Shox2 and temporomandibular joint formation

描述(申请人提供)：颞下颌关节强直(TMJ)在临床上被定义为由于髁突头部和关节窝之间的纤维或骨性结合而导致的有限的张口。虽然大多数TMJ强直事件发生在创伤或感染之后，但也有先天性病例的报告。目前对TMJ的形态发生和潜在的遗传、细胞和分子机制知之甚少。特别是，对导致先天性TMJ强直的基因改变一无所知。同源异型盒基因Shox2在上颌骨和下颌骨交界处的间充质细胞中特异表达，随后在TMJ的未成熟软骨细胞中表达。在脑神经脊源性细胞中，Shox2的条件失活会导致TMJ发育不良和强直。因此，Shox2突变小鼠为研究哺乳动物TMJ发育及其潜在机制提供了独特的模型系统。我们推测Shox2通过直接调控Runx2的表达在TMJ的发育过程中发挥重要作用。我们还假设Shox2a的相扑修饰对于TMJ的形成是必不可少的。为了检验这些假说，本文提出了四个具体目标。在目标1中，我们将通过报告实验、EMSA和芯片实验来确定Runx2是否是Shox2的直接下游靶点。我们还将通过创建模拟结构性磷酸化状态或结构性去磷酸化状态的Shox2a突变形式来确定磷酸化是否损害Shox2a的反式激活能力。在目标2中，我们将定义Shox2a的相扑修饰及其对Shox2a转录能力调节的后果。为此，我们将进一步确定Shox2a和组蛋白3.3之间的相互作用，这可能会增强Shox2a的转录能力。人类SHOXa将被并行地包括在拟议的研究中。在目标3中，我们将通过在内源性Shox2表达区域表达Shox2a的突变形式来确定Shox2a的相扑修饰在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在胚胎发育中是否在功能上是多余的。公共卫生相关性：TMJ强直是TMJ疾病的主要症状之一，该疾病在人类中发病率很高。TMJ强直的临床特征是关节部件的骨或纤维粘连的形成，这限制了颌骨的运动，导致进食、吞咽和呼吸困难。先天性TMJ强直已被报道，但对潜在的遗传改变知之甚少。在小鼠的TMJ细胞中，同源盒基因Shox2的条件失活会导致TMJ的异常形成和强直。因此，条件Shox2突变小鼠为研究TMJ发育和TMJ强直提供了一种独特的动物模型。本研究采用体外和体内方法研究了Shox2在TMJ发育中的作用及其作用机制。这项研究的结果将为我们理解人类这一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.