Margaret Hollyday - US grants

The normal functioning of an animal's limb depends on a highly ordered pattern of connections between motoneurons in the spinal cord and the various muscles of the limb. This pattern is established during development by specific axonal growth. Motoneuron axons normally make a series of pathway choices as they grow from the spinal cord to their target muscle. These choices require axons to recognize and respond appropriately to multiple growth cues associated with cells whose precise location and identity are presently unknown in any vertebrate. A combination of various surgical manipulations and several anatomical techniques will be used to localize and to identify the cell populations which provide the environmental cues to guide growing motor axons to their target muscles. All of the experiments will be done on chick or quail embryos. The first question is whether the cues for the two axes of the limb are separately located along the nerve pathways. Other experiments will determine whether the presence of muscle cells, the targets of motoneurons, are required for accurate pathfinding, or whether connective tissue cells are sufficient. The final series of experiments will explore the role of presumptive sheath cells, or Schwann cells, in assuring normal innervation of limb muscles. An increased knowledge of the location and cellular identity of guidance cues used by growing axons during normal development should aid efforts to understand neuromuscular disorders as well as to promote specific regrowth of adult nervous tissue following damage due either to disease or injury.

health science research support;

biomedical equipment resource; biomedical equipment purchase;

This is an application for a Career Advancement Award to support the applicant while she learns modern molecular biological techniques in the laboratory of another investigator. The objective is to enable the applicant, who has considerable experience with neuroanatomical and experimental embryological approaches to developmental neurobiology, to expand the range of techniques available to her for continued research on fundamental problems in nervous system development. The proposed project will teach her a variety of molecular biological methods including in situ hybridization and some associated recombinant DNA techniques, including PCR, subcloning, gel electrophoresis, plasmid preparation, restriction mapping, sequencing, and transcription of labelled RNAs. In the future, the applicant expects to use these methods in her own laboratory in combination with other traditional approaches for continued studies of patter formation in the central nervous system.

DESCRIPTION (provided by applicant): The large question motivating this specific project is to understand how developmental processes responsible for patterning the dorso-ventral axis of the vertebrate neural tube intersect with those regulating proliferation and neurogenesis. In the spinal cord neuron production and differentiation follows a ventral to dorsal gradient with motoneuron generation from basal plate precursors preceding differentiation of dorsal interneurons from alar precursor cells. Preliminary results suggest differences in cell generation time of the spinal cord neurepithelium that correlate with both developmental stage and position on the dorso-ventral axis. Experiments are proposed to further establish these differences and to explore regulation of these events at both embryological and molecular levels. The applicant proposes to test whether signals from the paraxial mesoderm and/or the ectodermal epithelium differentially influence cell generation time and neurogenesis in the dorsal and ventral regions of the spinal cord using experimental embryological methods. Cumulative BrdU labeling and immunocytochemistry will be used to estimate changes in cell cycle parameters and neuronal differentiation in normal and surgically manipulated embryos. Possible effects of surgical manipulations on cell death will be addressed using TUNEL assays. Molecular regulation of neurogenesis in the spinal cord will be studied by evaluating the expression patterns of four Id family members using in situ hybridization. Id family members are known to be negative regulators of the positive acting basic helix-loop-helix transcription factors, and they function in other systems as inhibitors of cell differentiation. Id proteins have also been implicated in cell cycle control and they are target genes of molecules involved in specifying the axes of the spinal cord. The applicant proposes to test the hypothesis that one or more of these genes is expressed in proliferating spinal cord neural epithelium, and that expression is down-regulated when cells withdraw from the cell cycle and begin to differentiate. The results of the initial studies will be used to guide future investigations on the patterned regulation of neurogenesis and its cessation, events that are critical for normal nervous system development. Deficiencies in neurogenesis may be underlying causes of human congenital brain abnormalities and/or mental retardation.