Regulation of Tendon Induction and Formation By TGFbeta Signaling

描述(申请人提供)：一个有功能的肌肉骨骼系统依赖于力量从肌肉到骨骼的传递，因此需要肌肉、软骨和肌腱的协调发展。然而，人们对肌腱形成的细胞和分子方面知之甚少。解开引导间充质细胞成为腱细胞的信号对于阐明塑造肌肉骨骼系统的组织和协调生长的早期事件具有特别重要的意义。此外，肌腱诱导活性对于改善肌腱损伤的治疗和从干细胞诱导肌腱形成的生物工程努力都具有相当重要的临床意义。最近的研究发现，肌腱细胞和肌腱前体细胞唯一的标志物是碱性成纤维细胞生长因子(SCX)，随后的一些研究表明，成纤维细胞生长因子信号在肌腱前体细胞的诱导中起作用。我们实验室的戏剧性结果现在证明了转化生长因子在肌腱诱导和分化中的更深层次的作用。胚胎中的转化生长因子(2)和转化生长因子(3)基因发生双突变，发育出四肢，四肢中没有肌腱。此外，在器官培养和组织培养实验中，转化生长因子信号是SCX的有效诱导剂，显著超过类似实验中成纤维细胞生长因子信号诱导SCX的程度和强度。我们建议通过三组不同的实验来探讨转化生长因子在肌腱形成中的作用。第一个目标将解决假设，即所有的肌腱组织依赖于转化生长因子(信号)。轴腱和头腱的表型将在转化生长因子(2和3个突变型)和转化生长因子受体(转化生长因子受体(RII)的突变型中进行评估，在这些突变型中，所有转化生长因子信号都被消除。我们将进一步研究这些突变体中肌腱表型的开始，以确定转化生长因子信号对肌腱形成至关重要的阶段。第二个目标是解决这样的假设，即转化生长因子(信号转导)在肌腱形成的后期也是必不可少的。可诱导性地消除转化生长因子(RII)将有助于探索转化生长因子(信号转导)的后期作用，并确定肌腱是从承诺的早期祖细胞池中产生的，还是通过转化生长因子(支持肌腱生长的信号)持续募集肌腱细胞而产生的。最后，我们将在组织培养和体内实验中探讨转化生长因子信号转导SCX的机制；比较转化生长因子信号转导和成纤维细胞生长因子信号转导的活性，并结合特定的拮抗剂和突变组织来探讨这两个信号转导通路之间协同或相互依赖的可能性。此外，还将使用逆转录病毒系统在子宫内应用转化生长因子(TGF)，以评估转化生长因子(TGF)诱导的细胞的能力(信号转导超过了SCX的表达，并导致异位或扩张肌腱的形成。

DESCRIPTION (provided by applicant): A functional musculoskeletal system depends upon the transmission of force from muscle to bone, thus requiring the coordinated development of muscle, cartilage and tendon. Little however, is known about the cellular and molecular aspects of tendon formation. Progress unraveling the signals that direct mesenchymal cells to become tenocytes is of particular importance in elucidation of the early events that shape the organization and coordinated growth of the musculoskeletal system. Moreover, a tendon inducing activity may be of considerable clinical importance both for improvements in the treatment of tendon injuries and in bioengineering efforts to induce tendon formation from stem cells. The recent finding that Scleraxis (Scx) a bHLH transcription factor is a unique marker of tenocytes and tendon progenitors facilitated studies of tendon development and a number of subsequent studies have implicated FGF signaling in the induction of tendon progenitors. Dramatic results in our laboratory now demonstrate an even more profound role for TGF( signaling in tendon induction and differentiation. Embryos doubly mutant for the TGF(2 and TGF(3 genes develop limbs in which no tendons can be detected. Moreover, TGF( signaling acts as a potent inducer of Scx, in organ culture and in tissue culture experiments, significantly exceeding the extent and intensity of Scx induction by FGF signaling in similar experiments. We propose to explore TGF( role in tendon formation in three separate groups of experiments. The first aim will address the hypothesis that all tendon tissues depend on TGF( signaling. The phenotype in axial and cranial tendons will be evaluated in TGF(2 & 3 mutants and in mutants for a TGF( receptor (TGF(RII), in which all TGF( signaling is eliminated. We will further study the onset of the tendon phenotype in these mutants to establish the stage in which TGF( signaling is essential for tendon formation. The second aim will be to address the hypothesis that TGF( signaling is essential for later aspects of tendon formation as well. Inducible elimination of TGF(RII will enable exploration of later roles for TGF( signaling and establish if tendons are generated from a committed pool of early progenitors or through continuous recruitment of tendon cells by TGF( signaling to support tendon growth. Finally, the mechanism of Scx induction by TGF( signaling will be explored in tissue culture and in vivo; the activities of TGF( and FGF signaling will be compared and the possibilities of synergism or interdependence between these two signaling cascades will be explored using a combination of specific antagonists and mutant tissues. In addition, TGF( will be applied in utero using a retroviral system to evaluate the capacity of the cells induced by TGF( signaling to go beyond expression of Scx and cause the formation of ectopic or expanded tendons.