Douglas J. Epstein - US grants

? DESCRIPTION (provided by applicant): The vertebrate central nervous system is formed with great precision into discrete yet interconnected structural and functional units that control anima behavior. Compartmentalization of the neuroepithelium into these functional units is facilitated by its exposure to signaling molecules secreted from localized organizing centers (Jessell, 2000; Hébert and Fishell, 2008; Scholpp and Lumsden, 2010). The zona limitans intrathalamica (zli) is one such signaling hub in the caudal forebrain that is the source of secreted morphogens, including Sonic hedgehog (Shh), that are essential for developing the thalamus into a vital processing and relay station for sensory and motor signals to the cerebral cortex (Epstein, 2012; Martinez-Ferre and Martinez, 2012). Mouse mutants that lack Shh in the zli and/or basal plate of the caudal diencephalon show significant alterations in thalamic progenitor identity and nucleogenesis (Szabó et al., 2009; Vue et al., 2009; Jeong et al., 2011; Bluske et al., 2012). Despite the importance of the zli for thalamic morphogenesis, our knowledge of how this narrow boundary between the thalamus and prethalamus is established as a major brain-organizing center remains unresolved. Central to the understanding of zli formation is the mechanism by which Shh transcription is activated within its prescribed domain. Studies from my lab have provided substantial insight to the regulatory landscape underlying Shh transcription in the CNS (Jeong et al., 2006; Jeong et al., 2008; Geng et al., 2008; Jeong et al., 2011; Zhao et al., 2012). Experiments in this proposal will address the hypothesis that enhancers with overlapping activity in key brain signaling centers share a common cis-regulatory signature. Our preliminary studies have identified six sequence motifs that are highly enriched in a set of enhancers that coordinate the expression of Shh and other co-regulated genes in the ventral midbrain, caudal diencephalon and zli. In the following three aims, we propose to systematically evaluate the requirement of candidate components of the cis and trans regulatory network underlying Shh/zli gene expression (Aims 1 and 2), clarify the evolutionary origin of the vertebrate zli (Aim1), and determine whether specific alterations in the regulation of Shh/zli expression contribute to neurodevelopment disorders of thalamic function (Aim3).

DESCRIPTION (provided by applicant): The auditory and vestibular structures of the inner ear mediate our senses of hearing and balance, respectively. Recent progress has been made in identifying some of the causes of hereditary forms of deafness and vestibular disease in humans; however, a detailed understanding of the genetic pathways coordinating inner ear development remains limited. By identifying the genetic networks regulating inner ear morphogenesis, our understanding of the association between otic development and disease should improve. The principal components for hearing (the cochlea) and balance (the semicircular canals, utricle and saccule) are formed from ventral and dorsal outgrowths, respectively, of a common bilateral structure, the otocyst. Organization of the inner ear into auditory and vestibular components is dependent on localized patterns of gene expression within the otic vesicle. Surrounding tissues are known to influence compartmentalization of the otic vesicle, yet the participating signals remain unclear. The notochord and floor plate are sources of the secreted protein Sonic hedgehog (Shh) that functions in both short and long range signaling events to promote growth and differentiation of progenitor cells in the ventral neural tube and paraxial structures. In the absence of Shh, ventral otic derivatives including the cochlear duct and cochleovestibular ganglia fail to develop. The origin of the inner ear defects in Shh -/- embryos can be attributed to alterations in the expression of a number of genes previously implicated in the specification of cochlear, neuronal and chondrogenic lineages. Although the effects of Shh signaling are detected in the otic epithelium, adjacent tissues including the periotic mesenchyme and neural tube are also targets of Shh action. This brings into question the relative contribution of Shh signaling in each of the tissues impacting on inner ear development. Experiments outlined in this proposal are aimed at elucidating the mechanism by which Shh specifies auditory cell fates in the ventral otocyst. The temporal and spatial requirements of Hedgehog (Hh) signaling in inner ear development will be addressed by the conditional inactivation of Smoothened, an essential transducer of all Hh signals, in each of the tissues impacting on the otic vesicle. Experiments to identity the downstream effectors of Shh signaling in otic development are also proposed. Finally, despite preliminary insights into how auditory cell fates are specified, little is known of the extrinsic cues that establish vestibular (dorsal) structures in the otic vesicle. Introduction of specific pathway inhibitors into the dorsal otocyst using transgenic approaches as well as the assessment of mouse mutants in candidate dorsalizing factors should contribute towards our overall understanding of how polarity is established along the dorsoventral axis of the inner ear.

DESCRIPTION: Extracellular signaling molecules provide the vertebrate inner ear with positional information at multiple stages of its development to produce the required patterns of growth and differentiation necessary for the formation of the vestibulum and cochlea, the two inner ear organs responsible for sensing, balance and sound, respectively. In previous work, we identified roles for the secreted factors, Shh and Wnt1/Wnt3a, in the polarization of the mouse otic vesicle along its dorsal-ventral axis. Our studies determined that Shh, secreted from the notochord, signals directly to ventral regions of the otic epithelium to direct the outgrowth of the cochlear duct. Whereas, Wnt1/Wnt3a, secreted from the dorsal hindbrain, signals to the dorsal otocyst to regulate vestibular morphogenesis. In addition to these early roles, Hedgehog (Hh) and Wnt/2catenin signaling pathways are also active at critical junctures later in ear development when sensory epithelial progenitors are undergoing their specification and differentiation into hair cells and support cells. We now propose to investigate the specific requirements of Wnt/2catenin and Hh signaling pathways at later stages of inner ear development in the mouse, using a conditional gene targeting strategy that inactivates essential mediators of these pathways at defined periods of cochlear and vestibular development. Our conditional gene targeting approach takes advantage of our recent finding that Wnt responsive cells originating in the dorsal otocyst extend ventrally over time and contribute to the sensory epithelium of the cochlear duct. Using a tamoxifen inducible form of cre recombinase, expressed under the transcriptional control of a Wnt responsive Top promotor (TopcreER), floxed alleles of 2catenin and Smoothened will be inactivated in sensory epithelial progenitors. Preliminary results indicate that Wnt/2catenin signaling is required for the specification and/or differentiation of hair cells and support cells in the organ of corti and cristae of the semicircular canals. Experiments in this proposal will further elaborate on the mechanisms by which Wnt/2catenin and Hh signaling pathways function to mediate sensory cell fates in the inner ear. We have also developed a genetic recombination based strategy to indelibly mark Wnt responsive cells in the dorsal otocyst in order to trace their fates over the course of inner ear development. Additional experiments described in this proposal will test the hypothesis that hair cells and support cells in the organ of corti derive from a population of Wnt responsive progenitors in the dorsal otocyst. Results from these studies should improve our fundamental understanding of sensory development in auditory and vestibular regions of the inner ear. PUBLIC HEALTH RELEVANCE The auditory and vestibular structures of the inner ear mediate our senses of hearing and balance, respectively. Progress continues to be made in identifying the causes of hereditary forms of deafness and vestibular disease in humans. Nonetheless, a detailed understanding of the genetic pathways coordinating inner ear development remains limited. By elucidating the genetic networks regulating cell fate decisions in the inner ear, our studies should not only improve our fundamental understanding of this intricate organ, but also the pathogenesis of congenital forms of deafness.

DESCRIPTION (provided by applicant): The mouse cochlea derives from the ventral extension of the otic vesicle. Over the course of several days during embryonic development, this outgrowth undergoes a complex sequence of morphogenetic changes resulting in its lengthening, coiling and differential patterning into sensory and nonsensory cell types that are essential for hearing (Groves and Fekete, 2012). Congenital malformations of the cochlea often lead to deafness, emphasizing the importance of a thorough understanding of its development (Jackler et al., 1987). We previously described a critical function of the Sonic hedgehog (Shh) signaling pathway in promoting ventral identity within the otic vesicle that is necessary for the subsequent outgrowth of the cochlear duct (Riccomagno et al., 2002; Bok et al., 2007b; Brown and Epstein, 2011). Mouse embryos lacking Shh, or carrying an ear conditional knockout of Smoothened (Smoecko), an essential Shh signal transduction component, exhibit cochlear agenesis. We also classified several transcription factors with key roles in cochlear development as either transcriptional targets (Pax2, Otx2, Gata3) or effectors (Gli2, Gli3) of Shh signaling within the ventral otic epithelium. However, despite these advances, a detailed understanding of the mechanism by which Shh dependent transcription factors promote cochlear duct outgrowth remains unclear, primarily since the genes acting downstream in this transcriptional cascade have yet to be determined. To identify novel targets of Shh signaling we compared the genome-wide expression profiles of control and Smoecko inner ears at E11.5, when the cochlea anlage is evident, and uncovered an intriguing set of Shh responsive genes with a combination of known and previously uncharacterized roles in cochlear morphogenesis. Interestingly, several of these genes maintain their expression at later stages of development within the prosensory domain of the cochlear duct, raising the possibility that Shh signaling is priming the presumptive sensory epithelium for subsequent steps in its development. We propose to characterize the ventral otic gene set according to the following experimental plan: classify their spatiotemporal patterns of expression and dependency on Shh signaling (Aim 1); decode their cis-regulatory architecture (Aim 2); and assess their functional contribution to cochlear development (Aim 3). The results of these experiments should advance our fundamental understanding of the molecular and cellular mechanisms underlying cochlear morphogenesis and cell fate specification within the inner ear.

This is a renewal application for training grant support for the Predoctoral Genetics Training Program at the University of Pennsylvania. The goal of the program is to provide broad-based training in cell and molecular biology and in-depth training in genetics in order to prepare young scientists for cutting-edge genetics research and careers in academia, private industry or government. The program brings together faculty from 16 different departments at the Schools of Medicine and Arts and Sciences, as well as the Children's Hospital of Pennsylvania. The combination of a research- oriented medical school and a strong university base provides an excellent environment for graduate education. The program obtains substantial financial support from Biomedical Graduate Studies, an institutional organization that coordinates student recruitment and oversight for many graduate groups. The Genetics and Epigenetics (GE) Program is part of the Cell and Molecular Biology Graduate Group, a multidisciplinary Graduate Group that administers the broad core curriculum for our predoctoral trainees. Within the CAMB framework, the GE Program offers advanced coursework and research training in genetics, and program-specific activities such as an annual Genetics Retreat, trainee seminars, journal cubs, technical workshops, career forums and social events. These courses and activities are made available to students from multiple graduate groups, expanding the breadth and reach of our training program. After coursework is completed, the student must pass an oral preliminary exam that consists of a defense of a written NRSA-style thesis research proposal. The student then carries out a significant genetics research project under the direction of a laboratory mentor and the advice of a thesis committee, with additional oversight and mentoring from the training program faculty. Students are selected for two years of training grant support after they have completed coursework, selected a thesis lab and passed their Preliminary exam. Thus, we support students who are highly motivated to carry out genetic research. New enhancements initiated within the last four years include the Penn Genetics Retreat, a requirement for coursework in Biological Data Analysis, increased emphasis on grant writing skills, and use of Individual Development Plans to guide career discussions and planning. We propose to continue this program at its current size of 8 predoctoral trainees per year.

The identification of gene mutations associated with hearing loss in humans and animal models has contributed greatly to our understanding of cell type specific functions in the inner ear. Despite this progress, the cause of inherited forms of hearing loss still remains unknown in many cases, suggesting that the discovery of new genes associated with sensorineural hearing loss (SNHL) is far from saturated. To fill this gap, we performed a screen for novel regulators of cochlear development and identified several genes that are predicted to play important roles in cochlear function. We generated a mouse knockout for one of these genes, Growth arrest specific 2 (Gas2), encoding a putative cytoskeletal regulatory protein. Gas2 knockout mice display severe hearing loss with no alterations in inner ear development at embryonic stages. Instead, we propose that the cause of hearing loss is due to a progressive destabilization of the microtubule cytoskeleton in pillar and Deiters? cells, two specialized support cells in the organ of Corti that are thought to provide tensile strength and a structural framework for transferring mechanical forces during sound-evoked vibrations. The experiments in this grant proposal are designed to transform our understanding of the role that support cells play in cochlear function. Firstly, we will test the hypothesis that Gas2 dependent stabilization of microtubule bundles in pillar and Deiters? cells is required for hearing. Secondly, we will determine the influence of Gas2 and a-tubulin detyrosination on cell surface mechanical properties and microtubule dynamics. Finally, we will pursue the exciting possibility that viral delivery of Gas2 to support cells might prevent hearing loss when administered prior to the onset of symptoms in neonatal Gas2 knockout mice, and even more provocatively, might repair the cytoskeletal defects and restore hearing when administered to adults. Taken together, these experiments will clarify the proposed role of Gas2 as a microtubule and actin cross-linking protein that is required to stabilize microtubule bundles in cochlear support cells and that this activity is necessary for auditory function.

PROJECT SUMMARY (GENETICALLY MODIFIED MOUSE CORE) The significant incidence of digestive, liver and pancreatic diseases in the American population demands continued exploration of a broad array of corresponding mechanistic pathways, pathophysiologic sequelae, and potential therapeutic approaches. In many cases these investigations can only be accomplished, or can be accomplished most efficiently and relevantly, using model systems established in intact animals. The use of mouse models in these pursuits is now well established for their power, feasibility, flexibility, and enormous potential. Creation of such models, by targeted alterations of the mouse genome, is an essential component of an overall research effort in understanding normal functions and pathologic perturbations in the digestive tract, liver, and pancreas. The Genetically Modified Mouse Core (GMMC) provides investigators of the University of Pennsylvania Center for the Molecular Study of Digestive and Liver Diseases (CMSDLD) with the ability to carry out these technologically-demanding studies in a cost effective and efficient manner and enhance the rigor and relevance of these approaches. The GMMC has a dedicated and highly skilled staff that applies state-of-the-art equipment and techniques, and the facility consists of a microinjection suite, an adjacent dedicated cage room, and an off-site and highly secure cryopreservation storage facility. Major services available to CMSDLD investigators include the generation of transgenic mice by DNA pronuclear injection, creation of chimeric mice by ES cell injection into blastocysts, and direct genome mutation, editing, and modification via the use of targeted endonuclease (TALEN and Crispr-CAS technologies); these are complemented by the genotyping of founder mice, assisted (in vitro) fertilization, cryopreservation, long-term cryostorage, and shipping of frozen embryos or sperm to/from other facilities. To provide these services, the GMMC utilizes multiple microinjection platforms, laser-assisted technologies, state-of-the-art cryopreservation approaches, and highly efficient line re-derivation pipelines. All functions, from ordering services, to following workflow, to storing and sending out lines, are on-line and can be monitored in real-time. These efforts by the GMMC contribute substantially to the overall productivity of CMSDLD investigators and enhance the rigor and relevance of their studies to the mechanisms of digestive, liver, and pancreatic diseases in physiologically- intact mammalian systems. Moreover, the GMMC enhances interactions and collaborations for CMSDLD investigators and lowers the technical and financial barriers that would otherwise impede the application of these approaches for individual investigators. Thus the Genetically Modified Mouse Core contributes greatly to the research efforts of the CMSDLD.