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

摘要 颅面疾病约占新生儿出生缺陷的1/3，是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的 发展过程。颅面发育的最早期事件之一是第一个 咽弓，面部和颈部的骨、软骨和结缔组织从咽弓产生。大部分 这是由内皮素受体A型（EDNRA）信号传导介导的，它建立了腹侧（未来） 下颌骨）和中间（未来的中耳/下颌关节）领域的弓。这是一个进化上的 保守的机制，因为EDNRA信号的丢失导致下颌的同源异型转化为 斑马鱼、老鼠和人类的上颌骨结构。虽然EDNRA调控的基因是很好的， 由于EDNRA信号转导的特点，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已经与 异常的EDNRA信号 EDNRA是一种G蛋白偶联受体，可激活多种G蛋白; 这些可能需要形成腹侧和中间区域的图案。这在Gq-/-/G11-/-中说明。 小鼠，其中仅近端下颌骨和颌关节（均为中间衍生物）受到影响。我 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骨结构的缺陷。基于这些发现，我 假设沿第一咽弓D-V轴沿着形成EDNRA介导的图案 由一种新的潜在G蛋白编码所控制为了开始剖析这段代码，本提案将研究Gq/11如何 机制驱动中间域模式化和发展的两个目标。在目标1中，我将定义 通过进行单细胞RNA-聚合酶链反应，在中间结构域NCC中由Gq/11控制的基因调控网络 对YM处理的胚胎进行测序。将通过转基因或基因工程对鉴定的基因进行功能测试。 在发育中的斑马鱼中失活。在目标2中，我将确定Gq/11介导的模式化是否 通过防止过早的软骨细胞分化来协调关节和软骨的形成， Gq/11在其他研究中负调节软骨细胞分化。这将通过以下方式进行测试： YM处理的胚胎中的延时成像和整体包埋免疫组织化学测定。综合这些 提出的目标将阐明Gq/11依赖的机制所需的中间域衍生 颌骨结构的形成，并提供深入了解新的G蛋白编码的模式。这个机制是 很可能在所有有颌脊椎动物中是保守的，并且在寻求靶向 在患有颅面出生缺陷的人中的信号传导的药理学调节。

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.