Song-Hai Shi - US grants

DESCRIPTION (provided by applicant): Proper formation of the cerebral cortex depends on an orderly production of a large number of neurons during embryonic development. Recent studies have convincingly shown that radial glial cells are a major population of neuronal progenitor cells in the developing cortex. In addition to their well-characterized role in guiding radial migration of newly born neurons, radial glial cells divide in the ventricular zone to generate neurons. Precise control of radial glial cell division in the developing cortex is likely a major factor in controlling the number of neurons in the mature cerebral cortex. Despite this fundamental role in cortical development, the mechanisms that regulate radial glial cell division are poorly understood. The long-term goal of this project is to elucidate the molecular and cellular processes underlying radial glial cell division and daughter cell fate specification. During peak neurogenesis, radial glial cells predominantly divide asymmetrically to self-renew and to generate neurons. Asymmetric cell division usually requires the dividing cells to be polarized so as to ensure differential inheritance of cell fate determinants by the two daughter cells. The objectives of this proposal are to uncover the molecular control of radial glial cell polarity and to define how the polarization of radial glial cells may regulate the mode of their division (i.e. being symmetric or asymmetric) in the developing cortex. Radial glial cells originate from epithelial cells that are highly polarized with distinct apical and basal subcellular compartments. This apical-basal polarity is controlled by a set of evolutionarily conserved protein complexes, among which the Par (partition defective) protein complex plays a central role. Moreover, the Par protein complex is essential for polarizing neural progenitor cells (i.e. neuroblasts and sensory organ precursors, SOPs) in the Drosophila nervous system and ensuring their asymmetric cell division. Based on these observations, the central hypothesis of this application is that the mammalian Par (mPar) protein complex controls the polarity and the division mode of radial glial cells in the developing cortex. Guided by strong preliminary data, this hypothesis will be tested by pursuing these three specific aims: 1) To determine the subcellular localization of the mPar protein complex in dividing radial glial cells;2) To define the function of the mPar protein complex in regulating radial glial cell division and daughter cell fate specification;and 3) To delineate the molecular and cellular pathways of the mPar protein complex in the developing cortex. The approach is innovative, because it combines advanced laser scanning microscopy with molecular genetics techniques. The proposed research will provide new insights concerning how neuronal progenitor cells in the developing cortex divide to give rise to neurons. Many human neurological and psychiatric disorders are associated with defects in cortical neurogenesis, ranging from severe malformations with mental retardation and epilepsy, to more subtle ones such as autism and maladaptive behavior associated with drug abuse. The results of this study may shed light on mechanisms relevant to the etiology of many of these disorders. PUBLIC HEALTH RELEVANCE: This study investigates the processes underlying how neuronal progenitor cells divide to give rise to neurons in the developing brain, an important and under-investigated area in neuroscience. It will advance and expand the understanding and treatment of a variety of neurological and psychiatric disorders caused through defects in cerebral cortex development, such as mental retardation, epilepsy, autism, and maladaptive decision- making behavior associated with drug abuse.

DESCRIPTION (provided by applicant): An essential step towards understanding the function of a brain structure is to understand the intricacies of its neuronal interconnections. Extensive evidence suggests that neurons in the cerebral cortex are interconnected into functional columns. Moreover, columns with similar physiological properties are arranged close to each other and lead to the formation of cortical maps. At the level of neuronal populations, cortical maps varied smoothly across the surface of the cortex. However, recent studies demonstrate that neurons next to each in the cerebral cortex can have drastically different physiological properties, arguing that the microcircuit in functional columns is highly specific at the level of individual neurons. Given the daunting complexity in the number and type of neurons in the cerebral cortex, the formation of this fine-scale microcircuit is a seemingly bewildering task. This raises an intriguing possibility whether the formation of precise microcircuits in the cerebral cortex depends on the highly regulated processes underlying the early cortical development, e.g. neurogenesis and neuronal migration. It has been previously suggested that the formation of functional columns is related to ontogenetic radial units, the construction units of the cerebral cortex that consist of individual radial glial progenitor cells and associated daughter cells - migrating cortical neurons. To test this hypothesis, it is essential to identify ontogenetic radial units in the development cortex and investigate their morphological and functional development. In this project, we will develop new experimental and computational tools to label ontogenetic units of excitatory and inhibitory neurons in the developing cortex, analyze their morphological development, and map their synaptic connectivity. Specifically, we will engineer retroviruses that express fluorescence proteins and use them to infect dividing radial glial progenitor cells in the developing cortex in vivo in a cell/tissue specific manner. The morphogenesis of individual ontogenetic radial units expressing fluorescent proteins will be analyzed using state-of-the-art imaging techniques and powerful imaging analysis tools. Furthermore, we will combine whole-cell electrophysiological recordings with the latest development of photostimulation techniques to investigate the microcircuit development among neurons in ontogenetic units at single-cell resolution. The proposed research promises a wealth of new insights into the developmental and functional organization of the cerebral cortex. It will also facilitate the understanding and treatment of various neurological and psychiatric disorders caused by the cerebral cortex malformation and malfunction. PUBLIC HEALTH RELEVANCE This project will develop new experimental and computational tools for labeling neurons in the cerebral cortex that share the same origin and for investigating their morphological and functional development. It will advance and expand the understanding and treatment of a variety of neurological and psychiatric disorders caused through defects in cerebral cortex development and function, such as mental retardation, epilepsy, autism, and maladaptive decision-making behavior associated with drug abuse.

DESCRIPTION (provided by applicant): Virtually all neuronal circuits in the mammalian neocortex are composed of glutamatergic excitatory neurons and GABAergic inhibitory interneurons. While excitatory neurons are responsible for generating the output, interneurons provide a rich variety of inhibitions through an extraordinary diversity in subtypes that often determine output. Extensive studies over the past decade have revealed key insights into the production and organization of excitatory neurons in the neocortex. In contrast, our knowledge of interneuron production and organization in the neocortex remains very limited. For example, it is unclear whether a single interneuron progenitor cell gives rise to different subtypes of interneurons and whether sister interneurons originating from the same progenitor cell are specifically organized and thereby provide potential anatomical substrates for the formation of functional circuits in the neocortex. To address these fundamental questions, we propose to perform clonal analysis of interneuron production, migration and structural and functional organization in the neocortex. To achieve our goals, we will develop innovative methods for effectively and selectively labeling interneuron progenitor cells in the ventral telencephalon - the ganglionic eminences - at clonal density. We will analyze the production, migration, and structural and functional organization of individual interneuron clones being labeled using state-of-the-art imaging (e.g. two photon lasers scanning microscopy) and electrophysiology (e.g. multi-electrode whole-cell patch clamp recording) approaches and link these processes to functional circuit formation in the neocortex. Interneuron malformation and dysfunction have been associated with many neurological and psychological disorders, such as epilepsy, schizophrenia and autism. Therefore, our research will not only provide fundamental insights into interneuron development and greatly advance our understanding of the functional organization of the neocortex, but will also shed light on the etiology of many devastating brain disorders. 1 PUBLIC HEALTH RELEVANCE: Interneurons are vital components of neural networks in the brain and are responsible for providing a rich variety of inhibition actions that restrain the brain activity. Malformation and dysfunction of interneruons have been linked to many neurological and psychological illnesses, including epilepsy, schizophrenia and autism. Our studies on interneuron production and organization in the mammalian brain will shed light on the etiology and thereby provide new ideas for the medical treatment of many of these devastating brain disorders. 1

DESCRIPTION (provided by applicant): Proper formation of the neocortex depends on the orderly production of a large number of neurons during embryonic development. Radial glial cells have been demonstrated to be a major population of neuronal progenitor cells in the developing neocortex. In addition to their well-characterized role in guiding radial migration of new-born neurons, radial glial cells divide in the ventricular zone to generate neurons. Precise control of radial glial cell division determines the number of neurons in the mature neocortex. Despite this fundamental role in neocortical development, the mechanisms that regulate radial glial progenitor cell division are poorly understood. The long-term goal of this project is to elucidate the molecular and cellular processes underlying radial glial progenitor cell division and neocortical neurogenesis. It is generally thought that radial glial progenitor cells initially divie symmetrically to amplify themselves and then divide asymmetrically to self- renew and give rise to neocortical neurons. A prevailing model of progenitor cell asymmetric division holds that proper orientation of the mitotic spindle relative to the axis of progenitor cell polarity ensures unequal segregation of critical cellular fate determinants between the two daughter cells. Recent studies from our lab and others have shown that the evolutionarily conserved partition defective (Par) protein complex regulates the symmetric versus asymmetric division of radial glial progenitor cells. Furthermore, we recently found that the duplicated mother and daughter centrosomes in asymmetrically dividing radial glial progenitor cells are differentially inherited b the two daughter cells dependent on the centrosome maturity and daughter cell fate specification. As the major microtubule-organizing center in animal cells, the centrosome is not only critical for the formation of the mitotic spindle, but also absolutely required for the formaton of a cilium, the cellular antennae that orchestrate important signaling pathways related to cell proliferation and differentiation. Based on these observations, the central hypothesis of this application is that the Par polarity complex regulates mitotic spindle orientation, mother versus daughter centrosome inheritance, and ciliogenesis in radial glial progenitor cells. Guided by strong preliminary data, this hypothesis will be tested by investigating the functions of the mPar protein complex in regulating: 1) mitotic spindle orientation in dividing radial glial progenitor cells, 2) mother versus daughter centrosome inheritance in dividing radial glial progenitor cells, and 3) ciliogenesis in dividing radial glial progenitor cells. With innovative approaches including high-temporal live imaging and molecular genetics techniques, the proposed research will provide fundamental new insights into the molecular and cellular regulation of radial glial progenitor cell division and neocortical neurogenesis. Many human neurological and psychiatric disorders are associated with defects in neocortical neurogenesis, ranging from the severe malformations of mental retardation and epilepsy, to more subtle ones such as autism and maladaptive behavior associated with drug abuse. The results of this application will shed light on the etiology of these disorders.

DESCRIPTION (provided by applicant): Proper development of the cortex, the command center of the brain, entails extensive production of neurons. This is largely achieved through the robust asymmetric division of radial glial progenitor cells (RGPCs) residing in the ventricular zone (VZ) of the developing cortex. While excellent progress has been made over the past twenty years in our understanding of RGPC division and neurogenesis in the developing cortex, our knowledge of the precise regulation and function of the centrosome, a key subcellular organelle for microtubule organization, cell division and ciliogenesis, during cortical neurogenesis remains limited. The goal of this application is to fill this knowledge gap, which wil also be crucial for understanding the etiology and pathophysiology of microcephaly, a neuro-developmental disorder that is characterized by small brain size as a result of deficient neuron production in the developing cortex. To date, at least eight autosomal recessive primary microcephaly (MCPH) loci and seven genes have been identified for autosomal recessive primary microcephaly (MCPH). Remarkably, all the defined MCPH genes encode centrosomal proteins, underscoring the unique importance of proper centrosome regulation and function in the production of normal neuron populations in the developing cortex. The central hypothesis is that the mature centrosome is essential for maintaining RGPCs in the VZ - a progenitor cell niche - and ensuring their proper division and survival in the developing cortex. This hypothesis has been formulated on the basis of the strong published and preliminary data produced in the applicant's laboratory. Guided by preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Reveal centrosome properties and behavior in interphase and mitotic RGPCs; 2) Define the functions of the centrosome in RGPCs; and, 3) Explore the mechanisms that regulate centrosome behavior in RGPCs. The experimental focus of this application is to provide a comprehensive understanding of the behavior, function and regulation of the centrosome in RGPCs as they proceed through the cell cycle to produce neurons and link it to microcephaly, using state-of-the- art imaging and mouse genetic approaches. As of this writing, few such studies have been reported. Accomplishing the aims in this project will not only provide important insights into RGPC division and cortical neurogenesis, but also help to define the cellular basis of microcephaly caused by genetic abnormalities, and thereby provide new ideas for early diagnosis and treatment.

DESCRIPTION (provided by applicant): There is a fundamental knowledge gap in understanding how precise functional neuronal circuits form in the neocortex, which is the most complex part of the brain and controls all aspects of behavior, from perception to emotion and cognition. The continuing existence of this gap represents a major problem because it severely hinders the understanding of malformation and malfunction of the neocortex. The long-term goal is to better understand the assembly of precise neuronal circuits in the developing neocortex. The objective in this particular application is to investigate the origin, basis and regulation of precise electrical synapse formation between neocortical excitatory neurons. The central hypothesis is that the lineage relationship guides precise electrical synapse formation between excitatory neurons in the developing neocortex. This hypothesis has been formulated on the basis of strong preliminary data produced in the applicant's laboratory. The rationale for the proposed research is that the processes of neurogenesis and neuronal migration regulate the formation of specific electrical synapses between excitatory neurons in the developing neocortex. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) to uncover the mechanisms that control the specificity of strong electrical synapse formation between sister excitatory neurons; 2) to determine the mechanisms that regulate the developmental removal of electrical synapses between sister excitatory neurons; and 3) to investigate the mechanisms that drive the preferential development of specific chemical synapses between electrically coupled sister excitatory neruons. The approach is innovative, because it combines a number of cutting-edge techniques including retroviral engineering, in utero labeling, mouse genetics, quadruple whole-cell patch clamp recording and two photon/confocal laser scanning microscopy. The proposed research is significant, because it is expected to fundamentally advance the understanding of precise neuronal circuit assembly and functional development of the neocortex. Ultimately, such knowledge has the potential to inform early diagnoses and the development of therapeutic treatments for many devastating brain disorders including schizophrenia, epilepsy, mental retardation and autism.

PROJECT SUMMARY Prenatal or neonatal exposure to drugs of abuse such as cocaine and ethanol has been shown to disrupt neurogenesis and/or gliogenesis in the developing cerebral cortex, and induce functional abnormalities late in life. Proper formation of the cortex depends on the orderly production of a large number of diverse neurons, as well as glial cells. Radial glial cells have been shown to be a predominant population of neural progenitor cells in the developing cortex. In addition to their well-characterized role in supporting neuronal migration, radial glial progenitors (RGPs) actively divide to proliferate and to generate neurons and glial cells either directly or indirectly. The division mode and dynamics of RGPs essentially determine the number and types of neurons and glia in the cortex; however, the precise behavior and lineage progression of RGPs and the underlying molecular regulation are poorly understood. The long-term goal of this project is to systematically delineate RGP behavior and progeny output at the cellular and molecular levels. RGPs are neither synchronized in division dynamics nor homogenous in division pattern and progeny output. This calls for a systematic and quantitative analysis of the precise mitotic behavior and progeny output of RGPs in vivo at the single cell resolution. Recently, we exploited the unprecedented resolution of mosaic analysis with double markers (MADM) on progenitor division and progeny output, and performed a systematic and quantitative clonal analysis of RGP division and lineage progression. We revealed, for the first time, that RGPs progress through a remarkably deterministic and orderly program in proliferation, neurogenesis, and gliogenesis. Based on strong published and preliminary data, the central hypothesis of this application is that the behavior and output of individual RGPs are highly programmed at the cellular and molecular levels to produce a correct number and type of neurons and glia in the cortex. This hypothesis will be tested by 1) systematically and quantitatively examine the number, type, and organization of astrocytes and/or oligodendrocytes generated by individual RGPs at different embryonic stages using MADM, and 2) elucidate the molecular programs that regulate RGP lineage progression in proliferation, neurogenesis, and gliogenesis by performing in-depth real time single-cell transcriptome analysis of RGPs across different embryonic stages using CEL-seq in conjunction with loss-of- function studies using mouse genetics and/or CRISPR/CAS9 approaches. By integrating a battery of cutting- edge techniques, the proposed research will provide fundamental new molecular and cellular insights into RGP behavior and cortical neurogenesis and gliogenesis. This contribution will be significant because it will not only advance the basic knowledge of cortical histogenesis, but will also expand our understanding of the underlying cause of drugs of abuse-induced brain damage or other devastating developmental brain disorders with cortical abnormalities such as microcephaly, macrocephaly, and autism, and thereby potentially identify important molecular and cellular targets for diagnosis and treatment.