IN SITU LOCALIZATION OF INFORMATIONAL EMBRYO RNAS

最近的研究表明，到了囊胚期，所有主要的细胞系 海胆胚胎(紫球海胆)致力于， 并且可以通过不同的基因表达模式来识别。我们的注意力 因此被非常早期的囊胚所吸引，因为那时 必须发生血统确定的关键事件。我们想要学习 表达仅限于早期囊胚和 使用谱系特异的mRNA作为标记来研究相互作用 细胞之间的差异影响它们对特定基因程序的承诺 表情。 编码mRNAs的重组DNA克隆 早期发育阶段将被分离并用作原位检测的探针 杂交分析用于确定不同发育阶段的mRNA分布 关于发展的问题。单个mRNA丰度的时间模式将 用RNA印迹和滴定方法进行分析。原位杂交将 用来显示单个mRNA是否也在 卵子发生。将选择用外胚层表达的序列的子集 用于详细研究和研究蛋白质的功能 它们代表着。为了寻找这些克隆发挥作用的线索， 将使用测序和适当的重组结构来产生 融合蛋白在大肠杆菌中的表达。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置 细胞蛋白质及其亚细胞分布。在适当的时候 病例抗体也将被用来尝试评估体内 阻断靶蛋白功能的效果。的潜力 还将探索其他替代遗传技术(例如反义RNA)。 如果可行的话，这些方法也将被用来探索 精选的蛋白质。 正常基因表达模式的建立往往取决于 细胞之间的感应相互作用，以及位置线索。这个 我们不断扩展的针对谱系特异性mRNAs的探针集合与 监测其表达的原位方法提供了一种评估 分离细胞编制正常基因程序的能力 并分析个体早期的发育能力 单独的和特定的组合中的卵裂球。这一分析还将 确定其表达需要细胞间相互作用的特定mRNAs。 将对早期胚胎的特定亚组分进行研究，以确定 实现基因表达的时间和空间调制 调节胚胎形态以产生正常胚胎。

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.