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-细胞外基质相互作用在胚胎发育期间肢体骨骼软骨模型的分化和模式中的作用。硫酸软骨素蛋白聚糖（versican）与肢体软骨形成有关，但其在这一过程中的确切作用尚未确定。先前的研究表明，在体内，versican在早期软骨形成区和关节形成区高度表达，并且在体外，来自hdf（心脏缺陷）突变小鼠的versican缺陷肢体间质中，软骨分化受到抑制。为了更好地了解versican在体内肢体发育过程中的功能，本研究将验证一种假设，即versican的特定结构域促进软骨骨骼模板的分化，并对滑膜关节形成所需的区间区域的建立至关重要。具体目的是评估：1)特异性基因域过表达对蛋鸡肢体发育的影响；2)在卵细胞中敲低成熟基因表达对鸡肢体发育的影响。这些研究将提供新的信息，以帮助确保未来儿童免于因阑尾骨骼正常发育中断而导致的骨骼/关节残疾。重组腺病毒构建体过表达versican透明质酸结合（G1）和选择蛋白样（G3）结构域，以及包含G1和G3结构域的全长versican亚型（V3），将在卵状肢体的不同阶段通过显微注射直接传递到肢体间质。在补充实验中，将通过将腺病毒编码的小干扰RNA结构物显微注射到肢体间质中来敲低卵细胞中versican的表达。对骨骼/关节模板形态发生的影响将通过原位杂交、免疫印迹和内源性或异位的免疫组织化学来评估，并通过花生凝集素结合、阿利新蓝组织化学以及II型胶原和透明质酸的定位来增强。拟议的研究通过提供对控制正常肢体骨骼发育的因素的新见解，与公共卫生具有重要的相关性。正是通过了解这些分子机制，才能预防或纠正影响发育中的阑尾骨骼的出生缺陷。

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.