Lisa V. Goodrich - US grants

DESCRIPTION (provided by applicant): The inner ear houses the sensory organs for hearing and balance. Sound is detected in the cochlea; linear acceleration and gravity are sensed in the otolith organs; and angular acceleration is detected in the three semicircular canals, oriented in three orthogonal dimensions. The entire structure arises during development from a simple ball of epithelium, the otic vesicle. Proper auditory and vestibular function therefore relies on the perfect execution of the genetic programs that transform the otic vesicle into the complex labyrinths of the mature inner ear. At a cellular level, morphogenesis of the inner ear involves regulated cell proliferation, apoptosis, and cell-cell interactions. Identification of the ligands and receptors that control these processes in mice may point to new candidate genes and/or therapeutic interventions for inherited inner ear defects in humans. Many forms of human congenital deafness and balance disorders are caused by malformations of the inner ear, which can range from a complete failure to progress beyond the otic vesicle stage to the loss of specific structures such as the cochlea or one semicircular canal. This study examines the contributions of an intriguing family of novel proteins, the Lrigs, to inner ear development. All three Lrigs are proposed to have extracellular domains largely consisting of protein interaction motifs, a single transmembrane domain, and divergent cytoplasmic tails. Lrig3 is required for formation of the lateral semicircular canal in mice. In order to understand the origin and nature of this defect experiments will be performed using mouse genetics, chick embryology, and molecular assays 1) to determine when and where the three Lrig genes are expressed in the ear, 2) to examine in detail the cellular nature of the Lrig3 mutant phenotype and 3) to define the molecular characteristics of this novel protein family, using both in vitro and in vivo assays. Results from these experiments may shed new light on the complicated series of events that are necessary for normal structure and therefore function of the inner ear.

DESCRIPTION (provided by applicant): Hearing begins with the detection of sound by hair cells in the cochlea of the inner ear. Spiral ganglion neurons provide the sole conduit for auditory information from hair cells to the central nervous system. Loss of auditory neurons occurs in response to injury, tumors, or hair cell degeneration, which are all common causes of human congenital and age-related deafness. The most effective treatment for deafness is the cochlear implant, which works by directly stimulating spiral ganglion neurons, emphasizing the need to maintain properly wired connections between the ear and the brain. Understanding how auditory neurons are patterned and wired during development will provide an important foundation for the design of therapies to protect inner ear neurons from degeneration and for the development of stem-cell based methods for neuronal replacement. A central question in auditory neuroscience is how spiral ganglion neurons acquire properties that are specific for the perception of sound. Spiral ganglion neurons originate together with vestibular ganglion neurons within a common neurogenic region of the otic vesicle. Precisely wired auditory circuits form through a series of events, including the extension of processes towards hair cells, bifurcation of projections in the cochlear nucleus, and the formation of specialized synapses with target neurons in the brainstem. Many of these events are highlighted by comparison with vestibular ganglion neurons, which underlie the perception of balance and therefore make a distinct series of wiring decisions within the same local environment. The transcription factor GATA3 is produced in auditory but not vestibular neurons. Based on its activity as a master regulator in other developing systems, GATA3 is hypothesized to coordinate auditory-specific programs of development in spiral ganglion neurons. There are three goals: 1) to compare gene expression profiles in highly purified spiral and vestibular ganglion neurons in order to define the auditory-specific programs of circuit assembly underlying specific wiring events;2) to understand how GATA3 exerts distinct effects on early and late wiring events by generating and analyzing conditional knock-out mice;and 3) to identify auditory-specific genes that act downstream of GATA3 to regulate multiple stages of circuit formation. Results from these experiments will provide key insights into the unique cellular and molecular properties of auditory neurons, and may shed light on the etiology of deafness associated with hypoparathyroidism, sensorineural deafness, and renal anomalies (HDR), which is caused by mutations in GATA3. Sounds are collected by the ear, but we only become aware of sounds because of the activity of complex networks of neurons that connect the ear to the brain. Auditory neurons can die due to traumatic injury or because other parts of the ear do not function properly. An effective treatment for deafness is the cochlear implant, which replaces the cochlea by directly stimulating auditory neurons. Understanding how specific sets of neurons become uniquely suited for the perception of sound is a crucial step towards improved cochlear implant technology, and could help identify new ways to keep neurons alive or to replace damaged neurons with new neurons that can re-establish precise connections between the ear and the brain.

DESCRIPTION (provided by applicant): Sound is detected and encoded by hair cells and neurons in the inner ear and processed by complex circuits in the central auditory system. Millions of people experience a degree of hearing impairment in either the detection or perception of sound, ranging from profound deafness to tinnitus and learning disabilities. A major objective in hearing research is to find the genes required for hearing in order to improve the diagnosis and treatment of this wide array of peripheral and central auditory disorders. Genetic screens in model organisms, together with positional cloning of human deafness loci, have uncovered many deafness genes and have greatly advanced our understanding of how the auditory system works. However, this knowledge remains incomplete, as highlighted by the fact that the mechanotransduction channel has yet to be identified. Moreover, little is known about the prevalence or etiology of central auditory processing disorders, due to inadequate diagnostic tools and a lack of knowledge of normal auditory circuit assembly and function. The long-term goal of this study is to develop a new method of gene discovery that will complement ongoing screens for deafness genes and expand our understanding of the molecular basis of hearing. We propose to create a new method for rapidly disrupting auditory gene function in vivo. This technique will use Cre-lox technology and RNA interference (RNAi) in the mouse to disrupt the activity of putative deafness genes in restricted cell populations of the inner ear. shRNA production will be linked to activation of a fluorescent marker, permitting easy visualization of neuronal morphology down to the level of the synapse. The method relies on a transgene that carries two sets of incompatible Cre recognition sites, a U6 promoter, a CAG promoter, and the DsRed and Venus coding sequences. These elements are configured such that Cre-mediated recombination results in expression of a gene-specific shRNA and a simultaneous switch from red to yellow fluorescence. The transgene will be targeted to a defined locus in embryonic stem cells, which will be used to establish lines of RNAi mice. These mice can then be crossed to inner ear-specific Cre drivers, circumventing pluripotent effects and lethality. The first aim is to create a Cre-RNAi vector that works effectively in vitro. The second aim is to validate the compatibility of this vector with an in vivo screen in the auditory system by targeting a known deafness gene, GATA3, which is mutated in HDR syndrome. To this end, we will compare the phenotypes of Gata3-RNAi and conventional conditional Gata3 knockout mice, taking advantage of the Venus fluorescence in knockdown mice to visualize changes in cochlear wiring. As well as providing a novel tool for finding genes required for hearing, this technique can be expanded to generate a resource of targeted ES cells that can be screened for function in any region of the nervous system. PUBLIC HEALTH RELEVANCE: Millions of people experience some form of hearing impairment, from profound deafness to central auditory processing disorders that contribute to tinnitus and learning disabilities. The identification of genes necessary for hearing will improve the diagnosis and treatment of a wide array of disorders. The goal of this project is to develop a new method of probing gene function in mice that will advance our understanding of how the auditory system normally functions and what happens when it doesn't in humans.

DESCRIPTION (provided by applicant): Brain function depends on the flow of information through precisely wired connections between axons and dendrites. Neurons within a circuit can vary widely in the number and arrangement of their dendrites, with some neurons extending only one primary dendrite into a well-defined neuropil and others developing multiple dendrites that extend symmetrically about the cell body. However, in contrast to excellent progress in uncovering mechanisms of axon specification and guidance, relatively little is known about the initial specification and outgrowth of dendrites. In vitro studies suggest that neurons initially extend multipotent neurites, one of which becomes an axon, leaving the remainder to differentiate as dendrites. These results suggest that many aspects of dendrite differentiation are intrinsically regulated. However, in vivo, dendrite development must also be coordinated with the surrounding tissue, such that dendrites are properly positioned to form the appropriate synaptic connections. How extracellular signals induce the intracellular rearrangements that drive the initial specification and subsequent morphogenesis of dendrites is unknown. In the past, this issue has been hard to tackle due to the lack of a suitable assay and the absence of any obvious molecular players. We have been addressing these problems by establishing a system for studying dendrite development in the amacrine cells of the retina. Amacrine cells are typically unipolar, extending a single apical dendrite into a discrete synaptic layer called the inner plexiform layer (IPL). However, in mice lacking the atypical cadherin Fat3, amacrine cells develop a second dendritic arbor that points away from the IPL. Since Fat3 is a cell surface receptor, these results suggest that Fat3 acts by inducing migrating precursors to retract their trailing processes in response to a signal encountered in the IPL. How Fat3 signaling ultimately promotes development of the apical dendrite is a mystery, with no known effectors or ligands. To establish a baseline of knowledge for more detailed analysis of dendrite development, two exploratory studies will be performed. First, we will develop a live imaging assay that can be used to describe the dynamic changes in neurite behavior and Golgi localization that occur as the leading process becomes a dendrite and the trailing process is retracted. Second, to work our way inside the dendrite, we will search for downstream effectors for Fat3, both by testing likely candidate proteins and by performing an unbiased screen for proteins that interact with the Fat3 intracellular domain. Together, these studies will define the salient events of dendrite specification and elucidate the signaling events that occur downstream of Fat3.

? DESCRIPTION (provided by applicant): Neurons exhibit diverse morphologies that influence how information is propagated and modulated through complex networks of connections. One key determinant of circuit function is the number and arrangement of dendrites. For instance, local interneurons extend multiple dendrites symmetrically from the cell body, whereas cerebellar Purkinje neurons elaborate a single huge dendritic arbor that is confined to the molecular layer. Like axons, dendrites develop from totipotent neurites that extend from the cell body of the differentiating neuron. One neurite becomes an axon. The remaining neurites are either retracted or retained to develop as dendrites. In vivo, these events are coordinated with the surrounding tissue, such that axons and dendrites develop in stereotyped locations where they are perfectly positioned to interact with appropriate synaptic partners. Our long term goal is to understand how extrinsic signals alter the intrinsic properties of naïve neurites, thereby ensuring that neurons acquire polarized morphologies that are correctly oriented with the rest of the circuit. To tackle this question, we will investigate mechanisms of dendrite specification in amacrine cells, which modulate the flow of information from the outer to the inner retina. Amacrine cells develop a single primary dendrite that points into a defined region of neuropil called the inner plexiform layer (IPL). Developing amacrine cells are bipolar as they migrate but become unipolar upon contacting the nascent IPL: the neurite that contacts the IPL is retained as a dendrite, but the neurite on the opposite pole of the cell is retracted. We have developed a time?lapse imaging system that allows us to document amacrine cells in the retina as they transition from a bipolar to unipolar morphology, both at the level of the overall cell shape and a the level of the cytoskeleton. We find that a key readout for this change in polarity is the positin of the Golgi apparatus, which moves into the nascent primary dendrite. In mice mutant for the atypical cadherin Fat3, this transition does not occur reliably, leading to the appearance of amacrine cells with two dendritic arbors and an ectopically placed Golgi apparatus. As a transmembrane receptor with a conserved intracellular domain harboring protein?binding motifs, Fat3 offers a potent entry point for understanding how extrinsic cues lead to intrinsic changes in neuronal morphology. Indeed, the Fat3 intracellular domain binds not only to known actin regulators (i.e. Ena/VASP family members) but also to proteins that control microtubule dynamics (i.e. CLASP1/2), suggesting that Fat3 coordinates dual effects on actin and microtubules. By pairing molecular and genetic studies of Fat3 and its effectors with time?lapse analysis of amacrine cell dendrite development in situ, we will gain new insights into the extrinsic and intrinsic mechanisms that govern dendrite specification.

? DESCRIPTION (provided by applicant): The sense of hearing depends on the perfect function of the cochlea, which is a highly organized structure made up of a wide array of cell types. Although hair cells are the primary detectors for sound, many other cells influence how wavelengths of sound travel through the cochlea and also establish the unique environment that is critical for hair cell activation and transmission of signals to spiral ganglion neurons. Hence, mutations that prevent the normal development or function of cells outside of the organ of Corti can also cause deafness, as exemplified by the prevalence of connexin-26 and pendrin mutations in the human population. Understanding how each of the specialized cell types in the cochlea develops to achieve its mature function will elucidate the diverse origins of deafness and improve methods of treatment. Among the least studied cells of the cochlea are the non-sensory cells that populate Reissner's membrane, the spiral limbus, and stria vascularis. To date, we know very little about how the early non-sensory epithelium is patterned to produce different types of cells, let alone how non-sensory cells might influence other cells in the cochlea. In fact, a number of secreted proteins, including neurotrophins, are produced by non-sensory cells, both during development and in the mature cochlea. We find that the secreted protein Netrin-1 (Ntn1) is produced by non-sensory cells in the roof of the developing cochlea. Surprisingly, extra neurons develop outside of the cochlear duct in mice completely lacking Ntn1 activity. Further, we discovered that non-sensory cells can be uniquely defined by expression of the immunoglobulin family member Lrig1. We therefore propose to use Lrig1 as a molecular handle to characterize the molecular and cellular properties of non-sensory cells and to investigate the role for non-sensory derived Ntn1 during cochlear development. These studies will allow us to explore the novel hypothesis that the non-sensory cochlea serves as a source for signaling molecules and will establish the resources needed to study and manipulate non-sensory cells and define their specific contribution to cochlear development, function, and the etiology of deafness.

Project Summary  The cochlea is innervated by two main classes of neurons: the spiral ganglion neuron (SGN) afferents, which  transmit information from the ear to the brain, and the olivocochlear neuron (OCN) efferents, which provide  feedback from the brain to the ear. Housed in the auditory brainstem, OCNs comprise two small populations  of  cholinergic  neurons  that  send  axons  along  the  eighth  nerve  and  into  the  cochlea.  One  subset,  the  medial  olivocochlear (MOC) efferents, extend myelinated axons that fasciculate with SGN afferents in radial bundles  and  terminate  on  outer  hair  cells  in  the  organ  of  Corti.  The  other  subset,  the  lateral  olivocochlear  (LOC)  efferents, develop thinner, unmyelinated axons that also follow along the radial bundles, but terminate instead  on the endings of Type I SGN afferents contacting the inner hair cells. Together, the LOC and MOC neurons  modulate the output of the cochlea, thereby improving binaural hearing and protecting the cochlea from the  effects  of  excess  noise  and  aging.  By  investigating  how  LOC  and  MOC  neurons  develop  and  establish  connections,  we  can  gain  valuable  insights  into  how  the  cochlea  is  wired  and  maintained  for  a  lifetime  of  hearing.  This  knowledge  will  improve  cochlear  implant  technology  and  identify  new  molecular  entry  points  for rewiring the damaged cochlea.  OCN axons develop in tight association with the SGN afferents, which appear to provide a scaffold for growth  within  the  cochlea.  In  turn,  OCN  efferents  influence  SGN  activity  both  indirectly,  by  forming  transient  synapses  with  the  IHCs  during  development,  and  directly,  by  forming  synapses  on  Type  I  peripheral  processes  that  can  regulate  mature  SGN  firing  properties.  Based  on  the  intimate  relationship  between  these  two  populations,  we  hypothesize  that  reciprocal  interactions  between  efferents  and  afferents  sculpt  the  final  wiring  pattern  of  the  cochlea.  To  investigate  this  idea,  we  propose  to  launch  a  new  research  project  aimed  at  defining how and when OCN axons interact with SGN afferents, both at the cellular level and at the molecular  level.  We  will  start  by  using  genetic  approaches  to  document  afferent?efferent  interactions  with  high  spatial  and  temporal  resolution.  In  parallel,  we  will  use  newly  available  molecular  biology  techniques  to  identify  genes  that  are  differentially  expressed  in  LOC  and  MOC  neurons,  including  those  that  might  direct  each  population towards distinct targets in the cochlea. These studies will be complemented with a focused analysis  of  the  transcription  factor  Gata3,  which  we  found  is  required  in  OCNs  for  proper  innervation  of  the  cochlea,  with  secondary  effects  on  SGN  afferent  growth  and  targeting.  Results  from  the  proposed  experiments  will  establish a framework for studying the development and function of OCNs and provide new insights into the  molecular pathways that guide the dual innervation of the cochlea by afferents and efferents.  

Project Summary A major breakthrough in neural development was the identification of attractive and repulsive molecules that guide neurons and their processes to their targets. Today, textbook models emphasize the importance of molecular gradients that act over long-range to steer neurons, epitomized by Netrin-1 (Ntn-1) and its receptor Deleted in colorectal cancer (DCC). Recently, this idea has come into question with the discovery that some of Ntn1's classic long-range effects may be better explained by its short-range, permissive functions. Additionally, the classic model does not adequately explain the remarkable reliability of guidance in vivo, where axons navigate through a complex and changing environment. Structural biological observations suggest a provocative new mechanism that reconciles conflicting observations and could enable greater fidelity than what is possible with a simple gradient. Our long-term goal is to define the molecular mechanism by which Ntn1 achieves both short and long-range functions, even via the same DCC receptor. The overall objective of this exploratory project is to create two new mouse lines so we can test structural predictions in vivo and determine whether a long- term, deeper investigation is warranted. Traditional models for chemoattraction are based largely on the activities of individual ligands and receptors and do not account for the ample cross-talk that also occurs. For example, Ntn-1 binds directly to the secreted protein Draxin, and both ligands bind to DCC, but through independent sites. Further, there are two isoforms of DCC, one short (DCCshort) and one long (DCClong), whose expression changes over development. By solving the structures of Ntn1 and Draxin bound to each other and to DCC, our collaborators determined that Ntn1 can participate in two distinct complexes: a Draxin-mediated 1:2 Ntn1/DCC complex and a DCClong- facilitated 1:1 Ntn1/DCC complex that triggers clustering and hence signaling. The realization that Ntn1's activity might be shaped both by the presence of Draxin and the nature of DCC offers a satisfying explanation for how Ntn1 can act permissively in some contexts and instructively in others, thereby steering cells and axons through complex environments with greater accuracy. We hypothesize that Ntn1 signals through different complexes to mediate short-range, adhesive interactions versus long-range guidance in response to a gradient. To begin to test this hypothesis, we will pursue the following three specific aims: 1) to create a mouse strain deficient for Draxin-DCC binding; 2) to create a mouse strain that cannot produce DCClong; and 3) to test whether predicted changes in the nature of the Ntn1/DCC complex differentially affect Ntn1's ability to act permissively or instructively in vivo. These studies will position us to define a conceptually novel mechanism for guidance and establish a new paradigm for Ntn1 signaling, with broad implications for neuroscience and cancer biology.

Project Summary Vision science is rapidly evolving to include increasingly interdisciplinary applications involving optics, electronics, and software. However, projects requiring the creation of custom software to run data analysis programs, engineering of hardware wired to provide visual stimuli in a controlled manner, and/or fabrication of optical devices for experimentation require expertise in engineering and electronics which many individual laboratories lack. The Neuroengineering Module has been established to meet current and future needs in these areas by providing integrated technical and customized engineering support to the Harvard vision research community. In addition to aiding in the design and fabrication of custom optical and electronic devices and their associated software, a second major focus of the Neuroengineering Module is to provide training in the design and implementation of these methods for the wider vision research community. The Neuroengineering Module, for example, hosts regular instructional classes on the use of electronics for neurobiology applications. Dr. Lisa Goodrich, a long-time faculty member and user of the Core, will serve as the Module Director, and Dr. Ofer Mazor, an accomplished engineer with postdoctoral experience in neuroscience, will provide additional staff expertise. By facilitating the transfer of tools and ideas among laboratories and providing critical training in these increasingly important areas of vision research, the Neuroengineering Module will serve a critical site of innovation and creativity for the Harvard vision research community.

PROJECT SUMMARY Neural circuit function depends on the precise organization of diverse types of synapses. In the vertebrate retina, key computations are performed by parallel networks of microcircuits that form highly ordered systems of synapses that are confined to discrete regions of neuropil. For instance, retinal amacrine cells integrate and compute inputs and then communicate this information to retinal ganglion cells via synapses in the inner plexiform layer (IPL). Although we have begun to identify the molecular mechanisms that dictate what type of synapse should form, we still know very little about how synaptic location is controlled. Our long term goal is to define a molecular pathway for synapse localization. The specific objective of this exploratory project is to test the new hypothesis that the atypical cadherin Fat3 determines where synapses will form by harnessing the activity of two known synaptogenic molecules, the WAVE Regulatory Complex (WRC) and the receptor tyrosine phosphatase protein PTP?. Data generated during the course of this work will allow us to update our model and develop a more focused investigation of this pathway in the future. Several observations suggest that Fat3 interacts with the WRC and PTP? to control synapse localization in the retina. Fat3 belongs to a family of atypical cadherins with known roles in planar polarity, a signaling system that creates and aligns asymmetries in neighboring cells by creating molecular subdomains (5). The Fat3 intracellular domain harbors multiple binding sites for diverse effectors, including known cytoskeletal regulators and synaptic components, such as the WRC and PTP?. Thus, Fat3 is well-suited to respond to signals in neighboring cells and then induce appropriate intracellular responses needed for synapse development. Consistent with this idea, in fat3 mutant mice, retinal amacrine cells show altered patterns of migration and retain extra processes outside of the IPL that go on to form an ectopic plexiform layer (4). Further, by creating and analyzing mice harboring deletions of various regions of the Fat3-ICD, we found that Fat3?s effects on migration and neurite retraction can be separated from its effects on synapse development. Importantly, Fat3-dependent synapse development appears to depend specifically on interactions with the WRC and PTP?. The WRC is a well-studied regulator of local changes to the actin cytoskeleton, including at the synapse (12), while PTP? is known to be important for synapse development elsewhere in the nervous system (13-15). To follow up on these observations, we will use a combination of biochemical and genetic approaches to characterize physical interactions among Fat3, WRC, and PTP?; test whether retinal synapse development in wild-type and fat3 mutant mice requires WRC function; and determine how Fat3 and PTP? influence each other?s distribution and function by examining single and double mutant mouse strains.

Project Abstract Understanding how neuronal computations build up a perception of the external world is fundamental to our understanding of how the brain works. This is particularly relevant to sensory systems, where heterogenous inputs representing distinct sensory features must be re-assembled to generate a perception. How individual neurons in early stages of sensory circuits process parallel inputs, and how these circuit elements later contribute to cortical computations that bind the inputs together is completely unknown. Studies have demonstrated that the timing, position and strength of a given input along the dendrite of a given neuron is a critical strategy used by the brain to encode sensory features. However, how such dendritic integrations of inputs in single neurons contribute to an animal's overall perception is not understood. To re-assemble diverse features from the same initial stimulus, the brain needs to determine which features occurred at the same time. Currently, little is known about how or where this timing information might be encoded. The auditory system offers an ideal system to tackle this question based on its tractability to interdisciplinary methods and its known ability to encode even miniscule differences in timing. Specifically, we will take advantage of a unique cell type in the auditory cochlear nucleus, called octopus cells, as a model to investigate the question of how small cell classes contribute to behavioral and perceptual circuits. Octopus cells are prominent in all mammalian species and are well known to encode temporal inputs with submillisecond precision through integration of primary sensory inputs along their large and extensive dendrites. We propose to carry out a multi- lab, integrated analysis of the molecular and biophysical properties of octopus cells and to track how these single cell computations are transformed along the auditory pathway to contribute to an animal's final auditory percept and hence behavior. Using the mouse as a model system, we will apply new sequencing methods together with high resolution brain imaging and single cell reconstructions to create a comprehensive wiring diagram of octopus cells and their auditory inputs. By generating mouse strains for selective access to octopus cells, we will be ideally positioned to investigate the in vitro and in vivo physiology of octopus cells and therefore bridge experimental and computational models for how timing information is encoded at the single cell level. Lastly, we will study how timing information propagates to higher auditory centers by recording from large populations of neurons in the midbrain, thalamus, and cortex and then assessing the functional relevance of temporal coding for auditory behavior. By leveraging molecular, biophysical, electrophysiological, behavioral, and computational approaches toward the study of this model cell type, these studies will allow us to extract general principles of single cell computations and their effects on systems-level circuit function, with broad implications for understanding how parallel streams of information are integrated to generate sensory perception.