Deciphering the Endothelin Receptor Type A Dependent G Protein Code that Patterns the Mandibular First Pharyngeal Arch

破译下颌第一咽弓的 A 型内皮素受体依赖性 G 蛋白密码

基本信息

批准号：
10222545
负责人：
Stanley Michinobu Kanai
金额：
$ 7.58万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10222545
关键词：
Affect Animal Model Antibodies Apoptosis Biological Assay Birth Cartilage Cells Cephalic Chondrocytes Chondrogenesis Cluster Analysis Code Complex Congenital Abnormality Connective Tissue Craniofacial Abnormalities Data Defect Development Developmental Process Disease Dorsal Drug Targeting Embryo Endothelin Receptor Endothelin-1 Event Exposure to Face Fertilization First Pharyngeal Arch Fluorescent in Situ Hybridization Future G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Gene Expression Regulation Gene Silencing Gene Transfer Techniques Genes Genetic Genetic Transcription Goals Health Hour Human Image Immunohistochemistry Infant Mortality Jaw Joints Ligands Link Mandible Mediating Molecular Morphogenesis Mus Mutant Strains Mice Neck Neural Crest Cell Newborn Infant Pathway interactions Pattern Pharmacology Process Proteins Regulator Genes Reporter Reporting Role Sampling Signal Transduction Structure Syndrome System Temporomandibular Joint Testing Transgenic Organisms Vertebrates Zebrafish base bone craniofacial craniofacial development craniofacial disorder design differential expression infant morbidity insight middle ear mutant novel premature prevent receptor single-cell RNA sequencing small molecule inhibitor time use zebrafish development

项目摘要

ABSTRACT Craniofacial disorders account for approximately 1/3 of birth defects in newborns and are a major cause of infant mortality. Preventing or mitigating craniofacial disorders requires a detailed understanding of normal developmental processes. One of the earliest events in craniofacial development is patterning of the first pharyngeal arch, from which the bone, cartilage and connective tissue of the face and neck arise. Much of this is mediated by Endothelin receptor type A (EDNRA) signaling, which establishes the ventral (future mandible) and intermediate (future middle ear/jaw joint) domains of the arch. This is an evolutionarily conserved mechanism, as loss of EDNRA signaling results in homeotic transformation of the lower jaw into upper jaw-like structures in zebrafish, mouse, and humans. Although EDNRA-regulated genes are well characterized, little is known about the mechanisms that link EDNRA signal transduction to gene regulation. This has human health implications because many facial birth defect syndromes have now been linked to aberrant EDNRA signaling. EDNRA is a G protein-coupled receptor that can activate multiple classes of G proteins; at least two of these are likely required to pattern the ventral and intermediate domains. This is illustrated in Gq-/-/G11-/- mice, in which only the proximal mandible and jaw joint (both intermediate derivatives) are affected. My preliminary data shows that zebrafish embryos exposed to YM-254890 (YM), a small molecule inhibitor for Gq/11, also only causes defects in intermediate-domain derived jaw structures. Based on these findings, I hypothesize that EDNRA-mediated patterning along the D-V axis of the first pharyngeal arch is implemented by a novel underlying G protein code. To begin dissecting this code, this proposal will investigate how Gq/11 mechanisms drive intermediate domain patterning and development in two Aims. In Aim 1, I will define the gene regulatory network controlled by Gq/11 in intermediate domain NCCs by performing single cell RNA- sequencing on YM-treated embryos. Identified genes will be functionally tested by transgenesis or gene inactivation in developing zebrafish. In Aim 2, I will determine whether Gq/11-mediated patterning coordinates joint and cartilage formation by preventing premature chondrocyte differentiation, as it has been shown that Gq/11 negatively regulates chondrocyte differentiation in other studies. This will be tested using time-lapse imaging and whole-mount immunohistochemistry assays in YM-treated embryos. Together these proposed Aims will elucidate the Gq/11-dependent mechanisms required for intermediate domain-derived jaw structure formation and provide insight into the novel G protein code of patterning. This mechanism is very likely conserved in all jawed vertebrates and would be a high value target in the quest for targeted pharmacological modulation of signaling in humans with craniofacial birth defects.

抽象的颅面疾病约占新生儿出生缺陷的 1/3，是导致新生儿出生缺陷的主要原因。婴儿死亡率。预防或减轻颅面疾病需要详细了解正常情况发展过程。颅面发育中最早的事件之一是第一个颅面的图案化咽弓，面部和颈部的骨、软骨和结缔组织均由咽弓产生。很多这是由 A 型内皮素受体 (EDNRA) 信号传导介导的，该信号建立了腹侧（未来下颌骨）和牙弓的中间（未来的中耳/下颌关节）区域。这是一个进化论保守的机制，因为 EDNRA 信号传导的丧失导致下颌同源异型转化为斑马鱼、小鼠和人类的上颌状结构。尽管 EDNRA 调控的基因很好然而，人们对 EDNRA 信号转导与基因调控之间的联系机制知之甚少。这对人类健康有影响，因为许多面部出生缺陷综合症现在都与异常的 EDNRA 信号。 EDNRA是一种G蛋白偶联受体，可以激活多类G蛋白；至少其中两个这些可能是形成腹侧和中间区域所需的。 Gq-/-/G11-/- 中对此进行了说明小鼠，仅近端下颌骨和颌关节（均为中间衍生物）受到影响。我的初步数据显示，斑马鱼胚胎暴露于YM-254890（YM），一种小分子抑制剂 Gq/11，也仅导致中间域衍生的颌结构的缺陷。根据这些发现，我假设 EDNRA 介导的沿着第一咽弓 D-V 轴的模式被实现由一种新颖的底层 G 蛋白密码实现。为了开始剖析这段代码，该提案将研究 Gq/11 如何机制驱动中间域模式化和发展有两个目标。在目标 1 中，我将定义通过执行单细胞RNA-中间域NCC中Gq/11控制的基因调控网络对 YM 处理的胚胎进行测序。鉴定出的基因将通过转基因或基因进行功能测试发育中的斑马鱼失活。在目标 2 中，我将确定 Gq/11 介导的模式是否通过防止软骨细胞过早分化来协调关节和软骨的形成其他研究表明 Gq/11 负向调节软骨细胞分化。这将使用进行测试 YM 处理胚胎的延时成像和整体免疫组织化学测定。一起这些拟议的目标将阐明中间域衍生所需的 Gq/11 依赖机制颌骨结构的形成并提供对新型 G 蛋白图案编码的深入了解。这个机制是很可能在所有有颌脊椎动物中都保守，并且将是寻找目标的高价值目标对患有颅面出生缺陷的人的信号传导进行药理学调节。