TEMPORAL REGULATION OF CRANIAL SKELETOGENESIS

描述（申请人提供）：精确控制间充质分化为骨和软骨的时间是颅面骨骼正常发育的必要条件。颅缝内过早分化会导致颅缝闭锁，而延迟分化则会导致与锁骨和脐部发育不良相关的囟门缺损。因此，确定控制骨骼分化时间的细胞和分子机制是预防出生缺陷的先决条件。在骨骼分化过程中起关键作用的两个分子是runx2和sox9，它们分别是骨骼和软骨所必需的。目前尚不清楚runx2和sox9的时间表达机制，以及骨和软骨分化的确切时间。该研究通过操纵间质相对年龄和改变runx2和sox9的调控来解决这一问题。鹌鹑和鸭胚胎的生长速度不同，神经嵴细胞原位移植形成颅面骨骼表明，鹌鹑供体细胞向骨和软骨分化的时间早于鸭宿主间质，runx2和sox9的表达证明了这一点。本文采用了三种方法来验证神经嵴间质通过调节runx2和sox9的表达和控制其自身对runx2和sox9信号的反应来确定骨骼分化时间的假设。每一种方法都涉及在相对年轻的宿主体内产生较老的供体间充质嵌合胚胎，或在相对年老的宿主体内产生较年轻的供体间充质嵌合胚胎。特异性目标1涉及体外实验，以确定骨和软骨形成何时需要组织相互作用，并评估神经嵴细胞控制这些相互作用的程度。Specific Aim 2鉴定了涉及FGF和TGFbeta家族成员及其靶点的神经嵴依赖性信号事件，这些信号事件调节runx2和sox9的表达，并控制骨骼分化的时间。具体目标3确定FGF和TGFbeta家族成员通过使用功能增益和功能丧失方法来调节骨和软骨形成来控制骨骼分化时间的潜力。一个重要的目标是“拯救”嵌合体中过早或延迟的骨骼分化，这对于设计基于分子的疗法来治疗影响骨骼分化时间的疾病具有明确的临床意义。

DESCRIPTION (provided by applicant): Precise temporal control of mesenchymal differentiation into bone and cartilage is essential for proper development of the craniofacial skeleton. Premature differentiation within cranial sutures produces craniosynostoses whereas delayed differentiation leads to fontanel defects associated with cleidocranial and campomelic dysplasias. Thus, identifying cellular and molecular mechanisms that control the timing of skeletal differentiation is a prerequisite for preventing birth defects. Two molecules that play critical roles during skeletal differentiation are runx2 and sox9, which are required for bone and cartilage respectively. What remain unclear are mechanisms that define the temporal expression of runx2 and sox9, and establish exactly when bone and cartilage differentiate. The proposed research addresses this issue by manipulating the relative age of mesenchyme and by altering the regulation of runx2 and sox9. Quail and duck embryos have divergent growth rates and orthotopic transplants of neural crest cells destined to form the craniofacial skeleton reveal that quail donor cells differentiate into bone and cartilage earlier than duck host mesenchyme as evidenced by expression of runx2 and sox9. Three approaches are taken to test the hypothesis that neural crest mesenchyme establishes the timing of skeletal differentiation by regulating the expression of, and governing its own response to, and signals that control runx2 and sox9. Each approach involves generating chimeric embryos with either older donor mesenchyme within a relatively younger host, or younger donor mesenchyme within a relatively older host. Specific Aim 1 involves in vitro experiments to determine when tissue interactions are required for bone and cartilage formation, and to assess the extent to which neural crest cells govern these interactions. Specific Aim 2 identifies neural crest-dependent signaling events involving FGF and TGFbeta family members and their targets, which regulate runx2 and sox9 expression, and govern the timing of skeletal differentiation. Specific Aim 3 ascertains the potential of FGF and TGFbeta family members to control the timing of skeletal differentiation by employing gain- and loss-of-function approaches to regulate bone and cartilage formation. One important goal is to "rescue" the premature or delayed skeletal differentiation in chimeras, which has clear clinical implications for devising molecular-based therapies to treat disorders that affect the timing of skeletal differentiation.