The role of fibulin-4 in development and homeostasis of elastic components of the skeletal connective tissue

fibulin -4是一种50 kDa的细胞外基质蛋白，属于纤维蛋白家族。最近的一项基因靶向研究表明，纤维蛋白-4是胚胎发育过程中弹性纤维组装的重要分子。缺乏纤维蛋白-4的小鼠由于心血管和肺部异常而导致围产期死亡。我们的初步数据显示，小鼠纤维蛋白-4缺乏也会导致骨骼异常。因此，一名纤维蛋白-4隐性错义突变的患者不仅表现出弹性发生缺陷，而且在出生时出现多处骨折，而两名患者表现出蛛形畸形。该项目的目标是阐明纤维蛋白-4在骨骼发育中的作用，特别是在骨骼细胞外基质成分的组装中。为此，我将使用形态学、免疫组织化学和原位杂交技术分析纤维蛋白-4缺陷小鼠胚胎的骨骼表型。对于出生后组织的分析，我将通过与Cola1- Cre缺失小鼠杂交产生条件Fbln4缺失小鼠。使用野生型和纤维蛋白-4缺陷小鼠分离的原代细胞，将在体外评估细胞分化、基质组装改变和TGF-β/BMP信号失调的潜在损害。将特别注意进一步鉴定纤维蛋白-4结合蛋白，以阐明纤维蛋白-4缺失导致小鼠和人类异常的机制。此外，在人类患者中发现的错义突变的病理后果将通过体外诱变重组产生的纤维蛋白4突变体进行分析。这些研究将为纤维蛋白-4在骨骼系统以及心血管组织的发育和稳态中的作用提供新的见解。

Fibulin-4 is a 50 kDa extracellular matrix protein belonging to the fibulin protein family. A recent gene targeting study has revealed fibulin-4 to be an essential molecule for the elastic fiber assembly during embryonic development. Mice deficient in fibulin-4 are perinatally lethal due to cardiovascular and lung abnormalities. Our preliminary data revealed that fibulin-4 deficiency in mice also results in skeletal abnormalities. Accordingly, a patient with a recessive missense mutation in fibulin-4 displays not only defects in elastogenesis, but also multiple bone fractures at birth, while two patients showed arachnodactyly. The goal of this project is to clarify the role of fibulin-4 in skeletal development, in particular in the assembly of extracellular matrix components of the skeleton. To this aim, I will analyze the skeletal phenotype of fibulin-4 deficient mouse embryos using morphological, immunohistochemical and in situ hybridization techniques. For the analysis of postnatal tissues, I will generate conditional Fbln4 null mice by crossing with Cola1- Cre deleter mice. Potential impairment of cell differentiation, alterations of matrix assembly, and dysregulation of TGF-β/BMP signaling will be assessed in vitro using primary cells isolated from wild type and fibulin-4 deficient mice. Particular attention will be paid to identify further fibulin-4 binding proteins in order to elucidate the mechanisms by which the absence of fibulin-4 leads to abnormalities in mice and human. Furthermore, pathological consequence of missense mutations found in human patients will be analyzed using mutagenized, recombinantly produced fibilin-4 mutants in vitro. The proposed studies will provide novel insights into the role of fibulin-4 in skeletal system as well as in the development and homeostasis of cardiovascular tissue.