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胚胎发育过程中细胞-细胞外基质相互作用在肢体骨骼软骨模型分化和形成模式中的作用。硫酸软骨素蛋白聚糖，多功能蛋白聚糖，已被牵连在肢体软骨形成，但其在这一过程中的确切作用尚未确定。先前的研究表明多功能蛋白聚糖在体内早期软骨形成和关节形成区域高度表达，并且在来自hdf（心脏缺陷）突变小鼠的多功能蛋白聚糖缺陷肢体间充质中，软骨分化在体外受到抑制。为了更好地了解多能蛋白聚糖在体内肢体发育过程中的功能，该提案将测试以下假设：多能蛋白聚糖的特定结构域促进软骨骨骼模板的分化，并且对于建立滑膜关节形成所需的区间区域至关重要。具体目的是评估以下的影响：1）特异性多功能蛋白聚糖结构域对卵内鸡肢发育的过表达;和2）成熟多功能蛋白聚糖表达对卵内鸡肢发育的敲低。这些研究将提供新的信息，以帮助确保在未来的儿童是免费的骨骼/关节残疾，由于破坏正常发育的apappropriate骨骼。用于过表达多功能蛋白聚糖透明质酸结合（G1）和选择素样（G3）结构域以及含有G1和G3结构域的全长多功能蛋白聚糖同种型（V3）的重组腺病毒构建体将通过卵内肢体显微注射直接递送至不同阶段的肢体间充质。在补充实验中，通过将腺病毒编码的小干扰RNA构建体显微注射到肢体间充质中来敲低卵中的多功能蛋白聚糖表达。将通过原位杂交、蛋白质印迹和内源性或异位多能蛋白聚糖的免疫组织化学评价对骨骼/关节模板形态发生的影响，并通过与花生凝集素结合、阿尔新蓝组织化学以及II型胶原和透明质酸的定位相关性进行增强。拟议的研究承担重要的相关性，公共卫生提供了新的见解，控制正常的肢体骨骼发育的因素。通过了解这些分子机制，可以预防或纠正影响发育中的无骨骨骼的出生缺陷。

DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to define the role of cell-extracellular matrix interactions in differentiation and patterning of the cartilage model of the limb skeleton during embryonic development. The chondroitin sulfate proteoglycan, versican, has been implicated in limb chondrogenesis, but its precise role in this process has yet to be defined. Previous work has shown that versican is highly expressed in early chondrogenic and joint-forming regions in vivo and that cartilage differentiation is inhibited in vitro in versican-deficient limb mesenchyme derived from the hdf (heart defect) mutant mouse. To better understand versican's function during limb development in vivo, this proposal will test the hypothesis that specific domains of versican facilitate differentiation of the cartilaginous skeletal template and are critical for establishment of interzone regions required for synovial joint formation. The specific aims are to assess effects of: 1) over-expression of specific versican domains on chick limb development in ovo; and, 2) knock down of mature versican expression on chick limb development in ovo. These studies will provide new information to help ensure that in the future children are free of skeletal/articular disability due to disruption of normal development of the appendicular skeleton. Recombinant adenoviral constructs for over-expression of versican hyaluronan-binding (G1) and selectin-like (G3) domains and a full length versican isoform (V3) containing both G1 and G3 domains will be delivered directly to limb mesenchyme at various stages by microinjection into the limb in ovo. In complementary experiments, knock down of versican expression in ovo will be performed by microinjection of adenovirally encoded small interfering RNA constructs into limb mesenchyme. Effects on morphogenesis of the skeletal/joint template will be evaluated by in situ hybridization, Western blotting, and immunohistochemistry for endogenous or ectopic versican, augmented by correlation with peanut agglutinin-binding, Alcian blue histochemistry, and localization of collagen Type II and hyaluronan. The proposed studies bear important relevance to public health by providing new insights into factors controlling normal limb skeletal development. It is through understanding these molecular mechanisms that prevention or correction of birth defects affecting the developing appendicular skeleton can be achieved.