STRUCTURE AND FUNCTION OF FRIZZLED-LIKE PROTEINS IN SKEL

Frzb是一种分泌型蛋白，最初是从纯化的软骨提取物中分离出来的，它与Wnt受体卷曲家族的半胱氨酸富含结构域(CRD)具有同源性。Wnt蛋白是一种分泌的信号分子，具有多种发育功能，包括骨骼发育和肿瘤发生中的功能障碍。已有研究表明，Frzb可以与Wnt结合并使其失活，从而推测其在改变Wnt诱导的发育和致癌事件方面具有潜在的治疗用途。我们已经证明Frzb在骨骼和颅面发育过程中在时间和空间上都有表达。为了了解Frzb在发育中的作用，我们使用Cre-loxP重组系统进行了条件性基因敲除。我们通过同源重组产生了Frzb基因两侧有loxP位点的“丛生”小鼠。这些小鼠随后在普遍存在的启动子的控制下与表达Cre重组酶的转基因小鼠品系杂交，以在发育早期删除Frzb。然而，零表型看起来是正常的。这很可能是由于功能冗余，因为现在已知有许多相关的Frzb样基因具有重叠的表达模式。因此，为了研究这些基因在发育过程中的作用，有必要对不同的家庭成员进行双重敲除。应该仍然有可能通过将FRZB小鼠与空白小鼠杂交来确定FRZB在肢体和面部发育中的作用，一旦有其他家庭成员的话。随后，在组织特异性启动子的控制下，通过将小鼠与表达Cre重组酶的转基因小鼠杂交，可以以组织特异性的方式删除Frzb。为了做到这一点，我们正在建立转基因小鼠系，以在肢体和面部间充质中特异性地表达Cre重组酶。这些小鼠目前正在接受测试。

Frzb is a secreted protein, initially isolated from purified cartilage extracts, which shares homology to the cysteine rich domain (CRD) of the frizzled family of Wnt receptors. The Wnt proteins are secreted signaling molecules having numerous developmental functions, including skeletal development, as well as dysfunction in oncogenesis. It has been shown that Frzb can bind to and inactivate Wnt activity, leading to speculation for its potential therapeutic use in modifying Wnt induced developmental and oncogenic events. We have demonstrated that Frzb is temporally and spatially expressed during skeletal and craniofacial development. In an attempt to understand the role of Frzb in development we conducted a conditional gene knockout, using the Cre-LoxP recombination system. We generated "floxed" mice, in which the Frzb gene is flanked by LoxP sites, by homologous recombination. These mice were subsequently crossed to a transgenic mouse line expressing Cre recombinase under the control of a ubiquitous promoter to delete Frzb early in development. The null phenotype, however, appears normal. This is most likely due to functional redundancy, as there are now known to be a number of related Frzb-like genes with overlapping expression patterns. Consequently, in order to study the role of these genes during development it will be necessary to carry out double knockouts of different family members. It should still be possible to determine the role of Frzb in limb and face development by crossing the floxed Frzb mice to null mice for other family members once they are available. Subsequently, Frzb can be deleted in a tissue-specific manner by crossing the mice with transgenic mice expressing Cre recombinase under the control of tissue-specific promoters. In order to do this we are generating transgenic mouse lines to express Cre recombinase specifically in the limb and facial mesenchyme. These mice, are currently being tested.