The question of how a single cell, the egg, becomes a multicellular organism with differing tissue types and form is a question basic to embryology and developmental biology. It has been speculated that localized cytoplasmic components (cytoplasmic determinants) may have a regulatory role in the initial stages of embryogenesis and it has also been speculated that RNA and/or protein are likely candidates for these regulatory molecules. However, few experiments exist which demonstrate this point. The existence of regionally localized molecules (RNA, protein, or other components) in the oocyte or embryo does not necessarily mean that the component is a cytoplasmic determinant; however, it is suggestive that the component may have a biologically important role (otherwise a uniform distribution of the component would be expected). In this proposal oocytes and embryos of Xenopus laevis will be used to study localized RNA and protein because: a) spatial maps of localized mRNAs exist in these oocytes (of all existing systems the Xenopus oocytes are best defined for poly(A)+RNA and mRNA); b) the synthesis of RNA and protein are relatively well understood during early development; c) oocytes and embryos are easily obtainable and their large size allows for easy manipulation. Their large size allows the spatial maps to be used as a guide for spatial fraction (division of the single-celled oocytes and embryos into regions) and then biochemical analysis of the RNA and protein in each region. The spatial fractionation will be performed by freezing, and sectioning the eggs in a cryotome, dehydration of eggs and """"""""cutting-out"""""""" of the desired region, and removal of regions of the egg cortex. In the mature oocyte the amount and type of RNA (or protein) in each region will be determined. The in vitro translation products of the extracted regional RNA will be compared with: 1) the steady-state proteins in the region, 2) the distribution of newly-synthesized proteins in each region. This will determine if a mechanism exists for the sorting-out of proteins. This analysis will be performed during all of oogenesis, meiotic maturation, and early embryogenesis. Antibody probes will be made to region-specific proteins to a) analyze the spatial distribution of the proteins, b) obtain polyribosomes for the proteins by precipitation of nascent chains in order to single out specific RNA's for future analysis. In addition, the mechanisms involved in maintaining the localized mRNA in Xenopus oocytes will be examined.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Modified Research Career Development Award (K04)
Project #
5K04HD000598-03
Application #
3073170
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1985-09-01
Project End
1990-08-31
Budget Start
1987-09-01
Budget End
1988-08-31
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Arizona State University-Tempe Campus
Department
Type
Schools of Arts and Sciences
DUNS #
188435911
City
Tempe
State
AZ
Country
United States
Zip Code
85287
Gallicano, G I; McGaughey, R W; Capco, D G (1992) Cytoskeletal sheets appear as universal components of mammalian eggs. J Exp Zool 263:194-203
Bement, W M; Gallicano, G I; Capco, D G (1992) Role of the cytoskeleton during early development. Microsc Res Tech 22:23-48
Bement, W M; Capco, D G (1991) Analysis of inducible contractile rings suggests a role for protein kinase C in embryonic cytokinesis and wound healing. Cell Motil Cytoskeleton 20:145-57
Gallicano, G I; McGaughey, R W; Capco, D G (1991) Cytoskeleton of the mouse egg and embryo: reorganization of planar elements. Cell Motil Cytoskeleton 18:143-54
Dersch, M A; Bement, W M; Larabell, C A et al. (1991) Cortical membrane-trafficking during the meiotic resumption of Xenopus laevis oocytes. Cell Tissue Res 263:375-83
Bement, W M; Capco, D G (1991) Parallel pathways of cell cycle control during Xenopus egg activation. Proc Natl Acad Sci U S A 88:5172-6
Capco, D G; Bement, W M (1991) Analysis of cellular signaling events, the cytoskeleton, and spatial organization of macromolecules during early Xenopus development. Methods Cell Biol 36:249-70
Bement, W M; Capco, D G (1990) Synthesis, assembly and organization of the cytoskeleton during early amphibian development. Semin Cell Biol 1:383-9
Smith, R C; Bement, W M; Dersch, M A et al. (1990) Nonspecific effects of oligodeoxynucleotide injection in Xenopus oocytes: a reevaluation of previous D7 mRNA ablation experiments. Development 110:769-79
Bement, W M; Capco, D G (1990) Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis. Cell Regul 1:315-26

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