Analysis of Ascidian Axis Development

一个复杂的有机体，如脊椎动物，从受精卵发育成一个多步骤的过程，涉及数千个基因的协调作用。在卵细胞形成成体的过程中，发现了细胞命运决定因素向发育中的胚胎特定区域分离的机制，以及诱导相互作用，在这种相互作用中，细胞之间的交流决定了邻近细胞的发育命运。在这个项目中，史密斯博士将寻求分离在脊索动物发育早期起作用的基因，这些基因与决定特定组织的命运有关。作为这些研究的模式脊索动物，他将使用海鞘（Ciona intestalis）。作为最原始的脊索动物之一，它为研究早期脊索动物的发育提供了许多有利条件。虽然海鞘保留了脊索动物的基本身体结构，但它们在早期发育中有固定的谱系，它们在幼虫阶段只有很少的细胞，它们的基因组只有脊椎动物的5%左右。为了确定对Ciona早期发育重要的基因，将使用表达克隆策略。质粒cDNA文库将从慈母原肠期胚胎中获得。这些文库的体外转录本将作为微注射池注入4细胞期非洲爪蟾胚胎。具有活性的池将被确定为那些给爪蟾胚胎带来发育变化的池。具有活性的单个cdna将通过将文库细分为越来越小的库来分离。通过这种方法分离的基因将通过核苷酸测序来确定，表达模式将通过原位杂交来确定分期的琼瑶胚。

Smith 9724094 The development of a complex organism, such as a vertebrate, from a fertilized egg is a multi-step process involving the coordinated actions of thousands of genes. In elaboration of the adult form from the egg, mechanisms involving both the segregation of cell fate determinants to specific regions of the developing embryo, and inductive interactions, in which cells communicate to determine the developmental fates of neighboring cells, are found. In this project Dr. Smith will seek to isolate genes that act early in chordate development and which are involved in determining the fates of specific tissues. As a model chordate in these studies, he will use the ascidian Ciona intestinalis. Ciona, which is one of the most primitive of the chordates, has many advantages for study of early chordate development. While ascidians retain the basic body plan common to chordates, they have a fixed lineage in early development, they have very few cells at the larvae stage ((2,300), and they have a genome that is only about 5% the size of a vertebrate. To identify genes which are important for early development in Ciona, an expression cloning strategy will be used. Plasmid cDNA libraries will be made from Ciona gastrula stage embryos. In vitro transcripts from the libraries will be microinjected as pools into 4-cell stage Xenopus embryos. Pools with activity will be identified as those that bring about developmental changes to the Xenopus embryos. Single cDNAs with activity will be isolated by sub-dividing the libraries into progressively smaller pools. The identity of genes isolated by this method will be determined by nucleotide sequencing, and the expression patterns will be determined by in situ hybridization to staged Ciona embryos.