Mesenchymal Regulation of Osteogenesis

描述（由申请人提供）：为了努力设计新的治疗影响颅面骨骼的疾病、损伤和出生缺陷的方法，需要做更多的工作来了解间充质细胞如何分化成骨细胞并形成骨。为了解决这个问题，我们在体内操作一个高度可接近的胚胎种群，颅神经嵴间充质（NCM），它产生面部和下颌骨骼的所有骨骼。在我们之前的R01奖发表的作品中，以及在初步研究中，我们观察到NCM自主同步和指导成骨诱导、增殖、分化、基质沉积、矿化和基质重塑。NCM如何完成如此复杂的任务，以及哪些因素足以复制这种现象，目前尚不清楚。可能的候选者可能包括转化生长因子- β （TGF¿）和骨形态发生蛋白（BMP）途径的成员和靶点，如Runx2， Dlx5和Msx1，因为已知它们影响成骨作用，并且它们在嵌合体中的表达发生改变。然而，没有邻近组织的BMP4治疗，或单独的Runx2过表达，不能产生过早骨，这意味着需要信号的组合。因此，我们假设NCM通过TGF¿和BMP通路的正调控和负调控来调控成骨事件的时间和顺序。为了验证我们的假设，我们利用了鹌鹑和鸭子不同的发育程序。我们将成熟较快的鹌鹑供体NCM移植到发育较慢的鸭子宿主体内，产生嵌合的鹌鹑；我们将较慢的鸭子供体NCM移植到相对较快的鹌鹑宿主体内，产生嵌合的双尾鸭。这提供了一种独特的方法来操纵NCM和邻近宿主组织之间的信号传导，并允许发现NCM依赖的过程。此外，所有的鹌鹑细胞都可以通过一种无处不在的核标记来检测，而这种标记在鸭子中并不存在。我们提出三个相互补充、互不依存的具体目标。特异性目的1将确定NCM在多大程度上利用TGF¿和BMP信号来控制成骨诱导、增殖和分化。具体目标2将确定NCM在多大程度上依赖于TGF¿信号来指导矿化时间。具体目标3将确定NCM在多大程度上利用TGF -信号靶点（包括RANKL和OPG）来时空调节破骨细胞、基质重塑和骨生长。我们采用功能增益和功能丧失技术来确定赋予NCM施加时间控制成骨能力的分子机制。每个特异性目标都具有特定的临床相关性，可以作为基于分子的治疗方法可以设计用于治疗影响成骨时间的疾病的原理证明。此外，确定供体NCM对宿主细胞（如破骨细胞）影响的传导机制，对创伤或骨质疏松症和骨坏死等疾病损伤的骨的修复和再生具有重要意义。我们希望我们的研究将为基于生物学的非手术方法提供基础，以治疗各种临床骨骼疾病。

DESCRIPTION (provided by applicant): In an effort to devise novel therapies for diseases, injuries, and birth defects that affect the craniofacial skeleton, more needs to be done to understand how mesenchymal cells differentiate into osteocytes and make bone. To address this issue, we manipulate in vivo a highly accessible embryonic population, the cranial neural crest mesenchyme (NCM), which produces all of the bones in the facial and jaw skeletons. In published work from our prior R01 award, and in preliminary studies, we observe that NCM autonomously synchronizes and directs osteogenic induction, proliferation, differentiation, matrix deposition, mineralization, and matrix remodeling. How NCM accomplishes such a complex task, and what factors are sufficient to replicate this phenomenon, is unknown. Likely candidates may include members and targets of the Transforming Growth Factor-Beta (TGF¿) and Bone Morphogenetic Protein (BMP) pathways such as Runx2, Dlx5, and Msx1, since they are known to affect osteogenesis and their expression is altered in chimeras. Yet BMP4 treatments without adjacent tissues, or Runx2 over-expression alone, cannot produce premature bone, implying that combinations of signals are needed. Therefore, we hypothesize that NCM elicits positive and negative regulation by the TGF¿ and BMP pathways to govern the timing and sequence of osteogenic events. To test our hypothesis, we exploit the divergent developmental programs of quail and duck. We transplant faster- maturing quail donor NCM into a slower-developing duck host, which creates chimeric quck; and we transplant slower duck donor NCM into the relatively faster quail host, generating chimeric duail. This provides a unique way to manipulate signaling between NCM and adjacent host tissues, and allows discovery of NCM-dependent processes. Also, all quail cells can be detected via a ubiquitous nuclear marker not present in duck. We propose three complementary and non-interdependent Specific Aims. Specific Aim 1 will determine the extent to which NCM uses TGF¿ and BMP signaling to control osteogenic induction, proliferation, and differentiation. Specific Aim 2 will determine the extent to which NCM relies on TGF¿ signaling to direct the timing of mineralization. Specific Aim 3 will determine the extent to which NCM enlists targets of TGF¿ signaling including RANKL and OPG to spatiotemporally regulate osteoclasts, matrix remodeling, and bone growth. We employ gain- and loss-of-function techniques to identify molecular mechanisms that endow NCM with the ability to exert temporal control over osteogenesis. Each Specific Aim has particular clinical relevance and can serve as a proof-of-principle that molecular-based therapies can be devised to treat disorders that affect the timing of osteogenesis. Moreover, identifying mechanisms through which donor NCM transduces its effects on host cells such as osteoclasts has implications for repair and regeneration of bones injured by trauma or diseases like osteoporosis and osteonecrosis. We are hopeful that our research will provide a foundation for biologically based, non-surgical methods to remedy a variety of clinical skeletal conditions. PUBLIC HEALTH RELEVANCE: How do cells learn when and where to make bone? Answering this question is important for preventing and treating birth defects, as well as for devising new therapies to repair or regenerate bones affected by injury or disease. The goal of this project is to identify genes and embryonic events that control bone formation.