Susan K. McConnell - US grants

A Presidential Young Investigator Award will support research focused on early development of the mammalian cerebral cortex. Heterochronic transplantation experiments will be performed to determine whether all cortical progenitor cells are developmentally equivalent, or whether the competence of ventricular cells to respond to environmental cues changes with increasing age. Monoclonal antibodies against cell surface increasing age. Monoclonal antibodies against cell surface molecules will be raised for the purpose of purifying neuronal precursors. The developmental potential of these purified populations of precursor cells will be studied in culture and following transplantation into the brain in order to identify environmental factors that are involved in the production of specific, committed neuronal phenotypes. These studies are fundamental to understanding the development of brain regions involved in higher cognition.

9350248 McConnell Neurons migrate to unique positions and form specific axonal connections within the developing cerebral cortex. This Presidential Faculty Fellow award will allow this investigator to explore the cellular and molecular mechanisms that control neuronal migration and axonal growth. Transplantation of developing cells in the cortex will be used to probe the cellular interactions that mold neuronal function. ***

DESCRIPTION (provided by applicant): The cerebral cortex is the brain structure that underlies our highest cognitive and perceptual abilities. During development, proliferating cells of the cortical neuroepithelium generate young neurons that migrate away from their site of origin into distinct positions within the cortex, from which they form specific axonal connections. Defects in the production and migration of cerebral cortical neurons have fundamental implications for mental health. An increase in the size of the cerebral cortex is observed in young children with autism, and migration disorders have been implicated in schizophrenia, bipolar affective illness, epilepsy, and dyslexia. We also note that of the genes known to be mutated in X-linked mental retardation, at least six involve signaling pathways that utilize Rho family GTPases, which are implicated in neurogenesis and migration. The goals of our research are to identify molecules that regulate neurogenesis, to determine how cortical progenitors decide whether to produce neurons or more progenitors, and to characterize the mechanisms by which young neurons migrate to appropriate positions in the brain. The following questions are addressed: 1) How do cilia influence the proliferation of cortical progenitor cells? We hypothesize that the cilia regulate signaling systems that control the proliferation of neural progenitor cells. 2) How does the spatial regulation of adhesion, tension, and endocytosis contribute to neuronal migration? We propose to visualize the establishment of adhesive contacts between migrating neurons and the extracellular matrix and to explore the hypothesis that these contacts are progressively weakened by clathrin-mediated endocytosis. 3) What role do Rho family GTPases play in regulating neural progenitor proliferation and the migration of young neurons? To explore the roles of Rac1 and Cdc42 at early stages of neuronal development, we propose to use conditional genetics to ablate Cdc42 or Rac1 in the developing mouse brain and to assess the roles of these genes in the proliferation of neural progenitor cells and in neuronal migration. PUBLIC HEALTH RELEVANCE: We are exploring the molecular mechanisms that control the production and migration of neurons in the cerebral cortex. An increase in the size of the cerebral cortex is observed in young children with autism, and migration disorders have been implicated in schizophrenia, bipolar affective illness, epilepsy, and dyslexia. Our studies focus in part on Rho family GTPases, which are implicated in X-linked mental retardation.

During development, young neurons within the nervous system must form specific connections to both local and long distance targets. Within the cerebral cortex, the majority of synaptic connections are formed by locally-projecting neurons; thus it seems likely that the malformation of such connections will be implicated in the generation of at least some of the epilepsies of childhood. Indeed, use of high resolution MRI has made it increasingly apparent that a number of the epilepsies are caused b cortical malformations. We need to understand much more about the rules by which connections form in the normal cortex, in order to understand and possibly treat the defects within cortical malformations that lead to epilepsy. The rules by which local connections are established during development appear to employ highly specific mechanisms of laminar recognition. The major aim of this research proposal is to understand the processes by which young neurons in the mammalian neocortex form local (intrinsic) axonal connections with appropriate sets of target neurons in restricted sets of layers. We will explore the development of the local projections of neurons the cells of cortical layer 2/3, which form extensive interconnections within layer 2/3 and also project to the deep layers 5 and 6; those of neurons in layer 5, which projects up to layer 2/3 and to the deep layers; and finally those of the cortical layer 6, which sends a feedback projection to layer 4. The following specific aims are outlined: 1) We will use organotypic cultured slices of the developing visual cortex to ask whether local circuit formation be recapitulated in slices, and whether axons require activity to establish their layer-specific patterns of axon collaterals. 2) By placing neurons in ectopic laminar positions, we will ask whether the formation of local projections an active process that involves the specific recognition of layer-specific cues. 3) Using membrane stripe assays in which neurons of the different layers are presented with a choice of growth substrates, we will explore the nature of the molecular cues that direct local axonal growth and sprouting in the cortex.

The Gordon Conference on Neural Development has evolved into one of the key meetings in the field. The subject of developmental neurobiology is very complex, requiring integration of hypotheses and information at the molecular, cellular, and systems levels. The conference is intended to bring together such a mixture of research groups in a format highly conducive to both formal and informal exchange. The emphasis will be on the discussion of cutting-edge, unpublished research. In addition, the breadth of the meeting provides an excellent opportunity for those who are beginning their careers or moving into a new subject area. The meeting is small (approximately 125 participants); however, the Chair and Vice-Chair will strive to ensure that a diverse group of both junior and senior investigators attends. The financial support requested will also be used to increase the numbers of women and minorities participating in this meeting. The speakers we have chosen represent not only some of the most active groups, but also individuals with the capacity to generate useful discussion of their own and other topics. The concept of the meeting has been not to try to cover the entire field thinly, but to focus on areas of exceptional activity or promise. Featured in this year's program are the following topics: neural induction and pattern formation, regionalization of the neural axis, stem cells, fate determination of neurons and glia, cell migration and axon outgrowth, axon guidance, synaptic and glial differentiation, and synaptic plasticity. Within these topics, we have included several speakers whose research has important clinical applications. The meeting is well-balanced, containing both promising young investigators as well as more senior leaders in the field, with a third of the speakers being women. Forty-five to fifty minutes will be allowed for each speaker's topic, of which one-third will be devoted to discussion. The afternoons are open for informal interactions. Several poster sessions in which conferees can present their work will be scheduled; these have been extremely well attended at previous conferences. Most participants will be expected to present a poster, thus this meeting will serve both a scientific and training function.

DESCRIPTION (provided by applicant): An early step in the creation of the nervous system is the generation of neurons and glial cells, the building blocks of all neural circuits. During development, some cells exit the cell cycle and differentiate into neurons or glia, while others reenter the cell cycle and remain as progenitors. Both extrinsic factors (such as peptide growth factors) and intrinsic factors (such as cell-surface receptors) play important roles in the regulation of proliferation, fate specification, and differentiation. The goal of the present proposal is to test the roles of these molecules in neurogenesis and patterning of the developing cerebral cortex. Such studies have been hampered by the realization that relatively small numbers of gene families are utilized over and over during development, with the outcome of a signaling event determined largely by the type of the responding cell and the developmental context in which a signal is presented. Because of the repeated utilization of a small set of signals at a number of different times, places, and stages of development, it can be difficult to use genetics explore the role of a particular signaling system in a particular tissue in vivo. We will use conditional molecular genetic techniques in the mouse to explore the role of extrinsic signaling molecules and their receptors in the production of cortical neurons in vivo, and to compare directly the roles of these signaling systems at distinct times during development. By using mice that express the bacteriophage recombinase Cre in telencephalic progenitor cells, we will generate conditional knockouts or express dominant negative forms of signaling molecules in the developing brain. We will focus on the roles of Bone Morphogenetic Proteins (BMPs) and Fibroblast Growth Factors (FGFs), in the control of patterning, cell number, phenotype, and differentiation in the developing cerebral cortex, Our first aim addresses the hypothesis that BMP signaling induces the development of the dorsal midline. We will perform conditional knockouts of Bmp4 or its major receptor Bmpr1a, either alone or in combination with a null mutation in Bmpr1b, at the earliest stages of telencephalic specification and analyze the effects of each mutation on cerebral patterning and dorsal midline development. The second aim is to examine the role of FGF signaling in the development of anterior-posterior patterning and the formation of cortical areas. We will generate conditional knockouts of the FGF receptors Fgfr1 and Fgfr2, alone or in combination with a null mutation in Fgfr3, to assess the roles of these signaling molecules in patterning cortical areas, Our third aim uses genetics to explore the opposing roles of BMP and FGF signaling in cortical cell proliferation (stimulated by FGFs), neurogenesis (promoted by BMPS), and the production of glia.

A major challenge in the study of mammalian brain development is to identify the extracellular ligands and receptors that direct the assembly of the complex wiring pattern of the brain. A recent modification of the gene trap technique in embryonic stem cells, the "secretory trap", allows the systematic trapping of genes encoding ligands and receptors, and the generation of mice harboring mutations in these genes, providing an important tool to elucidate the functions of these genes. However, the problem with studying brain wiring is that it can be extremely difficult to identify changes in wiring resulting from experimental perturbation because of the difficulty of tracing the axonal projections of neurons. In particular, it is usually extremely difficult to identify wiring defects in mutant mice, whether the mutation arose spontaneously or was generated by gene targeting or insertional mutagenesis. We have conceived a further modification of the gene trap technique that should now dramatically facilitate the identification and characterization of receptors involved in wiring the mammalian nervous system. In our method, a histochemical axonal marker is targeted to neurons that normally express the trapped gene. This enables direct visualization of the connections made by these neurons, and direct visualization of wiring defects in homozygous mutant animals, through a simple histochemical stain. In this way the role of the trapped gene in brain wiring can be rapidly assessed. Of equal importance, and of great benefit to the Neuroscience community at large, the method will also simultaneously result in the generation of a bank of mice expressing the histochemical marker in different populations of axons, which will provide an important tool for researchers interested in elucidating the normal pattern of neuronal connections in the mammalian brain. A request is made here to support the isolation of a large number of secretory trap ES cell lines using this modified vector. It is proposed that 80 of these lines per year will be put through the germ line for expression and phenotypic analysis to identify those involved in wiring the nervous system, and to provide a resource of mice expressing the axonal marker in different populations of neurons for use by the Neuroscience community. We expect that this method will help greatly accelerate the pace of discovery of mechanisms that direct the wiring of the mammalian nervous system.