DISSECTING THE GENE REGULATORY NETWORK FOR EPIDERMIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 项目说明 表皮是动物与环境之间的主要界面。表皮的发育依赖于控制其分化和形态发生的基因调控网络。这一调控网络的破坏导致了许多人类发育和疾病综合征。本研究旨在以同源盒转录因子基因D11B为起点，对一种新兴的模式生物--海鞘幼虫的表皮发育基因调控网络进行解剖。本研究计划利用本实验室开发的转基因试剂改变表皮细胞规范CiD11B的关键调控因子的表达，并测量对表皮发育的基因调控网络的影响。其具体目的是：1)检测由外胚层到内胚层细胞中CiD11B错误表达所引起的胚胎中假定的靶基因表达水平的变化；2)完成CiD11B显性负向转基因并检测胚胎表型和对可能的靶基因表达水平的影响；3)预测先前克隆的CiD11B调控元件中的结合位点，并通过定点突变分析上游调控；4)建立卵裂抑制方法，通过卵裂球分离分离外胚层和内胚层细胞，以发现外胚层谱系中差异表达的基因。这些目标将使我们能够制定和验证适用于我们正在开发的更大项目的提案的基因调控网络发现方法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. PROJECT DESCRIPTION The epidermis is the principal interface between an animal and its environment. Development of the epidermis is dependent on a gene regulatory network that controls its differentiation and morphogenesis. Disruption of this regulatory network causes a number of human developmental and disease syndromes. This proposal aims at the dissection of the gene regulatory network for epidermal development in an emerging model organism, the ascidian Ciona intestinalis, taking the homeobox transcription factor gene DllB as a starting point. The research plan is to use transgenic reagents developed in our laboratory to alter expression of the pivotal regulator of epidermal cell specification CiDllB, and measure effects on the gene regulatory network for epidermal development. The specific aims are 1) measure changes in putative target gene expression levels in embryos altered by misexpression of CiDllB from ectodermal to endomesodermal cells; 2) complete a CiDllB dominant negative transgene and examine the embryonic phenotype and effects on putative target gene expression levels; 3) predict binding sites in previously cloned CiDllB regulatory elements and analyze upstream regulation by site-directed mutagenesis; 4) develop methods for cleavage arrest and to separate ectodermal from endomesodermal cells by blastomere isolation in order to discover genes differentially expressed in the ectodermal lineage. These aims will allow us to work out and validate methods for gene regulatory network discovery applicable to proposals for larger projects which we are developing.