VERTEBRATE PRIMARY MOUTH FORMATION

描述（由申请人提供）：本提案的主要目标是以蛙非洲爪蟾为模型，了解脊椎动物初级口形成的潜在机制。初级口（也称为口道）是外部和发育中的咽之间的第一个开口，是颅面发育的最早元素。它对食物摄取至关重要，并可能对舌头和牙齿有贡献。我们以前定义的多个步骤，发生在爪蟾初级口的发展，并分离出一组调控基因，其表达是在这个地区丰富。原发性口腔形成的早期基本步骤是基底膜的局部降解，其通过2-连环蛋白介导的Wnt途径的局部抑制介导。有两个具体目标。目的1分析原发口区基底膜代谢的调控。重点是降解基底膜的基质金属蛋白酶（MMPs）的作用。将通过反义功能丧失测定和功能获得测定来分析发育中的原发性口腔中表达的MMPs的功能，其中使用“面部移植”和时间调节启动子构建体来定位效应。基底膜合成和降解是平衡的，并且通过Wnt信号传导协调激活的假设将被检验。如果基底膜不破裂，初级口腔形成的步骤将被定义。目的2研究指导初级口腔形成的信号系统。一个焦点是激肽-激肽释放酶途径，这是初级口腔形成所需的，但在胚胎发生过程中的特点很差。将通过反义功能丧失和体外测定来测试该途径中的酶、受体和调节剂在初级口形成期间的功能。初步数据将被扩展以分析BMP和非经典Wnt信号传导在初级口腔区域的作用。非洲爪蟾是一个杰出的系统，因为组织移植可以很容易地用于本地化基因扰动的影响，而在哺乳动物模型中的等效分析是困难的。由于青蛙胚胎发育迅速，因此检测速度很快;而且青蛙的基因与哺乳动物的基因是保守的。在小鼠和小鸡的初级口形成过程中发生的步骤似乎类似于我们在非洲爪蟾中观察到的，这表明我们的分析将是有用的，建议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，在1/700活产，但大多数的病因是未知的，和原发性口腔扰动一般不被认为是因果关系。基底膜重塑过程中Wnt和MMP信号的相互作用以及激肽-激肽释放酶途径在其他胚胎器官中可能具有重要意义。 公共卫生相关性：该提案分析了初级口腔的形成，这是从胚胎外部到肠道的基本第一次开口。我们将识别和研究基因的功能，并控制正常胚胎中初级口的形成。这项研究将揭示为什么人类胚胎中的初级嘴不能正确形成，并有助于解释颅面出生缺陷的分子原因。

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. PUBLIC HEALTH RELEVANCE: This proposal analyzes formation of the primary mouth, the essential first opening from the outside of the embryo to the gut. We will identify and study the function of genes and that control primary mouth formation in the normal embryo. This study will suggest why the primary mouth fails to form correctly in a human embryo, and help explain the molecular causes of craniofacial birth defects.