Scott E. Fraser, Ph.D. - US grants

The overall aim of the proposed projects is to develop, refine, and make available to the neuroscientific community a body of computer software for rigorously based simulation of the behavior of neuronal systems. The programs embody a compartmental description of neuronal morphology and include such mechanisms as voltage-dependent ion channels, kinetics of calcium-ion binding by a number of substrates, mobilization and packaging of neurotransmitter in vesicles, calcium-triggered release of transmitter quanta into the synaptic cleft, receptors for neurotransmitter that produce time- and voltage-dependent conductance changes or a time-varying concentration of second messenger, and modulation of ion channels by calcium ion or by second messenger. Available manipulations include steady or time-varying current injection, deterministic or stochastic stimulation of axons, single- or dual- electrode voltage clamps, and intracellular injection of calcium ion. Among the variables that can be recorded and plotted are membrane potentials, individual ionic and synaptic currents, individual and summed conductances, calcium-ion concentrations, free and bound neurotransmitter, and receptor states. The programs will be interactive and will make extensive use of high-resolution color graphics. They will enable neurobiologists to organize data collection and will provide theoretical frameworks for testing hypotheses and comparing prediction with experiment. Use of such models enriches the information obtained from animal experiments and eventually will make possible more explicit and quantitative determination of therapeutic neuropharmacological regimes.

The long-term goal of our laboratory is to investigate the interactions between cells important for the patterning of neural connections. The specific goal of this proposal is to explore the interactions important in the patterning of the retinotectal projection of the frog, Xenopus laevis. The retinotectal system of this lower vertebrate is studied because the eye forms an easily-studied, topographic projection to the optic tectum. Embryological techniques have been well worked out that permit the cells that give rise to the eye or the tectum to be experimentally manipulated. This permits unique experiments in which the very early events in the formation of a topographic neuronal projection can be studied. The proposed studies employ a recently-developed technique that permits the developing retinotectal projection to be studied in living animals in a non-invasive and non-deleterious way. Some of the cells of the eye rudiment are labeled with a fluorescent dye that persists in the cells for weeks, and the pattern of growth of the labeled cells is followed in the intact animal with an epifluorescence microscope. The experiments will: (1) follow the anatomy of individual developing optic nerve fibers, (2) study the effects of blocking neuronal activity and cell adhesion on the anatomy of single optic nerve fibers, and (3) investigate the nature of the positional cues important for the patterning of the developing projection. The results of the experiments will have implications for the understanding of the early events in the formation of ordered neural connections, and the events important in the re-establishment of connections following injury.

A central question of developmental neurobiology is how the descendants of an apparently homogeneous neuroepithelium give rise to the beutiful and complex patterns observed in the mature nervous system. Cell migration of cell lineage are two major processes involved in the formation of proper neuronal patterning. Because these patterns play an important role in the functioning of neuronal systems, a knowledge of their genesis is central to an understanding of neuronal function. The long term goals of these experiments is to employ newly developed techniques to follow neuronal migration and cell lineage in the vertebrate nervous system. These techniques permit single neuroblasts to be uniquely labeled in the intact embryo. The goal of the proposed experiments is to explore cell lineages and movements in the vertebrate hindbrain, which is subdivided into a series of metameric epithelial units termed rhombomerases. In the chicken embryo, the rhombomeres first become apparent on the secon day of development as a periodic pattern of ridges dividing the hindbrain rudiment into seven rostrocaudal segments. The segmental pattern is reflected in the pattern of neurogenesis, with even-numbered segments developing recognizable neurons before the odd-numbered segments. The proposed experiments will explore the functional consequences of the rhombomers by performing a prospective lineage analysis of neural tube cells using microinjected cell autonomous vital dyes. Only the descendants of the injected cell, which inherit the dye at mitosis, will be labeled. By analyzing the phenotypes of the labeled descendants of a single precursor, a direct analysis of cell lineage is possible. Furthermore, because the vital dye can be observed in the living intact embryo, comparison of the position of the original dye-injected cell and the eventual positions of its progeny offer a direct measure of cell migration of cell mixing. In particular, the experiments will determine: 1. The spatial patterns of neurogenesis in the developing hindbrain, directly assessing if the populations of cells comprising individual rhombomeres are segregated by lineage restriction boundaries. 2. The phenotypes of the descendants of the labeled precursors in the hindbrain to determine if the apparent early seeregation of the hindbrain is also manifest in an early segregation of cell fate. 3. The spatial patterns and fates of the descendants of single neural tube cells in the trunk region of the embryo to determine if there is a parallel of the neuroblasts even at spinal levels of the neuroaxis, where there are no noticeable boundaries.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

Description: This project seeks to define spatio-temporal relationships between head mesoderm, neural crest, and CNS tissue in the developing mouse, using MRI microscopic techniques. The cranial neural crest of the mouse shows non-uniformities in its migration that may depend on segmental differences in the neural tube from which it originates and/or the environment through which it migrates. Studies from several laboratories have defined genes that are expressed in a non-uniform fashion, suggestive of a role in the segmentation of the cranial mesoderm, neural crest and CNS. However, little is known about the exact spatial or temporal relationships of these genes and developmental events. The proposal lists three specific aims. MRI microscopy "atlases" of mouse craniofacial development will be acquired (aim 1). These digital three-dimensional (3D) atlases will be obtained with MRI microscopy of craniofacial structure precursors in the developing mouse, both in vivo and after fixation. A subset of the animals imaged will be processed by conventional histology and the three image data sets compared. Vital dye fate-mapping of mouse craniofacial development will be done (aim 2). Current MRI contrast agents, especially ones based on Gd-DTPA- dextran and Gd-tagged lipid, will be refined so that small groups of cells can be indelibly marked. After characterizing new agents in fixed specimens, these MRI dyes will be used to follow directly cell and tissue movements within the mouse embryo by repeated MR imaging of the same animal as embryogenesis proceeds. Parallel studies will employ light microscopy (both laser confocal and conventional histological methods) to fate map selected regions and correlate the movements of the cells with molecular domains defined by the expression of lacZ transgenes or by in situ hybridization. MRI microscopy of developmental gene expression will be further developed by synthesis of targeted MRI contrast agents applied to determine patterns of gene expression (aim 3). Three dimensional images of gene expression domains will be compared with developmental events in normal and mutant mice. These experiments are expected to better define spatiotemporal relationships between developmental events such as tissue movements and causal gene expression patterns.

Funds are requested for partial Support of the 1995 Gordon Research Conference on Developmental Biology (Cynthia Kenyon and Scott Fraser, co- chairs). This Gordon Conference has long been recognized as the major meeting in Developmental Biology, bringing approximately 150 outstanding senior and junior scientists for discussions of the recent advances in the field of development and cell differentiation. The major advantage in the Gordon Research Conference on Developmental Biology over its nearly three decade history is that it intentionally spans a wide variety of experimental systems. The speakers chosen represent not only some of the most active groups, but also individuals with the capacity to generate useful discussion of their own and other topics. The concept of the meeting has been not to try to cover the field thinly, but to pick areas of exceptional activity or promise. Each of the major sessions are designed to present specific classes of phenomena important in the development of a wide variety of different organisms. By bringing together scientists working with different development developmental system and experimental approaches, the presentations and discussions naturally lead to a more critical analysis of the results, as well as new questions about the underlying mechanisms. A second major advantage of the Gordon Conference is it's geographical isolation, keeping participants in close proximity for five days of discussion without the distractions of nearby ski-slopes or cities. The conference format will consist of short talks (15-20 minutes) followed by discussion (10 minutes minimum), on topics ranging from cell fate determination and morphogenesis to cell signaling and gene regulation. A significant fraction of the session time will be kept uncommitted until near the time of the session to assure that the latest- emerging work can be presented at the meeting. By maximizing both formal discussion and informal interactions the Gordon Conference on Developmental Biology should help to define both the present state and future of the field.

DVD /CD ROM; bioimaging /biomedical imaging; fluorescent dye /probe

The long term goal of this work is to better define the emergence of pattern in the developing brain as well as the changes in these patterns with the progress of development, of aging and pathology. The goal of the proposed project is to develop and apply the tools needed tot image cells and axons within the intact brain as it develops. Because no one imaging technique offers the combination of field of view, depth of penetration and resolution, imaging neural development will require combination multiple imaging modalities at multiple resolutions. Our approach is based upon our previous successes in labeling cells with fluorescent dyes so that their movements and the navigation of their labeled axons can be followed with light microscopy. It is also based upon recent advances both in MRI microscopy and in contrast agents that permit labeled cells to be followed in both the MRI and fluorescence microscopes. Together, these advances offer the promise of following changes in the nervous system as they take place within individual animals by combining data obtained at multiple resolutions and multiple imaging modalities. This approach poses several challenges, ranging from the labeling of the cells and acquisitions of the individual images to the registration of the multiple images into a single rendering and the databasing of the findings. Preliminary experiments and test computer programs suggest that this combined approach is feasible. The proposed project will combine the talents of a computational biology laboratory and a biological imaging laboratory to develop the needed experimental and computer tools. In particular the project will: . Develop the tools for acquiring, aligning and merging 3-D data sets from confocal and 2-photon light microscopes. . Develop the tools for acquiring, aligning and merging 3-D data sets from light and MRI microscopes. . Utilize these tools for examining the refinement of patterned neuronal projects and the migrations of neuronal precursors within living animals. The approach offers a new paradigm for following dynamic changes in the nervous system at resolutions spanning brain areas to individual synapses.

DESCRIPTION: (Verbatim from the Applicant's Abstract) Although the symptomology of multiple sclerosis (MS) has been described for decades, an understanding of the mechanisms and causes of the disease are only now emerging. Early signatures of multiple sclerosis (MS) often involve degradation of visual acuity, sometimes resulting in episodes of double vision and even blindness. Visual evoked potentials (VEPs) are widely used to asses patients suspected have having MS because of their high sensitivity for detecting even clinically silent lesions of the visual pathway. Although increased VEP latency and broadened waveforms are taken to indicate lesions, little is known of the exact relationship (s) between the VEP and the sizes, locations or histories of lesions. Investigating such relationships would require some means to perform longitudinal histopathological studies of the optic nerve. The proposed experiments will draw upon recent advances in magnetic resonance imaging microscopy (uMRI) to perform longitudinal in vivo imaging studies of the optic nerve in mice. These uMRI methods are based on qualitative measurements of local water diffusion. Pilot experiments, in which the techniques have been applied to fixed spinal cords from nice demonstrate that the approach offers unprecedented sensitivity tot the trajectories and myelination of nerve tracks. The goal here is to harness the potential of these new uMRI methods to perform longitudinal, in vivo studies of the demyelination of the optic nerve and correlate these with the VEPs, performed on the same animals, as a functional assay for visual function. The studies will employ a strain of transgenic mice which experience spontaneous demyelination of the central nervous system (CNS), with episodic weakness and demyelination similar to MS, making it an appropriate model system for the technical developments and experiments proposed here. Quantitative MRI measurements of water diffusion will be performed hind-in-hind with theoretical modeling of the diffusion though neural tissues to develop a sensitive and high-resolution probe of myelination in vivo. By performing quantitative uMRI measurements on the same animal, and correlating these images with longitudinal measurements of the VEP and with conventional histopathalogical analyses of the same animal, the goal is to permits a one-to-one comparison between the anatomy, physiology and symptomology of a demyelinating disease, modeling and the longitudinal imaging studies described in this proposal.

Recent advancements in the development of fluorescent indicator dyes, the isolation of proteins that naturally fluoresce, and the refinement of techniques for in vivo microscopy offer unprecedented opportunities to study the cellular and molecular events within living, intact embryos. In parallel, there has been dramatic progress in the study of gene regulation during the embryonic development of the sea urchin and the ascidian, culminating in the proposal of Gene Regulatory Networks for developmental patterning. Because of their size, shape and transparency, the sea urchin embryo and ascidian embryos are ideal systems for study with light microscopy. The proposed research plan will combine these recent advances in imaging science and developmental biology to attack three related goals: first, to better define the spatio-temporal aspects of gene activity within intact embryos; second, to refine tools for the imaging and perturbation of factors thought to be involved in the patterned gene expression; third, to characterize the mobilization and activity of transcription factors during key developmental and gene regulatory events. Two-photon laser-scanning microscopy (TPLSM) will be used as the major imaging tool in these experiments, because of its high sensitivity, low photo-toxicity and deep penetration into tissues. The first experimental goal will be achieved by combining TPLSM with reporter genes that encode green fluorescent protein (GFP) and its color variants to yield quantitative assays of gene activation in intact embryos. Parallel experiments will employ fluorescent in situ hybridization technologies to assay gene activation in fixed embryos. The second goal will involve TPLSM combined with GFP fusions and specific antibodies to better define the regional and temporal redistribution of transcription factors, and caged morpholinos to permit photoactivatable perturbation of the gene regulatory network. The third goal will involve fusions of transcription factors with green fluorescent protein mutants to permit transcription factor mobilization and docking to be followed by advanced fluorescence techniques. The long-term goal of this project is use a combination of molecular and optical techniques to define the cellular events that drive patterned gene regulation and the developmental events that are in turn driven by patterned gene regulation. This will provide the tools needed to test the genetic regulatory networks proposed in the other components of this Program Project.

We are requesting financial support for the 2002 Santa Cruz Meeting on Developmental Biology (SCDB). The meeting is designed as a stimulating meeting ground for approximately 50 speakers and 120 Poster presenters. The meeting offers ample opportunity for both formal and informal discussion among participants. The sessions are organized with substantial time for discussion after each session, and the speakers have been warned that they will not be allowed to run over into their discussion period; the lengthy discussion period was cited as one of the best features of the previous SCDB. Informal discussion will be fostered by the many social hours and poster sessions held each afternoon. The conference site has been arranged to bring the posters, lectures and meals into close proximity, resolving one of the few problems in past SCDB meetings. This year, the meeting will incorporate a significant number of talks from plant researchers and a growing number of talks at the interface of evolutionary and developmental biology. These talks are not segregated into their own sessions, but instead are integrated throughout the meeting to maximize interactions and emphasize interconnections. The 2002 Santa Cruz Meeting on Developmental Biology, to be held August 15-19, 2002, builds upon the success of previous meetings in this series (held in 1992, 1994, 1996, 2000). As before, the meeting will be held on the University of California, Santa Cruz campus in Northern California. The setting is very cost effective, and meeting logistics are simplified by the UCSC conference center. Travel from nearby airports is straightforward, but the meeting site is sufficiently isolated and pleasant to keep participants and speakers in residence at the meeting. Thus the SCDB combines many of the positive features of the Developmental Biology Gordon Conference (held in alternative years from the SCDB), but with significant advantages that have made it one of the favorite conferences of previous attendees.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] In this proposal we address the hypothesis that hemodynamic forces, such as wall shear-stress, are central to normal vascular formation and remodeling in mouse embryonic development. The proposal that shear stress plays an important role has been made many different times and there has been some support for this theory; however, few quantitative experiments have tested it in vivo in an intact embryo. To this end, the proposed work will develop image-based quantitative indices of morphological remodeling which allow statistical comparison with measured hemodynamic properties in vivo. [unreadable] [unreadable] The proposal requires technical development of the non-invasive imaging techniques, particularly magnetic resonance microscopy (MRM) imaging. MRM will be used to measure blood plasma velocity, blood volume and regional flow across the whole embryo using endogenous image contrast alone. Confocal laser scanning microscopy (CLSM) will address direct time-of-flight measurements of erythrocyte flow and endothelial motion over more limited regions of the forming embryonic vasculature. Use of two imaging modalities allows complementary flow quantitation and cross-validation. [unreadable] [unreadable] Experimental perturbations of flow, pulsatility and hematocrit result in changes in hemodynamic forces on the developing vasculature. Perturbations will be characterized with both MRM and CLSM; the changes in hemodynamic forces will be correlated with morphological remodeling indices. Overall, these approaches will define the dynamic changes in morphology that occur in normal development and when blood flow is disrupted. Morphological changes will be related to quantitative data revealing how flow and development are interlinked. These experiments will establish a model system to study fluid effects on vascularization. Once this model has been established, studies to understand the interplay between mechano-sensory signals and genetic signaling pathways can begin. [unreadable] (End of Abstract) [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The research plan will refine and deploy a set of advanced tools for the imaging of tissue structure, gene expression domains and cellular dynamics during craniofacial development. Volumetric imaging tools will be used td create accurate 3D atlases that can be digitally dissected to permit the tissue interactions and cellular events to be better understood in the forming faces of normal, mutant and perturbed mouse and avian embryos. Molecular imaging agents, optimized for imaging intact tissues, will be employed to create atlases of the molecular correlates of these embryos. Finally, intraviral imaging tools will be used to study cell and tissue interactions as they take place, offering a view into the dynamic events that execute craniofacial development. The data will be assembled into atlases that will offer unprecedented tools for exploring the cellular, tissue and molecular correlates of craniofacial development. These atlases will be made widely available for the use of others in the Face Base consortium and the broader community of craniofacial researchers through an online resource created locally. In addition, they will be linked to other Face Base resources through established hubs, so that the data in our atlases can be used in concert with the molecular and structural data provided by other members of the consortium. The data acquisition pipeline established here in close collaboration with the laboratories of Dr. Yang Chai (University of Southern California) and Dr. Marianne Bronner-Fraser (Caltech) will create a model that can be expanded to phenotype and analyze other experimental systems. PUBLIC HEALTH RELEVANCE: Cleft palate, with or without cleft lip. is one of the more common birth defects, affecting approximately one in 2500 live births. The basic events by which cells of different origin come together to form the face have long been known, but the exact tissue movements and the signals that guide them remain undefined. The goal of this work is to help fill this void by imaging the morphology of normal craniofacial development, the abnormal development seen in cleft palate, and providing a means to experimentally test the roles of signaling pathways in the tissue interactions that build the face. This work offers a powerful platform for devising and testing potential therapeutic approaches.

DESCRIPTION (provided by applicant): Since its first use in the retina in 1961, fluorescein angiography (FA) has been the gold standard for objective characterization of retinovascular disease (e.g. diabetic retinopathy, retinal vein occlusions, and choroidal neovascularization associated with age-related macular degeneration (AMD)). Despite its widespread use, FA is costly, invasive, and time consuming, limiting the usefulness as a screening tool. Optical coherence tomography (OCT) and its faster variation called spectral domain OCT (SDOCT) are interferometric imaging techniques which allow three-dimensional imaging of the retinal structure. This technique has enabled clinical imaging of retinal morphology and pathologic alteration with resolution nearly comparable to histologic sections. Because OCT is not capable of rendering most retinovascular abnormalities, it is frequently performed with FA to diagnose and manage patients with macular disease. As a potential alternative to FA in diagnosing retinovascular disease, we have developed an imaging method called phase contrast optical coherence tomography (PC-OCT) which uses specialized software analysis of data acquired from clinically available SDOCT imaging systems to provide the additional functionality of high resolution imaging of retinal vessels and choroidal neovascularization. To develop PC-OCT imaging as a non-invasive alternative for fluorescein angiography, we have the following goals: (1) improve the speed, accuracy and automation of PC-OCT imaging to allow for general clinical usage, and apply this developed technique towards disease targets of (2) retinal capillary non- perfusion in diabetic retinopathy and (3) choroidal neovascularization (CNV) in age-related macular degeneration (AMD). The visualization capabilities of PC-OCT imaging will be compared directly against fluorescein angiography images for subjects of the target diseases. Successful completion of the Phase I aims will lead to further development of PC-OCT imaging techniques and analysis for detection of vascular leakage and screening for sub-clinical CNV in asymptomatic fellow eyes. Developing motion contrast capabilities of PC-OCT to be used with clinically available SDOCT systems can establish a new non-invasive screening tool for retinovascular diagnosis. Earlier detection through non-invasive screening of AMD patients may enable earlier detection of subclinical CNV and initiation of therapy before central vision is lost. PUBLIC HEALTH RELEVANCE: This proposal describes a new software product that enhances the diagnostic capability of optical coherence tomography (OCT), the most commonly performed retinal diagnostic test used in the US for evaluation of macular degeneration and diabetic retinopathy. By non-invasively providing high resolution retinovascular imaging, the product, phase contrast OCT (PC-OCT), may potentially replace fluorescein angiography, the current invasive and costly gold standard for retinovascular imaging. The proposed research plan improves the capabilities of PC-OCT and then does a head-to-head comparison with fluorescein angiography in evaluation of patients with wet macular degeneration and diabetic retinopathy.

DESCRIPTION (provided by applicant): This Center of Excellence in Genomic Science (CEGS) assembles a multidisciplinary group of investigators to develop innovative technologies with the goal of imaging and mutating every developmentally important vertebrate gene. Novel "in toto imaging" tools make it possible to use a systems-based approach for analysis of gene function in developing vertebrate embryos in real time and space. These tools can digitize in vivo data in a systematic, high-throughput, and quantitative fashion. Combining in toto imaging with novel gene traps permits a means to rapidly screen for developmentally relevant expression patterns, followed by the ability to immediately mutagenize genes of interest. Initially, key technologies will be developed and tested in the zebrafish embryo due to its transparency and the ability to obtain rapid feedback. Once validated, these techniques will be applied to an amniote, the avian embryo, due to several advantages including accessibility and similarity to human embryogenesis. Finally, to monitor alterations in gene expression in normal and mutant embryos, we will develop new techniques for in situ hybridization that permit simultaneous analysis of multiple marker genes in a sensitive and potentially quantitative manner. Our goal is to combine real time analysis of gene expression on a genome-wide scale coupled with the ability to mutate genes of interest and examine global alterations in gene expression as a result of gene loss. Much of the value will come from the development of new and broadly applicable technologies. In contrast to a typical technology development grant, however, there will be experimental fruit emerging from at least two vertebrate systems (zebrafish and avian). The following aims will be pursued: Specific Aim 1: Real-time "in toto" image analysis of reporter gene expression. Specific Aim 2: Comprehensive spatiotemporal analysis of gene function of the developing vertebrate embryo using the FlipTrap approach for gene trapping. Specific Aim 3: Design of quantitative, multiplexed 'hybridization chain reaction'(HCR) amplifiers for in vivo imaging with active background suppression. Specific Aim 4: Data analysis and integration of data sets to produce a "digital" fish and a "digital" bird. The technologies and the resulting atlases will be made broadly available via electronic publication.

DESCRIPTION (provided by applicant): The research plan will refine and deploy a set of advanced tools for the imaging of tissue structure, gene expression domains and cellular dynamics during craniofacial development. Volumetric imaging tools will be used td create accurate 3D atlases that can be digitally dissected to permit the tissue interactions and cellular events to be better understood in the forming faces of normal, mutant and perturbed mouse and avian embryos. Molecular imaging agents, optimized for imaging intact tissues, will be employed to create atlases of the molecular correlates of these embryos. Finally, intraviral imaging tools will be used to study cell and tissue interactions as they take place, offering a view into the dynamic events that execute craniofacial development. The data will be assembled into atlases that will offer unprecedented tools for exploring the cellular, tissue and molecular correlates of craniofacial development. These atlases will be made widely available for the use of others in the Face Base consortium and the broader community of craniofacial researchers through an online resource created locally. In addition, they will be linked to other Face Base resources through established hubs, so that the data in our atlases can be used in concert with the molecular and structural data provided by other members of the consortium. The data acquisition pipeline established here in close collaboration with the laboratories of Dr. Yang Chai (University of Southern California) and Dr. Marianne Bronner-Fraser (Caltech) will create a model that can be expanded to phenotype and analyze other experimental systems. PUBLIC HEALTH RELEVANCE: Cleft palate, with or without cleft lip. is one of the more common birth defects, affecting approximately one in 2500 live births. The basic events by which cells of different origin come together to form the face have long been known, but the exact tissue movements and the signals that guide them remain undefined. The goal of this work is to help fill this void by imaging the morphology of normal craniofacial development, the abnormal development seen in cleft palate, and providing a means to experimentally test the roles of signaling pathways in the tissue interactions that build the face. This work offers a powerful platform for devising and testing potential therapeutic approaches.

DESCRIPTION (provided by applicant): Since its introduction in the 1960's, fluorescein angiography (FA) has been the gold standard for retinal vascular diagnosis. However, FA is costly, invasive, and time-consuming, which limits its usefulness as a screening tool. As a potential non-invasive alternative to FA for diagnosing retinovascular disease, we have developed an imaging method called phase contrast optical coherence tomography (PC-OCT). Our technology uses specialized software analysis of data acquired from clinically available optical coherence tomography imaging systems to provide an additional functionality of three-dimensional angiography. The majority of eye care in the US is currently performed by optometrists, and in many underserved communities, they are the sole providers. Unfortunately, most optometrists cannot perform FA due to its requirement for intravenous injection. Therefore, retinal vascular diagnostics are limited to clinical exam and fundus photography. Patients receiving optometric care would benefit from a non-invasive alternative to FA for improved screening of retinovascular diseases, especially in these underserved communities. PC-OCT has the potential to provide a low-cost and convenient vascular screening method, which could result in timely referrals to retinal specialists, as well as improved visual outcomes. To advance PC-OCT as a retinovascular imaging tool, we have the following goals: (1) develop PC-OCT software into a faster automated platform for application to commercial spectral domain (SD-OCT) systems; (2) optimize optical scanner performance for imaging both dilated and non-dilated pupils; and (3) demonstrate the feasibility of PC-OCT as a wide-field screening diagnostic for diabetic retinopathy. The visualization capabilities of PC-OCT imaging will be compared directly against fundus photography, the current diagnostic standard for eye care professionals. Successful completion of Phase II will result in an automated software package capable of producing PC-OCT microvascular images from the raw data of a commercial SD-OCT system, allowing for convenient vascular imaging accessible to all eye care practitioners. This could lead to better-quality detection capabilities for retinovascular disease and potentially improved visual outcomes.

DESCRIPTION (provided by applicant): Here we propose to develop an experimental paradigm to allow dynamic monitoring of the strength and location of every glutamatergic and GABA/Glycinergic synapse within the brain of a living organism. In combination with behavioral manipulation of the organism this paradigm will allow for study of how the brain encodes information in synaptic structure. This paradigm will involve combining three technologies: 1. Recombinant probes with which postsynaptic excitatory and inhibitory sites can be labeled in vivo, allowing the location and strength of synaptic connections to be monitored in parallel. 2. 2P-SPIM microscopy, which can image large volumes very quickly, without bleaching and, potentially, with isotropic resolution. 3. Software to calculate and store the location and strengt of each synapse in such a manner that it can be easily manipulated and analyzed. Experiments will be performed in zebrafish, as they have semi-transparent brains that are relatively small, yet they are capable of relatively complex behaviors. Experiments will be used both to establish the viability of the paradigm and to answer fundamental questions about how synapses are modulated during sleep, as well as how they are changed in learning paradigms such as sound habituation and place preference/aversion conditioning.

This is an INSPIRE award that was co-funded by the Biosciences Directorate, Division of Molecular and Cellular Biosciences (MCB), Systems and Synthetic Biology (SSB) program, and the Engineering Directorate, Division of Civil, Mechanical, and Manufacturing Innovation (CMMI), Dynamics, Control and System Diagnostics (DCSD) program.

Beneficial and pathogenic bacteria alike, commonly interact with host cells along mucosal epithelia. These surfaces are often lined with dense fields of motile cilia that serve both a biomechanical function for generating mucociliary flows, and a biochemical function to detect and present molecular signals. The goal of this project is to investigate a the dynamic association of healthy and diseased ciliated tissues. The project posits that cilia-generated flows influence bacteria-host interactions, thereby challenging the conventional view in biology that attributes bacterial recruitment mostly to active bacterial behavior and passive diffusion, ignoring the effect of cilia-generated flows on both motility and mass transport. The broader impacts of this study are that this project provides excellent educational and training opportunities at the intersection of disciplines, while also generating new insight in microbial host colonization that are likely to reveal avenues for impactful strategies to block pathogenic bacteria from colonizing the host, while enhancing the colonization potential of beneficial organisms.

The approach incorporates interdisciplinary methods, ranging from cutting-edge imaging and genetic tools to novel microfluidic technologies, all combined with powerful mathematical and computational framework, to investigate this fundamental problem in bacteria-host associations, namely, the role of cilia-generated flows in shaping these associations. The project will build a quantitative and predictive model that is informed by experimental assays in two complementary model systems:
1. In vivo invertebrate model: the squid-vibrio system, an intact animal host operating in its natural fluid environment, will be used to study how the host's ciliated epithelium initiates contact with its native, flow-borne bacterial community. Events in the squid-vibrio association share remarkable similarities with host responses to human-relevant pathogens, while still being accessible to imaging and experimental manipulation.
2. In silico microfluidics: properly designed microfluidic channels will be used as a tool to analyze the response of bacterial cells to carefully controlled microenvironment. This approach is aimed at complementing the squid-vibrio studies by providing a platform that isolates the chemical gradients and flow shear gradients observed in the in vivo model, thus allowing to unravel the mechanisms dictating bacterial behavior and possibly triggering the changes in bacterial gene expressions causing the transition from free-swimming to biofilm state.

PROJECT SUMMARY The formation of a malignant tumor from a benign tumor has been strongly linked to the existence of heterogeneous cellular features within the tumor. Thus, elucidating the mechanisms underlying generation of tumor cell heterogeneity will be an important milestone to understand malignant tumor formation. We believe two applications of our work could be 1) earlier, more quantitative detection of malignancy based on molecular markers and 2) therapeutics that can freeze tumor development before metastasis or even revert back to a benign state. Our approach is to answer the following two questions: 1. What is the impact of the tumor-initiating cell?s identity on generating tumor cell heterogeneity? In experiments to date, the inability to avoid contamination of different cell origins, including tissue stem cells and non-stem cells into the resultant tumor mass has prevented identification of the precise molecular mechanisms. We address the contamination problem by establishing an experimental system that enables precise tracing of tumor cell lineage derived from single cells. Our method is the non-invasive, in vivo imaging of endogenous, single cell-derived tumor development in adult transparent zebrafish. This experimental system allows us to visualize individual tumor cells, and, more importantly, also selectively label and isolate the cells of interest for detailed cell characterization. Using this system, we have already imaged the tumors arising from single cells in the skin epithelium. Importantly, we have found some major differences in tumor cell proliferation between tumorous cell clusters and in tumor cell differentiation that lead to acquisition of invasive features. Our results identifying a subset of tumor cells displaying invasive features is nicely representing the generation of tumor cell heterogeneity within a tumor. We will perform long-term clonal analysis of tumor development and transcriptome analysis of isolated individual tumors to determine their cellular identity. Retrospective imaging analysis will be used to study the effect of a tumor?s microenvironment on generating tumor cell heterogeneity. 2. How is heterogeneity within a tumor established? Identification of the cell types of tumor-initiating cells will be followed by the investigation of the mechanisms driving a single tumor-initiating cell to become heterogeneous during multiple rounds of cell divisions. Our ability to distinctively label and isolate invasive tumor cells from non-invasive tumor cells enables us to investigate the gene expression modulations responsible for a subset of tumor cells acquiring invasive features during tumor development. We will further perform functional experiments in vivo to define the roles of specific molecular pathways in tumor invasion.

This BRAIN EAGER will support an interdisciplinary team of investigators to jointly develop and deploy optimized light sheet microscopy and novel genetically encoded probes to image synaptic function in the intact nervous system. This team will optimize a novel 3D imaging microscope, in which multi-photon light sheet illumination is combined with light field microscopy to permit a single snapshot to capture the full 3D image, enabling the team to visualize these probes with unprecedented speed and coverage. They will also develop new genetically encoded glutamate and calcium probes, targeted to defined synaptic compartments, to optimize signal magnitude and report synaptic activity with high sensitivity and fidelity. These innovations will be exploited to synergistically visualize synaptic structure and monitor glutamate and calcium dynamics in the intact Drosophila central nervous system.

Although these tools could be used in a variety of settings in the vertebrate or the invertebrate nervous system, these will be first applied to address the fundamental relationship between sleep and synaptic plasticity. Although sleep is ancient, the essential biological function of this behavior remains a great mystery of science. This project will explore the exciting possibility that a fundamental function of sleep, operating at the level of individual neurons and synapses, is the homeostatic modulation of synaptic strength. Addressing this hypothesis has been beyond our capabilities because visualizing neural activity in the central nervous system during sleep-wake behavior has been limited in both speed and resolution. Through a combination of new genetically encoded probes reporting synaptic structure and activity and cutting-edge imaging approaches, this project will permit the imaging of synapses over time without perturbing the nervous system or the sleep-wake cycle. These test experiments will advance our knowledge of the complex, fundamental, and poorly understood signaling systems that orchestrate the homeostatic control of synaptic strength, and their modulation during sleep behavior. The education and outreach activities of the research team will be intimately linked with their research programs, and will include a research project with local inner-city Los Angeles high school students investigating sleep and circadian behavior. In addition, a new undergraduate course will be developed exploring the biological functions of sleep.

DESCRIPTION (provided by applicant): Here we propose to develop an experimental paradigm to allow dynamic monitoring of the strength and location of every glutamatergic and GABA/Glycinergic synapse within the brain of a living organism. In combination with behavioral manipulation of the organism this paradigm will allow for study of how the brain encodes information in synaptic structure. This paradigm will involve combining three technologies: 1. Recombinant probes with which postsynaptic excitatory and inhibitory sites can be labeled in vivo, allowing the location and strength of synaptic connections to be monitored in parallel. 2. 2P-SPIM microscopy, which can image large volumes very quickly, without bleaching and, potentially, with isotropic resolution. 3. Software to calculate and store the location and strengt of each synapse in such a manner that it can be easily manipulated and analyzed. Experiments will be performed in zebrafish, as they have semi-transparent brains that are relatively small, yet they are capable of relatively complex behaviors. Experiments will be used both to establish the viability of the paradigm and to answer fundamental questions about how synapses are modulated during sleep, as well as how they are changed in learning paradigms such as sound habituation and place preference/aversion conditioning.

Classical conditioning has been studied in many different animal models, and even in humans. However, the larval zebrafish with its transparent brain offers a unique opportunity to observe large scale changes in synaptic structure that accompany this form of learning. Accordingly, we have developed a novel paradigm for visualizing synaptic changes that occur during classical conditioning in larval zebrafish. Using this paradigm we have observed striking region-specific changes in the distributions of synapses that drive the rewiring of neural circuits that mediate threat responses. In this grant we will expand this paradigm by monitoring neuronal activity through imaging of genetically encoded calcium indicators throughout the pallium (the homolog of the amygdala) before, during and after classical conditioning and extinction. This will allow us to identify cells that comprise the circuits that control threat and safety and explore their connectivity using optogenetics. We will investigate how different sensory inputs can cause changes in the activity of those cells leading to synapse change, and the formation or extinction of associative memories. A crucial component of these studies will be the recording of field potentials to capture rhythmic activity throughout the pallium and high speed SPIM imaging of genetically encoded voltage indicators to record rhythms in individual cells. By understanding the precise timing of signals that impinge on individual cells we will uncover mechanisms that underlie synaptic plasticity. Our goal is to develop a theoretical model describing the neural circuits that underlie threat detection and how they can change as a result of associative memory formation and extinction.