Eva Anton - US grants

Radial glial cells play a critical role in the construction of the mammalian brain, initially, by providing a permissive and instructive scaffold for neuronal migration and eventually, by contributing to the formation of diverse glial cell lineages in the mature brain. Abnormalities in radial glial development, differentiation, and neuron-radial glial interactions lead to aberrant placement and connectivity of neurons and disordered lamination in human brain, an underlying cause of many congenital brain disorders such as developmental dyslexia, epilepsy, microencephaly (small brain), schizencephaly (split brain hemispheres), lissencephaly (smooth cerebrum, without convolutions), macrogyria (large convolutions), polymacrogyria (small cerebral convolutions), and tuberous sclerosis. The aim of this proposal is to elucidate the mechanisms that determine how radial glial cells are established and maintained during embryonic cortical development and transformed into astrocytes once the generation and migration of cortical neurons are completed. To examine the molecular signals regulating this process, we have focused on glial growth factor (GGF) and its receptors (erbB2, 3, and 4). Our earlier findings demonstrate that GGF and their receptors play a crucial role in radial glial cell function in the developing cerebral cortex. GGF expressed by developing cortical neurons promotes neuronal migration on radial glia by promoting its maintenance and its function as substrate for neuronal migration and differentiation. In the absence of GGF signaling via erbB2 receptors, radial glial development is abnormal. Based on these preliminary results we hypothesize that the GGF-erbB signaling system is a crucial modulator of radial glial cell development. The proposed studies will test this hypothesis by analyzing (1) the role of GGF and its receptors, erbB2, erbB3, and erbB4, in the establishment, maintenance and transformation of radial glial cells and (2) whether developmental changes in GGF-erbB2 signaling system trigger the transformation of radial glial cells into astrocytes. Together, these studies on radial glial development and differentiation will help in deciphering the basic mechanisms guiding normal cerebral cortical development and in unveiling the pathogenesis of various developmental brain disorders where abnormal radial glial development and differentiation results in defective cerebral cortical organization.

Description (Verbatim from the applicant's abstract): Neuronal migration on radial glia and other neural substrates enables the deployment of accurate numbers of neurons at the right time into appropriate locations, where they interact with various inputs to produce the characteristic synaptic connectivity and laminar organization of the mammalian cerebral cortex. Abnormalities in neuronal migration on radial glia, and radial glial development lead to aberrant placement and connectivity of neurons in human brain, an underlying cause of brain disorders such as schizophrenia. Thus, elucidation of mechanisms that determine how neurons reach their appropriate positions in the developing cerebral cortex would help in understanding normal cortical development as well as in deciphering the pathogenesis of various developmental brain disorders. To study radial glial cell surface molecules that function to signal cortical neurons to stop at the appropriate laminar location in the developing cerebral cortex, we identified a putatively novel radial glial cell surface molecule (Radial Glial Stop Signal molecule: RAGS 1) whose temporal and spatial distribution during cortical development in regions where neurons terminate their migration suggest that it may function as a positional indicator to migrating neurons, signaling them that they have arrived at the appropriate location in the developing cortical plate. Here, we propose to elucidate (1) its functional importance, primarily by studying the outcome of interference with the biological activity of this molecule during cortical development, and (2) decipher its molecular characteristics. In combination, the functional and molecular characterization of this radial glial antigen will help in understanding the role neuron-glial interactions play in the emergence of laminar organization of the cerebral cortex as well as in deciphering the pathogenesis of various developmental brain disorders affecting mental health.

DESCRIPTION (provided by applicant): Polarized radial glial cells provide a template for the formation of cerebral cortex. Initially, they function as a source of new neurons and provide a permissive and instructive scaffold for neuronal migration. Subsequently, they contribute to the formation of glial cell lineages in the mature brain. Abnormalities in radial glial development, differentiation, and neuron- radial glial interactions lead to aberrant generation, placement and connectivity of neurons and glia in human brain, an underlying cause of many developmental brain disorders such as schizophrenia, and gross brain malformations that accompanies mental retardation and seizure disorders (reviewed in Marin and Rubinstein, 2003; Ayala et al., 2008; Ghashghaei et al., 2008). The aims of this proposal are to elucidate the molecular mechanisms regulating the polarity of radial glia and how radial glial polarity is translated into distinct radial glial functions. Our preliminary results show that Adenomatous Polyposis Coli (APC) serves an essential function in the development of polarized radial glial scaffold during brain development. APC signalling thus provides a unique avenue to examine the mechanisms that determine radial progenitor polarity and its contribution to the formation of cerebral cortex. Analysis of these processes, using APC signalling as a molecular model, assumes additional significance due the known effects of APC mutations in mental retardation, autism, and brain tumors (Attard et al., 2007; Barber et al., 1994; Finch et al., 2005; Gsmez Garcma and Knoers, 2008). Based on these findings, we hypothesize that APC is an essential regulator of distinct aspects of radial glial polarity and is critical for the construction of cerebral cortex. The proposed studies will test this hypothesis by examining the following three related questions: (1) What is the role of APC in the emergence and maintenance of radial glial polarity and the resultant formation of cerebral cortex?, 2) What are the signaling pathways mediating APC function in radial glial progenitors?, and (3) What is the function of APC in neuronal progeny of radial glia? Together, these studies will significantly advance our understanding of the role radial progenitors and their derivatives play in the emergence of cerebral cortex. Further, elucidating the role of APC signalling in cerebral cortical formation will help to delineate the biological basis of neurodevelopmental brain disorders and brain tumorogenesis. PUBLIC HEALTH RELEVANCE: Public Health Relevance Statement Abnormalities in radial progenitor development, differentiation, and neuron- radial glial interactions lead to aberrant generation, placement and connectivity of neurons in human brain, an underlying cause of many developmental brain disorders such as schizophrenia, and gross brain malformations, such as lissencephaly, polymicrogyria, and heterotopias (reviewed in Marin and Rubinstein, 2003; Ayala et al., 2008; Ghashghaei et al., 2008). Characterization of signaling mechanisms controlling the polarity and differentiation of radial progenitors and their progeny, as outlined in this proposal, will significantly advance our understanding of the role radial progenitors and their derivatives play in the emergence and maintenance of the cerebral cortex. The use of APC signalling as a molecular model in these studies has significant additional human health relevance since germ-line mutations of the APC gene results in familial adenomatous polyposis (FAP) (Kinzler et al., 1991). The significance of APC in cerebral cortical development is evident in recent studies demonstrating that APC mutations in humans cause brain tumor polyposis (Attard et al., 2007). Mental retardation or autism is also evident in individuals with APC mutations (Barber et al., 1994; Finch et al., 2005). Therefore, elucidating the role of APC signalling in radial progenitors and their derivatives will help in deciphering the basic mechanisms guiding normal cerebral cortical development as well as in unveiling the pathogenesis of various developmental brain disorders.

Genome wide linkage studies, case-control association studies, and functional data suggest a role for Neuregulin 1 (NRG1) in the e'tiology of schizophrenia. Developmental studies indicate that defects in NRG1 signaling mediated through its receptor, ErbB4, could lead to the incorrect generation, placement, differentiation, and function of neurons in the developing brain (Corfas et al., 2004). The resultant changes in neural circuitry in the cerebral cortex may thus lead to neurodevelopmental disorder such as schizophrenia. Recent evidence indicating that altered NRG1-erbB4 interactions may enhance susceptibility to Schizophrenia (Norton et al.,2005;Hahn et al.,2006) and that NRG1(I) expression is deregulated in dorsolateral prefrontal cortex and hippocampus in schizophrenia (Hashimoto et al 2004;Law et al., 2006) further support this hypothesis. Therefore, an understanding of the functions of NRG1 and erbB4 receptors in the developing cerebral cortex, especially during interneuronal development, is essential to delineate the neurodevelopmental pathways whose disruption is likely to be integrally related to the development of schizophrenia. We propose to accomplish this by examining the following three related questions: (i) Determine the patterns of migration and positioning of interneurons in the embryonic cerebral cortex, (2) Determine whether the development and differentiation of interneurons, during early embryonic development and postnatally, depends on NRG1- ErbB4 interactions, and (3) Determine the role of ErbB4 in the function of cortical interneurons. .

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (08) Genomics and specific Challenge Topic, 08-MH- 102 Schizophrenia Interactome. Schizophrenia is thought to be a complex neurodevelopmental disorder with many genetic factors contributing to its pathology, possibly in an interrelated or convergent manner. Two of the strongest susceptibility genes for Schizophrenia are neuregulin 1 (NRG1) and DISC1. Although they are broadly expressed in the developing brain and are thought to play important roles in neurodevelopmental processes, it remains unclear if these molecules are functionally related to each other and how this interaction affects cerebral cortical development. We hypothesize that changes in the functional interactions between these genetic factors and the resultant changes in the formation of neural circuitry in the cerebral cortex may lead to neurodevelopmental disorders such as schizophrenia. An understanding of the functional interactions between these important SZ susceptibility genetic factors in the developing cerebral cortex will be essential to delineate the pathophysiological processes that culminate in schizophrenia. To accomplish this, we will (1) determine the effects of NRG1 on DISC1 expression and the underlying signaling mechanisms in the developing cerebral cortex and (2) define the functional significance of NRG1-DISC1 interactions in the formation of cerebral cortex. This integrated analysis of how strong susceptibility genetic factors for schizophrenia interact during cortical development will help to decipher some of the key neurodevelopmental pathways whose disruption can lead to the development of schizophrenia. PUBLIC HEALTH RELEVANCE: Schizophrenia is a complex neurodevelopmental disorder with many genetic factors contributing to its pathology in an interrelated or convergent manner. Two of the strongest susceptibility genes for Schizophrenia are neuregulin 1 (NRG1) and DISC1. Changes in the functional interactions between these genetic factors and the resultant changes in the formation of neural circuitry in the cerebral cortex may lead to schizophrenia. Therefore, we aim to define how susceptibility genetic factors for schizophrenia interact during cortical development. This will help to decipher the key neurodevelopmental pathways whose disruption can lead to the development of schizophrenia.

DESCRIPTION (provided by applicant): The Neuroscience Center at the UNC-Chapel Hill School of Medicine requests funding for the purchase of an Olympus Fluoview FV1000-MPE multiphoton imaging system dedicated to in vivo functional imaging of the developing and regenerating nervous system. The ability to observe the dynamics of neuronal proliferation, migration and differentiation in real time and in situ revolutionizes our ability to study the function of specific genes underlying neurodevelopmental disorders such as autism or schizophrenia. The requested Olympus Fluoview FV1000-MPE system represents a new "state of the art" as it allows outstanding fluorescence imaging hundreds of microns deeper into living tissues than previously possible, providing the highest penetration depths of all commercially available multiphoton laser scanning microscopes. This system will be part of an established Core. This Core is dedicated to provide effective access and training to the UNC Neuroscience community in advanced nervous system imaging, was established 6 years ago with strong institutional support from the UNC-Chapel Hill School of Medicine, funds from the Neurodevelopmental Disorders Research Center at UNC and funds from the National Institutes of Neurological Disorders and Stroke. Although this facility has supported the imaging needs of more than 40 neuroscience researchers at UNC, the lack of a dedicated multiphoton imaging station for in vivo imaging has restricted the progress of projects related to visualizing the development of the mammalian brain or regrowth of injured neurons in vivo. For example, visualization of cortical neuronal layer formation and development of synaptic connections in the brains of intact developing rodents is now feasible but requires the ability to visualize fluorescently labeled structures beyond depths which can be visualized by conventional confocal microscopes. The requested equipment will enable a cadre of internationally recognized young researchers at the UNC Neuroscience Center to establish and disseminate innovative imaging technologies relevant to the study of neurodevelopmental disorders and to expand research programs that have recently resulted in publications in the top biomedical research journals. Our main goals with the requested equipment are to provide a platform for in vivo functional studies using mouse genetic models and use it to innovate and implement new imaging technologies to probe neural function during development.

DESCRIPTION (provided by applicant): The appropriate wiring of neurons in the brain requires appropriate migration and placement of cortical neurons. Neuronal migration and the resultant placement of neurons provide the framework for the emergence of neuronal connectome or functional wiring. Area specific and neuronal type specific defects in neuronal migration and the subsequent changes in neuronal connectivity is an underlying cause of a wide spectrum of neurological disorders, including autism, schizophrenia, epilepsy, mental retardation, and malformations such as lissencephaly, schizencephaly, microencephaly, and macro/microgyria. However, very little is known about how distinct classes of neurons coordinately navigate from their sites of birth to their final laminar and areal specific destinations in the developing cerebral cortex. Understanding how the different types of cortical neurons migrate at the right time in right numbers to appropriate cortical areas will be essential to understand the pathogenic mechanisms that underlie neurodevelopmental and neuropsychiatric disorders. We therefore aim to define how cortical neurons (i.e., projection neurons and interneurons) achieve their appropriate placement in the cerebral cortex. Towards this goal, we will develop methods for real time, 4-D confocal and multiphoton imaging of genetically defined subtypes of cortical neurons in the developing brain. Using this approach, we will map how distinct classes of cortical neurons navigate through the developing cerebral wall to arrive at their appropriate areal and laminar positions. Together, this comprehensive mapping of coordinated migration and placement of interneurons and projection neurons during cerebral cortical development will define the neuronal blueprint used to form brain connectome, facilitate the development of new in vivo live embryonic brain imaging methods, help delineate the basis of brain abnormalities in a spectrum of neurodevelopmental disorders, and thus will generate transformative insights into normal and aberrant brain development.

? DESCRIPTION (provided by applicant): Disrupted cilia function in humans results in profound brain abnormalities and cognitive impairments. However, little is known about the molecular mechanisms underlying the brain malformation in this class of disease, called ciliopathies. Recessive mutations in ARL13B or INPP5E cause Joubert Syndrome and Related Disorders (JSRD), a human ciliopathy defined by a specific hindbrain abnormality, the molar tooth sign. Here, we propose to use mouse models of JSRD causing genes (Arl13b, Inpp5e) and their JSRD-causing human mutations to systematically delineate the mechanistic underpinnings of the brain malformations in JSRD. Towards this goal, we will functionally characterize the cilia-dependent and/or cilia-independent signaling mechanisms triggered by ARL13B or INPP5E gene mutations that lead to hindbrain abnormalities. The outcomes of this work will define the role of primary cilia signaling during neuronal development and connectivity. Importantly, delineation of molecular cascades and neurodevelopmental pathways, whose disruptions are integrally related to the development of brain malformations in ciliopathies will enable us to devise optimal diagnostic and therapeutic strategies for these brain disorders.

Principal Investigator (Last, First, Middle): Anton, Eva S. PROJECT SUMMARY Radial progenitors serve as an instructive matrix to coordinate the generation and placement of appropriate number and types of neurons in the developing cerebral cortex. Radial progenitors divide asymmetrically to generate neurogenic intermediate progenitors (IPs) and the symmetric proliferation of IPs serves to rapidly expand cortical neuronal population. The dynamic maintenance of the balance between radial and intermediate progenitors is of fundamental importance to the generation of right number and types of projection neurons at the right time in the cerebral cortex. Once generated, growth and connectivity of cortical neurons enables the formation of basic neuronal circuitry in the cerebral cortex. The balanced diversity of cortical progenitors and the resultant generation, placement, and connectivity of projection neurons thus serve as a blueprint to guide the formation of an appropriately wired cerebral cortex. Disruptions in these essential features of the developing cerebral cortex are at the core of many human neurodevelopmental disorders including microcephaly, macrocephaly, lissencephaly, epilepsy, schizophrenia, and autism spectrum disorders. However, the molecular logic that instructs progenitor balance and projection neuronal connectivity remains an enigma. This proposal aims to remedy this gap in our understanding of cerebral cortical formation. In particular, (1) we will discover how the developmental balance between radial and intermediate progenitors, vital for the production of right number and types of cortical neurons at the right time, is achieved, (2) define hitherto uncharted, primary cilia-mediated mechanisms guiding projection neuronal growth and connectivity, and (3) determine how changes in these developmental processes can cause cortical malformations underlying human neurodevelopmental disorders. We aim to make these goals attainable by using an innovation driven approach that involves combined application of latest advances in progenitor or neuron type specific mouse genetic models, live imaging, lineage tracing, mapping of signaling interactomes, optogenetic and chemogenetic manipulation of primary cilia signaling, single cell genomics, and functional evaluation of human mutations associated with neurodevelopmental disorders. Understanding how progenitors and neurons are assembled, organized, and connected appropriately to facilitate cerebral cortical formation, offers us the opportunity to rethink and redraw the rules of corticogenesis in the service of better diagnostic and therapeutic insights into neurodevelopmental disorders.

Regularly interspaced, non-overlapping, tiled organization of polarized radial glial cells (RGCs) serves as an instructive framework to generate and organize neurons in the developing cerebral cortex. Disruptions in this fundamental cellular feature of the developing cerebral cortex lead to a spectrum of neurodevelopmental disorders (e.g., autism, schizophrenia, and epilepsy) and brain malformations (e.g., lissencephaly, schizencephaly, microencephaly, and macro/microgyria). However, little is known about the molecular logic of radial glial tiling and how it drives the appropriate formation of the cerebral cortex. We discovered that RGC tiling is dependent on Memo1 (Mediator of cell motility 1). Genetic mutations in MEMO1 lead to autism. Memo1 thus provides a window into the mechanisms that instruct radial glial tiling and the resultant assembly of the cerebral cortex. Leveraging the latest advances in progenitor specific mouse genetic models of Memo1, MADM based profiling of radial glial differentiation, mapping of Memo1 interactome, mechanistic dissection of cellular functions of Memo1, live imaging of Memo1 deficient radial glial cell functions, and functional analysis of MEMO1 mutation from autism probands, we aim to (a) discover the role of Memo1 in the tiling of RGCs and the resultant formation of the cerebral cortex, (b) identify the Memo1 interactome that contributes to and determines RGC tiling, and (c) interrogate the contributions of MEMO1 to cortical malformations associated with autism. Collectively, the outcome of this work will reveal the molecular logic underlying radial glial tiling, the vital relevance of this process for cortical development, and how changes in this process can cause brain malformations and neurodevelopmental disorders. Importantly, understanding how radial glial cells are assembled and organized appropriately to facilitate cerebral cortical formation offers the opportunity to redraw the rules of corticogenesis in the service of better diagnostic and therapeutic insights into neurodevelopmental disorders.