IN SITU LOCALIZATION OF INFORMATIONAL EMBRYO RNAS

最近的研究表明，在囊胚阶段，所有主要的细胞谱系 的海胆胚胎（Strongylocentrotus purpuratus）致力于， 并通过不同的基因表达模式来识别。 我们的注意力 因此被吸引到早期的囊胚， 必须发生谱系特化的关键事件。 我们希望研究 其表达仅限于早期囊胚的基因和基因产物， 使用谱系特异性mRNA作为标记， 影响它们对特定基因程序的承诺 表情 编码mRNA的重组DNA克隆，所述mRNA的表达限于 早期发展阶段将被隔离，并作为探针，在原位 杂交分析以确定不同阶段的mRNA分布 发展质量和 单个mRNA丰度的时间模式将 通过RNA印迹和滴定方法进行分析。 原位杂交将 用于显示单个mRNA是否也在 卵子发生 将选择外胚层中表达的序列的子集 用于详细研究和调查蛋白质的功能 他们代表。 为了寻找这些克隆人的线索 测序的和适当的重组构建体将用于产生 融合蛋白E.杆菌 针对这些融合制备的抗体 蛋白质将允许确定表达的时间和位点， 细胞蛋白及其亚细胞分布。 以适当 抗体也将用于尝试评估体内 阻断靶蛋白功能的作用。 的潜力 将探索其他替代遗传技术（如反义RNA） 如果可行的话，这些方法也将用于探测 精选蛋白质 正常基因表达模式的建立通常依赖于 细胞间的感应相互作用，以及位置线索。 的 将我们针对谱系特异性mRNA的扩展探针组与 用于监测其表达的原位方法提供了一种评估 分离的细胞产生正常基因程序的能力 表达和分析个体早期的发展能力 单独的卵裂球和特定的组合。 该分析还将 鉴定其表达需要细胞-细胞相互作用的特定mRNA。 将研究早期胚胎的特定亚组分，以确定 基因表达的时间和空间调节 调节胚胎模式以产生正常胚胎。

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.