Transcriptome Analysis of Identified Cells in Developing Xenopus laevis CNS

描述（申请人提供）：非洲爪蟾（Xenopus laevis）一直是研究神经发育的重要模型系统。近年来，由于缺乏分子和细胞操作所需的遗传工具，它的效用已被其他模型系统所掩盖。尽管如此，非洲爪蟾的许多特征使其成为研究、操纵和测试体内神经元发育模式的独特和不可替代的模型。拟议研究的重点有三个方面：1)为爪蟾群落提供急需的遗传信息，包括爪蟾大脑多个区域、几种已鉴定的神经细胞类型和发育阶段的基因转录本和microrna的序列和表达；2)为已鉴定的转录本和信号通路（包括反义morpholino构建物）的分子/遗传操作提供试剂；siRNA和感兴趣基因的细胞型特异性表达；3)利用试剂探测候选调控基因在完整动物神经发生中的功能。拟议的工作将产生丰富的试剂，这将大大提高非洲爪蟾作为生物医学研究的实验模型的使用，并允许跨物种比较研究，这将丰富其他实验系统的研究。

DESCRIPTION (provided by applicant): Xenopus laevis has been a valuable model system for studying neural development. In more recent years its utility has been eclipsed by other model systems simply because it lacks the genetic tools required for molecular and cellular manipulations. Nevertheless, many features make Xenopus a unique and irreplaceable model for studying, manipulating and testing neuronal developmental paradigms in vivo. The focus of the proposed studies is threefold: 1) to provide the Xenopus community with much needed genetic information on the sequence and expression of gene transcripts and microRNAs in several regions of the X. laevis brain, in several identified neural cell types and at several developmental stages, 2) to generate reagents for molecular/genetic manipulations of identified transcripts and signaling pathways, including antisense morpholino constructs, siRNA and cell-type specific expression of genes of interest and 3) to use the reagents to probe the function of candidate regulatory genes in neurogenesis in the intact animal. The proposed work will generate a wealth of reagents that will significantly enhance use of Xenopus as an experimental model for biomedical research and permit cross-species comparative studies that will enrich research in other experimental systems. PUBLIC HEALTH RELEVANCE: This project will characterize the transcriptome of identified cells in the brain of developing Xenopus laevis tadpoles using whole transcriptome sequencing to identify genes that are differentially expressed in targeted neural progenitor cells. Using the sequencing data we will develop appropriate expression constructs, siRNA constructs and antisense morpholinos to probe the role of candidate regulatory genes in neurogenesis in vivo. These studies will provide new genomic resources for the Xenopus community and promote the use of Xenopus as an experimental system to study developmental brain disorders.