Elizabeth A. Grove - US grants

neurogenetics; peptides; neurogenesis; developmental neurobiology; telencephalon; neuroanatomy; gene expression; cell type; biological signal transduction; hippocampus; beta galactosidase; tissue /cell culture; immunocytochemistry; laboratory mouse; histology; polymerase chain reaction; in situ hybridization;

DESCRIPTION (provided by applicant): My long term goal is to understand the mechanisms that initiate development of the mammalian cerebral cortex and control the formation of the cortical area map, the basic functional organization of the cortex. Findings should be relevant to understanding a wide variety of cortical birth defects, and diseases with later onset that stem from early cortical abnormalities. I propose here to continue a fruitful line of research in which my research group found that the secreted signaling molecule FGF8 regulates patterning of the cortical area map along the anterior/posterior (A/P) axis of the cortical primordium. This proposal has three aims. The first aim is to understand the A/P patterning signal better. At present we do not know if FGF8 and members of the same FGF subfamily form a signaling gradient to impart positional values to the cortex, or if they trigger a relay of other patterning mechanisms. Using mouse genetics we will generate mice with progressively lower levels of FGF8 subfamily ligands to determine if the cortical area map shows increasing shifts. If so, this would provide support for a gradient model, and discount the simplest relay model. To determine which FGF receptors relay the patterning signal, mice that lack combinations of FGF receptors will be analyzed to determine if their cortical maps show defects similar to an FGF8 deficiency. Because FGF/FGFR binding requires heparan sulfate (HS), we will evaluate the cortical area map in mice that lack HS in the cerebral cortex. Although other growth factors require HS, we want to know how loss of FGF signaling will affect area patterning. Will the map be homogenized, or will a default pattern be present? In Aim 2 we will use in utero electroporation of dominant negative FGFRs to determine if cortical cells detect levels of FGF signaling at a distance from the FGF8 source, and, when these levels change, respond by adopting a new area fate. We will also introduce a second source of FGF8 by electroporation and determine if multiple areas are duplicated, and if so, whether and how duplicate maps are ordered along a secondary A/P axis. In Aim 3, we will test the hypothesis that FGF signaling is also involved in the primary division of the telencephalon into the dorsal cerebral cortex and the nuclei of the basal forebrain. We will use mouse genetics, in utero electroporation, and attempted rescue of mouse mutants with excess FGFs to test whether FGF signaling suppresses the cortical fate and promotes ventral telencephalic fates.

DESCRIPTION (provided by applicant): The long-term goal of these studies is to understand how the basic functional organization of the mammalian cerebral cortex - the cortical area map - arises in development. We are testing a model in which the area map is set up by discrete signaling centers. One such center is the cortical hem, generating Wingless-lnt (WNT) and Bone Morphogenetic Protein (BMP) proteins. Our primary hypothesis is that the cortical hem regulates development of the area map along the medial/lateral (M/L) axis. In addition, preliminary findings indicate new roles for the hem via its generation of a rich variety of cell types, transient and permanent, neuronal and non-neuronal. In aim 1, we will fate map the hem using the WNT3a locus to direct Cre expression to the hem. The resulting mouse line will be crossed with reporter mice, carrying marker genes flanked by IoxP sites. In the offspring, WNT3a-expressing hem cells and their progeny will be permanently marked. Hem-derived cells will be definitively characterized with markers of cell division, cell type, cell death and connectivity. Their development and striking migratory behavior will be followed in fixed tissue and living slices; cues that direct their migration will be explored. In aim 2, we will ablate the cortical hem utilizing the WNT3a locus and Cre-lox recombination to direct expression of a cellular toxin to the hem. Cortices in which the hem is entirely or partially ablated, or in which hem cells are ablated mosaically ("hem hypomorphs") will be analyzed at embryonic and postnatal ages. Gene expression patterns, neurochemistry, cytoarchitecture and functional connectivity will be used to identify shifts or loss of M/L patterning in the cortex. With similar techniques, we will determine the effects of the loss of hem-derived cells. A possibility is defective cortical lamination, which could indicate that the hem directs cortical patterning along two axes. Aim 3 will focus more specifically on the contributions of WNT and BMP signaling from the hem. Using classic mouse genetic approaches, we will evaluate the effects of different, lower levels of BMP signaling on M/L cortical patterning. Next, we will attempt to rescue particular defects that result from hem loss by replacing WNT and BMP signals, utilizing in utero electroporation-mediated gene transfer. Together these studies should clarify normal development of the cerebral cortex, and shed light on the possible causes of human cortical malformations.

The long-term goal of these studies is to understand how the basic functional organization of the mammalian cerebral cortex - the cortical area map - arises in development. We are testing a model in which the area map is set up by discrete signaling centers. One such center is the cortical hem, generating Wingless-lnt (WNT) and Bone Morphogenetic Protein (BMP) proteins. Our primary hypothesis is that the cortical hem regulates development of the area map along the medial/lateral (M/L) axis. In addition, preliminary findings indicate new roles for the hem via its generation of a rich variety of cell types, transient and permanent, neuronal and non-neuronal. In Aim 1, we will fate map the hem using the WNT3a locus to direct Cre expression to the hem. The resulting mouse line will be crossed with reporter mice, carrying marker genes flanked by IoxP sites, in the offspring, WNT3a-expressing hem cells and their progeny will be permanently marked. Hem-derived cells will be definitively characterized with markers of cell division, cell type, cell death and connectivity. Their development and striking migratory behavior will be followed in fixed tissue and living slices;cues that direct their migration will be explored. In Aim 2, we will ablate the cortical hem utilizing the WNT3a locus and Cre-lox recombination to direct expression of a cellular toxin to the hem. Cortices in which the hem is entirely or partially ablated, or in which hem cells are ablated mosaically ("hem hypomorphs") will be analyzed at embryonic and postnatal ages. Gene expression patterns, neurochemistry, cytoarchitecture and functional connectivity will be used to identify shifts or loss of M/L patterning in the cortex. With similar techniques, we will determine the effects of the loss of hem-derived cells. A possibility is defective cortical lamination, which could indicate that the hem directs cortical patterning along two axes. Aim 3 will focus more specifically on the contributions of WNT and BMP signaling from the hem. Using classic mouse genetic approaches, we will evaluate the effects of different, lower levels of BMP signaling on M/L cortical patterning. Next, we will attempt to rescue particular defects that result from hem loss by replacing WNT and BMP signals, utilizing in utero electroporation-mediated gene transfer. Together these studies should clarify normal development of the cerebral cortex, and shed light on the possible causes of human cortical malformations.

DESCRIPTION (provided by applicant): Longterm goals of the proposed research are to understand the mechanisms that initiate development of mammalian cerebral cortex, and control formation of the neocortical area map. Findings should be relevant to a range of human disorders that stem from developmental defects in cerebral cortex. This proposal, which has three aims, is based on evidence that the signaling molecule Fibroblast Growth Factor (FGF) 8 acts as a graded morphogen in the embryonic mouse neocortical primordium (NP), thereby initiating basic NP patterning. Strikingly, ectopic FGF8 in the NP can induce duplicate areas, and even complex maps. Aim 1 tests the hypothesis that FGF8 initiates a series of gene regulatory events that specifies the area map. A systematic search for genes downstream of FGF8 that have a role in cortical regionalization will use next generation deep sequencing (RNA-Seq) to compare transcriptomes of NP tissue exposed to different concentrations of FGF8 in vitro, mimicking the natural gradient of FGF8, or freshly dissected parts of the NP, representing different presumptive types of neocortex. Secondary screening will look for candidates with regional NP expression. Aim 2 will investigate how FGF8 interacts with FGF receptors (FGFRs), and a potential endogenous inhibitor, to control development of the normal area pattern. Wild type or mutant FGFRs will be introduced into different regions of the embryonic NP with in utero electroporation. After appropriate survival times, brains will be examined for effects on FGF8 diffusion and signaling, and mature area map organization. Some prefrontal areas derive from progenitors inside the source of FGF8, and, most likely, inside a larger source of FGF17, an FGF8 family member. Fate-mapping experiments will investigate if the nested sources of FGF8 and FGF17 give rise to nested areas of prefrontal cortex. Aim 3 builds on a new finding, namely that a major class of cortical projection neurons, mitral cells of the olfactory bulb (OB), is also generated by progenitors inside the FGF8/17 source. Experiments are proposed to determine if FGF8/17 signaling induces the OB primordium, a bounded cortical region, confirm that FGF8/17 drive mitral cell development, and identify transcription factors downstream of FGF8/17 that specify mitral cells.

Project Summary/Abstract My overall goal is to understand how the neocortical area map develops in the embryonic and early postnatal mammalian brain. The prevailing model of how area patterning is initiated in the embryo is based on research in the smooth-brained or lissencephalic mouse, yet studies that support this model are aimed at revealing how the area map is established in mammals in general. Recent discoveries regarding the generation of cortical neurons in gyrencephalic mammals, however, suggest problems with applying the mouse model to these species. A major concern in the field, therefore, is that there can be no general model of neocortical patterning. I propose here to test whether the current model holds for area map development in the gyrencephalic cortex of the ferret. First, I will verify that the ferret cortical primordium (CP) has similar signaling centers to those that pattern the cortex in the mouse. I predict this will be so, given that one center has already been identified in humans. Further, I will identify which of the gene and protein expression patterns that delineate areas in the postnatal mouse cortex also delineate area boundaries in ferret. In the mouse telencephalon, a rostral patterning center (RPC) releases Fibroblast Growth Factor (FGF) 8, a morphogen that disperses in a gradient across the CP supplying positional values to cortical progenitor cells. I expect the embryonic ferret cortex to have an RPC, which patterns both cortical and subcortical structures. In utero microelectroporation (IUME) will be used in live ferret embryos to manipulate levels of FGF8 at the RPC, and to introduce new sources of FGF8 in the CP. The area map will be examined in the pups at 3 weeks, using gene and protein expression patterns to identify area boundaries. If FGF8 controls the ferret area map, as in the mouse, then augmenting the rostral FGF8 source will shift area boundaries caudally, enlarging rostral areas, and shrinking caudal areas. Depleting the source will have opposite effects, and introducing a new source of FGF8 will duplicate areas. This project is designed to address a major stumbling block in the field of cortical development, and to (re)introduce the ferret as a species in which to investigate the mechanisms that pattern the neocortical area map. A full appreciation of human cortical development in the embryo will aid in understanding several major human neurological and psychiatric disorders with a strong developmental component.

PROJECT SUMMARY/ABSTRACT Sensory thalamic nuclei relay information from sensory surfaces such as the retina and the vibrissae on rodent's face to the cerebral cortex, connecting the cortex with the outside world. In turn primary sensory areas of neocortex project to sequentially connected higher order neocortical areas that mediate such functions as perception, memory and movement control. Knowing the mechanisms that direct thalamocortical axons to the correct neocortical target is therefore important for understanding the development of brain function, and aspects of abnormal development underlying human brain disorders. Although an abundance of studies has uncovered many cellular and molecular signals that control the growth of thalamic axons through subcortical structures, as well as how axons are precisely sorted along their route, little attention has been paid to guidance signals inside the cortex itself. Yet substantial evidence indicates that intra-cortical cues exist. The proposed work will utilize a mouse line in which axons from sensory thalamic nuclei have been genetically labeled with fluorescence. This creates a visible map of sensory areas on the surface of the brain. The neocortical area map will be altered in these mice by manipulating the neocortical morphogen Fibroblast Growth Factor 8 (FGF8), which will shift and duplicate sensory areas. Axonal tracers will be placed in different parts of the altered area map, using the fluorescent map to identify the desired targets. After the tracer has had time to travel from cortex to thalamus, the brain will be fixed and sectioned. The trajectories of labeled thalamocortical axons will be viewed and reconstructed in 3D using a confocal microscope. These trajectories will shift dramatically at points of normal thalamic guidance in the cortex because the cortical map itself has been disrupted. The study will test the hypothesis that positional cues for thalamic axons are present in the cortical subplate and in layer 4, the major recipient of thalamic input, and will be a preface to investigating the molecular identity of such cues.

The proposed research focuses on development of the olfactory bulb (OB), the most rostral division of cerebral cortex. The OB has a single layer of mitral cell projection neurons. Mitral cells receive olfactory information from olfactory receptor neurons, and send their axons along one major output pathway, the lateral olfactory tract (LOT), directed in part by guidepost cells, called ?lot? cells. The LOT carries olfactory input to olfactory cortex, and to a variety of brain structures concerned with motor behavior, memory and mood. The OB develops adjacent to an embryonic source of Fibroblast Growth Factors (FGFs) in the rostral telencephalon. In Preliminary Studies, introducing FGF8 ectopically into the lateral cortical primordium (CP) created OB-like structures (OBLSs) in the lateral wall of the cerebral hemisphere. An FGF8-induced OBLS was composed of mitral cells, but also attracted interneurons characteristic of the OB. The highly ectopic mitral cells sent compact axon bundles (tributary LOTs or ?tLOTS?) ventrally to join the LOT. Meanwhile, lot guidepost cells migrated rostrally from their source at the telencephalic/diencephalic boundary to surround the OB, diverging from their main pathway to surround each OBLS as well. Axons in the tLOTs grew out to the main LOT through funnels of ectopic lot cells. These preliminary findings prompt Aims 1 and 2 of the present proposal, to determine the role of FGF8 in inducing the OB. In Aim 3, the function of lot cells in guiding axons of the LOT will be explored, testing, in particular, the hypothesis that the OB itself guides the migration of lot cells. These studies should provide a wealth of new information on the development of the OB and its major output pathway. Reduction or loss of the sense of smell in humans causes depression, anxiety and diminished pleasure in life. Understanding how the OB is induced and how it communicates with the rest of the brain is a topic of basic and clinical significance. Notably, the combination of an underestimated frequency of olfactory dysfunction in the human population, and the rarity with which clinicians image central olfactory structures in anosmic patients, suggests the role of central developmental defects may be greatly underappreciated. Investigation of the normal development of the OB therefore has the potential to drive new discoveries concerning OB development and function in human syndromes.

The division of the neocortex into distinct areas is the fundamental way in which neocortical functions and connectivity are organized. How the neocortical area map forms during development is thus a basic question in developmental neurobiology. Mistakes in the map cause behavioral deficits in mice and may underlie human mental health disorders. A major question is when, and how, distinct areas appear in development. Evidence that area-specific signals provide final guidance to thalamocortical afferents (TCAs) suggests distinct areas exist before TCAs enter the NP. The proposed work is designed to test this hypothesis, and potentially resolve a long debate concerning the relative importance of afferent axons versus mechanisms intrinsic to the cortex in setting out the neocortical area map. First I propose to investigate area pattern formation in an unusual mouse mutant brain. Mice that constitutively lack the transcription factor gene, Barhl2, completely lack a thalamus. This mouse represents a striking new resource for investigating the influence of the thalamus on neocortical development. I propose to investigate area boundaries in the Barhl2 null mouse using gene expression that marks out area boundaries in wildtype mice, and by examining cortical connectivity. Determining whether areas develop in the Barhl2 null mouse will provide fresh evidence that the area map does, or does not depend on thalamic innervation for its formation. Prompted by strong evidence that embryonic areas attract appropriate thalamic input, I further propose to search for genes selectively expressed in presumptive areas before TCA entry into neocortex. Sample tissue will be collected from a mouse in which TCAs heading for primary sensory areas express green fluorescent protein (GFP), which marks out primary sensory areas, including primary sensorimotor cortex (S1). In a pilot study, presumptive S1 was detectable at the required age by a thick bundle of GFP-positive axons beneath the cortex proper, waiting to enter S1. Primary motor cortex (M1) is rostral to S1 and markedly lacked GFP- positive TCAs beneath it. Samples of S1 and M1 tissue will be prepared for RNA-Seq and delivered to the Genomics Core Facility at the University of Chicago. Members of the Core will carry out paired-end sequencing at a depth of 80 million reads. This depth will assure detection of transcripts with low expression levels, and RNAs encoding different protein isoforms. I chose this depth to increase sensitivity over previous searches for area-specific genes in developing mouse neocortex. Differentially expressed genes will be validated with qPCR and expression of genes of interest will be examined in both wildtype mice and Barhl2 mutants. Expression in the Barhl2 mutant mouse NP would verify the independence of expression from TCA contact. Identifying genes selectively expressed in presumptive areas would be a major step towards understanding how the area map develops, and set the stage for investigating the functions of these genes, and how their expression is confined to particular areas.