Development of the Vertebrate Primary Mouth

描述(申请人提供)：这项提案的目标是以热带非洲爪蛙为模型，确定脊椎动物初级口腔形成的分子机制。初级口腔(也称为口器、颊咽膜)是从胚胎外部到肠道的初始开口。初级口腔形成是颅面部发育的重要步骤，但目前尚不清楚。在脊椎动物中，神经脊在初级嘴巴周围生长，形成颌骨和面部，因此必须形成次级张嘴。然后，主要的嘴巴就变成了咽口。初生口形成于胚胎最前面的一个独特区域，在那里外胚层和内胚层直接并列。为了确定这一过程中涉及的分子机制，我们开始在莱氏X.laevis中对这一过程进行表征。我们绘制了推测的初级口腔图，并确定了诱导所需的组织。为了确定原始口腔形成所需的基因，我们使用数据库挖掘和表达微阵列分析，以确定在假定的原始口腔区域中表达丰富的基因。从这项分析中得出的两个基因是Fgf8和Wnt途径抑制物frzb1，初步数据表明这两个基因对初级口腔形成都是必不可少的。我们建议将这一项目扩展到热带黄蜂。与莱维氏X.laevis相比，该物种具有二倍体基因组的优势，使反义敲除更有效，并提供快速制备稳定转基因株系的能力。最初的数据表明，莱维氏X.laevis和热带X.c.的初级口腔形成非常相似。我假设初级口腔的形成过程是高度保守的。我进一步假设，初级口腔形成需要从几个诱导区域表达的多个调控基因的协调和顺序输入。我们将使用表达可诱导的显性负性或RNAi结构的转基因品系和移植试验来研究在初级口腔形成过程中对成纤维细胞生长因子和Frzbl功能的时间和空间需求。我们将开始使用反义方法检查对其他基因的需求。这项建议是探索性的，因为这项研究是新的和未发表的，因为它建议推广到一个新的物种，而且它需要试剂开发。开发的试剂不仅对这个项目很重要，对热带按蚊群体也很重要。初级口腔是正常进食和说话所必需的基本结构。原始口腔异常或缺失会严重影响颅面发育，但这一过程的分子机制尚不清楚。这项研究将深入了解颅面畸形的遗传基础，重点放在一个关键的结构上。

DESCRIPTION (provided by applicant): The goals of this proposal are to define the molecular mechanisms involved in vertebrate primary mouth formation, using the frog Xenopus tropicalis as a model. The primary mouth (also called the stomodeum, buccopharyngeal membrane) is the initial opening from the outside of the embryo to the gut. Primary mouth formation is an essential step in cranoiofacial development, but is not well understood. In vertebrates, the neural crest grows around the primary mouth to form the jaws and face and a secondary mouth opening must therefore form. The primary mouth then becomes the pharyngeal opening. The primary mouth forms from a unique region at the extreme anterior of the embryo where ectoderm and endoderm are directly juxtaposed. In order to define the molecular mechanisms involved in this process, we began to characterize this process in X. laevis. We fate mapped the presumptive primary mouth, and defined tissues required for its induction. In order to define genes required for primary mouth formation, we used database mining and expression microarray analysis, to identify genes whose expression is enriched in the presumptive primary mouth region. Two genes that came out of this analysis are fgf8 and the Wnt pathway inhibitor, frzb1, and preliminary data indicates that both are essential for primary mouth formation. We propose to extend this project to X. tropicalis. Compared to X. laevis, this species has the advantage of a diploid genome, making antisense knockdowns more effective, and offering the ability to rapidly prepare stable transgenic lines. Initial data suggests that primary mouth formation in X. laevis and X. tropicalis is very similar. I hypothesize that the process of primary mouth formation is highly conserved. I further hypothesize that primary mouth formation requires the coordinate and sequential input of multiple regulatory genes expressed from several inducing regions. We will examine the temporal and spatial requirement for Fgf and Frzbl function during primary mouth formation using transgenic lines expressing inducible dominant negative or RNAi constructs, and transplant assays. We will begin to examine the requirement for other genes using antisense approaches. This proposal is exploratory since the study is new and unpublished, since it proposes extension into a new species and since it requires reagent development. Reagents developed will be important not only for this project, but for the X. tropicalis community. The primary mouth is an essential structure required for normal eating and speaking. Craniofacial development is severely impacted by an abnormal or absent primary mouth, however the molecular mechanisms underlying this process are not understood. This study will add insight into the genetic basis of craniofacial abnormalities, focusing on a crucial structure.