Effects of Thyroxine on the Craniosynostotic Phenotype

描述(申请人提供)：颅缝早闭(CS)是颅骨的一个或多个纤维关节过早融合，通常允许扩张的脑颅生长。在美国，CS的发病率是每2500名活产儿中就有一名。CS的病因是多因素的，对于大多数临床病例来说，遗传或环境因素往往是未知的。一般的基因-环境模型认为，如果遗传易感性与特定的环境暴露相结合，其影响可能是相加的，甚至是乘性的，导致严重的表型异常。遗传变异和环境暴露之间的相互作用已经被研究了几种颅面畸形，包括口面部裂开。然而，目前对基因-环境相互作用在调控CS表型中的作用知之甚少。疾病控制中心的国家出生缺陷预防研究已经确定了几个与CS相关的环境因素，包括循环中的甲状腺激素水平。虽然这些药物在流行病学上与CS有关，但其机制尚不清楚。我们建议在一个已知突变的CS小鼠模型中探索这些外源因素对细胞、分子和形态的影响。当Twist-1靶向突变小鼠与对照C57BL小鼠饲养时，产生类似Saethre-Chotzen CS综合征的表型。与人类的情况类似，特征表达是高度可变的，这使得它对于建模如何修改因素很有用 可能与CS表型的范围有关。由于CS在时间和严重程度上的表达范围，我们可以专门研究侮辱对细胞形态、分子表达、组织中某些活性蛋白的存在以及生物体发育的影响。在目标1中，我们建议在Twist-1 CS小鼠模型原代细胞中确定致畸挑战对细胞形态、蛋白质、表观遗传学和分子表达的下游影响。具体地说，我们将用甲状腺素对细胞进行传统的液态刺激，并通过Q-PCR评估成骨的各种标志物的表达。此外，全基因组微阵列将在这些条件下分析TWIST下游的靶点和与异常骨形成或CS相关的途径。在目标#2中，我们建议在Twist-1模型中确定宫内暴露于甲状腺激素的体内形态效应。结果指标将通过骨和缝线微结构的3D成像、颅面形态计量学、免疫组织化学、缝线组织形态计量学和整体颅骨分析的组合进行评估。通过确定基因致畸作用的具体途径，本研究将更好地了解CS的遗传-表观遗传-环境相互作用，并有助于头面部畸形的诊断和治疗。这一模式可以应用于其他已知与CS相互作用的致畸药物，如选择性5-羟色胺再摄取抑制剂、低氧或促排卵药物。

DESCRIPTION (provided by applicant): Craniosynostosis (CS) is the premature fusion of one or more of the fibrous joints of the skull, which normally allow for the growth of the expanding neurocranium. In the United States, the incidence of CS is one in every 2500 live births. The etiology of CS is multifactorial, and whether genetic or environmental is often unknown for the majority of clinical cases. The general gene-environment model proposes that if a genetic predisposition is coupled with certain environmental exposures, the effects can be additive or even multiplicative, resulting in severely abnormal phenotypes. The interaction between genetic variants and environmental exposures has been studied for several craniofacial anomalies, including orofacial clefting. However, at present, very little is known about the role of gene-environment interactions in modulating CS phenotypes. The Centers for Disease Control National Birth Defects Prevention Study has identified several environmental factors associated with CS, including circulating levels of thyroid hormone. Although these agents have been epidemiologically associated with CS, the mechanism is not understood. We propose to explore the cell, molecular, and morphological effects of these exogenous factors in a CS mouse model with a known mutation. The Twist-1 targeted mutation mouse when bred with control C57BL mice produces a phenotype similar to Saethre- Chotzen CS syndrome. Similar to the human condition, trait expression is highly variable, making it useful for modeling how modifying factors might relate to the range of CS phenotypes. Because of the range of expression in timing and severity of CS, we can specifically study the effects of the insults on cellular morphology, molecular expression, presence of certain active proteins in the tissues, and development of the organism. In Aim #1 we propose to determine the downstream effects of teratogenic challenges to cell morphology, protein, epigenetic and molecular expression in the Twist-1 CS mouse model primary cells. Specifically, we will subject cells to traditional liquid-phase stimulation wih thyroxine and various markers of osteogenesis will be evaluated for expression via q-PCR. In addition, genome wide microarrays will be analyzed for targets downstream of TWIST and for pathways linked to aberrant bone formation or CS under these conditions. In Aim #2 we propose to determine the in-vivo morphological effects of in utero exposure to thyroxine in the Twist-1 model. Outcome measures will be assessed by a combination of 3D imaging for bone and suture microarchitecture, craniofacial morphometrics, immunohistochemistry, suture histomorphometry and whole mount calvaria analysis. By determination of the specific pathway of teratogenic action upon the gene, the proposed studies will provide a better understanding of the genetic-epigenetic-environment interaction for CS and aid in the diagnosis and management of craniofacial anomalies. This paradigm can then be applied to other teratogens with known interaction with CS, e.g. selective serotonin reuptake inhibitors, hypoxia, or ovulation stimulatin drugs.