Shox2 and temporomandibular joint formation

描述（由申请人提供）：颞下颌关节（TMJ）强直在临床上被定义为由于髁突头部与关节盂窝之间的纤维或骨愈合而导致的开口受限。虽然大多数TMJ强直发生在创伤或感染后，但先天性病例也有报道。目前对颞下颌关节的形态发生及其潜在的遗传、细胞和分子机制知之甚少。特别是，对导致先天性颞下颌关节强直的基因改变一无所知。同源盒基因Shox2在上下颌交界处的间充质细胞中特异表达，随后在颞下颌关节髁突的未成熟软骨细胞中表达。颅神经嵴来源细胞中Shox2的条件性失活导致TMJ发育不良和强直。因此，Shox2突变小鼠可作为研究哺乳动物TMJ发育及其潜在机制的独特模型系统。我们假设Shox2通过直接调节Runx2的表达在TMJ的发展中起着至关重要的作用。我们还假设Shox2a的SUMO修饰对TMJ的形成至关重要。提出了四个具体目标来检验这些假设。在Aim 1中，我们将通过报告基因实验、EMSA和ChIP实验来确定Runx2是否是Shox2的直接下游靶点。我们还将通过创建模拟组成性磷酸化状态或组成性去磷酸化状态的Shox2a突变体来确定磷酸化是否会损害Shox2a的反激活效力。在Aim 2中，我们将定义Shox2a的SUMO修饰及其对Shox2a转录能力调节的影响。为此，我们将进一步确定Shox2a与Histone 3.3之间的相互作用，这可能会增强Shox2a的转录能力。人类SHOXa将同时包括在拟议的研究中。在Aim 3中，我们将通过表达内源性shox2表达域中不能被转录或模拟组成性转录状态的Shox2a突变形式，来确定Shox2a的SUMO修饰在TMJ发育中的作用。在最后一个目标中，我们将通过将人类SHOX基因定向插入小鼠SHOX2等位基因来测试人类SHOX和SHOX2在胚胎发育中是否具有功能冗余。公共卫生相关性：颞下颌关节（TMJ）强直是人类频繁发生的TMJ疾病的主要症状之一。TMJ强直的临床特征是关节部件形成骨或纤维粘连，限制下颌运动，导致进食、吞咽和呼吸困难。先天性颞下颌关节强直已有报道，但对潜在的遗传改变知之甚少。小鼠TMJ细胞中同源盒基因Shox2的条件失活可导致TMJ异常形成和强直。因此，条件Shox2突变小鼠为研究TMJ发育和TMJ强直提供了独特的动物模型。本研究通过体外和体内方法研究Shox2在TMJ发育中的作用及其功能机制。从这项研究中获得的结果将为我们了解人类这种独特关节的形成和功能提供基础信息。

DESCRIPTION (provided by applicant): The temporomandibular joint (TMJ) ankylosis is clinically defined as limited mouth opening due to either a fibrous or bony union between the head of the condyle and the glenoid fossa. Although most incidents of TMJ ankylosis occur after trauma or an infection, congenital cases have been reported. Currently little is known about the TMJ morphogenesis and the underlying genetic, cellular and molecular mechanisms. Particularly, nothing is known about genetic alteractions that cause congenital TMJ ankylosis. The homeobox gene Shox2 is expressed specifically in the mesenchymal cells of the maxilla-mandibular junction and later in the immatured chondrocytes of the condyle of the TMJ. A conditional inactivation of Shox2 in cranial neural crest derived cells leads to dysplasia and ankylosis of the TMJ. Thus, Shox2 mutant mice serve as a unique model system for the studies of the mammalian TMJ development and its underlying mechanisms. We hypothesize that Shox2 plays a crucial role in TMJ development by regulating Runx2 expression directly. We also hypothesize that SUMO modification of Shox2a is essential for the TMJ formation. Four specific aims are proposed to test these hypotheses. In Aim 1, we will determine if Runx2 is a direct downstream target of Shox2 by reporter assay, EMSA and ChIP assay. We will also determine if phosphorylation impairs Shox2a's transactivating potency by creating mutant forms of Shox2a mimicking constitutively phosphorylation state or constitutively dephosphorylation state. In Aim 2, we will define SUMO modification of Shox2a and its consequences on the modulation of the Shox2a's transcriptional capacity. In this aim, we will further define the interaction between Shox2a and Histone 3.3, which may enhance the transcription capacity of Shox2a. The human SHOXa will be included in parallel in the proposed studies. In Aim 3, we will determine the role of SUMO modifcation of Shox2a in the TMJ development by expressing the mutated forms of Shox2a that either can not be sumoylated or mimic constitutively sumoylated status in the endogenous Shox2-expressing domains. In the last aim, we will test if human SHOX and SHOX2 are functionally redundant in embryonic development through targeted insertion of the human SHOX gene into the mouse Shox2 allele. PUBLIC HEALTH RELEVANCE: The temporomandibular joint (TMJ) ankylosis is one of the major symptoms of TMJ disorders that occur highly frequently humans. TMJ ankylosis is clinically characterized by the formation of bone or fibrous adhesion of the joint components, which restricts the jaw movement and causes difficulty in feeding, swallowing, and breathing. Congenital TMJ ankylosis have been repored, but very little is known about the underlying genetic alteractions. A conditional inactivation of the homeobox gene Shox2 in the TMJ cells in mice leads to abnormal formation and ankylosis of the TMJ. Thus the conditional Shox2 mutant mice provide a unique animal model for studying TMJ development and TMJ ankylosis. This proposal studies the role of Shox2 in the TMJ development and its functional mechanisms using in vitro and in vivo approaches. The results obtained from this study will provide fundamental information for our understanding of the formation and function of this unique joint in humans.