Genetic control of skeletal development by Pbx1

描述(申请人提供)：细胞增殖的程度和时间与终末分化的完美协调是一个受基因控制的过程，对所有组织和器官系统的发育至关重要。放松对这一严格监管过程的监管可能会导致人类出生缺陷和肿瘤转化。Pbx1是一种同源结构域蛋白，它与Hox蛋白协同结合DNA，调节它们的DNA结合特异性，是果蝇牙外环(EXD)的同源物，其在形成苍蝇身体平面的功能已被基因证明。我们正在进行的努力和长期目标是利用转基因小鼠模型来评估PBX辅因子家族对哺乳动物模式和形态发生的贡献。在胚胎中，Pbx1(Pbx1-L-)的缺失导致妊娠晚期死亡，轴向和附件骨骼广泛的图案化缺陷，第二鳃弓神经嵴细胞来源的骨骼结构的同源性转化，显著抑制软骨细胞的增殖，伴随着早熟软骨细胞肥大和骨的过早成骨。与Pbx1-L不同，Pbx2-L-和Pbx3-L-小鼠在图案或骨骼发育/成熟方面都没有明显的异常。尽管如此，Pbx1-L-；Pbx2-L-突变体在宫内死亡更早，并表现出严重的骨缺陷恶化。这项建议的目的是通过以下具体目标精细剖析同源框基因Pbx1对图案和骨骼发育的遗传控制：1)通过利用现有的Cre小鼠，通过产生Pbx1在神经脊和软骨细胞中以组织特异性的方式灭活Pbx1，从基因上分离Pbx1在图案形成中的早期作用和其在软骨增殖、分化和软骨内成骨中的后期作用；2)通过使用转基因(Pbx1-L-)间充质细胞在培养中表征Pbx1在软骨细胞增殖中的作用，如小鼠胚胎成纤维细胞(MEF)和大量间充质培养；3)确定Pbx1与相关家族成员Pbx2在骨骼发育中的独特和重叠的功能，以及在软骨形成的遗传控制中的独有功能。这些研究的完成将促进我们对Pbx1图案化和骨骼发育的遗传调控的理解。在更广阔的视角下，这项工作将阐明骨骼发育扰动的戏剧性影响，并有望影响我们对影响颅面、枢椎和附件骨骼发育的人类出生缺陷的发病机制的理解。

DESCRIPTION (provided by applicant): Perfect coordination of the extent and timing of cellular proliferation with terminal differentiation is a genetically controlled process, fundamental for the development of all tissues and organ systems. Deregulations of this tightly regulated process can cause human birth defects and neoplastic transformation. Pbx1 is a homeodomain protein that collaboratively binds DNA with Hox proteins to modulate their DNA binding specificities and is a homolog of Drosophila extradenticle (EXD), whose function in patterning the fly body plan has been demonstrated genetically. Our ongoing efforts and long-term goals utilize genetically modified mouse models to assess the contributions of the Pbx family of Hox cofactors to mammalian patterning and morphogenesis. In embryos, the lack of Pbx1 (Pbx1-l-) results in late gestational lethality, widespread patterning defects of the axial and appendicular skeleton, homeotic transformation of second branchial arch neural crest cell-derived skeletal structures, markedly diminished chondrocyte proliferation, accompanied by precocious chondrocyte hypertrophy, and premature ossification of bone. Unlike Pbx1-l-, both Pbx2 -l-and Pbx3-l- mice do not display gross abnormalities either in patterning or in skeletal development/maturation. Nonetheless, Pbx1-l-; Pbx2-l- mutants die earlier in utero and show drastic exacerbation of the skeletal defects. The goal of this proposal is to finely dissect the genetic control of patterning and skeletal development by the homeobox gene Pbx1, through the following specific aims: 1) genetically uncouple the early roles of Pbxl in patterning from its later roles in cartilage proliferation, differentiation and endochondral ossification, through the generation of knockout mice where Pbxl is inactivated in a tissue-specific manner in neural crest and chondrocytes, by utilizing available Cre mice; 2) characterize the role of Pbx1 in chondrocyte proliferation by using genetically modified (Pbx1-l-) mesenchymal cells in culture, such as Mouse Embryonic Fibroblasts (MEFs), which show a striking growth defect, and Micromass Mesenchyme Cultures; 3) identify unique and overlapping functions of Pbx1 with the related family member Pbx2 in skeletal development and also exclusively in the genetic control of chondrogenesis. Completion of these studies will advance our understanding of the genetic regulation of patterning and skeletal development by Pbx1. Under a broader perspective, this work will shed light on the dramatic effects of the perturbations of skeletal development and hopefully impact on our understanding of the pathogenesis of human birth defects that affect the development of the craniofacial, axial and appendicular skeleton.