Richard S. Nowakowski - US grants

The goals of this research are to reach a better understanding of the sequence of events which lead a young neuron from the proliferative zones to its proper position in the mature brain, to begin to understand how specific genes might contribute to (and/or disrupt) the migratory process, and to analyze the capacity of neurons which are in the wrong position to make adjustments in their connectivity while differentiating. The approach to be taken will be to exploit the existence of mutations in the mouse which disrupt the migration of neurons in the developing central nervous system and thereby produce malformations of the adult brain in which specific populations of neurons reside in abnormal positions. Specific aims of this project are: 1) to determine the development processes modified by four different genetic defects which affect the position ultimately assumed by young neurons during their migration from the proliferative zone to their final position during the development of the central nervous system, and 2) to analyze in the mature central nervous system the changes in the axonal and dendritic arborizations of neurons which migrate to the wrong position during the developmental period. Experiments will be conducted which will: 1) explore the relative contributions of the migrating neurons themselves, of radial glial cells, of previously generated neurons, and of axons from adjacent structures in influencing the "stopping" point along the migratory trajectory of a young neuron, 2) analyze the morphological differences between neurons which have reached a "wrong" position and those which have assumed the "correct" position, and 3) clarify some aspects of the genetics of the experimental situations. This work is significant because of its contribution to the understanding of cell migration during the development of the central nervous system.

Rapid technical developments in electronics and computing have recently made it possible to collect and analyze data concerning the three-dimensional structure of cells. These advances are particularly important to the field of neurobiology because of the three-dimensional complexity of neurons and the relationship of this complexity to the function of the nervous system. it is now possible to obtain accurate, reproducible measurements and reconstructions of the complex shape of neurons. The availability of this technology is changing the way neurobiologists conceive of cell-cell interactions in both the adult and developing nervous system and is contributing greatly to an improved understanding of the functional significance of this three-dimensional complexity. The equipment requested has three purposes: 1) three-dimensional reconstruction of the dendritic trees of neurons impregnated by the Golgi method or filled by an intracellular dye, 2) three-dimensional reconstruction of electron microscopically obtained serial sections, and 3) the enhancement and digitization of images stained with fluorescent dyes or visualized by differential interference contrast or other optical enhancement methods. This equipment or other equipment with similar capabilities is not available on this campus or on the adjoining campus of Rutgers University. The equipment requested in this application will provide a diverse group of neuroscientists with modern morphological capabilities that are essential to the contemporary neurobiology.

The regulation of cell number and, more specifically, neuron number in the developing CNS is a largely unexplored question. Any answer to this question must consider two important developmental issues: 1) the regulation of neuronal production and 2) the phenomenon of naturally occurring cell death. This project is concerned with the first of these issues. We will determine: 1) how the relative number of proliferative cells changes during the development of a structure, 2) how frequently the proliferative cells divide, and 3) what proportion of the proliferative population becomes permanently post-mitotic at each pass through the cell cycle. We will examine this issue in four different proliferative zones in the developing hippocampal region of the mouse: 1) the ventricular zone of the hippocampus and subiculum, 2) the ventricular zone of the periallocortex (presubiculum, parasubiculum and entorhinal area, 3) the subventricular zone of the periallocortex, and 4) the intrahilar proliferative zone of the dentate gyrus. We will measure the length of the cell cycle (Tc) and the DNA-synthetic phase (Ts) for all of the proliferative population and also for that subpopulation which will produce neurons. For several ages, the proportion of the daughter cells that leave the proliferative zones to become permanently post-mitotic will be determined to test the hypothesis that during developing that proportion increases gradually such that the proliferative population becomes self-exhausting several generations before cell proliferation for that structure ceases. The output of the proliferative zones will be measured by determining the distribution of cells that leave each of the four proliferative populations within a one-hour period (i.e., a "one-hour cohort"). The pattern of distribution of labeled cells from retroviral infections for progeny from three of the four different proliferative populations will be determined. We will develop three probabilistically- driven cytogenetic and histogenic models, a cytokinetic model, and output (or cell proliferation) model, and a cell dispersion model. The model will be used to determine if the results of the various experiments are consistent internally and with each other and to make specific testable predictions. The major methods to be used are: 1) bromodeoxyuridine immunohistochemistry and tritiated thymidine autoradiography both alone and in a series of double labeling experiments, and 2) retroviral transfection of clonally related populations.

DESCRIPTION: The applicant proposes to examine the effects of space flight and microgravity on the cells of the developing cerebral cortex. These studies will examine three cellular processes: 1) cell proliferation, 2) neuronal migration, and 3) neuronal differentiation, including dendrite/axon orientation and acquisition of appropriate neuronal circuitry. Two markers of cell proliferation (bromodeoxyuridine and tritiated thymidine) will be administered to pregnant rats on selected days during the development of the cerebral cortex. Short term analyses (sacrifice fetuses 2.5 hrs post-injection) will examine the responses of cell proliferation to space flight by examining changes in the number of proliferating cells or the length of the cell cycle. Intermediate-term effects will be assessed by sacrificing the fetal animals 1-3 days post-injection. The migratory fates of cells within the developing cerebral cortex will be examined. Long-term effects will be assessed by sacrificing animals that were treated as fetuses at different ages through 30 days after birth. The final position of cells born at particular ages will analyzed.

DESCRIPTION (provided by applicant): Mammals, including humans, lacking pigment in the retinal pigment epithelial cells have a number of abnormalities of the retina and visual system. Similar abnormalities occur in albinos that lack pigment in the entire body including the eye and in ocular albinos that lack pigment only in the eye. We propose that the lack of pigment affects cell proliferation early in the development of the neural retina and that these abnormalities in cell proliferation affect both cell number and cell class in the adult retina. We describe a hypothetical set of behaviors for the proliferating cells of the retina in pigmented vs non-pigmented eyes and then propose specific experiments to test this hypothesis. For this project mice with two different types of non-pigmented retinas will be used: C57BL/6J-tyrc2J/tyrc2J, which are albino, and Oa1tm1Inc/Y which are ocular albinos. These mice have mutations at different locations in the pigment processing pathway, and both have phenotypes that are substantially similar to their human counterparts. The Oa1tmInc mouse is a murine homolog of human Oa1 (also known as Nettleship-Falls Type Ocular Albinism). The pigmented mice that we will use are chosen to have the same genetic background as the non-pigmented mice. The project has 3 specific aims which will examine at high spatial and temporal resolution: 1) the cell cycle, 2) the proportion of daughter cells leaving vs remaining in the cell cycle, and 3) the generation of cells of specific retinal classes. We will achieve both high spatial and temporal resolution for this project using methods developed in this laboratory that exploit double labeling with two S-phase markers, bromodeoxyuridine and tritiated thymidine. The results obtained will tell us the earliest differences in the production of cells in the developing retina for pigmented and non-pigmented animals including information about regional differences within the retina. Such detailed information is important for understanding how the lack of retinal pigment produces abnormalities and for the rationale development of treatments and therapies.

[unreadable] DESCRIPTION (provided by applicant): The mouse and human genomes have now been sequenced, but the task of linking genes to functions is just beginning. We will combine old and new technologies as a generalized method of linking genetic loci to functions and also as a method of linking disparate functions to each other. The proposed project depends on the existence of resources that have been produced by mouse geneticists over the past century, including the numerous inbred, recombinant inbred (RI), consomic, and congenic strains. These resources are a rich source of genetic diversity that remains essentially untapped for studies of the central nervous system. In this project we will exploit this genetic diversity to begin to define the set of traits that comprise the "hippocampal phenotype," i.e., what might be termed the hippocampal "phenome." We call this approach a "phenomic analysis." This project will focus on traits that involve inter-strain variation in adult neurogenesis and in hippocampal morphology and development. The general strategy will be to: 1) Identify multiple genetic traits that vary among a set of inbred strains and to use network construction (i.e., a novel approach consisting of a collection of statistical methods and data visualization techniques) to generate hypotheses about the potential relationships among these diverse traits (Specific Aim 1); 2) exploit existing mouse genetic resources, i.e., RI strains and consomic strains, to test the hypotheses generated from the network analysis and simultaneously to map the genetic loci responsible for the identified traits (Specific Aim 2); and 3) to determine the developmental basis for the traits identified as significant and/or possible related from the network analysis of the traits identified in adult animals (Specific Aim 3). As described in Preliminary Results, we have made some progress towards each of these steps, and it is clear that there are numerous genetic traits that we will be able to study and map simultaneously. This "moderate throughput" approach, in effect, provides a genetic dissection of hippocampal structure, function, and development [unreadable] [unreadable]