TEMPORAL REGULATION OF CRANIAL SKELETOGENESIS

描述(申请人提供)：精确的间质分化为骨和软骨的时间控制对于颅面骨骼的正常发育是必不可少的。颅缝内的过早分化会导致颅缝融合，而分化延迟则会导致与锁骨颅骨发育不良和钟状骨发育不良相关的颧骨缺陷。因此，确定控制骨骼分化时机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，这两个分子分别是骨骼和软骨所必需的。目前尚不清楚的是哪些机制定义了Runx2和Sox9的临时表达，并准确地确定了骨和软骨分化的时间。这项拟议的研究通过操纵间充质的相对年龄和改变Runx2和Sox9的调节来解决这个问题。鹌鹑胚胎和鸭胚胎的生长速度不同，原位移植形成颅面骨架的神经脊细胞表明，供体细胞分化为骨和软骨的时间早于鸭宿主间充质细胞，Runx2和Sox9的表达证明了这一点。采用三种方法来检验这一假说，即神经脊间充质通过调节Runx2和Sox9的表达和控制其自身的反应和信号来确定骨骼分化的时间。每种方法都涉及产生嵌合胚胎，要么在相对年轻的宿主中产生较年长的供体间充质细胞，要么在相对较老的宿主中产生较年轻的供体间充质细胞。具体目标1包括体外实验，以确定骨和软骨形成何时需要组织相互作用，并评估神经脊细胞控制这些相互作用的程度。特异性目标2识别涉及成纤维细胞生长因子和转化生长因子β家族成员及其靶点的神经峰依赖信号事件，这些信号事件调节Runx2和Sox9的表达，并控制骨骼分化的时机。具体目的3确定成纤维细胞生长因子和转化生长因子β家族成员通过功能的获得和丧失来调节骨和软骨的形成，从而控制骨骼分化的时机。其中一个重要的目标是挽救嵌合体中过早或延迟的骨骼分化，这对设计基于分子的疗法来治疗影响骨骼分化时机的疾病具有明显的临床意义。

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.