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.

抽象的 颅面疾病约占新生儿出生缺陷的1/3，是主要原因 婴儿死亡率。防止或减轻颅面疾病需要详细了解正常 发展过程。颅面发展中最早的事件之一是对第一个 咽弓，面部和颈部的骨骼，软骨和结缔组织从中出现。大部分 这是由内皮素受体A型（EDNRA）信号传导介导的，该信号建立了腹侧（未来 下颌骨）和中间（未来的中耳/下颌关节）拱门。这是进化 保守的机制，因为埃德拉信号传导的丢失导致下颌骨的同源转化 斑马鱼，小鼠和人类的上颌骨状结构。尽管EDNRA调节的基因很好 特征是将Ednra信号转导与基因调节联系起来的机制知之甚少。 这具有人类健康的影响，因为现在许多面部出生缺陷综合症已与 异常的Ednra信号传导。 Ednra是G蛋白偶联受体，可以激活多种类别的G蛋白。至少两个 这些可能需要对腹侧和中间域进行图形。这在GQ - / - /G11 - / - 中进行了说明。 小鼠，其中仅影响近端下颌骨和下颌关节（两个中间衍生物）。我的 初步数据表明，暴露于YM-254890（YM）的斑马鱼胚胎是一种小分子抑制剂 GQ/11，也仅导致中间域衍生的颌骨结构中的缺陷。根据这些发现，我 假设沿着第一个咽弓的D-V轴ednra介导的图案已实施 通过新颖的基础G蛋白质代码。为了开始解剖此代码，该提案将调查GQ/11 机制以两个目标推动了中间域的模式和开发。在AIM 1中，我将定义 通过执行单细胞RNA- 对YM处理的胚胎进行测序。鉴定的基因将通过转基因或基因进行功能测试 在发展斑马鱼中失活。在AIM 2中，我将确定GQ/11介导的图案是否 通过预防过早的软骨细胞分化来坐标和软骨形成，因为它已经 表明在其他研究中，GQ/11负调节软骨细胞分化。这将使用 在经YM处理的胚胎中进行延时成像和整个安装免疫组织化学测定。在一起 拟议的目标将阐明中间域衍生所需的GQ/11依赖性机制 下颌结构的形成，并洞悉新型G蛋白质代码。这种机制是 在所有颚式脊椎动物中，很可能是一个高价值的目标 颅面先天缺陷的人类信号传导的药理调节。