Robert F. Hevner, MD PhD - US grants

DESCRIPTION (Adapted from applicant's abstract): Disorders of forebrain development have been implicated in the etiology of psychiatric illnesses including autism and schizophrenia. Previous studies have shown that homeobox genes Dlx and Emx are expressed in restricted domains of the mouse forebrain (the basal forebrain and cortex, respectively) during periods of neurogenesis, migration, and differentiation. This proposal tests two hypotheses: (1) that Dlx genes control phenotypic differentiation of the basal forebrain; (2) that Emx genes control phenotypic differentiation of the cortex. The hypotheses will be tested by gene targeting to alter Dlx and Emx expression in mice. The following approaches will be taken: (1) Null mutants of Dlx and Emx genes (generated previously) will be characterized phenotypically. (2) Re-specification of regional phenotypes will be attempted by ectopic expression of Dlx and Emx. (2a) To test whether ectopic expression of Dlx redirects cortical precursor cells to a basal forebrain phenotype, the Dlx-2 coding sequence will be placed under control of the Emx-1 promoter by gene substitution ("knockin"). (2b) To test whether ectopic expression of Emx redirects basal forebrain precursors to a cortical phenotype, the Emx-1 coding sequence will be placed under control of the Dlx-2 and Dlx-5 promoters. (3) The roles of these and related genes in normal and abnormal human brain development will be studied by northern blotting, in situ hybridization and immunohistochemistry of surgical and autopsy tissue specimens.

DESCRIPTION (provided by applicant): The cerebral cortex is a higher brain region with critical motor, sensory, language, and cognitive functions. Developmental abnormalities of the cortex can cause neurologic and psychiatric diseases including epilepsy, mental retardation, and autism. In this proposal for an Independent Scientist Award, the mechanisms that control the laminar fates of cortical neurons (including molecular expression) will be studied. Dr. Hevner is a neuropathologist/neuroscientist who studies cortical development. His immediate career goals are to: 1) establish a vigorous independent research program, 2) expand the repertoire of techniques in his laboratory through collaboration with a more senior expert (Dr. David Price), 3) to increase productivity through enhanced interactions with colleagues at the University of Washington, and 4) to continue enhancing his skills as a neuropathologist and teacher. His long-term career objective is to apply advances in basic research to the understanding, diagnosis, and treatment of diseases affecting the cortex. Dr. Hevner's laboratory and office are located in a modern, well-equipped research facility with animal housing at the Harborview Medical Center (HMC), near the main campus of the University of Washington (UW). HMC and UW support a large community of accomplished developmental neuroscientists, and offer an abundance of available courses, seminars, journal clubs, and presentations. Previous studies have shown that each layer of the cortex contains projection neurons related by similarities of cell size, axonal connections, and molecular expression. The expression of these properties is closely correlated with cell birth date, but mechanisms are unclear and some properties may be regulated post-mitotically. This proposal tests the hypothesis that layer-specific molecular expression can be regulated post-mitotically. Newly generated post-mitotic neurons will be transplanted into the cortex of control (same age) embryos, heterochronic (different age) embryos, or Rein mutant embryos, which lack Reelin and have a disorganized cortex. The molecular expression of transplanted cells will then be examined using a panel of layer-specific markers. If molecular fates are aEered in heterochronic or Rein mutant cortex, this would suggest that certain aspects of laminar fate can be regulated post-mitotically.

[unreadable] DESCRIPTION (provided by applicant): Malformations of cortical development are a major cause of epilepsy, mental retardation, autism, and related neurological disorders. Malformations arise from defects of cortical cell migration, axon tract formation, and differentiation. These fundamental processes are necessary to produce the characteristic laminar structure, precise neural circuitry, and specialized neuron types in the cortex, respectively. Tbr1 is a T-domain transcription factor that is necessary for fundamental developmental processes in the neocortex (the largest part of the cortex). Tbr1 deficient mice have a severe migration disorder (which resembles a form of human lissencephaly), as well as defects of axon pathfinding (affecting the callosal, corticospinal, corticothalamic, and thalamocortical projections) and neuronal differentiation. The broad goal of this project is to elucidate underlying mechanisms of cortical development by studying the role of Tbr1. Our hypotheses postulate that Tbr1 regulates specific aspects of glutamatergic neuron migration, axon pathfinding, and differentiation. This project has three specific aims. Aim 1 is to define the role of Tbr1 in cortical cell migration. This will be accomplished by transplantation studies. Aim 2 is to define the role of Tbr1 in formation of corticothalamic and thalamocortical axon connections. Using a novel in vitro assay, we will determine if Tbr1 regulates cortical and thalamic axon responses, guidance cues, or both. Also, by overexpressing Tbr1 in embryos, we will resolve whether Tbr1 specifies cortical axon connections cell autonomously. Aim 3 is to define the role of Tbr1 in glutamatergic differentiation and layer-specific fate choices. Gain-of-function assays will be used to determine if Tbr1 induces glutamatergic differentiation, suppresses GABAergic differentiation, and (at high levels) induces phenotypes of deep-layer neuron types. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This project revision extends our analysis of hippocampal neurogenesis from adulthood, the focus of the parent grant, into postnatal ages in mice. The overall goal of the parent project is to gain greater understanding of adult hippocampal neurogenesis, its mechanisms and regulation. Adult neurogenesis has important implications for designing strategies of neural replacement therapy, which could be used to treat neurodegenerative disorders and other conditions of neuronal loss. The goal of this revision project is to gain insights into postnatal development of the hippocampus, especially the dentate gyrus. Development of the dentate gyrus not only lays the foundation for adult neurogenesis, but also utilizes unique developmental mechanisms that may be sensitive to perturbation in disease pathogenesis. Indeed, abnormalities of postnatal hippocampal development have been implicated in the pathophysiology of depression, schizophrenia, epilepsy, and addiction. This project will particularly focus on the role of transcription factors, especially Tbr2, in postnatal hippocampal development. Preliminary studies for this project demonstrate that Tbr2 is essential for development of the dentate gyrus: conditional Tbr2 inactivation causes severe hypoplasia of the dentate gyrus, with defects of cell migration and differentiation. To further investigate the mechanisms affected, we will pursue the following 2 specific aims: (1) First, we will analyze and compare the migrations of different progenitor cell types and neurons in normal and Tbr2 conditional null postnatal hippocampus using a battery of cell type specific markers. (2) Second, we will use multiphoton time-lapse imaging to directly observe and characterize the migrations of different progenitor types in normal and Tbr2 conditional null postnatal hippocampus. Upon completion of these studies, we will have defined mechanisms of cell migration in the postnatal dentate gyrus and their regulation by transcription factor Tbr2. These insights will provide a more detailed scientific basis for understanding diseases involving abnormal hippocampal development. This project fulfills Recovery Act goals by accelerating the tempo of progress in this research area, and by providing for the retention and employment of scientists, through increased effort as well as creation of a new job position for a research technician. PUBLIC HEALTH RELEVANCE: Abnormalities of neurogenesis in the postnatal and adult dentate gyrus (DG) of the hippocampus have been implicated in the pathophysiology of several neuropsychiatric diseases, including depression, schizophrenia, epilepsy, and addiction. This revision project extends our analysis of adult neurogenesis in the DG, to further investigate basic mechanisms of dentate gyrus development in the postnatal period. In particular, defects of cell migration, differentiation, and hippocampal morphogenesis will be studied in targeted genetic mutant mice deficient in the transcription factor Tbr2. These studies will not only improve our understanding of disease mechanisms but also enhance approaches to neuroregeneration as a potential therapeutic approach.

DESCRIPTION (provided by applicant): The cerebral cortex is critically important for higher motor, sensory, and cognitive functions of the brain, such as social interactions and communication, and together with subcortical areas of the limbic system, regulate complex emotional and motivated behaviors. Disorders of cortical development underlie neuropsychiatric illnesses such as autism, schizophrenia, and depression, and the abnormal developmental trajectories of cortical and limbic circuits may impact adaptive behaviors such as drug abuse. The objective of this proposal is to investigate the molecular and cellular mechanisms regulating the normal production of neurons in the cerebral cortex, which arise from a heterogeneous population of stem/progenitor cells located in the ventricular zone of the neocortex and essential for correct neural circuit trajectories. Cell-cell interactions mediated by Delta-Notch signaling are required for both producing the correct number of neurons and maintaining the progenitor pool during corticogenesis, although essential details remain unknown. Importantly, the specific cellular and molecular interactions between different cell types have not been delineated. We hypothesize that different cell types signal to each other through evolutionary diversified components of Delta-Notch gene families, and that a novel Delta-mediated interaction between a subset of progenitors controls the balance between neuron production and progenitor maintenance. We will first determine the cortical cell types that express different Delta-Notch signaling components using combined in situ hybridization/immunolabeling techniques in conjunction with transgenic markers of defined cell types. We have developed a novel 2-color 2-photon live-cell imaging technique that serves as major innovation, allowing us to determine the cellular basis of Delta-Notch signal transduction in dynamically behaving cells that will be tested in functional experiments. The results of this exploratory R21 grant will provide the foundation of a sophisticated new model we propose that encompasses multiple sources of cell-cell interactions regulated by diverse Delta-Notch signaling in the developing cortex. Future studies will examine this mechanism in developing subcortical regions, and in adolescent and adult neurogenesis. PUBLIC HEALTH RELEVANCE: Neurological and behavioral diseases and disorders, such as autism, schizophrenia and susceptibility to drug abuse are thought to arise from developmental deficits in the generation/trajectory/connectivity of neurons in the brain. If we know how these neurons normally develop, then we will be better able to develop strategies aimed at repairing or replacing them when their normal development goes awry.

DESCRIPTION (provided by applicant): Intermediate neurogenic progenitors (INPs) are a relatively new type of neocortical progenitor cells whose properties, daughter neuron fates, and functions in the regulation of neurogenesis remain unclear. This project will characterize molecular and cellular properties of INPs, define their contribution to cortical areas and layers, and study the regulation of INPs by intrinsic and extrinsic factors. The central hypothesis of this project is that INPs regulate not only overall neurogenesis, but also more subtle aspects of neuron differentiation such as regional identity and laminar fate. These studies will be accomplished using a variety of mouse alleles, including a novel genetic lineage tracing system to identify cortical neurons derived from INP cohorts. The first Aim of this project is to characterize the INP transcriptome, and define proliferative and clonal properties of INPs. The second Aim is to determine the contribution of INP daughter neurons to cortical areas, layers, and cell types. The third Aim is to determine how Eomes, a transcription factor that is specifically expressed in INPs, regulates molecular and developmental properties of INPs. The fourth Aim is to determine how fibroblast growth factor signaling affects INP proliferation and differentiation. Together, these focused studies will provide a coherent understanding of INPs, their roles in corticogenesis, and their possible contributions to neurodevelopmental disorders and therapies. Ultimately, our understanding of diseases such as autism and intellectual disability will be advanced, as will our ability to better diagnose and treat these disorders.

DESCRIPTION (provided by applicant): Boys and girls show differential susceptibility to diseases such as dyslexia, autism and attention deficit hyperactivity disorder, in part due to differences in brain development. Previous studies suggest that sexual dimorphisms of brain structure and gene expression reflect not only the influence of gonadal hormones, but also direct effects of sex chromosome genes. This project examines differential expression of specific X chromosome genes in male and female mice and their effects on brain development, especially in the cerebral cortex. This objective of this project is to test the hypothesis that differential expression of Xlr3 genes (which encode chromatin binding proteins) regulates neural precursor proliferation, differentiation, migration, downstream gene expression, and morphology. This objective will be accomplished through 3 Specific Aims: (1) analyze the expression of Xlr3 genes in developing male and female brains; (2) identify downstream genes regulated by differential Xlr3 expression; and (3) characterize neurodevelopmental functions of Xlr3 genes through gain- and loss-of-function assays in embryonic cerebral cortex in vivo. This project will thus elucidate gender differences in brain development that may contribute to disease susceptibility. .

? DESCRIPTION (provided by applicant): The dentate gyrus (DG) is a region of cerebral cortex in the medial temporal lobe that is frequently involved in epilepsy and other neurodevelopmental disorders. Interestingly, DG neurogenesis is extremely prolonged relative to other cortical areas, and depends on migrations of progenitor cells within the dentate migration stream (DMS) and other transient neurogenic niches. Previous studies from the PI's lab have shown that one type of cortical progenitor cells, known as intermediate progenitors (IPs) or transit-amplifying cells, specifically express Tbr2, a T-box transcription factor, during DG development as well as adult neurogenesis. New preliminary data show that Tbr2+ IPs plays major roles in transient neurogenic niches and dentate migration streams that are essential to morphogenesis of the dentate gyrus. For example, Tbr2+ IPs appear to pioneer the DMS and enhance the subsequent migration of neural stem cell (NSC)-like radial glial progenitors (RGPs). Aims 1 and 2 of this project will analyze transient DG niches and cell migrations in mice, and determine the roles of RGPs and IPs in gyrogenesis (development of convoluted cortex). Aim 3 extends these approaches to characterize DG malformations in mutant mice with defects of IP or radial glial progenitor (RGP) differentiation.