Effect of Versican Mutation on Limb Development In Vitro

描述（由申请人提供）：拟议研究的长期目标是确定细胞-细胞外基质（ECM）相互作用在软骨、肌肉和神经分化中的作用，以及它们在诱导胚胎肢体发育的适当空间关系。最近，在转基因小鼠hdf（心脏缺陷）中发现了Cspg2基因的隐性致死插入突变。Cspg2编码硫酸软骨素蛋白聚糖、versican的核心蛋白，hdf突变导致成熟蛋白聚糖的表达缺失。Versican已被认为在软骨形成和细胞模式中发挥作用。然而，关于胚胎ECM中versican功能的细节知之甚少。hdf突变提供了一个独特的机会来探索肢体的变异功能。本研究的具体目的是首先分析hdf小鼠肢体发育过程中versican的时空表达。LacZ报告组织化学、原位杂交和免疫组织化学将被用于确定半合子hdf和野生型小鼠肢体发育过程中versican的表达，以将其定位与其假设的功能联系起来。其次，将评估成熟的多聚糖蛋白聚糖在体外肢体软骨形成过程中不稳定性聚集中的作用。HDF肢体间质的微团培养将用于确定这种突变是否导致软骨前间质凝聚和随后的软骨分化的抑制。

DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to define the role of cell-extracellular matrix ECM) interactions in differentiation of cartilage, muscle, and nerve in their proper spatial relationships luring embryonic limb development. Recently, a recessive lethal insertional mutation in the Cspg2 gene was identified in a transgenic mouse line, hdf (heart defect). Cspg2 encodes the core protein of the chondroitin sulfate proteoglycan, versican, and hdf mutation results in loss of expression of the mature proteoglycan. Versican has been suggested to play a role in chondrogenesis and cellular patterning. However, little is known regarding details of versican function in the embryonic ECM. The hdf mutation offers a unique opportunity to explore versican function in the limb. The specific aims of this proposal are to first analyze the spatial and temporal expression of versican during limb development in the hdf mouse. LacZ reporter histochemistry, in situ hybridization, and immunohistochemistry will be utilized to determine versican expression during limb development in carefully staged hemizygous hdf and wildtype mice to correlate localization with its hypothesized functions. Second, the role of the mature versican proteoglycan in precartilage aggregation during limb chondrogenesis in vitro will be evaluated. micromass cultures of hdf limb mesenchyme will be utilized to determine if this mutation results in inhibition of prechondrogenic mesenchymal condensation and subsequent cartilage differentiation.