The brain of most vertebrate embryos initially grows by expansion of its fluid-filled cavity much like a balloon expands. This rapid growth is under the direct control of internal fluid pressure. For such pressure to be generated, the early brain must become a closed system, a relatively little understood process that involves occlusion of the spinal cord lumen. The occluded spinal cord exhibits close apposition of the internal walls lining the lumen which serves to seal off the cavity from the outside and to confine fluid eventually to the brain. Occlusion occurs just before the onset of rapid brain enlargement, is short lived, and exhibits a definite repeatable pattern both temporally and positionally in chick and human embryos. Studies with chick embryos have shown what occlusion looks like at the light and EM level, where and when it occurs along the spinal cord, and to a limited extent, how it is maintained. This proposal focuses on identifying in greater detail the mechanisms initiating and maintaining occlusion.
Our specific aims are to determine: (1) what factors intrinsic (cell shape change) and/or extrinsic (somites, perineural ECM, notochord, embryo elongation) to the neuroepithelium initiate occlusion; and (2) whether occlusion is initiated and/or maintained by cytoplasmic contractile proteins (actins). If so, the route by which calcium modulates contraction will be assessed (calmodulin/cAMP). Putative extrinsic and intrinsic mechanisms responsible for the initiation and/or maintenance of occlusion will be isolated by applying appropriate chemical or sugical probes to living chick embryos to selectively eliminate a particular factor. Such inhibitions or deletions will be carried out prior to the time in embryogenesis when occlusion is known to occur (stage 8). Ten to fifteen embryos will be used for each control and treatment group. The treated embryos along with appropriate control embryos will be allowed to develop to the period in embryogenesis when occlusion is most prominent (stage 12) and at that time occlusion will be assessed physiologically (injection of dye into brain) and anatomically (examination of serial cross sections). This approach will identify some factors responsible for initiating occlusion which will aid understanding of the mechanisms of normal brain development in vertebrates. Isolating mechanisms of normal development of the central nervous system may provide insight into the mechanisms of genesis of certain congenital malformations such as microcephaly and hydrocephaly.
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