IN SITU LOCALIZATION OF INFORMATIONAL EMBRYO RNAS

点击翻译按钮获取中文摘要

Studies have recently shown that by blastula stage all major cell lineages of the sea urchin embryo (Strongylocentrotus purpuratus) are committed to, and identifiable by, distinct patterns of gene expression. Our attention is therefore drawn to the very early blastula because it is then that critical events of lineage specificiation must occur. We wish to study genes and gene products whose expression is confined to early blastula and to use lineage-specific mRNA as markers to investigate how interactions among cells affect their committments to specific programs of gene expression. Recombinant DNA clones encoding mRNAs whose expression is restricted to early stages of development will be isolated and used as probes for in situ hybridization analyses to determine mRNA distributions at different stages of development. The temporal pattern of abundance of individual mRNAs will be analyzed by RNA blot and titration methods. Hybridization in situ will be used to show whether individual mRNAs also are expressed during oogenesis. A subset of sequences expressed in ectoderm will be selected for detailed study and for investigation of the function of the proteins they represent. To search for clues to function these clones will be sequenced and appropriate recombinant constructions will be used to produce fusion proteins in E. coli. Antibodies prepared against these fusion proteins will allow determination of time and site of expression of the cellular proteins, and their subcellular distribution. In appropriate cases antibodies will also be used in attempts to assess the in vivo effects of blocking the function of target proteins. The potential of other surrogate genetic techniques (e.g. antisense RNA) will be explored and, if feasible, such methods will also be used to probe the function of selected proteins. The establishment of normal patterns of gene expression often depends on inductive interactions among cells, as well as positional cues. The combination of our expanding set of probes for lineage-specific mRNAs with the in situ method for monitoring their expression provides a way to assess the capacity of separated cells to elaborate normal programs of gene expression and to analyze the developmental capacity of individual early blastomeres alone and in specific combinations. This analysis will also identify specific mRNAs whose expression requires cell-cell interactions. Specific subfractions of the early embryo will be studied to deterine the temporal and spatial modulations of gene expression that implement regulation of embryonic pattern to produce normal embryos.