Dennis DM O'Leary - US grants

The long term objectives of this work are to contribute to the elucidation of the normal structural organization and development of the vertebrate visual system, with an emphasis on characterizing the mechanisms that govern the development of connections between the eye and central visual centers. The issues to be addressed by the proposed studies are of broad significance. The experiments will be carried out principally using fetal and postnatal rats, and in chick embryos, combining in vivo and in vitro approaches. As experience has shown, though, the developmental mechanisms identified and characterized in the avian and rodent visual system also operate in the development of the primate visual system. The first of the 4 major aims of this proposal concerns the process of target selection by developing retinal axons. Aims 2 - 4 characterize the mechanisms employed by retinal axons to develop topographically ordered projections within their principal target structures, the superior colliculus, and its avian homologue, the optic tectum. AlM 1. To characterize in vivo the mode of in growth of rat retinal axons in to the dorsal lateral geniculate nucleus and test in vitro the hypothesis that a target-derived chemoattractant controls the process by which retinal axons select this nucleus for innervation. AIM 2. To define for the rat retinocollicular projection the temporal sequence of emergence of topographic order from an initially diffuse projection and determine whether topographically aberrant arbors transiently form synaptic contacts. AIM 3. To characterize in rodents the early targeting of developing retinal axons along the rostral-caudal axis of their primary target, the superior colliculus, and assess using an in vitro assay whether this targeting and subsequent arborization of retinal axons reflect their response to regional-distinctions in molecular cues present on the surfaces of collicular cells. AIM 4. To evaluate in vivo the growth behavior of developing chick retinal axons to determine their response to putative molecular cues that encode position along the rostral-caudal and medial-lateral axes of their primary target, the optic tectum.

American scientists and students will participate in an international conference on brain development entitled 'Specificity in Neural Development.' Normal function of the nervous system depends on a precise pattern of connections between nerve cells. In the adult brain, this pattern of connections is very complex, with some nerve cells communicating with other nerve cells over a meter away. The mechanisms by which this complex pattern of connections develops are unknown, but considerable insight into these mechanisms has been gained through recent investigations. This conference will bring together many of the world's scientists working on this problem. These scientists will share their knowledge and through discussion chart the future directions of the field.

DESCRIPTION (provided by applicant): The neocortex, the largest and most complex brain structure, is unique to mammals. It is responsible for sensory perception and cognition, as well as control of our motor systems. In its tangential dimension, the neocortex is organized into subdivisions referred to as areas that are distinguished from one another by major differences in their cytoarchitecture and chemoarchitecture, thalamocortical axon (TCA) input and layer 5 and 6 output connections, and patterns of gene expression. These attributes form a specific combination of properties unique for each area, and together with unique combinations of gene expression, determine the functional specializations that characterize and distinguish areas in the adult. The first transcription factor shown to potentially influence arealization was only identified a decade ago. Although of inarguable importance, the mechanisms controlling arealization of the neocortex remain sketchy and controversial, and are largely limited to generalized axial changes in the size and position of primary areas. Here we propose a series of aims to address specific hypotheses on the requirements of certain regulatory genes to specify the identities and properties of cortical areas in the progenitors that generate them. These studies will break new ground, generate novel insights, and revise and correct misconceptions. The studies include determining the specific mechanisms and transcription factors that are required to specify the primary visual area, V1, redundancy in their function, and limits in their action across the cortical hemisphere. We will also address for the first time the genetic mechanisms involved in the specification of higher order areas and test specific hypotheses on these mechanisms. We will reassess roles for Pax6 is arealization, and use its function as a model for studying the effects of specification of area field size on the representation of the sensory periphery within a cortical area, and the importance of graded expression of transcription factors on their function in establishing sensory maps and representations. Finally, we will investigate the influence of cortex-intrinsic genetic changes to area patterning on the principal sensory thalamic nuclei that relay sensory input to cortex, and define mechanisms of reverse plasticity that serve to systems-match thalamic nuclei to their target areas, and to re-pattern them through a retrograde interaction with their target area. These studies will be carried out using conditional loss- and gain-of-function genetics in mice, using numerous mouse lines that we have made for this proposal, and will make use of new gene markers developed for higher order areas. Further, the considerable amount of preliminary findings that we present support our hypotheses, and indicate that our findings will lead to the development of new concepts, in some cases challenging and replacing dogma, that will have implications for neocortical development and plasticity.

We propose to study the mechanisms that control distinct phases of the development of cortical layer 5 and layer 6 efferent projections to the brainstem and spinal cord, including their pathfinding and the molecular control of their recognition and innervation of targets. We will test the hypothesis that the pioneering of the major path in and out of the cortex by cortical subplate axons through this path. This will be done by creating transgenic mice engineered to have stunted subplate axons, or lack subplate neurons altogether, and by analyzing Mash-1 mutant mice that fail to form a thalamocortical projection. To test the idea that the axonal chemoattractant, Netrin-1, molecularly defines the subcortical path of layer 5 corticospinal axons through the forebrain, midbrain and hindbrain, we will correlate this axonal path with the pattern of Netrin-1 expression, and will analyze mutant mice deficient for Netrin-1 or its receptor, DCC, for aberrant pathfinding by corticospinal axons. The remaining aims focus on target recognition by corticospinal axons, which is characterized by a de novo formation of branches along the axon shaft and their directed growth into targets. We will study the roles of BMP-3 and a novel secreted CAM-like protein with 6 Ig domains and a MAM domain (temporarily named "Ig6M"), which were isolated using a differential display PCR screen to identify candidate target recognition molecules for corticospinal axons. We will use in vitro axon guidance and branching assays, as well as gain-of-function and loss-of-function genetic experiments in mice, to determine the sufficiency and requirement of BMP-3 and Ig6M in controlling the formation and directed growth of corticospinal axon branches.

DESCRIPTION (provided by applicant): A fundamental principle that underlies the anatomical and functional organization of the adult mammalian forebrain is the patterning of the neocortex into distinct areas and the parcellation of the dorsal thalamus (dTh) into nuclei. The developmental patterning of the neocortex into areas, i.e. the process of arealization, is controlled by both intrinsic mechanisms, defined here as regulatory genes that specify positional or area identities of cortical neurons, and extrinsic influences, mainly considered to be area-specific thalamocortical axon (TCA) input, or the information being relayed by it, that arises from dTh nuclei and is established in part by the genetic framework intrinsic to the neocortex. Evidence for the intrinsic genetic regulation of arealization has only begun to emerge over the past few years and thus far has implicated the homeodomain transcription factor EMX2 and the paired-box transcription factor PAX6 in controlling arealization. Less is known about the genetic mechanisms that pattern the dTh into nuclei and specify nuclei-unique properties. We hypothesize that the LIM-homeodomain (LIM-HD) transcription factors LHX2 and LHX9 specify area identities in the cortex and nuclei identities in the dTh through a direct mechanism, based on the known functions of LIM-HD genes, the uniquely graded expression of LHX2 in the neocortex, and the combinatorial expression of LHX2 and LHX9 in subsets of dTh nuclei. We also propose that they indirectly, but critically, influence neocortical arealization by controlling patterning of the dTh into nuclei and specifying properties autonomous to dTh projection neurons that determine the development of their TCA projections to specific neocortical areas, which in turn influence cortical patterning. To study these roles for LHX2 and LHX9, we will employ a range of approaches including conditional gain- and loss-of-function analyses using tissue specific transgene expression and targeted gene inactivation in mice designed to survive to adulthood. We will analyze area- and nuclei-specific phenotypes of cortical and dTh neurons, including expression of markers of identity, specific input and output projections, and the organization of neocortex into areas and dTh into nuclei. The multilevel analyses and alternative strategies proposed will help circumvent potential problems associated with more limited approaches, provide complementary findings to support interpretations, and help establish a hierarchy of regulation of forebrain patterning.

by an NSF Shared Instrumentation Grant and 40% were 'matching" funds from the Salk Institute. In the past year, funds were obtained by Chris Kintnerto purchase a Biorad confocal fitted on an inverted Zeiss microscope. We have both confocal systems in a dedicated room specifically designed to house the confocal facility. This facility is centrally located within the East wing of MNL, immediately adjacent to the labs of Goulding, Lemke and O'Leary, and a short walk from the Pfaff lab. These systems, particularly the Zeiss system, are very heavily used by all of the labs of the program, and our work is completely dependent upon this microscopy. We have also obtained an additional computer workstation with similar software packages (and keys!) that allow for image analysis freeing the computer systems directly operating the confocal microscopes for image acquisition. We also have dedicated laboratory space assigned to the second component of the core - a facility for the analysis of gene expression. This facility includes space for warming trays, an embedding station, perfusion equipment, and incubators for in situ hybridizations and immunohistochemistry of whole embryos, dissected tissues, and sections (slides). A storage facility and log for cDNA and antibody reagents frequently used by all component laboratories of the program project will also be housed in the Analytical core, and managed by the core technician. Dennis O'Leary (5% effort) will supervise the operation of the Analytical Core, which will be directly managed by an experienced technician, Ms. Berta Higgins (50% effort), who has the management skills and experience appropriate for this position. This technician's responsibilities include: 1. Management and supervision of a central facility for confocal and conventional microscopy. This room contains the microscopes, bench and shelf space, and computers for the support of an LSM 510 confocal microscope that is outfitted with both Ar/Kr (488nm and 568nm) and HeNe2 (633nm) lasers. The u.v. laser and an u.v. scan head for the confocal were purchased with a funding supplement to the program project obtained from a one-time NINDS equipment grant program several years ago. This part of the core will accommodate individual users from component projects in addition to the core technician, who will help operate the microscope, maintain equipment, update software for image analysis, manage data storage, organize time for individual members of the component labs to use the facility, schedule and oversee service contract maintenance, and importantly, train personnel in the proper use and care for this sophisticated, costly, and delicate equipment. 2. Management of a core facility for the analysis of gene expression. The bench space for this facility is also centrally located (immediately outside of the room that houses the confocal microscopes), in a broad corridor space that connects the Lemke, Goulding and O'Leary labs. These benches were previously used for manual DMAsequencing, and are therefore adjacent to a hood now used for histological procedures. The facility includes space for warming trays, an 248

PROJECT 3: THALAMIC PATTERNINGAND INFLUENCE ON CORTICAL AREALIZATION Thalamocortical axon (TCA) input from the principal sensory nuclei of dorsal thalamus (dTh) to the primary areas of neocortex define the modality-specific functions of areas in the adult, and we hypothesize that during development TCA input plays a critical role in patterning the neocortex into areas. This patterning process, referred to as arealization, is controlled by intrinsic mechanisms, i.e. regulatory genes that specify positional or area identities of cortical neurons, and extrinsic influences, e.g.area-specific TCA input, or information relayed by it. Recent studies have begun to define genetic mechanisms intrinsic to the developing neocortex that regulate arealization. Relatively little, though, is known about mechanisms that pattern dTh into nuclei, and the degree to which dTh patterning, presented through TCA input, influences arealization. Our aims fall into two related sets: the first set addresses genetic mechanisms that specify nuclei-specific properties of dTh neurons and pattern the dTh into the principal sensory nuclei;the second set addresses the influence of dTh patterning and TCA input on the patterning of the neocortex into primary sensory areas. To accomplish our aims, we will first examine roles for regulatory genes in specifying nuclei- specific identities of dTh neurons and the differentiation of dTh nuclei, by conditional gain- and loss-of- function analyses using selective gene activation / inactivation in mice designed to survive to adulthood. We will then select mice in which dTh nuclei and their TCA input are disproportionately expanded and reduced by these genetic manipulations selective for dTh, and analyze the effects of changes in TCA input on key features of cortical area patterning, including area-specific input and output projections and gene expression. We will complement these studies by analyzing mice in which TCA input is deleted or substantially diminished using different genetic strategies. As an alternative to change relative sizes of dTh nuclei and TCA input, we will selectively expand progenitors that generate different subsets of dTh nuclei by expressing in them a modified beta-catenin. These studies will define the mechanisms and plasticity of neuronal specification and forebrain patterning, as well as help to provide insights into genetic and environmental causes of neurological disorders, particularly those of a developmental and cognitive nature such as autism.

DESCRIPTION (provided by applicant): The cerebral cortex is the largest and most complex component of the mammalian brain, reaching its pinnacle in humans. The neocortex is the largest region of the cerebral cortex and is organized into areas that are functionally unique subdivisions distinguished by differences in cytoarchitecture, connectivity, and patterned gene expression. The specification of neocortical areas is controlled by an interplay between genetic regulation intrinsic to the neocortex, characterized by transcription factors (TFs) expressed by cortical progenitors, and extrinsic influences such as thalamocortical (TCA) input that relays sensory information to cortical areas. Proper area patterning of the cortex is a critical developmental event, because cortical areas form the basis for sensory perception, the control of our movements, and mediate our thoughts and behaviors. Although of undeniable importance, relatively little is known about the genetics of arealization. Current findings indicate a regulatory hierarchy that begins with patterning centers at the perimeter of the cerebral cortex that secrete morphogens, which in turn establish the graded expression of TFs in cortical progenitors that specify their area identities as well as those of their neuronal progeny. The major goal of this grant is to determine the TFs that control arealization, and define their roles in specifying area identities. The major issues to be addressed include: (1) defining the TFs that control the patterning of frontal / motor areas, and caudal / sensory (C/S) areas, as well as the interactions between these TFs to balance the rostral-caudal area patterning of the cortex, and (2) to distinguish roles for these TFs in the intrinsic genetic specification of area-specific properties in the cortical plate versus roles for TCA input in controlling the differentiation of area-specific properties and specializations that distinguish areas. Surprisingly, the size of each primary area in human neocortex varies by as much as two- to three-fold within the normal population. In mice, the sizes of a primary area can also vary significantly between individuals. These variations in area size can have dramatic effects on behavior. For example, genetic manipulations during embryonic development that result in proportional decreases or increases in the sizes primary areas in adults result in significant deficiencies at modality-specific behaviors. These findings indicate that areas have an optimal size, and underscore the importance of establishing during development the appropriate expression levels of TFs that specify area identities, as changes in them can result in a proportional change in area size, and thereby these early developmental events can have a prominent influence on behavior later in life, affecting performance and likely underlying many forms of cognitive dysfunction and neurological disorders. Therefore, the third major goal of this proposal is to establish the mouse as a model for relating differences in area patterning to variations in TF expression, and after validating this relationship, to use it as a basis to define roles for these TFs in area patterning in humans. PUBLIC HEALTH RELEVANCE: The neocortex is the largest and most complex component of the mammalian brain, reaching a pinnacle in humans. This proposal addresses the genetic mechanisms that control the patterning of the neocortex intro areas-- anatomically and functionally distinct subdivisions responsible for sensory perception, voluntary movements, thinking and behaviors. The findings from the proposed aims will form a basis of understanding of cognitive dysfunction and neurological disorders.

The Salk Institute for Biological Studies requests support for a new Ceriter Core Grant for Neuroscience Research. Since the founding ofthe Institute in the 1960s, neuroscience has grown to be the major research emphasis of Salk faculty. Over half of the faculty, heading.32 Institute laboratories, are currently engaged primarily in neuroscience research. The major research interests ofthe faculty are grouped into several broad programmatic areas: Clinical and Translational Neuroscience, Molecular and Cellular Neuroscience, Neural Development, and Systems Neuroscience. Seventeen research projects, supporting twelve faculty as principal investigators, are currently funded by NINDS. These awards include nine independent R01/R37 research projects, an active RC2 (Grand Opportunities) award, a K99, and a POI program project. Nine Salk investigators are either current or past Jacob Javits awardees. The specific aim of this Center Core Grant application is to provide support for key resources that are needed for current and future NINDS-funded research projects. Three new core resources are proposed: 1) a Genome Manipulation Core, providing services to produce gene-targeted ES cell lines for in vivo and in vitro models for functional analysis of genes and neurologic disease, 2) an Imaging Core, focusing on dedicated support for TEM ultrastructural imaging and fostering integrated structural analysis across multiple advanced imaging platforms, and 3) a Behavior Core, providing both dedicated expertise in small animal behavioral analysis and consistency across different testing modalities. The management of these cores will focus first on providing support for the eight Salk Institute investigators with current NINDS-funded R01/R37 research programs. Any excess core capacity will be made available to other investigators at The Salk Institute who have research projects that are consistent with the mission of NINDS. This infrastructure program will provide core services that will facilitate application of continually evolving advanced technologies to the NINDS-funded research projects at the Salk Institute, and leverage existing funding as a resource multiplier to better advance these projects in addressing important problems relevant to the NINDS mission.

The Salk Institute for Biological Studies requests support for a new Center Core Grant for Neuroscience Research. Since the founding of the Institute in the 1960s, neuroscience has grown to be the major research emphasis of Salk faculty. Over half of the faculty, heading 32 Institute laboratories, are currently engaged primarily in neuroscience research. The major research interests of the faculty are grouped into several broad programmatic areas: Clinical and Translational Neuroscience, Molecular and Cellular Neuroscience, Neural Development, and Systems Neuroscience. Seventeen research projects, supporting twelve faculty as principal investigators, are currently funded by NINDS. These awards include nine independent R01/R37 research projects, an active RC2 (Grand Opportunities) award, a K99, and a P01 program project. Nine Salk investigators are either current or past Jacob Javits awardees. The specific aim of this Center Core Grant application is to provide support for key resources that are needed for current and future NINDS-funded research projects. Three new core resources are proposed: 1) a Genome Manipulation Core, providing services to produce gene-targeted ES cell lines for in vivo and in vitro models for functional analysis of genes and neurologic disease, 2) an Imaging Core, focusing on dedicated support for TEM ultrastructural imaging and fostering integrated structural analysis across multiple advanced imaging platforms, and 3) a Behavior Core, providing both dedicated expertise in small animal behavioral analysis and consistency across different testing modalities. The management of these cores will focus first on providing support for the eight Salk Institute investigators with current NINDS-funded R01/R37 research programs. Any excess core capacity will be made available to other investigators at The Salk Institute who have research projects that are consistent with the mission of NINDS. This infrastructure program will provide core services that will facilitate application of continually evolving advanced technologies to the NINDS-funded research projects at the Salk Institute, and leverage existing funding as a resource multiplier to better advance these projects in addressing important problems relevant to the NINDS mission.