VERTEBRATE PRIMARY MOUTH FORMATION

脊椎动物的初级口腔形成

• 批准号: 8043545
• 负责人: Hazel L Sive
• 金额: $ 47.29万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8043545
• 关键词: Apoptosis; Basement membrane; Biological Assay; Biological Models; Cell Death; Cell Polarity; Comparative Study; Congenital Abnormality; Data; Development; Elements; Embryo; Embryonic Development; Enzymes; Equilibrium; Etiology; Financial compensation; Food; Frequencies; Gene Expression; Genes; Goals; Heat-Shock Response; Human; In Situ Nick-End Labeling; Ingestion; Kallikrein-Kinin System; Kininogenase; Kinins; Live Birth; Mammals; Mediating; Membrane Proteins; Metabolism; Metalloproteases; Modeling; Molecular; Monitor; Mus; Oral cavity; Organ; Pathway interactions; Pharyngeal structure; Play; Rana; Regulation; Regulator Genes; Role; Signal Pathway; Signal Transduction; Staining method; Stains; Stomodeum; System; Systems Analysis; Testing; Tongue; Tooth structure; Transgenic Organisms; Transplanted tissue; Xenopus; Xenopus laevis; craniofacial; facial transplantation; gain of function; gene function; immunocytochemistry; in vitro Assay; loss of function; membrane synthesis; promoter; public health relevance; receptor

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.

描述（由申请人提供）：该提案的主要目标是使用非洲爪蟾作为模型，了解脊椎动物主口形成的机制。初级口（也称为食道）是外部和发育中的咽部之间的第一个开口，并且是颅面发育的最早元素。它对于食物摄入至关重要，并且可能对舌头和牙齿有影响。我们之前定义了非洲爪蟾初级口腔发育过程中发生的多个步骤，并分离了一组其表达在该区域丰富的调节基因。初级口腔形成的早期重要步骤是基底膜的局部降解，这是通过 2-连环蛋白介导的 Wnt 通路的局部抑制来介导的。有两个具体目标。目标 1 分析初级口腔区域基底膜代谢的调节。重点是基质金属蛋白酶 (MMP) 降解基底膜的作用。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初级口腔中表达的MMP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，并且由 Wnt 信号传导协调激活的假设将得到检验。如果基底膜不破裂，初级口腔形成的步骤将被定义。目标 2 检查指导初级口腔形成的信号系统。一个焦点是初级口腔形成所需的激肽-激肽释放酶途径，但在胚胎发生过程中很少被表征。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测定来测试。初步数据将被扩展以分析 BMP 和非规范 Wnt 信号在主要口腔区域中的作用。非洲爪蟾是该项目的一个杰出系统，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的测定却很困难。由于青蛙胚胎发育迅速，检测速度很快；青蛙的基因与哺乳动物的基因一样保守。小鼠和雏鸡主嘴形成过程中发生的步骤与我们在爪蟾中观察到的步骤相似，这表明我们的分析将有助于建议在哺乳动物中进行比较研究。这项研究具有广泛的意义。颅面出生缺陷的出现频率很高，占活产婴儿的 1/700，但大多数的病因尚不清楚，而且原发性口腔干扰通常不被认为是因果关系。基底膜重塑期间 Wnt 和 MMP 信号传导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。 公共健康相关性：该提案分析了初级口腔的形成，初级口腔是从胚胎外部到肠道的第一个重要开口。我们将鉴定和研究正常胚胎中控制初级口腔形成的基因的功能。这项研究将揭示为什么人类胚胎中主口无法正确形成，并有助于解释颅面出生缺陷的分子原因。