Marla B. Luskin - US grants

The mammalian cerebral cortex is an elaborately organized structure containing a constellation of neuronal and glial cell types arranged in laminae. The long term goal of these studies is to understand the mechanisms that govern how this complex organization arises during the formation of the mammalian visual cortex. The role of lineage-derived information in the determination of cell phenotype and establishment of cortical cytoarchitecture will be investigated using as lineage tracers, recombinant retroviruses, which have inserted into their genome exogenous, easily detectable genes, that can be passed on to all the progeny of an infected progenitor cell. The proposed research will combine retroviral lineage tracers with cell type specific antibodies to distinguish the phenotype and neurotransmitter content of clonally related cells, and video microscopy to image over time identified lineally related cells in living slices of developing telencephalon. The specific aims are to : (1) Analyze the lineage relationships among the different subtypes of neurons and glia comprising the cortex. The phenotypic composition of clonally related cells in the cortex will be examined following injections of retroviral lineage tracers into the telencephalon of rodent embryos, to determine which cell types originate from a common precursor at different times during development and when decisions about cell commitment are made. 2) Investigate the distribution of clonally related cells in the mature an immature cortex in order to reveal the positional relationships among clonally related cells. To determine whether clonally related neurons travel to the cortical plate aligned with a group of radial glial fibers and the extent to which the progeny of a single progenitor cell disperse, the arrangement of clonally related cells will be compared to the distribution of radial glial fibers. (3) Study the dynamic aspects of stem cell proliferation and cell migration within cultured slices of developing cortex to unequivocally identify the progeny of an identified progenitor cell and their properties. The analysis of cell lineage in the visual cortex will serve as a foundation for the ultimate identification of genes that are differentially expressed as cells become committed to a specific pathway of differentiation. In addition, these studies are important for understanding the cause of congenital malformations of human cerebral cortex, believed to result from abnormal cell proliferation, migration and gene expression.

The investigators will follow the proliferative and migratory behavior of individual precursor cells and their progeny in mouse telencephalon. They will accomplish this by monitoring the spatial and temporal properties of development dynamically in cultured slices of cerebral cortex, which has never been attempted before. Precursor cells in the ventricular zone of the telencephalon will be marked by either intracellular injection of fluorescein dextran, by extracellular labeling of small numbers of precursor cells with a lipophilic dye, or with a retroviral lineage marker. Subsequently, at selected time intervals, the individual labeled cells as well as their progeny will be visualized by video-enhanced confocal fluorescence microscopy.

The olfactory bulb, as well as the olfactory sensory epithelium, retain the ability to generate new neurons throughout life. The major sources of neurons destined to populate the olfactory bulb, as with other telencephalic structures, are the ventricular and subventricular zones surrounding the lateral ventricles. We have previously identified a region of the anterior part of the subventricular zone, the SVZa, that consists of purely neuronal progenitor cells whose progeny migrate to the olfactory bulb where they differentiate into GABAergic and dopaminergic interneurons. In the proposed studies we will examine the spatial- temporal features that establish the SVZa as a unique germinal zone, and identify signals that control the proliferation and differentiation of its cells. In Specific Aim 1 we will perform in situ studies using cell- type specific antibodies and the cell proliferation marker BrdU to examine the anterior-posterior axis of the ventricular-subventricular zone complex as a function of age. The results will determine if changes in the underlying ventricular zone presage the appearance of the SVZa as a distinct region, or whether the SVZa arises de novo from the subventricular zone. To further determine the place and timing of SVZa genesis, complementary in vitro studies will examine the mitotic behavior and phenotype of SVZa cells from different developmental stages and regions of the ventricular-subventricular complex. In Specific Aim 2 we will determine if epigenetic factors resulting from cell-cell interactions regulate the proliferation and differentiation of SVZa cells. SVZa cells will be cultured either alone or in combination with olfactory bulb and/or olfactory epithelial cells. The proliferation of SVZa cells will be assessed by BrdU-incorporation and their neurotransmitter phenotype determined by immunohistochemical labeling for GABAergic and dopaminergic markers. In Specific Aim 3 we will determine if previously identified growth factors, which are known to influence the development of other neural populations, also influence SVZa cell development. The proliferative behavior of treated SVZa cells will be measured as above, and their neurotransmitter phenotype determined by immunocytochemical labeling. The factors to be tested include: neurotrophins (NGF, BDNF, NT3, NT4/5), ciliary neurotrophic factor, EGF, basic FGF, heregulin, and glial-derived neurotrophic factor. The results of these studies will identify molecular signals that influence the proliferation and differentiation of olfactory bulb interneurons.