VERTEBRATE PRIMARY MOUTH FORMATION

描述（由申请人提供）：本提案的主要目标是了解脊椎动物初级口腔形成的机制，以非洲爪蟾为模型。初级口（也称为口）是外部和发育中的咽之间的第一个开口，是颅面发育的最早组成部分。它对食物的摄入至关重要，可能对舌头和牙齿有影响。我们先前定义了爪蟾初级口腔发育过程中的多个步骤，并分离出一组在该区域表达丰富的调控基因。初级口腔形成的早期重要步骤是基底膜的局部降解，这是由2-catenin介导的Wnt途径的局部抑制介导的。有两个具体目标。目的1分析初口区基底膜代谢的调控。重点是降解基膜的基质金属蛋白酶（MMPs）的作用。在发育中的初级口腔中表达的MMPs的功能将通过反义功能丧失测定和功能获得测定来分析，其中使用“面部移植”和暂时调节的启动子构建来定位效果。基底膜的合成和降解是平衡的，并由Wnt信号协调激活的假设将被验证。如果基膜不被破坏，初级口腔形成的步骤将被定义。目的2检查直接初级口腔形成的信号系统。其中一个重点是激肽-钾likrein通路，这是初级口腔形成所必需的，但在胚胎发生过程中却很少被描述。该途径中酶、受体和调节剂在初级口腔形成过程中的功能将通过反义功能丧失和体外实验进行测试。初步数据将扩展到分析BMP和非规范Wnt信号在初级口腔区域的作用。非洲爪蟾是该项目的杰出系统，因为组织移植可以很容易地用于定位基因扰动的影响，而在哺乳动物模型中进行等效分析是困难的。检测快速，因为青蛙胚胎发育迅速；而且青蛙的基因和哺乳动物一样保守。在小鼠和小鸡的初级口腔形成过程中发生的步骤与我们在非洲爪蟾中观察到的相似，这表明我们的分析将有助于在哺乳动物中进行比较研究。本研究具有广泛的意义。颅面出生缺陷出现的频率很高，每700个活产婴儿中就有1个出现颅面出生缺陷，但大多数的病因尚不清楚，而且通常不认为原发性口腔紊乱是导致颅面出生缺陷的原因。基底膜重构过程中Wnt和MMP信号的相互作用以及激肽-钾likrein通路可能在其他胚胎器官中也具有重要意义。

DESCRIPTION (provided by applicant): The broad goals of this proposal are to understand the mechanisms underlying formation of the vertebrate primary mouth, using the frog Xenopus laevis as a model. The primary mouth (also called the stomodeum) is the first opening between the outside and the developing pharynx, and is the earliest element of craniofacial development. It is critical for food ingestion, and may contribute to the tongue and teeth. We previously defined multiple steps that occur during Xenopus primary mouth development, and isolated a set of regulatory genes whose expression is enriched in this region. An early, essential, step in primary mouth formation is local degradation of the basement membrane, which is mediated by local inhibition of the 2-catenin-mediated Wnt pathway. There are two Specific Aims. Aim 1 analyses regulation of basement membrane metabolism in the primary mouth region. Focus is on the role of matrix metalloproteases (MMPs) that degrade the basement membrane. Function of MMPs expressed in the developing primary mouth will be analyzed by antisense loss of function assays, and gain of function assays, where effects are localized using "face transplants" and temporally regulated promoter constructs. The hypothesis that basement membrane synthesis and degradation are balanced, and coordinately activated by Wnt signaling will be tested. Steps in primary mouth formation that are altered if the basement membrane does not break down will be defined. Aim 2 examines the signaling systems that direct primary mouth formation. One focus is on the kinin-kallikrein pathway that is required for primary mouth formation, but is poorly characterized during embryogenesis. The function during primary mouth formation of enzymes, receptors and modulators in this pathway will be tested by antisense loss of function and in vitro assays. Preliminary data will be extended to analyze the role of BMP and non-canonical Wnt signaling in the primary mouth region. Xenopus is an outstanding system for this project since tissue transplants can readily be used to localize effects of gene perturbation, whereas equivalent assays are difficult in mammalian models. Assays are rapid, as frog embryos develop rapidly; and frog genes are conserved with those of mammals. Steps occurring during formation of the mouse and chick primary mouth appear similar to those we have observed in Xenopus, indicating that our analyses will be useful for suggesting comparative studies in mammals. This study has broad significance. Craniofacial birth defects appear with high frequency, in 1/700 live births, however the etiology of most is unknown, and primary mouth perturbations have not generally been considered as causal. Both the interplay of Wnt and MMP signaling during basement membrane remodeling, and the kinin-kallikrein pathway are of likely significance in other embryonic organs.