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

Fibulin-4是属于fibulin蛋白家族的50 kDa细胞外基质蛋白。最近的基因靶向研究表明，fibulin-4是胚胎发育过程中弹性纤维组装的重要分子。由于心血管和肺异常，缺乏fibulin-4的小鼠在围产期是致命的。我们的初步数据显示，小鼠的fibulin-4缺陷也会导致骨骼异常。因此，1例fibulin-4隐性错义突变患者不仅表现出弹性蛋白生成缺陷，而且在出生时出现多处骨折，而2例患者表现出蛛网膜囊肿。该项目的目标是阐明fibulin-4在骨骼发育中的作用，特别是在骨骼的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fibulin-4缺陷小鼠胚胎的骨骼表型。为了分析出生后的组织，我将通过与Cola 1- Cre删除小鼠杂交来产生条件性Fbln 4缺失小鼠。将使用从野生型和纤蛋白-4缺陷小鼠分离的原代细胞在体外评估细胞分化的潜在损伤、基质组装的改变和TGF-β/BMP信号传导的失调。将特别注意鉴定进一步的纤蛋白-4结合蛋白，以阐明纤蛋白-4的缺乏导致小鼠和人类异常的机制。此外，将使用诱变的、重组产生的纤蛋白-4突变体在体外分析在人类患者中发现的错义突变的病理后果。这些研究将为fibulin-4在骨骼系统以及心血管组织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.