Mesenchymal Regulation of Osteogenesis

描述(申请人提供)：为了努力为影响头面部骨骼的疾病、损伤和出生缺陷设计新的治疗方法，需要做更多的工作来了解间充质细胞如何分化为骨细胞和形成骨。为了解决这个问题，我们在体内操纵了一个高度可访问的胚胎群体，即脑神经脊间充质(NCM)，它产生面部和颌骨中的所有骨骼。在我们之前的R01奖发表的工作中，在初步研究中，我们观察到NCM自主地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(TGFβ)和骨形态发生蛋白(BMP)通路的成员和靶点，如Runx2、Dlx5和Msx1，因为它们已知影响成骨，并且它们的表达在嵌合体中发生变化。然而，没有邻近组织的BMP4治疗，或者仅有Runx2的过度表达，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通过对转化生长因子和骨形态发生蛋白途径的正负调节来控制成骨事件的发生时间和顺序。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。我们将成熟较快的供体NCM移植到发育较慢的鸭子宿主中，从而产生嵌合体QUCK；我们将较慢的供体鸭子NCM移植到相对较快的鹌鹑宿主中，产生嵌合二重体。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递，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在鸭子中不存在的普遍存在的核标记来检测到。我们提出了三个相辅相成、互不依赖的具体目标。具体目标1将确定NCM使用转化生长因子和BMP信号来控制成骨诱导、增殖和分化的程度。具体目标2将决定NCM在多大程度上依赖转化生长因子信号来指导矿化的时间。具体目标3将确定NCM在多大程度上招募包括RANKL和OPG在内的转化生长因子信号靶标，在时空上调节破骨细胞、基质重塑和骨生长。我们使用获得和丧失功能的技术来确定赋予NCM对成骨施加时间控制能力的分子机制。每个特定的目标都有特定的临床相关性，并可以作为一种原则的证明，即可以设计出基于分子的疗法来治疗影响成骨时机的疾病。此外，识别供体NCM转导其对破骨细胞等宿主细胞的作用的机制，对于修复和再生因创伤或疾病(如骨质疏松和骨坏死)而受伤的骨骼具有重要意义。我们希望我们的研究将为以生物学为基础的非手术方法治疗各种临床骨骼疾病提供基础。 公共卫生相关性：细胞如何学习何时何地制造骨骼？回答这个问题对于预防和治疗出生缺陷，以及设计修复或再生受伤害或疾病影响的骨骼的新疗法都很重要。该项目的目标是确定控制骨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.