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

• 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
• 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

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.

摘要 颅面部疾病约占新生儿出生缺陷的三分之一，是导致新生儿死亡的主要原因 婴儿死亡率。预防或减轻头面部疾病需要对正常的 发育过程。颅面发育最早的事件之一是第一个头骨的图案化 咽弓，面部和颈部的骨骼、软骨和结缔组织从这里形成。大部分 这是由内皮素受体A型(Ednra)信号介导的，它建立了腹侧(未来) 下颌)和中间(未来的中耳/颌关节)区域。这是一种进化上的 保守机制，因为Ednra信号的丢失导致下颌骨的同源异型转化为 斑马鱼、老鼠和人类的上颌状结构。尽管Ednra调控的基因很好 目前，人们对Ednra信号转导与基因调控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征现在都与 异常的Ednra信号。 Ednra是一种G蛋白偶联受体，可以激活多种类型的G蛋白；至少有两种 这些可能是形成腹侧和中间区域的模式所必需的。这一点在GQ-/-/G11-/-中说明 小鼠，其中只有近端下颌骨和颌关节(这两个中间衍生品)受到影响。我的 初步数据显示，斑马鱼胚胎暴露于YM-254890(YM)，一种小分子抑制物 GQ/11也只在中间区域派生的颌骨结构中造成缺陷。基于这些发现，我 假设沿第一咽弓的D-V轴实现Ednra介导的图案形成 通过一种新的潜在G蛋白密码。为了开始剖析这一代码，该提案将调查GQ/11 机制驱动中间领域模式的形成和发展有两个目标。在目标1中，我将定义 由GQ/11调控的中间结构域NCC中的基因调控网络 YM处理的胚胎的测序。已确定的基因将通过转基因或基因进行功能测试 斑马鱼发育过程中的失活。在目标2中，我将确定GQ/11介导的模式 通过防止软骨细胞过早分化来协调关节和软骨的形成，就像过去一样 其他研究表明，GQ/11负向调节软骨细胞的分化。这将使用以下工具进行测试 YM处理的胚胎的时间推移成像和整体安装免疫组织化学分析。把这些放在一起 提议的AIMS将阐明中间域派生所需的依赖GQ/11的机制 颌骨结构的形成，并提供了对图案的新G蛋白密码的洞察。这个机制是 很可能在所有有颌骨的脊椎动物中保存，并将成为寻找目标的高价值目标 人类头面部出生缺陷信号的药理学调节。