LASER SCANNING CONFOCAL MICROSCOPE
Donna M Fekete
Purdue University West Lafayette, 155 S Grant Street, West Lafayette, In 47907-2114
Grant 1S10RR023734-01A1 from National Center For Research Resources
Abstract: Nineteen researchers in the Department of Biological Sciences and the Purdue Cancer Center at Purdue University seek support for advanced live cell imaging with a Zeiss LSM 710 confocal microscope on an inverted microscope platform equipped with an incubation chamber. Live cell imaging is at the forefront modern cell biology and lack of access to these methods cuts off Purdue investigators from key advances. Support for this microscope provides essential research infrastructure to a successful cadre of NIH-funded researchers with a sound record of productivity, reporting significant advances in biomedical research. Acquisition of this microscope by this user group will open new research avenues for proven researchers who are poised to move strongly and successfully into advanced live cell imaging using the LSM 710. A sampling of the NIH-funded research that purchase of this microscope will support includes the development of the vertebrate eye and ear in an effort to discover new genes underlying blindness (Leung) or deafness (Fekete) in humans; the mechanism by which defects in axonal transport of mitochondria give rise to neurodegenerative disease (Hollenbeck); cell lineages involved in pancreatic cancer initiation (Konieczny); cellular details of replication and assembly of human pathogen alphaviruses and flaviviruses (Kuhn); the function of TRP channels that are important in a vast array of biological functions, including sensory perception, especially pain (Pak); development and maintenance of healthy photoreceptors (Chang, Ready), regulation of neuronal growth cone motility and guidance critical to nervous system growth and repair (Suter); how Salmonella exploits the host acting cytoskeleton network to promote Salmonella entry and induce diarrhea in humans (Zhou). The requested inverted microscope complements the recent acquisition of an upright Zeiss LSM 710 by the Purdue Life Sciences Fluorescent Imaging Facility. Having both platforms will allow Purdue investigators to move seamlessly between upright and inverted microscopes so as to choose the best tool for each enquiry. This will also provide economies in training, administration and support. The microscope will be a nexus of interdisciplinary exchange of ideas and methods. In addition to serving established investigators, it will provide a cadre of outstanding young researcher´s state of the art instrumentation at the critical outset of their careers. Lack of advanced live cell imaging methods is a significant research bottleneck at Purdue and investment in this microscope will stimulate experimental and intellectual success in biomedical research
Keywords: Alpha Virus; Alphavirus; Arbovirus, Group B; Arboviruses, Group A; Arts; Axonal Transport; Axoplasmic Transport; Biologic Sciences; Biological Function; Biological Process; Biological Sciences; Biomedical Research; Blindness; Cell Lineage; Cell Locomotion; Cell Migration; Cell Movement; Cellular Matrix; Cellular Migration; Cellular biology; Communicating Junction; Complement; Complement Proteins; Cytoskeletal System; Cytoskeleton; Deafness; Defect; Degenerative Diseases, Nervous System; Degenerative Neurologic Disorders; Development; Diarrhea; Ear; Ear structure; Electromagnetic, Laser; Eye; Eyeball; Flavivirus; Funding; Gap Junctions; Generalized Growth; Genes; Growth; Growth Cones; Human; Human, General; Image; Infrastructure; Instrumentation, Other; Investigators; Investments; Lasers; Life; Life Sciences; Low-resistance Junction; Maintenance; Maintenances; Malignant Pancreatic Neoplasm; Malignant neoplasm of pancreas; Man (Taxonomy); Man, Modern; Methods; Microscope; Mitochondria; Motility; Motility, Cellular; NRVS-SYS; Nervous System; Nervous system structure; Neurodegenerative Diseases; Neurodegenerative Disorders; Neurologic Body System; Neurologic Degenerative Conditions; Neurologic Diseases, Degenerative; Neurologic Organ System; Nexus; Nexus Junction; Pain; Painful; Pancreas Cancer; Pancreatic Cancer; Perception; Photoreceptor Cell; Photoreceptors; Photosensitive Cell; Productivity; Purdue Cancer Center; Radiation, Laser; Regulation; Reporting; Research; Research Infrastructure; Research Personnel; Researchers; Salmonella; Sampling; Scanning; Sensory; Sound; Sound - physical agent; TRP channel; TRP protein; Tissue Growth; Training; Universities; Visual Receptor; cancer initiation; career; cell biology; cell imaging; cell motility; cellular imaging; imaging; imaging modality; instrumentation; intracellular skeleton; mitochondrial; neurodegenerative illness; neuronal growth; ontogeny; pathogen; repair; repaired; sound; success; tool
Project start date: 2010-05-06
Project end date: 2011-05-05
Budget start date: 6-MAY-2010
Budget end date: 5-MAY-2011
PFA/PA: PAR-09-028
1S10RR023734-01A1 (2010): $499610
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Donna M Fekete
DEVELOPMENT STUDIES OF THE INNER EAR
Donna M Fekete, Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 7R01DC002756-03 from National Institute On Deafness And Other Communication Disorders IRG: HAR
Abstract: The sense of hearing is fundamental to man s ability to communicate, and is particularly important in children as they acquire language. The sensory receptor cells of the inner ear play a pivotal role their absence due to birth defects (congenital deafness), or their loss later in life, results in total deafness. To combat congenital deafness and the resulting deficits in human communication, it is necessary to understand the normal development of the inner ear. As the ear develops, the cells that comprise the ear must undergo a series of key decisions as to what cell type(s) to become (i.e., fate determination), and what spatial arrangements they must make with neighboring cells to generate the appropriate three-dimensional structure (i.e., pattern formation). The specific aims of this project are (1) to describe the kinds of cell fate decisions made by individual progenitor cells in the vertebrate inner ear through lineage analysis and (2) to test one candidate molecule for a role in cell fate determination and/or pattern formation. The methods for accomplishing these two specific aims primarily involve using retrovirus vectors as a highly efficient gene transfer technique to introduce genes into the developing inner ear of the chicken. For Specific Aim 1 (lineage analysis), replication-defective retrovirus vectors will be used to deliver a marker gene encoding either alkaline phosphatase or beta- galactosidase to individual progenitor cells as a way of labeling them and all of their progeny. Clonal analysis will be performed at a stage when regional and cell type distinctions are readily apparent. For Specific Aim 2 (gene perturbation), replication-competent retrovirus vectors that encode various forms of a homeobox-containing gene, SOHo-1, will be used to misexpress this protein in inner ear progenitor cells. SOHo-1 is proposed to play a role in the cell fate decision involving whether or not to become a sensory organ based on preliminary descriptions of its mRNA expression pattern. The purpose of this study is to further define the normal expression pattern of SOHo-1, and to alter this expression pattern during critical stages of ear formation. Alterations in the formation of the ear as a result of SOHo-1 misexpression would indicate that this gene does indeed play a functional role in establishing the normal anatomy of the ear. The gene perturbation methods established during the course of this study can be used in the future to test many other candidate genes for a role in ear development. The phenotypes obtained from gene perturbation experiments may mimic, and in some cases correlate with, human congenital malformations of the inner ear.
Keywords: developmental genetics, developmental neurobiology, homeobox gene, labyrinth, Retroviridae, alkaline phosphatase, beta galactosidase, ear hair cell, epithelium, gene mutation, genetic marker, neuroanatomy, neurogenetics, transfection vector, chick embryo, chicken, immunocytochemistry, in situ hybridization, polymerase chain reaction
Project start date: 1995-08-01
Project end date: 2000-07-31
7R01DC002756-03 (1997): $168680
5R01DC002756-05 (1999): $241832
5R01DC002756-04 (1998): $174441
3R01DC002756-04S1 (1998): $53499
2R01DC002756-06 (2000): $277643
5R01DC002756-12 (2007): $419871
5R01DC002756-11 (2006): $422376
5R01DC002756-10 (2005): $422559
2R01DC002756-09A1 (2004): $376465
Sponsored Links Excellgen http://Excellgen.com
MOUSE WHOLE EMBRYO CULTURE PARADIGM OF EAR MORPHOGENESIS
Donna M Fekete, Professor
Biological Sciencespurdue University West Lafayette
302 Wood St
west Lafayette, In 479072108
Grant 5R21DC004490-02 from National Institute On Deafness And Other Communication Disorders IRG: ZDC1
Abstract: Efforts to understand the genetic mechanisms that underlie congenital inner ear defects are now focused on both the normal processes of development and on the cascade of events that can arise in response to a single gene mutation. Because the generation of animals with a targeted gene defect has become commonplace in mice, this species has emerged as a powerful model system in which to pursue the relationship between gene expression and morphogenesis. Counteracting the power of mouse genetics is the inaccessibility of the mouse embryo throughout the critical stages of organogenesis, which begins in utero during the second week after fertilization. Because of its inaccessibility, one key experimental approach, fate mapping of an organ primordium, is rarely performed in the mouse embryo. At the present time, it is not known in any species which parts of the otic placode and early otic vesicle give rise to the different parts of the ear. Such information can be obtained by fate mapping, and is critical for interpreting how gene expression domains get converted into patterning information. The ability to superimpose the expression domains of the greater than 40 genes expressed in the ear with a high-resolution fate map of the otocyst could have a major impact on the field of ear development. Furthermore, fate mapping a mouse inner ear that is abnormal due to a known genetic mutation promises to provide insights that are simply not possible by descriptive analysis alone. For example, it may provide information about whether a specific genetic mutation is causing a change in cell fate that can explain the mutant phenotype. The first specific aim is to fate map the mouse otic cup in both the wild-type mouse and in a mouse mutant, kreisler, that develops with gross abnormalities in the inner ear. This will be accomplished by small focal injections of lipophilic carbocyanine dyes directed into the developing ear epithelium of mouse embryos grown in culture. The labelled cells will be mapped to see where they reside after otic vesicle closure (after 24 hours) or otic vesicle morphogenesis (after 48 hours). The second specific aim is to pilot methods to facilitate focal gene transfer into the otocyst of the cultured mouse embryo. This will be accomplished by injection of retrovirus stocks or small numbers of retrovirus-producing cells. The study will be performed with green fluorescent protein as a marker for the purpose of piloting the methods. The long-term goal driving the development of an in vitro paradigm for mouse ear development is that it may lead to intervention strategies (such as virus-mediated gene transfer) designed to rescue the inner ear defects arising from known genetic mutations. If the mouse whole embryo culture paradigm proves successful, its impact is likely to extend far beyond the proposed studies, given that the number of mutant and knockout mice generated as potential models of human deafness genes will continue to rise
Keywords: ear, embryo /fetus culture, gene mutation, histogenesis, labyrinth, mammalian embryology congenital ear disorder, technology /technique development, transfection green fluorescent protein, laboratory mouse, microinjection
Project start date: 2000-08-01
Project end date: 2003-07-31
5R21DC004490-02 (2001): $76000
1R21DC004490-01 (2000): $73400
A GENE-TRAP SCREEN FOR HEARING AND BALANCE
Donna M Fekete
Purdue University West Lafayette, 155 S Grant Street, West Lafayette, In 47907-2114
Grant 5R21DC008997-02 from National Institute On Deafness And Other Communication Disorders
Abstract: Congenital forms of sensorineural hearing loss can arise from perturbations in development of either peripheral- or central-nervous-system components. Gene discovery approaches that can identify new genes expressed during development of the auditory or vestibular systems in animal models should assist in revealing genetic causes of congenital deafness in humans. We have devised a new Gal4-UAS-based gene- trap screen for zebrafish embryos that should facilitate not only gene discovery, but also both loss-of-function and gain-of-function approaches for testing candidate genes involved in complex processes such as development of the auditory and vestibular systems. Furthermore, our gene-trapping strategy should be applicable to any developing organ or system in the zebrafish, thereby enhancing the versatility of zebrafish as an important model organism for understanding the genetic causes of various human birth defects. We propose two Specific Aims. (1) To generate new otic- or neural-specific gene-trap lines in zebrafish. Pseudotyped retroviral vectors or Tol2 transposases will be used to insert a gene-trap construct into the zebrafish germline. The trapping construct will use a GAL4-UAS system to transactivate the expression of a reporter gene that can be screened by fluorescence imaging of live embryos. Lines showing relatively specific expression in peripheral or central components of mechanosensory systems will be created and trapped genes will be cloned. (2) To use gene-trapped Gal4-driver lines for targeted cell ablation in vivo. One major advantage of our gene-trap design is its potential for targeting bioactive molecules to specific cells in vivo without requiring the isolation of cell- or tissue-specific promoters. This can be accomplished by crossing a particular Gal4-trap line (i.e., the activator line) with a transgenic line carrying a target gene placed downstream of a UAS sequence (i.e., the effector line). Only when both the activator and effector are active in the same cells is the effector protein expressed. An inducible form of Gal4 (GeneSwitch) will permit even more control over the onset of effector protein expression. As proof-of-principle, an effector line will be created with UAS upstream of a toxin gene. When crossed to any of the driver lines, we expect the toxin will specifically kill only those cells expressing the trapped gene. This should prove especially powerful for selective ablation of subsets of CNS neurons to assess their role in development and/or in behavior. Our novel gene-trap screen should facilitate gene discovery in zebrafish, and readily allow tests of candidate genes for their involvement in development of the auditory system. Our long-term goal is to determine whether any of the newly discovered hearing-related genes in zebrafish also correspond to genes underlying congenital deafness in humans
Keywords: Ablation; Animal Model; Animal Models and Related Studies; Antimorphic mutation; Auditory; Auditory system; Behavior; Birth Defects; Body Tissues; Brachydanio rerio; Breeding; Candidate Disease Gene; Candidate Gene; Cell Isolation; Cell Lineage; Cell Segregation; Cell Separation; Cell Separation Technology; Cells; Cellular Mechanotransduction; Central Nervous System; Complex; Congenital Abnormality; Congenital Anatomic Abnormality; Congenital Anatomical Abnormality; Congenital Defects; Congenital Deformity; Congenital Malformation; Corynebacterium Diphtheriae Toxin; DNA Transposable Elements; Danio rerio; Deafness; Defect; Detection; Development; Diphtheria Toxin; Disease; Disorder; Dominant Negative; Dominant-Negative Mutant; Dominant-Negative Mutation; Ear, Internal; Embryo; Embryonic; Equilibrium; Event; Fishes; Funding Mechanisms; Gene Action Regulation; Gene Expression Regulation; Gene Regulation; Gene Regulation Process; Gene Targeting; Genes; Genes, Reporter; Genetic; Genetic Alteration; Genetic Change; Genetic defect; Goals; Hearing; Hearing Loss; Hearing Loss, Sensorineural; Human; Human, General; Hypoacuses; Hypoacusis; Image; Injection of therapeutic agent; Injections; Killings; Labyrinth; Lead; Life; Live Birth; Man (Taxonomy); Man, Modern; Maps; Mechanosensory Transduction; Mechanotransduction, Cellular; Methods; Modeling; Molecular Genetic Abnormality; Mutation; Nature; Nerve Cells; Nerve Unit; Nervous; Nervous System, CNS; Neural Cell; Neuraxis; Neurocyte; Neurons; Numbers; Organ; Pb element; Peripheral; Pilot Projects; Population; Process; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Proteins; RNA Splicing; Reporter Genes; Retroviral Vector; Retroviridae; Retrovirus Vector; Retroviruses; Risk; Role; Sensorineural Deafness; Sensorineural Hearing Loss; Sensory Hearing Loss; Signal Transduction, Mechanical; Site; Splicing; System; System, LOINC Axis 4; Targetings, Gene; Testing; Tissues; Toxin; Transgenic Organisms; Transposable Elements; Transposase; Virus-Retrovirus; Zebra Danio; Zebra Fish; Zebrafish; adult animal; balance; balance function; base; cell sorting; congenital deafness; design; designing; disease/disorder; fluorescence imaging; gain of function; gene discovery; gene product; genome mutation; hearing impairment; hearing perception; heavy metal Pb; heavy metal lead; imaging; in vivo; inner ear; loss of function; mature animal; model organism; mutant; neural; neuronal; novel; pilot study; protein expression; relating to nervous system; response; social role; sound perception; transgenic; vector
Project start date: 2007-06-15
Project end date: 2010-05-31
Budget start date: 1-JUN-2008
Budget end date: 31-MAY-2010
PFA/PA: PA-06-365
5R21DC008997-02 (2008): $0
1R21DC008997-01 (2007): $211984
DEVELOPMENT STUDIES OF THE INNER EAR
Donna M Fekete, Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01DC002756-08 from National Institute On Deafness And Other Communication Disorders IRG: ZRG1
Abstract: Adapted from the Investigator s ) The goal of this research is to understand the molecular mechanisms that control patterning and cell fate determination in the developing inner ear. The inner ear, unique to vertebrates, is remarkable for the complex three-dimensional arrangement of its constituent cells, which include neurons, sensory receptors and non-sensory cells organized into tubules, ducts and other specialized tissues. It is likely that the morphogenetic mechanisms required to form such structures will be shared by vertebrates. In humans and animal models, disruption of the precise morphology of the inner ear due to congenital anomalities or disease can result in deafness, and/or difficulties with balance and equilibrium, often accompanied by profound vertigo and nausea. The PI s efforts to understand the fundamental defects that result in inner ear abnormalities are focused on both the normal processes of development and on the cascade of events that can arise as a result of a specific gene defect. The aims are to (1) undertake a lineage analysis of the progenitor cells in the early chick otocyst to reveal when distinct cell lineages diverge, such as neurogenic vs. non-neurogenic or sensory vs. non-sensory; (2) undertake a lineage analysis of the mouse organ of Corti to determine whether hair cells and supporting cells share a common progenitor; (3) generate a fate map the chick otic cup to understand the relationships between gene expression and morphogenetic movements; and (4) force both focal and global perturbations of gene expression domains to help define the rules governing pattern formation in the eveloping inner ear. The studies will employ focal dye injections as well as infection with pseudotyped replication-defective retroviral vectors to limit gene transfer to a small number of otic cells and their progeny. The fourth aim will use replication-competent viruses to generate widespread misexpression of patterning genes. Together, the proposed studies should provide insight on the divergence of inner ear lineages and what role, if any, morphogenic movements and patterning genes play in the process. Their studies are designed to test a model of inner ear patterning that is based on the establishment of compartments and boundaries. The information provided by these animal studies may aid in understanding the molecular-genetic basis of human birth defects that cause deafness and vestibular dysfunction in humans.
Keywords: congenital deafness, developmental neurobiology, ear hair cell, otocyst /otolith, vertebrate embryology, Retroviridae, Vesiculovirus, epithelium, gene expression, gene mutation, genetic marker, histogenesis, transfection /expression vector, chick embryo, confocal scanning microscopy, cyanine, fluorescence microscopy, laboratory mouse
Project start date: 1995-08-01
Project end date: 2004-08-31
5R01DC002756-08 (2002): $255604
5R01DC002756-07 (2001): $277781
5R01DC002756-02 (1996): $166072