Role of Muscle Connective Tissue in Muscle and Tendon Development

描述（由申请人提供）：脊椎动物肌肉骨骼系统的发育需要肌肉、肌肉结缔组织、肌腱和骨骼的协调形态发生。在肢体中，肌肉来源于体节的迁移前体，而肌肉结缔组织、肌腱和骨骼成分来源于新兴的侧板衍生的肢芽的中胚层。随着肌肉前体迁移到肢体中，它们分化成肌纤维，正确地形成不同的肌肉，并与肌肉结缔组织，肌腱和骨骼组装成功能性肌肉骨骼。肌肉和肌腱如何与肌肉结缔组织形成图案和组装在很大程度上是未知的，在多种人类遗传综合征中有缺陷，也是本提案的主题。肌肉和肌腱与结缔组织的密切发育关系表明，这些组织之间的相互作用可能对其发育至关重要。然而，由于缺乏标记结缔组织成纤维细胞的分子标记物和遗传试剂，阻碍了对肌肉结缔组织的研究。我们发现，转录因子Tcf 4（Tcf 7 L2）在成人结缔组织成纤维细胞和胚胎中的前体细胞中强烈表达。在上一个资助期间，我们设计了Tcf 4GFPCre和Tcf 4CreERT 2小鼠，这是第一种允许对这些成纤维细胞进行遗传操作的试剂。我们的Tcf 4功能的初步研究表明，结缔组织调节肌肉和肌腱的形态发生在发展中的肢体。此外，我们和我们的合作者发现Lmx 1b，Tbx 3，Tbx 4和Tbx 5是建立肢体背侧/腹侧，前/后和前/后肢不对称并确定肢体肌肉和肌腱模式的关键转录因子。初步研究表明，这些转录因子通过其在肌肉结缔组织中的功能，非细胞自主地调节肌肉和肌腱的形态发生。在这里，我们提出测试的假设，结缔组织，通过Tcf 4，Lmx 1b，Tbx 3，Tbx 4和Tbx 5功能，决定了四肢肌肉和肌腱的模式。这些实验将阐明协调和组装一个完整的功能性肌肉骨骼系统所必需的细胞-细胞和分子相互作用。此外，Lmx 1b、Tbx 3、Tbx 4和Tbx 5的突变已被确定为人类指甲-髌骨、尺骨-乳房、小髌骨和Holt-Oram综合征的遗传原因，其特征在于肢体肌肉、肌腱和骨骼的严重异常。这些基因突变如何导致这些肌肉骨骼缺陷尚不清楚。我们对这些基因的小鼠突变体的分析将阐明导致这些肢体肌肉骨骼异常的细胞和分子缺陷。

DESCRIPTION (provided by applicant): Development of the vertebrate musculoskeletal system requires the coordinated morphogenesis of muscle, muscle connective tissue, tendon, and skeleton. In the limb, muscle derives from migratory precursors originating from the somites, while the muscle connective tissue, tendons, and skeletal elements derive from the mesoderm of the emerging lateral plate-derived limb bud. As muscle precursors migrate into the limb, they differentiate into myofibers, become correctly patterned into distinct muscles, and are assembled with muscle connective tissue, tendons, and bones into a functional musculoskeleton. How muscles and tendons are patterned and assembled with muscle connective tissue is largely unknown, defective in multiple human genetic syndromes, and the subject of this proposal. The close developmental association of muscle and tendon with connective tissue suggests that interactions between these tissues may be critical for their development. However, study of muscle connective tissue has been hindered by the lack of molecular markers and genetic reagents to label connective tissue fibroblasts. We identified that the transcription factor Tcf4 (Tcf7L2) is strongly expressed in adult connective tissue fibroblasts and in their precursors in the embryo. During the previous grant period, we engineered Tcf4GFPCre and Tcf4CreERT2 mice, the first reagents to allow for genetic manipulation of these fibroblasts. Our preliminary studies of Tcf4 function suggest that the connective tissue regulates muscle and tendon morphogenesis in the developing limb. In addition, we and our collaborators have found that Lmx1b, Tbx3, Tbx4, and Tbx5 are key transcription factors that establish the dorsal/ventral, anterior/posterior, and fore/hindlimb asymmetries of the limb and determine the pattern of limb muscles and tendons. Preliminary studies suggest that these transcription factors regulate muscle and tendon morphogenesis non-cell autonomously via their function in muscle connective tissue. Here we propose to test the hypothesis that connective tissue, via Tcf4, Lmx1b, Tbx3, Tbx4, and Tbx5 function, determines the pattern of limb muscles and tendons. These experiments will elucidate the cell-cell and molecular interactions necessary to coordinate and assemble an integrated and functional musculoskeletal system. Furthermore, mutations in Lmx1b, Tbx3, Tbx4, and Tbx5 have been identified as the genetic causes of human Nail-Patella, Ulnar-Mammary, Small Patella, and Holt-Oram syndromes, characterized by severe abnormalities in limb muscle, tendon, and skeleton. How these genetic mutations cause these musculoskeletal defects is unknown. Our analysis of mouse mutants of these genes, which phenocopy the human syndromes, will elucidate the cellular and molecular defects that lead to these limb musculoskeletal abnormalities.