Gunther S. Stent - US grants

A central problem in developmental neuroscience is how individual neurons acquire a constellation of idiosyncratic properties, such as their location, shape, function and synaptic connections, that define their uniqueness. This issue will be addressed in the nervous system of the leech embryo, a species that offers the advantages of having relatively few, stereotyped cells that are easily accessible to experimental manipulation. The mechanisms by which segmentally repeated patterns of neuronal development are modulated differentially among the 32 body segments to provide for the specialized functions of the various body regions, such as the head, the reproductive organs and the tail, will be studied. The hypothesis to be tested is that the segment-specific modulation of the developmental pathway is the consequence of an intracellular subdivision of an early embryonic precursor cell into sectors that are differentially labeled with indicators of the future longitudinal body axis and are parcelled out in an orderly manner to the daughter cells that found successive body segments. Cells in the embryo will be marked by intracellular injections and marking of the cell surface with cell lineage tracers. Molecular cell cloning technology will also be used to identify homeotic genes in the leech that may be involved in specifying neurons to specific body segments. The study of the mechanisms underlying neurogenesis in this simple nervous system promise to provide insights of general relevance to the understanding of nervous system development in more complex organisms.

The long-term objectives of this project are to arrive at an understanding of how the nervous system generates behavior and how its neuronal components and circuitry arise during ontogeny. To this end, we have been studying the structure, function and development of the nervous system of leeches for the past ten years. The research plan proposed in the application concerns the development of the nervous system in embryos of the neurologically and embryologically highly favorable leeches Haementeria ghilianii, Helobdella triserialis and Theromyzon rude. Although leeches are phyletically remote from mammals, it can be expected that this project will provide insights of general relevance for understanding developmental mechanisms, including those governing normal and abnormal human development. Our specific aim are: 1) To investigate the developmental mechanism of mutual avoidance of growing axon branches belonging to the same neuron that was found to be operative in the establishment of sensory fields in the embryonic leech skin and to obtain experimental support for the hypothesis that this recognition is based on the detection of coherent electrical activity. 2) To examine the possible guidance role of identified peripheral neuronal cell bodies and of identified early muscle fibers in the stereotyped outgrowth pattern of axon branches from embryonic neuronal cell bodies by obsserving the effects on axon outgrowth pattern of photoablation of putative embryonic guidance cells, rendered specifically photosensitive by inheritance of a novel fluorescent cell lineage tracer from their precrusor blastomere. 3) To investigate the conditions which are necessary for the biochemical differentiation by which each postmitotic neuroblast gains the capacity to synthesize its cell-specific neurotransmitter, by exposing embryos to anti-mitotic agents at various stages prior to the overt appearance of neurotransmitters. 4) To secure a sset of monoclonal antibodies capable of binding specifically to antigens carried by some but not all of the embryonic neuronal precursor cells, in the hope that some of these antigens are, or are products of, the determinants of cell fate which can be inferred to segregate asymmetrically in successive cleavages, in accord with the stereotyped cell-lineage relations that govern neuronal development in the leech.

The long-term objective of this project is to ascertain the relative roles of cell lineage and cell interactions in the determination of the course of normal and abnormal embryonic development. This project, which uses the embryos of leeches as its working material, is related to health, in that its results are likely to provide insights into the causes of human birth defects. The proposed research plan concerns the embryo of the leech Helobdella triserialis. It addresses the general problem of the commitment to their specific developmental fates of the four bilateral pairs of embryonic blast cell bandlets, designated as n, o, p and q, whose descendant cell clones give rise to the ectodermal tissues of the 32 bilaterally symmetric, serially homologous segments of the adult leech. One main focus of interest is the commitment of cell clones descended from the initially uncommitted, pluripotential primary o and p blast cells to one of two alternative developmental pathways. An empirical test for the commitment of blast cell clones is provided by the phenomenon of "transfating": upon ablation of the p bandlet, an as yet uncommitted o bandlet cell will follow the P developmental pathway, whereas an already committed o bandlet cell will follow the O pathway. The particular developmental pathways taken will be identified by means of cell lineage tracers. The specific aims of this project include a direct demonstration that each of the three successive commitment steps to the O pathway is associated with an asymmetric division in the o blast cell line and that the elements of the fate for which joint commitment occurs in each of the three successive steps are clonally related. The specific aims include also an elucidation of the possible role in the commitment process of gap junctions linking blast cells of adjacent bandlets and of the nature of the pathway guidance which the committed blast cells of the p and q bandlets receive in their centripetal migration from their lateral points of origin in the germinal plate towards the ventral midline, to the positions in which they, or their daughter cells, eventually differentiate into neurons or glia.

The long-term objectives of this project are to arrive at an understanding of how the nervous system generates behavior and how its neuronal components and circuitry arise during ontogeny. To this end, we are studying the structure, function and development of the nervous system of leeches. The research plan proposed in this application concerns the development of the nervous system in embryos of the neurologically and embryologically highly favorable leeches Haementeria ghilianii, Helobdella triserialis and Theromyzon rude. Although leeches are phyletically remote from mammals, it can be expected that this project will provide insights of general relevance for understanding developmental mechanisms, including those governing normal and abnormal human development. Our specific aim is to identify the elements of the metameric mesodermal cell clone descended from each primary m blast cell that are necessary for laterad migration of neuroblasts within the ganglionic rudiment and that provide topographic cues for the orthotopic placement of neuroblasts and axon outgrowth in ganglion morphogenesis. Although circular and longitudinal muscle fibers are prime candidates for the elements of the mesodermal cell clone that guide neuroblast migration, the mesodermal tissues in contact with the ganglionic rudiment will be examined for the presence of other cell types which might perform that morphogenetic function. Various partial disruptions of the normal mesodermal cell pattern or function will be produced and these disruptions will be correlated with the resulting abnormalities of neuronal positioning or axonal projection patterns. Embryos will be exposed to oligopeptides known to interfere with binding of fibronectin-like adhesion proteins in order to interfere with migration of neuroblasts by inhibition of their adhesion to the substrate provided by mesodermal cell types.