the knockout mice. First, using the knock-out mice, the investigator has obtained evidence for a role of contactin in regulating the number of granule cell precursors in the external germinal layer (EGL). The project will test several hypotheses, which will address if contactin-mediated cellular interactions are required for the proliferation, survival and/or maintenance of the undifferentiated state of granule neuron precursors in the EGL. Second, additional preliminary data have revealed a later role of contactin in the fasciculation of parallel fiber axons in the cerebellar molecular layer. The investigator proposes to investigate the mechanism by which contactin regulates this function, and will determine in vitro the ligands with which contactin interacts in controlling axon-axon interactions. These studies will move forward the understanding of the molecular basis of cerebellar development, and, in particular, provide an opportunity to dissect the function of an IgSF cell adhesion/recognition molecule in a biological setting. Thus, this work may set an example for future studies on a host of similar proteins.

DESCRIPTION (provided by applicant): The Cell Imaging and Histology Shared Resource provides the equipment and expertise for three types of services: 1) High resolution microscopic techniques to obtain information about the morphology of cells, and sophisticated imaging techniques to analyze the distribution of molecules and their interactions within their cellular environment; 2) Histological and immunohistological techniques to study the morphology of normal and pathological specimens and the distribution of molecules in tissues; and 3) electron and immunoelectron microscopy to analyze the ultrastructure of of cells and the association of molecules with subcellular organelles. The Resource operates four light micsroscopes for confocal and standard imaging and an electron microscope for ultrastructural analysis. Equipment and services are provided for tissue embedding, sectioning, laser capture dissection, staining, and microscopic analysis of specimens. The goals for the next five years include expansion of existing services and introduction of new services. An increased effort will be placed on assisting users with the design and set-up of experiments, including sample collection and processing. New advanced optical imaging strategies for visualizing molecular events in living cells will be implemented, such as: 1) Optical imaging based on the principle of fluorescence resonance energy transfer (FRET) and fluorescent lifetime imaging (FLIM); 2) Total internal reflection fluorescence microscopy (TIRF) to register real-time interactions and trafficking of fluorescent molecules, and to study the dynamics of protein-protein interactions and enzymatic reactions in live cells; and 3) Fluorescence recovery after photobleaching (FRAP) to examine the lateral diffusion and mobility of fluorophore-labeled molecules into an area that has been photobleached. Overall the Cell Imaging and Histology Shared Resource provides essential microscopic and histological services and will accommodate the increased use and requirement for new services in response to new research initiatives at the Cancer Center.

INTRODUCTION TO REVISED APPLICATION The reviewers of the A1 application considered the Core essential for the successful accomplishment of all the projects in the Program. Discrepancies between the budget and the grant text have been corrected in the current revision. The letter from Ms. Karin Eastham, Vice President and Chief Operating Officer of the Burnham Institute, is enclosed at the end of section II and also in the Appendix. This letter states that the Burnham Institute will provide, as part of its commitment to this Program, two rigs for electrophysiological recording from slices and cell cultures. It is also now clearly stated that both Research Associates, Dr. Hadieh Badie-Mahdavi and Dr. Barbara Fredette, will contribute their expertise to the work in Core A starting in year 1. Their effort has been reduced to 50%, as recommended by the reviewers. This also reflects decreased needs due to elimination of the previous Project 1. Dr. Ranscht's effort, on the other hand, has been increased to 15% due to her ability to dedicate increased effort to the preparation of mixed neuron-glia cultures and also due to the need for her consultation to evaluate the effects of heparan sulfate proteoglycans and NG2 at the nodes of Ranvier, as recommended by the reviewers. The revised portions of the application are indicated with a line on the left margin. The overall editorial modifications introduced to unify the style of the application are not marked. Objectives The goal of this Program is to analyze the molecular signals exchanged between neurons and glia at synapses and in myelinated axons. The program has identified cell surface components implicated in neuron-glia communication and now intends to study the function of these molecules in vivo and in tissue culture models that closely mimic the in vivo interactions. Core A of this Program Project will provide the infrastructure and the expertise to accomplish this goal. The Core will offer support for two aspects of the work, analysis of neuronal function by electrophysiology and modeling neuron-glia interactions in suitable culture systems. The Electrophysiology component adds a new research dimension that will provide the Program with the tools and know-how for functional and activity-dependent studies designed to acquire knowledge of the electrophysiological changes occurring in neurons in response to glial cells or glial-derived ligands. It is now apparent that neuron-glia interactions contribute to the functional properties not only of myelin but also of synapses. Addition of the electrophysiology component to the Program will overcome previous limitations and enable the Program to functionally analyze functional defects resulting from the genetic disruption of neural cell surface proteins and their associated signal transduction pathways. The electrophysiological approach is geared towards the analysis of functional neuron-glia interactions at the level of nerve impulse conduction along axons, as well as synaptic function and plasticity. Electrophysiological recordings will assess the modifications in neuronal membrane properties resulting from changes in the expression or function of glial proteoglycans. The Program will employ transgenic and knockout mice that are already in hand and have a sufficiently long survival time for conducting the electrophysiological analyses. Electrophysiological recordings will be conducted on brain slices, single cells, and isolated nerves. Slice recordings will focus on determining the role of the glial protein ephrin-A3 in synaptic efficacy changes during LTP and LTD. Single-cell recording will assess the contribution of ephrin- A3 to neuronal excitability and plasticity through its neuronal receptor, EphA4. Whole nerve recording will detect conduction velocity changes in the compound action potential caused by malfunction of the myelin sheath. Recordings at these different levels are necessary for gaining insights into the molecular interactions that underlie neuron-glia crosstalk as outlined in the individual projects. The second critical aspect of the proposed work is to probe neuron-glia interactions at the cellular level using material from genetically manipulated mice. This is effectively accomplished using suitable systems of primary neural cultures that mimic specific in vivo interactions. Specifically, Core A will provide the co-cultures of neurons and myelin-forming Schwann cells or oligodendrocytes for studies of myelinogenesis. The use of hippocampal cultures for studies on the influence of glial ephrin-A3 in regulating synaptic function and plasticity will be a continuation of the current Core.

Our study using a conditional knockout of EXT1, te gene encoding an enzyme essential for heparan sulfate biosynthesis, has demonstrated the crucial ole of heparan sulfate proteoglycans (HSPGs) in multiple CMS morphogenetic evets (Inatani et al., Science 302:1144-1146, 2003). Yet the role of HSPGs in the nervous system is not limited to the CNS. There is evidece that HSPGs also play critical roles in the development and physiology of the peripheral nervous systm (PNS) by mediating molecular communications between axons and gia cells. This Component will focus on the role of heparan sulfate and HSPGs in neuron-glia interactions using Schwann cell-axon interactio as a model system. Aim 1. Role of heparan sulfate in Schwann cell development: We have found that in ur EXT1 conditional knockout mice, the number of Schwann cells in peripheral nerves is greatly reduced. This suggests that HSPGs play a crucial role in the proliferation and/or migration of Schwann cells, the developmental events in which axon-derived factors and adhesive interactions with axons play significan roles. We will employ the EXT1 conditional knockout mice, cultures of Schwan cells, and neuregulin-1 knockout mice to investigate the role of HSPGs in Schwann cell development and their involvement in neuregulin-ErB signaling. Aim 2. Role of heparan sulfate in myelin formation and function: Molecules that bind SPGs, such as laminins and neuregulins, are known to play criticalroles in myelination, and our preliminary studies have demonstrated aberrant myelination in the EXT1 conditional knockout mice. There are also clinicl reports suggesting that HSPGs are involved in the development of neuropathies. In this aim, by enetic, cell biological, and physiological experiments, we will investigate the role of HSPGs in developmental and physiological aspects of PNS myelin ensheathment. Overall, this Component will generatenew insight into the function of HSPGs in various types of neuron-glia interactions. Furthermore, through a series of collaborative studies, we will explore new lines of research at the interface between Components, obtaining answers to important questions beyond the immediate reaches of each Component.

Observations of molecular changes in patient tissues or caused by genetic or experimental manipulations in mice and cancer cells in culture critically depend on histological and immunohistological analyses in cells and tissues through advanced imaging techniques. The mission of the Cell Imaging and Histopathology Shared Resource is to provide state-of-the-art imaging and histopathology tools, training, and services to Cancer Center researchers. The Shared Resource is divided into two adjacent Facilities: 1) The Cell /mag/ng facility houses four confocal microscopes and five additional fluorescence microscopes. All the microscopes are equipped with advanced digital cameras and image analysis software. Available systems include laser scanning confocal microscopes with single and multi-photon lasers, as well as a spinning disk confocal microscope for extended live cell imaging. Time-lapse imaging systems are available to study cell motility, proliferation, and differentiation, with FRET and TIRF systems to image intracellular dynamic interactions. A full range of wide-field imaging approaches are also supported. The facility transmission electron microscope service provides analysis of cell and tissue ultrastructure. Facility staff maintains the imaging equipment and provides investigator training and support for imaging and image analysis, as well as imaging service, and active development of new imaging tools. 2) The Histopathology facility offers the preparation, sectioning, staining and analysis of tissue samples for histological and histopathological analyses and probe-specific detection of cellular and molecular changes in tissue sections. For analysis of identified cells or cell groups within tissue sections, the facility offers Laser Capture Microdissection services. An automated slide scanning system affords efficient histological data acquisition, image archiving, analysis and quantitation. A staff pathologist with expertise in both human and mouse specimens provides consultation, as well as assistance with tissue microarrays. The Facility staff provides consultation, user training, development and dissemination of imaging-, histology- and immunohistochemical protocols, and assistance with instrumentation for efficient histology data documentation, analysis and storage. The Shared Resource supported the studies of 35 Cancer Center Members in the past year, and is essential to advance the understanding of molecular and cellular events leading to cancer development and spreading. Overall, $185,234 in CCSG support is requested in the first year, representing 18.4% of the total projected annual operating budget.

5.2 Abstract - CELL IMAGING AND HISTOLOGY (Core Group A) The Cell Imaging & Histology Shared Resource (Core Group A) provides Cancer Center researchers with modern imaging tools and histology services, facilitates microscope-acquired data documentation and analysis, and enables small and large volume electronic data sharing and storage. The Core supports studies of pathological and molecular changes in human cancers as well as in experimental animal and cell culture models that require histological examinations and probing of molecular changes in tissues and cells through advanced imaging techniques. The Cell Imaging Facility provides state-of-the-art microscopy resources. Data acquired using three confocal and six additional microscopes, supported 130 publications during the past grant cycle. In the past year, the imaging capabilities of the Facility were utilized by 26 Cancer Center laboratories. Expanded imaging capabilities, added through acquisition of a Zeiss LSM710 NLO multi-photon Laser Scanning Microscope in 2011, permits deep laser scanning confocal microscopy, and extended live cell imaging. High-resolution imaging and confocal microscopy, in combination with other imaging techniques, including FRET, TIRF and time-lapse imaging, permit a spectrum of applications, including studies of cancer cells motility, proliferation, and differentiation, protein localization and dynamic molecular interactions in cells, time-lapse visualization of three-dimensional cancer cell accumulation, and studies of cellular responses to drugs. Facility personnel maintain the microscopes, train investigators in optimal microscopy and imaging techniques, and in advanced image analysis. Full-service imaging is also available. The Histology Core supports histological and immunohistochemical analysis of cancer tissues, performs tissue preparation, fixation and processing, paraffin- and cryosectioning, and histological and immunological stains to monitor morphological and molecular alterations in cancer tissues. Last year, the Facility produced over 20,000 slides for 25 Cancer Center laboratories, and processed over 5,000 for histology or immunological stains. The Facility is taking a central role in archiving and sharing microscope-acquired data by scanning slides into the Aperio XT- and -FL scanners, and supports automated data analysis and quantitation. The availability of easily shared high-resolution scans of histological samples aids in utilizing an extended network of specialized consulting pathologists who assist the Cancer Center. The core's web site provides contact information for a number of pathologists, and the Facility Manager assists in matching a project with specific expertise. The Histology Facility, which supported over 125 publications in the previous granting period, is currently launching a service to assist investigators in obtaining human tissues. Addition of a Tissue Procurement Specialist will help investigators locate sources for tumor tissues, facilitate inter-institutional agreements and IRB approvals, coordinating and managing the entire procurement process. Centralized and expert assistance with tissue procurement will greatly enhance the efficiency of connecting basic research with early translational studies.