Keith C. Cheng - US grants

Cheng 9319817 Abstract Zebra fish have many advantages as an experimental organism for developmental studies. However, its use is limited unless it can be developed as a genetic system also. It is proposed that this project will demonstrate the feasibility of using zebra fish as experimental genetic organisms. Mutations that increase genomic instability, e.g. by increasing the rate of chromosome loss or mutation, are expected to increase the frequency of loss of heterozygosity. That loss can be used as a phenotypic screen in that a recessive pigment mutation which can be quantitatively assessed by screening the number of nonpigmented cells in an otherwise pigmented retinal epithelium of three-day old unhatched embryos can be used as a tracer. The mutant candidates can be generated as the progeny of mutagenized gol-1+ males and non- mutagenized gol-1(b1) females. The recessive mutant screen proposed involves inspection under a dissecting microscope of the progeny. Clutches of progeny yielding a potential aggregate of more than 100,000 cells will be scored in situ under a dissecting microscope at three days. A simpler dominant screen will be evaluated in parallel primarily with males after standard crosses. When possible, complementation analysis of recessive mutations, outcrosses and quantitation of the effect of mutations will be begun. %%% The feasibility of developing zebra fish as useful genetic organisms will be determined. The strains necessary for the projected screen for mutant progeny will be generated. Genomic instability mutants, both dominant and recessive, will be generated. When possible, the mutants will be characterized and karyotyping will be attempted. ***

The histopathology of cancer suggests a progressive loss of differentiation, where mature cells are replaced by less-differentiated counterparts with embryonic behaviors such as high rates of growth and cell migration. Some of the genes controlling these functions may act in a tissue-specific manner and potentially function as tumor suppressor genes. It is further hypothesized that new tissue-specific tumor suppressors may be found by the histopathologic examination of mutant embryos of the zebrafish (Danio rerio), for abnormal differentiation in specific, mature embryonic tissues. The experimental features of the zebrafish make it a unique vertebrate model to find mutants defective in differentiation of specific tissues. First, each female is capable of producing an average of about 100 rapidly developing, 1 x 1 x 3 mm, transparent embryos per week, making it possible to readily produce and examine thousands of mutant candidates ex vivo, at a relatively low cost. Parthenogenesis can be used to unmask recessive mutations within one generation in heterozygous parents. Lethal mutations can also be studied, since the heterozygous parents allow one to generate additional mutants for further study or genetic mapping. Recent progress in zebrafish genetics makes positional cloning possible. The production of many early embryonic developmental mutants by other investigators indicates that the zebrafish is a practical vertebrate model for generating interesting genetic mutations. These studies are expected to reveal genes important to differentiation of specific tissues in the zebrafish. Since human cancers show abnormal differentiation, homologues of some of these genes may be affected in human cancer. It is hoped that work on such genes, their products, and expression, will lead to prognostic and therapeutic tools for patient care.

DESCRIPTION: (Applicant's Abstract) This is a proposal, in response to RFA HD-OO-0004, to perform a histological screen for ENU-induced mutations causing either organ-specific or general histological phenotypes in 7 day old parthenogenetic haif-tetrad larvae of zebrafish (Danio rerio). Subgoals include confinnation of each mutation as an inheritable trait, preliminary characterization, and mapping of each mutation to a chromosome arm. Community functions will include posting of mutant phenotypes on the web, and distribution of mutants to the Zebrafish Stock Center. We expect to find mutants with defective tissue differentiation, and to produce models for aspects of human disease, including cancer and diseases of the eye and gastrointestinal tract. The motivation for the screen is the power of histologic analysis in the characterization of pathological changes in human tissues. Hundreds of cell types and pathological changes are distinguishable from the light microscopic study of stained tissue sections. For example, the histological changes common to all cancers include defects in tissue organization and in the cytological appearance of cancer cells. A genetic dissection of these changes can be accomplished through the generation and study of mutants with histological phenotypes. The experimental features of the zebrafish make it an ideal vertebrate model to identify mutants with histological phenotypes. Each female can produce hundreds of embryos per week at a relatively low cost. Recessive mutations carried by females may be unmasked in parthenogenetic half-tetrad progeny. A pilot histologic screen 01 half-tetrad families of 72 carrier females produced two mutations affecting the neural retina, two affecting the gastrointestinal tract, and two with multi-organ effects (Cheng et al.,submitted). One mutant showed a strong cytological phenotype in multiple organs that would be diagnostic of severe atypia or cancer in a human cytology specimen. The screen's success establishes the ability of this laboratory to perform all required steps in the proposed screen, including mutagenesis, parthenogenesis, histologic screening, and mapping. This project will contribute to an elucidation of both organ-specific and general pathways of cell differentiation in vertebrates.

DESCRIPTION (provided by applicant): NIH-sponsored zebrafish and Aquatic Models working groups have recommended the creation of web based anatomy, histology and pathology atlases as a community resource. The principal investigators propose to generate and digitize reference glass slides for web-based virtua/microscopy and 3D at as of the zebrafish. This atlas will be available through the Zebrafish Information Network (ZFIN) of the University of Oregon (http://zfin.org/ZFIN/). In virtual microscopy, any computer with a web browser and fast internet connection can function as a microscope, using annotated virtual slide files on a remote server. Virtual slides are GB size, compressed composites of high magnification image tiles or strips; image packets are sent to the remote user according to user-chosen field-of-view and magnification. In the 3D anatomy atlas, selected anatomical structures can be viewed and rotated. The atlases will include normal, and some abnormal, embryos, larvae, juveniles and adults. The experimental features of the zebrafish have made it a premier model for functional genomics. High-throughput mutant, antisense knockdowns ("morphants"), fluorescent transgenic, and chemical screens have already yielded thousands of potential models of human disease affecting a variety of biological functions at a variety of stages of growth and development. A web-based, comprehensive reference atlas of normal zebrafish would greatly facilitate this work. Remote annotation of and access to such atlases will facilitate the growth of the atlas as a resource, as well as individual and collaborative investigations. Specific Aim 1 is to generate a registry of serial, stained paraffin and plastic sections of normal zebrafish embryos, larvae, juveniles, and adults. Specific aim 2 is to scan these sections to create virtual slides. Illustrative pairs of annotated fields will be generated. Specific Aim 3 is to digitally outline histologically identifiable structures in zebrafish embryos, larvae, juveniles, and adults for 3D reconstructions. The resultant infrastructure will be complementary to, adaptable to, and coordinated with those of other model systems. The atlas will support the zebrafish community across multiple disciplines, and address the missions of nearly all Institutes of the NIH.

DESCRIPTION (provided by applicant): Cells containing melanin pigmentation aid vision, protect against UV radiation, and can transform into neoplasms. This proposal is based upon three related discoveries in our lab. 1) The zebrafish hypopigmentation mutant, golden, shows a melanosomal phenotype similar to that seen in light-skinned people. 2) The affected gene in this mutant is slc24a5, which encodes a putative K-dependent-Na/Ca exchanger. 3) A coding polymorphism in the human orthologue SLC24A5 is an important determinant of the difference in human skin color between Africans and Europeans. Our long-term goal is to elucidate the role and mechanism of the SLC24A5 protein in melanogenesis. We propose a collaborative, multidisciplinary approach that includes ultrastructural analysis, functional genomics, and transport studies. We are testing the hypothesis that slc24a5 is a calcium exchanger located in the membranes of melanosomes or their precursors, and that the organellar ionic milieu established by its transport activity is important in melanosome morphogenesis and pigment formation. Specific Aim 1 seeks to determine the subcellular distribution of the slc24a5 protein among melanosomes and their precursors using double-label, confocal immunofluorescence microscopy, subcellular fractionation, and immunoelectron microscopy. Specific Aim 2 combines ultrastructural analysis with antisense morpholino knockdown to define the morphogenetic roles of slc24a5. Specific Aim 3 seeks to establish the transport properties of the slc24a5 protein using a combination of calcium imaging, electrophysiology, and Ca-45 transport studies. Specific Aim 4 addresses the functional consequences of the common human polymorphism in SLC24A5. Findings from the proposed work will enhance our understanding of melanosome morphogenesis and melanin pigmentation in vertebrates, including humans. Melanin-containing cells aid vision, protect us from UV radiation, and when transformed, become one of the deadliest cancers in humans, malignant melanoma. This work will clarify how a newly-discovered sodium- calcium exchanger protein, first characterized in zebrafish, affects pigmentation, and lays a foundation for drugs to treat skin pigmentation problems, and perhaps malignant melanoma.

[unreadable] DESCRIPTION (provided by applicant): Abstract: One of the remaining unanswered mysteries in human biology is the identity of the genes are responsible for the lighter skin of East Asians. The goal of this revision application of AR052535, entitled Genetic analysis of genomic instability and cancer in zebrafish is to work towards finding one or more of those genes. Solving this mystery will be a major contribution towards understanding basic mechanisms of human pigmentation, a phenotype of profound influence to human welfare. This work will also be important to our understanding of human skin cancer, since the light skin mechanisms of Europeans confer greater susceptibility to cancer than the mechanisms in East Asians and Amerindians. This collaborative and interdisciplinary project combines the power of a developmental model, the zebrafish, with the power of systems biology. This will be accomplished through the coordinated work of a team that includes the lab of a second NIH-funded investigator, Dr. Ross Hardison, whose expertise is the development of bioinformatics tools for discovery of functional, noncoding sequences. This work will be accomplished in three related specific aims that are extensions of ongoing work in both labs. Specific Aim 1 is to take a systems approach to identifying coding and noncoding sequences of potential functional importance in East Asians. This aim will be accomplished using tools 1) developed in the course of discovery of a functionally important coding single nucleotide polymorphism (SNP) in SLC24A5, which appears to have played an important role in the evolution of light skin in European populations, 2) developed and being developed in the Hardison lab for studying noncoding sequences. The results of these analyses will correlate SNPs of high frequency distinction between East Asian (CHB and JPT) and African (YRI) HapMap populations that are associated with large regions of reduced heterozygosity. Specific Aim 2 is to map East Asian pigment genes using whole-genome SNP and copy number analysis. The first part of this aim will be to obtain human skin reflectance measurements and DNA from the saliva of individuals in families and unrelated individuals from populations of East Asian (or Amerindian)/African admixture. Initial whole genome SNP/copy number analysis will be done using the highly reliable Illumina platform, to map candidate regions defined by regions of diminished heterozygosity in individuals of the most extreme skin color variation, focusing on SNPs whose ancestral allele is in the African HapMap population. The relevant populations have been found by the PI and specific communities were found by collaborators in the University of West Indies and Oxford University. Specific Aim 3 will be to test the most promising candidates for pigmentary function by tissue localization (whole mount in situ) and antisense morpholino knock-down analysis in zebrafish. Within the year, we will have completed collaborative bioinformatics analysis, find candidate East Asian genes by genetic mapping, and test candidate genes using functional studies in zebrafish. Even early discussion has led the investigators to realize that the work will greatly enhance both of the NIH-funded projects in the course of fine tuning and validating a new systems genetics approach for the study of quantitative traits in humans. This work can also be expected to contribute towards the missions of multiple Institutes at the National Institutes of Health. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Cancer results from an accumulation of genetic and epigenetic changes in cancer genes, including tumor suppressor genes; these changes cause normal somatic cells to evolve into cancer cells. In Knudson's two-hit paradigm, loss of tumor suppressor gene function occurs in two steps. The second of those steps can occur by multiple mechanisms including chromosome loss, recombination, insertion, deletion, point mutation, or epigenetic modification. In order to find vertebrate genes involved in those steps, which serve to maintain genome stability, we have used the zebrafish (Danio rerio) to perform a screen for genomic instability mutants that models this second hit. This screen used the mosaic eye assay, in which pale cells appear in a black background of retinal pigmented epithelial (RPE) cells that are heterozygous for a recessive pigment mutation, goldenb1. Twelve ENU- induced genomic instability (gin), or somatic mutator mutations were isolated in this screen at a rate suggesting the potential existence of 200 such genes. Most of the gin mutations showed weak dominance in heterozygotes, and all showed a stronger phenotype in homozygotes. The strongest mutant, gin-10, showed a striking (~10-fold) phenotypic enhancement over homozygous phenotype when inherited maternally in trans with other gin mutations (the "gin-10 interacting group"); gin-10 carriers show a 9.6-fold increase in susceptibility to spontaneous cancer. These findings represent first steps in our long-term goal of developing the zebrafish as a vertebrate model for finding genes involved in the control of genomic stability in vertebrates. Now that we have shown that the zebrafish can be used to find somatic mutators that are susceptible to cancer, we propose to elucidate the nature of the striking interactions between the genes comprising the gin-10 interacting group, and to better-characterize the associated tumor susceptibility. This will be accomplished in three Specific Aims. For Specific Aim 1, we will clone members of the gin-10 interacting group using positional cloning for gin-10 and gin-12, and using a sensitized insertional screen with a novel "gene breaking" To12 transposon-based vector. Specific Aim 2 is to characterize the mechanism of genomic instability using microsatellite, interphase FISH analysis, and exon sequencing of the golden gene in golden RPE cells from mosaic eyes and by similar analysis of p53 in gin-10-associated tumors. Specific Aim 3 is to determine the tumor susceptibility of the strongly mosaic gin-10/gin-12 trans-heterozygotes. The proposed studies will contribute to our understanding of gene interactions involved in somatic loss of gene function, and to clarify how they contribute to cancer susceptibility. It is our hope that such understanding will lead to novel ways to prevent or treat human cancer. PUBLIC HEALTH RELEVANCE: Cancer is a disease in which normal cells accumulate changes in their DNA as they evolve into cancer cells. We now know that the genes controlling the stability of our DNA play a key role in cancer. We are using the unique power of the zebrafish as a vertebrate model system to find and study genes that are important in genetic stability and cancer, in the hope that our increased understanding will lead to measures to treat and prevent human cancer. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The zebrafish is now a common, non-mammalian model of human disease due to experimental features such as small size, transparency, and powerful genetic tools. In response to NIH-sponsored Zebrafish and Aquatic Models working groups' recommendations, an important piece of American scientific infrastructure is being established in the form of a web-based atlas of anatomy, histology, and pathology. This atlas will be accessible from www.zfatlas.psu.edu and the Zebrafish Information Network (ZFIN) at the University of Oregon (www.zfin.org). Normal anatomy will serve as critical context for the study of abnormal anatomy caused by genetic deficiencies and disease. The atlas' personnel, imaging and computer hardware and web site comprise the infrastructure for users to view mutant/morphant/diseased zebrafish in the context of normal, in both 2D and 3D. Specific Aim 1 is to integrate abnormal phenotypes into the atlas starting with an established mutant collection, to create a nondestructive mechanism for remote labeling of web-based, state-of-the-art virtual slides that allows viewing of histological tissue sections at different powers, to integrate the zebrafish atlas with other web-based resources including the home of zebrafish web-based resources (www.zfin.org), and to use the web interface to display a range of mutant phenotypes. Specific Aim 2 is to develop mechanisms for analyzing and displaying 3D zebrafish data. We will segment and label different organs from digital 3D representations of zebrafish, and explore, test, and implement integrated interfaces between virtual slides and 3D representations of zebrafish created by microCT imaging at subcellular resolutions. Specific Aim 3 is to build a foundation for structural and functional integration across models systems. This aim will utilize zebrafish, mouse, and human as the organisms of interest, and use specific skeletal, eye, and skin color mutations as a paradigm for describing the roles of model systems in understanding a biological function and disease. This aim will focus on normal tissues shared by all three species followed by pathology shared by all three species. The integrations of this community resource will serve as a model for all model system web sites, serving multiple missions across the NIH. PUBLIC HEALTH RELEVANCE (provided by applicant): Compelling experimental features have made the zebrafish a model for a wide range of human maladies, including cancer, heart disease, and infection. The atlas (www.zfatlas.psu.edu) contains high-resolution 2D and 3D representations of normal and, as proposed, abnormal zebrafish. By cross-referencing with other model systems, the atlas will also provide infrastructure for integrative, multidisciplinary projects.

DESCRIPTION (provided by applicant): We propose to modernize, expand, and consolidate our current zebrafish capabilities through the creation of a Zebrafish Functional Genomics Core (ZFGC). This core will have high impact, because it will enrich and connect three of our new and growing institutional health research investments: The Institute for Personalized Medicine, the Drug Discovery Development and Delivery Core (D4), and a university-wide Systems Biology cluster hire. Penn State investigators have been pioneers in the use of zebrafish as a model system for the study of a variety of conditions; the ZFGC will allow us to expand our research capabilities. Our first aim will be to increase and centralize housing space, quarantine space, and procedural space through renovation of four existing, underutilized animal rooms totaling over 1,200 net square feet. Our second aim will be to improve the efficiency and cost-effectiveness of the care and use of zebrafish through the installation of a state-of-the art zebrafish husbandry system. Our third aim will be to establish a dedicated procedure room for shared operations including microinjection, mass breeding, and photography. Accomplishing these aims will facilitate the use of zebrafish in currently funded projects involving human cancer, cardiovascular disease, metabolic disorders, neurodevelopment and skin pigmentation. A pilot project program will ensure lasting use of this new core, as well as integration of research activities between cores. Newly targeted directions of research utilizing the zebrafish model will include personalized medicine, disease modeling, and drug discovery.

Project Summary Each major human disease is associated with a specific range of morphological changes to cells and tissues in the micron scale. Normal and abnormal structure was discovered and is still characterized using histology - a microscopic technique that depends on physical tissue slices. Presently, histology?s use in systems biology is limited by its largely descriptive and two-dimensional nature. Making histology quantitative and three-dimensional would be potentially transformational for research and diagnostics, but has been impractical. Accordingly, we have now created a 3D form of histology by customizing X-ray microtomography (micro-CT) of fixed and stained, millimeter-scale, whole organisms and tissue samples. We used fixed and metal-stained, whole zebrafish because they contain a full range of tissues within the size range currently studied histologically. The result is the first practical way to create virtual histology-like ?sections? in any plane. Three-dimensional, complete histological phenotyping has potential use in genetic and chemical screens, and in clinical and toxicological tissue diagnostics. Here, we propose the next steps needed to enable high-throughput, quantitative, 3D histological phenotyping of whole, millimeter-scale animals. The proposed work applies the principles of chemistry, physics, and computer science to improve image resolution, throughput, and analytics, organized into three specific aims. Specific Aim 1 will build on our developments in this project and further improve imaging volume and resolution by upgrading imaging array, optics, and sub-pixel shifting, and to throughput by changes in sample embedding, loading geometry and mechanics, helical CT scanning, scintillator material, and to data sharing by improvements to the ViewTool infrastructure and user interface. Specific Aim 2 will yield reference images to define the range of normal phenotypic variation and to obtain samples related to a range of potential applications. Specific Aim 3 will apply the power of machine learning to segmentation, annotation, and analytics. Together, this work will establish a practical foundation for large-scale genetic and chemical screens involving mm-scale, whole organisms based on 3-dimensional, quantitative, histological phenotyping. The instrumentation and analytics will be state-of-the-art in its combination of resolution, field-of-view, pancellularity, image quality, analytical potential, throughput, sample stability, and reproducibility and largely usable with both tube and synchrotron X-ray sources. The voxel resolution will be at least 0.5 ?m across fields-of-view of up to 1 cm. Representation of every cell type make the images suitable for cross-referencing across imaging modalities. Potential applications will be explored, ?wild-type? will begin to be defined, and training sets for automated segmentation generated. The potential impact will encompass the missions of most NIH Institutes and Centers. The whole-animal genetic and chemical screens enabled are expected to impact drug development, diagnostics, and our basic understanding of how genes and environment define phenotype.