Donna Fekete - US grants

The evaluation of retinal structure as determined by photographic documentation at the three month follow-up examination has indicated a favorable outcome in eyes treated in the CRYO-ROP trial. This outcome makes long-term follow-up of infants in the CRYO-ROP trial essential to evaluate the long-term safety and efficacy of the procedure, to determine the optimal disease threshold for application of the therapy, and to define the indications for routine follow-up of infants with mild retinopathy of prematurity. The primary outcome measure of the CRYO-ROP study was the status of the retinal structure, as determined by photographic documentation at one year after cryotherapy. During the time since the intake of patients for the trial began in 1986 the technology for measuring visual function in infants has evolved to the point that it has been possible to add a second outcome measure at one year after cryotherapy. This second outcome measure is grating visual acuity, as assessed monocularly by the Teller Acuity Card method. We propose to continue to evaluate retinal structure and visual function in infants in the CRYO-ROP trial for an additional five year follow-up period in order to attempt for the first time to correlate the structural changes observed in both the acute proliferative and the scarring phases of ROP with the eventual visual function of the eye. We will also evaluate the long term risk/benefit ratio of recommending cryotherapy in infants with differing degrees of ROP and document the long term eye status and visual function of very low birthweight infants with varying degrees of treated or untreated ROP. Three specific questions will be examined: 1. Are there long term structural or functional ocular sequelae in eyes treated with cryotherapy that could require re-evaluation of the risk/benefit ratio of recommending cryotherapy? 2. Do long term data on structural and functional sequelae in untreated eves with ROP indicate that the "threshold" for cryotherapy should be lowered? 3. Is the incidence of structural or functional ocular abnormalities any different in infants with mild ROP than in infants with no ROP? If not, future resources could be focused on follow-up of infants who have moderate or severe ROP.

DESCRIPTION (provided by applicant): The goal of our research is to understand the timing and molecular mechanisms that control patterning and cell fate specification 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 morph genetic 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 anomalies or disease can result in deafness and/or to difficulties with balance and equilibrium. Our 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 genetic defect. In this study, we aim to: (1) undertake lineage analysis of the progenitor cells in the ready chicken otocyst to reveal when distinct cell lineages diverge, such as sensory vs. non-sensory or neurogenic vs. nonneurogenic; (2) undertake lineage analysis in the mouse inner ear to determine whether hair cells and supporting cells share a common progenitor and whether there are cell lineage (compartment) boundaries in the organ of Corti; and (3) explore the role of the Wnt signaling pathway in cell fate specification in the ear, particularly with respect to the auditory vs. vestibular cell fate decision. Our studies will employ replication defective retroviral vectors to limit gene transfer to a small number of otic cells and their progeny. To study the Wnts, we will use replication-competent viruses to generate widespread misimpression for both gain-offunction and loss-of-function experiments. Together, the proposed studies should provide insight on the divergence of inner ear lineages. Our studies are designed to test a model of inner ear patterning that is based on the establishment of compartments and boundaries within the otic epithelium. Our data to date reveal that the auditory-vestibular fate decision can be manipulated through Wnt/b-catenin signaling. These findings, and new data generated from this study, may provide baseline data for a therapeutic strategy to direct stem cells along different developmental fates according to which sensory organ cell types may need to be replaced.

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.

[unreadable] DESCRIPTION (provided by applicant): 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. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): 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.

The Zika virus (ZIKV) of the flaviviridae family of viruses poses a significant threat to the health of newborn infants and young children. ZIKV infection predominantly affects the development of the nervous system and its neurotopism has been well-established. In September 2016, ZIKV was linked to sensorineural hearing loss in newborn infants that were prenatally exposed, but essentially nothing is known about where the developmental pathologies lie in the auditory pathway. Reports of abnormal otoacoustic emissions in ZIKV-exposed infants suggest that at least some of the sensorineural defects may originate within the cochlea, the auditory organ of the inner ear. This study will focus on correlating the timing of ZIKV exposure at embryonic and perinatal stages to the types of inner ear cells that become infected, using two animal models (chicken and mice). Preliminary data show that ZIKV can infect the cochlea, in particular the sensory domain, the stria vascularis (tegmentum vasculosum in the chicken cochlea) and the auditory ganglion. The proposed studies will involve delivery of ZIKV directly to the embryonic inner ear and the cochlea to assess cellular tropism and otic pathogenesis associated with ZIKV. Aim 1 will determine the cellular tropism and timing of ZIKV infection in ovo by direction injection of virus into the chicken otocyst from embryonic day (E) 3?5. Virus distribution, cell proliferation, apoptosis, and gene expression related to innate immune responses will be assayed several days after infection. Aim 2 will determine the cellular tropism and timing of ZIKV infection in vitro in embryonic (E12.5, E15.5) and neonatal (postnatal day 1) mouse cochlea cultures. Genetic labeling of hair cells, supporting cells, or intermediate cells of the stria vascularis will aid in following the fates of these cell types for up to 6 days in culture. Virus distribution, cell proliferation, apoptosis, hair cell ultrastructure, and hair cell mechanotransduction will be evaluated several days after ZIKV exposure. Antibody blocking experiments will be used to explore the role of the periotic mesenchyme as a direct target of ZIKV. These studies should aid in understanding how ZIKV infection of the developing sensory periphery might contribute to sensorineural hearing loss in human infants.

PROJECT SUMMARY/ABSTRACT There is a national need to advance the understanding of hearing in both healthy patients and those with various causes of hearing loss. The objective of the proposed program is to train the next generation of faculty who could populate colleges of science, engineering, and health sciences, as well as to send graduates into in order to advance auditory neuroscience training, this new graduate program will leverage faculty expertise in basic hearing science and technology development, from three Purdue University colleges (Science, Engineering, and Health & Human Sciences) and 6 doctoral admissions programs. Two types of investigators are included in the training program: 10 hearing scientists with focused research programs related to auditory system neuroscience, and 7 technology innovators who are trained in other disciplines (electrical, computer and biomedical engineering, and chemistry). Collectively and collaboratively, the program will expand knowledge about mechanisms at the molecular, cellular and systems levels that underlie auditory information processing. This fundamental knowledge can then be applied to better understand the changes that lead to pathologies of the auditory system due to damage, disease, aging, and congenital disorders, as well as understanding how hearing evolved and influences behavior and natural selection. Technological approaches to these questions include, but are not limited to, fluorescent sensors to detect purinergic signaling in the intact nervous system, biological implants for neuromodulation, high-resolution four-dimension calcium imaging deep in the mammalian brain, optogenetics and robotics (automated patch-clamping) for brain circuit analysis, and industry prepared to work toward creative solutions for treating hearing loss. Specifically, This training program is unique in that it is specifically designed to serve students with undergraduate degrees in the disparate disciplines of life science, physical science or engineering, and merge them into a unified cohort focused on auditory neuroscience. Students will be selected for a 2-year term on the training grant, beginning at the start of their second year. The training curriculum includes 3 core courses (one each in neuroscience, the auditory periphery, and signal processing), several recommended courses (e.g., in neurosurgery or neuroscience), a weekly Hearing Science seminar series, and yearly attendance at extramural hearing-related courses and/or auditory neuroscience conferences. In addition to administrative support for the program, the Purdue Institute for Integrative Neuroscience will provide students with additional resources, such as supervised grant writing, hands-on training in animal behavior and human stem cells, annual neuroscience retreats, and access to in-house competitions for travel grants and pilot funding for collaborative projects. Further, this program builds on Purdue's extensive history in training graduate students in collaborative research (particularly in hearing science and technology development), and preparing these students for successful research careers in academia, industry and the clinic. multimodal brain imaging methods.