The signaling role of Qsulf in embryonic neural tube

描述（由申请人提供） 该提案的目的是描述两个 候选硫酸乙酰肝素蛋白聚糖（HSPG）修饰酶，鹌鹑 葡糖胺6-O-硫酸酯酶1（QSulf 1）和QSulf 2在神经管发育中的作用。细胞 表面HSPG在调节分布和/或 信号分子如Wnt、Shh、FGF和BMP的活性。QSulf1，the 第一个已知的细胞外基质G6-硫酸酯酶，是体节MyoD所必需的 通过其酶活性激活以调节Wnt途径。QSulf 2是 家族中第二个功能未知的成员。QSulf 1和QSulf 2具有 胚胎神经管中的重叠和离散表达域。的 该提案将检验QSulf 1和QSulf 2具有独特功能的假设 在细胞外信号通路中， 由它们不同的亲水结构域控制。首先，我们将描述 QSulf 1和QSulf 2基因在整胚胚胎发生中表达模式 原位杂交结合免疫组化。结果从这个 实验将作为功能分析的初始步骤， QSulfs在神经管发育中的作用。第二，在 神经管，我们将操纵每个基因的表达， 鸡电穿孔介导的过表达和反义抑制 第12期的胚胎神经管形成的分子标记物和神经 嵴细胞将通过免疫组织化学表征。从这些 实验将提供QSulfs在胚胎发育中的信号作用的信息。 神经管第三，免疫共沉淀和酵母双杂交筛选将 进行鉴定，以确定不同亲水性的结合蛋白， QSulfs的结构域被假设为控制 QSuIf 1或QSuIf 2。这项研究将提供有关机制的见解， 在胚胎中定位QSulf功能。

DESCRIPTION (provided by applicant) The objective of this proposal is to characterize the signaling roles of two candidate heparan sulfate proteoglycan (HSPG) modifying enzymes, quail glucosamine 6-O-sulfatase 1 (QSulf1) and QSulf2 in developing neural tube. Cell surface HSPGs play important roles in modulating the distribution and/or activity of signaling molecules such as Wnts, Shh, FGFs and BMPs. QSulf1, the first known extracellular matrix G6-sulfatase, is required for somite MyoD activation through its enzymatic activity to regulate Wnt pathway. QSulf2 is the second member of the family with unknown function. QSulf1 and QSulf2 have overlapping and discrete expression domains in embryonic neural tube. The proposal will test the hypothesis that QSulf1 and QSulf2 have unique functions in extracellular signaling pathways and the functional distinction is controlled by their divergent hydrophilic domains. First, we will characterize the expression pattern of QSulf1 and QSulf2 during embryogenesis by whole mount in situ hybridization combined with immunohistochemistry. Results from this experiment will serve as the initial steps for the functional analysis of QSulfs in developing neural tube. Second, to define QSulf function in the neural tube, we will manipulate the expression of each gene both by electroporation-mediated overexpression and antisense inhibition in chick embryos at stage 12. Molecular markers for neural tube patterning and neural crest cells will be characterized by immunohistochemistry. Results from these experiments will provide information in signaling roles of QSulfs in embryonic neural tube. Third, co-immunoprecipitation and yeast two-hybrid screen will be conducted to identify the binding proteins for the divergent hydrophilic domains of QSulfs that are hypothesized to control the localized functions of QSuIf1 or QSulf2. This study will provide insights on the mechanisms for localized QSulf function in the embryo.