Mesenchymal Regulation of Osteogenesis

描述（由申请人提供）：在努力为影响颅面骨骼的疾病、损伤和出生缺陷设计新疗法的过程中，需要做更多的工作来了解间充质细胞如何分化为骨细胞并制造骨骼。为了解决这个问题，我们在体内操作一个高度可访问的胚胎群体，颅神经嵴间充质（NCM），它产生了面部和下颌骨骼中的所有骨骼。在我们先前的R 01奖的已发表工作中，以及在初步研究中，我们观察到NCM自主地调节和指导成骨诱导、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 β）和骨形态发生蛋白（BMP）途径的成员和靶点，如Runx 2，Dlx 5和Msx 1，因为已知它们影响骨生成，并且它们的表达在嵌合体中改变。然而，没有邻近组织的BMP 4治疗，或单独的Runx 2过表达，不能产生过早的骨，这意味着需要信号的组合。因此，我们假设NCM通过TGF β和BMP途径的正性和负性调节来控制成骨事件的时间和顺序。为了验证我们的假设，我们利用鹌鹑和鸭的不同发育程序。我们将更快成熟的鹌鹑供体NCM移植到发育较慢的鸭宿主中，这产生了嵌合的鹌鹑;并且我们将较慢的鸭供体NCM移植到相对较快的鹌鹑宿主中，产生了嵌合的双胞胎。这提供了一种独特的方式来操纵NCM和相邻宿主组织之间的信号传导，并允许发现NCM依赖性过程。此外，所有的鹌鹑细胞可以通过一个无处不在的核标记不存在于鸭检测。我们提出了三个相辅相成、互不相关的具体目标。具体目标1将确定NCM使用TGF β和BMP信号传导来控制成骨诱导、增殖和分化的程度。具体目标2将确定NCM依赖TGF β信号传导来指导矿化时间的程度。具体目标3将确定NCM在多大程度上招募TGF？信号传导的靶点，包括RANKL和OPG，以时空调节破骨细胞、基质重塑和骨生长。我们采用获得和丧失功能的技术来确定赋予NCM对骨生成施加时间控制的能力的分子机制。每个特定目标都具有特定的临床相关性，并且可以作为基于分子的疗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，鉴定供体NCM转导其对宿主细胞如破骨细胞的作用的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，以补救各种临床骨骼疾病。 公共卫生相关性：细胞如何学习何时何地制造骨骼？解决这个问题对于预防和治疗出生缺陷以及设计新的治疗方法来修复或再生受损伤或疾病影响的骨骼非常重要。该项目的目标是识别控制骨骼形成的基因和胚胎事件。

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.