Robbert J. Creton - US grants

Calcium signaling in the developing zebrafish brain

Robbert Creton

The long-term objective of this project is to better understand the mechanisms regulating brain regionalization during early development, using zebrafish embryos as a model system. Large-scale mutagenesis screens in zebrafish have revealed a network of genes, which play a role in patterning of the brain. However, the dynamics of this network is only partly understood. New insights may be obtained by combining ideas and approaches from developmental genetics and neurophysiology. The central hypothesis of this project is that calcium signals play a key role in the signaling pathways that pattern the brain. Calcium signals can spread rapidly over large distances within the embryo and are capable of regulating a variety of cellular events including post-translational modification of signaling proteins, secretion, contraction, and gene expression. Moreover, various lines of evidence suggest a role of calcium in the midline signaling pathway, which induces the neural tube floor plate and ventral brain regions. To further test the role of calcium in midline signaling, calcium patterns will be manipulated and effects on neural tube and brain development will be characterized. In addition, the interaction between calcium and components of the midline signaling pathway will be examined. Obtained results will give a first view of calcium interacting with signaling pathways in the developing forebrain. A broader impact of this project is the establishment of full-time research within the Bioimaging Facility at Brown University. This multi-user facility provides training in microscopy to undergraduate students, graduate students, post-docs, staff, faculty, and visiting scientists. Local high schools regularly tour the facility, and for many high school students this is their first visit to a university, and their first contact with scientific research. In addition, the facility provides free consultation on microscopy and image analysis to the local community including other universities, small businesses, and nearby hospitals. A full-time research program at the Bioimaging Facility will affect the outreach potential of this facility, in part by enhancing the infrastructure of this facility, and in part by bringing in investigators who will teach microscopy and image analysis.

bioimaging /biomedical imaging; confocal scanning microscopy; microscopy; biomedical facility;

bioimaging /biomedical imaging

DESCRIPTION (provided by applicant): Pharmaceuticals that modulate calcium signaling are successfully used for treating high blood pressure, heart arrhythmias, angina pectoris, and migraine. However, their widespread use in medicine and subsequent discharge in the environment is concerning, since studies in animal model systems have shown that subtle calcium manipulations during embryonic development can induce specific brain defects. The potential risk for human brain development is difficult to evaluate because of the large number of pharmaceuticals that can affect calcium signaling either directly or indirectly, a lack of basic information on the sensitive developmental times, and the potentially pleiotropic effects on brain development and behavior. Our long-term goal is to better understand how modulation of calcium signaling affects brain development and behavior. This long-term goal will be pursued using zebrafish as a model system. Zebrafish embryos develop rapidly and externally, are accessible to genetic and experimental manipulation, and develop predictable neural patterns and behaviors, which have been described in detail. The specific hypothesis that guides this project is that modulators of calcium signaling induce laterality defects in the brain by changing specific developmental processes during a limited window of sensitivity. This hypothesis will be tested in three specific aims that integrate approaches in cell biology, developmental biology, neuroscience, and ethology to provide an overview of the mechanisms by which modulators of calcium signaling affect development of the brain. 1) The first aim is to identify modulators of calcium signaling that induce laterality defects in the brain. We will determine the dose-response curves for defects in neural patterning and behavior and will examine potential synergistic effects. 2) The second aim is to identify calcium patterns that predict the development of specific laterality defects in the brain. Calcium patterns will be imaged in untreated embryos and in embryos exposed to modulators of calcium signaling that induce defects in brain development and behavior. 3) The third aim is to identify calcium-sensitive gene expression patterns that play a role in brain development. We will examine three sets of candidate genes that were selected based on their role in bilateral division of the brain, their role in development of left-right asymmetry in the brain, and their sensitivity to calcium modulation. The obtained results will provide a better understanding of calcium-sensitive mechanisms that play a role in the development of laterality in the brain, which is important for risk assessment and the development of preventative strategies. PUBLIC HEALTH RELEVANCE: This project is focused on brain defects and behavioral defects caused by embryonic exposures to pharmaceuticals that modulate calcium signaling. The obtained results will provide a better understanding of the basic mechanisms by which modulators of calcium signaling affect brain development. These mechanisms are important, since human embryos may inadvertently be exposed to modulators of calcium signaling by maternal use of medicine during early pregnancy or by trace concentrations of pharmaceuticals in the environment and drinking water.

? DESCRIPTION (provided by applicant): Visual impairments affect 285 million people worldwide: 39 million people are blind and 246 million people have low vision. Novel treatments are urgently needed and several lines of evidence suggest that it may be possible to restore vision by regenerating photoreceptors and neural connections. The interest in such regenerative processes is highlighted by the audacious goal of the National Eye Institute at the NIH to `regenerate neurons and neural connections in the eye and visual system'. However, as novel methodologies are developed, a critical question will need to be addressed: how do we monitor in vivo for functional success? An effective approach to monitor for functional success in animal model systems is the analysis of behavior, since behavioral analyses can reveal subtle functional defects, even if the visual system appears normal by morphological criteria. The current project is focused on the automated analysis of behavior in response to visual stimuli, using zebrafish larvae as a model system. Zebrafish larvae are ideally suited for such studies, since high-throughput analyses of behavior can be combined with genetics, high-resolution imaging and experimental manipulations. The long-term goal of the project is to contribute to the prevention and treatment of visual defects, through an in-depth understanding of behavior in response to visual stimuli. The project is guided by the overall hypothesis that the automated analysis of zebrafish behavior is an effective and sensitive approach to identify specific visual defects and monitor the recovery from such defects. This hypothesis will be tested in three specific aims. The first aim is to further improve the automated analysis of zebrafish larval behavior in response to visual stimuli, using a custom-built imaging system. Novel algorithms and assays will be developed and the behavioral responses to visual stimuli will be examined at different developmental stages. The second aim is to identify changes in behavioral profiles caused by specific defects in the visual system. We will analyze behavior in larvae with genetic mutations that affect development of the optic stalk, photoreceptors, retinal pigment epithelium, hyaloid vasculature and the lens. The third aim is to identify behavioral profiles which indicate a recovery from visual defects. Zebrafish have a remarkable capacity for regeneration of the visual system and the behavioral responses to visual stimuli constitute an in vivo monitor for the recovery of visual function. The project will also examine if functional recoveries can be stimulated by small molecules, which have been successful in stimulating regeneration in other systems. The expected outcome from this work includes a better basic understanding of behaviors that are influenced by visual stimuli and high-throughput tools to evaluate treatments of blindness and low vision. The developed tools may be used in future research to screen for a broad range of genetic and environmental factors that cause visual defects and to screen small molecule libraries for novel treatments of blindness and low vision.

CORE SUMMARY/ABSTRACT The Molecular Pathology Core provides researchers in the Superfund Research Program with equipment and technical expertise necessary for the evaluation of molecular and morphological changes in cells, tissues, and organs following exposure to complex environmental contaminants. The Core houses state- of-the-art equipment, including an automated tissue processor, a paraffin embedding center, two automated microtomes, a cryostat, a vibratome for soft-tissue sectioning, a multiheaded light microscope with projection capabilities, a system for laser capture microdissection, and a slide scanner with analysis software for the identification and quantification of morphological structures. The Molecular Pathology Core is staffed by a director, a histologist and a histotechnician, who have expertise in histopathological and immunocytochemial methods, including fixation, dehydration, embedding, sectioning, histological staining, immunolabeling, high- resolution imaging, and quantitative image analysis. Facility staff provides all-inclusive services in sample preparation, offers assistance in imaging and image analysis and provides consultation for ongoing or future research projects. In addition, the core established an extensive training program for students and investigators who use the centrally available equipment. The broad, long-term objective of the Molecular Pathology Core is to contribute to the detection, diagnosis, and prevention of disease caused by exposures to complex environmental contaminants. This long- term goal will be achieved through the following specific aims: Specific Aim 1. Provide high-quality, cost- effective services in molecular pathology for the research projects in the Superfund Research Program. Specific Aim 2. Further integrate methodologies in the Molecular Pathology Core and the Leduc Bioimaging Facility, which houses electron, fluorescence, and confocal microscopes. Specific Aim 3. Identify the bottlenecks in specific protocols and design innovative solutions to improve the pipeline from the proposed experiments to publication of the results. Specific Aim 4. Provide training in tissue processing, sectioning, histological staining, immunolabeling, high-resolution imaging, and quantitative image analysis. The expected outcome from this work includes the identification of molecular markers and specific pathological changes in cell and tissue morphology following exposure to complex environmental contaminants. The results and the developed methodologies will be essential to the individual research projects in the Superfund Research Program and contribute to evidence-based risk assessment of environmental contaminants and mixtures. In addition, students who are trained in the Molecular Pathology Core will be equipped with interdisciplinary tools and approaches to tackle future problems in the environmental sciences.

Summary Brown University requests support for a FEI Teneo VS variable pressure Scanning Electron Microscope (SEM) for serial block face imaging in the life sciences. Ultrathin sections are cut inside the microscope and series of images are acquired from the freshly cut block face, while the sections are discarded. The automated system is capable of imaging large z-stacks at isotropic 10x10x10 nm resolution. The series of images can be processed for 3D reconstruction of cells and tissues, with sufficient resolution to identify small vesicles, organelles and neural connections. The user group requested the basic Teneo VS system. This system includes equipment essential for serial block-face imaging and 3D reconstruction of the data sets, but does not include additional features or accessories. The requested system will replace a 25-year old SEM in the Division of Biology and Medicine at Brown University. The Teneo VS will be the only SEM in the Division of Biology and Medicine and the only variable pressure SEM for serial block-face imaging in the State of Rhode Island. The microscope will be used immediately by 15 investigators at Brown University and Rhode Island Hospital for high-resolution 3D imaging of cellular and sub-cellular structures ranging from germ plasm in embryos to neural circuits in the retina. Collectively, these 15 users are funded by 26 NIH grants. The Major Users have a substantial need for the requested SEM for serial block-face imaging. As an indication of this substantial need, all Major Users have obtained preliminary results with various out- of-state SEMs for serial block-face imaging. While these preliminary results demonstrate the Major User group's need and expertise, the major users had limited access to the SEMs for serial block-face imaging and could only image a subset of samples that were important to their research programs. The Minor Users were unable to gain access to SEMs for serial block-face imaging, but have research projects that would immediately benefit from the proposed equipment. The impact of the proposed imaging system will extend beyond the projects of the Major and Minor Users, since the microscope will be made be made available to the broader scientific community in Rhode Island, which has more than 600 active NIH-funded projects with a total annual budget of $184 million. The microscope will be set up in the Leduc Bioimaging Facility at Brown University, which has the technical expertise to maintain the instrument, train its users and provide consultation on planned and ongoing research projects. In addition, the users will have access to assistance in sample preparation and data analysis. The Leduc Bioimaging Facility, supported by a broadly-based Advisory Committee, has an excellent track-record maintaining various high-end imaging systems and has strong institutional support, which guarantees that the instrumentation will be used to its maximum potential for years to come.

Project Summary Calcineurin is a phosphatase with broad clinical significance. Calcineurin inhibitors are used as immunosuppressants to prevent rejection of organ transplants and calcineurin signaling is thought to play a critical role in Down syndrome and Alzheimer?s disease. However, very little is known about the effects of modulated calcineurin signaling in the brain. Animal model systems are available, but changes in neural networks are easily missed when studying an organ as complex as the brain. Analyses of behavior offer a potential solution, since subtle changes in brain structure and function can be detected. The laboratory of the principal investigator developed an imaging system for automated analyses of behavior in zebrafish larvae. This imaging system can measure larval responses to moving visual stimuli in microplates, which is not possible with commercially-available imaging systems. The capabilities of this imaging system will be further expanded for automated analyses of complex behaviors in a 384-well format. The project is guided by the central hypothesis that novel imaging technologies for high-throughput analyses of vertebrate behavior will contribute to the discovery of novel treatments for neural dysfunction. This hypothesis will be tested in three specific aims. The first aim is to optimize imaging technologies for high-throughput analyses of complex behavior. The second aim is to use the optimized technologies to image calcineurin-dependent behaviors. The third aim is to screen for novel treatments of calcineurin-related neural dysfunction. Overall, successful completion of these aims will expand the methodologies for high-throughput analyses of complex behaviors, provide a better basic understanding of calcineurin signaling in the brain, and contribute to the design of novel treatments for calcineurin-related neural dysfunction. The project will benefit from the vibrant and collegial research environment at Brown University. The principal investigator has a primary appointment in the Department of Molecular Biology, Cell Biology and Biochemistry and is a faculty member in the Carney Institute for Brain Science and the Center to Advance Predictive Biology. The department offers expertise in developmental biology and cell signaling and the two centers provide a dynamic intellectual environment for research in behavioral neuroscience and the analysis of signaling networks using high-content imaging technologies.