Genetic Study of Heparan Sulfate Function in Development

说明(申请人提供)：硫酸乙酰肝素蛋白多糖(HSPGs)参与多种生物学过程，如生长因子信号转导。许多人类疾病被发现与HSPG生物合成的缺陷有关。硫酸乙酰肝素(HS)链具有明显的多相结构，主要由N-、2-O-、6-O-和3-O-硫酸盐基团的调节引入而产生。证据表明，HS的这些“精细结构”控制着细胞表面的离散信号事件。然而，这种控制的分子基础在很大程度上是未知的。我们的目标是利用一种遗传上易驯化的模式生物--黑腹果蝇，了解特定的HS精细结构在形态发生中的作用。我们最近发现果蝇HS 6-O-SutfoTransfer ase(DHS6ST)是气管系统发育过程中成纤维细胞生长因子信号的正向调节因子。这与最近的生化数据一致，这些数据表明6-O-硫化是脊椎动物系统中激活成纤维细胞生长因子信号的原因。然而，HS的这种特定的硫化作用控制生长因子信号的分子机制仍有待阐明。我们还研究了HS的3-O-位上的硫酸盐化，这是已知对哺乳动物的血液凝固和病毒感染至关重要的。然而，人们对3-O-硫化在发育中的作用知之甚少。在我们的初步研究中，果蝇HS 3-O-磺基转移酶-b(dHS3ST-b)参与了Notch信号和许多组织的组装，参与了发育过程中的3-O-硫化。 在拟议的研究中，我们将研究果蝇HSST的三个基因dHS6ST、dHS3ST-a和-b在发育过程中的体内功能。目的：1.探讨dHS6ST在果蝇发育过程中生长因子信号转导中的分子功能。目的2.确定dHS3ST-b基因在Notch信号通路中的分子功能。目的3.在果蝇组织中定位配体特异性HS。这项研究将使用两种最新开发的技术：转基因RNA干扰(AIM 2)和原位配体结合分析(AIM 3)。

DESCRIPTION (provided by applicant): Heparan sulfate proteoglycans (HSPGs) are involved in a variety of biological processes such as growth factor signaling. Many human diseases have been found to be associated with defects in the HSPG biosynthesis. The heparan sulfate (HS) chains have markedly heterogeneous structures that are mainly produced by the regulated introduction of N-, 2-O-, 6-O-, and 3-O-sulfate groups. Evidence suggests that these "fine structures" of HS control discrete signaling events at the cell surface. The molecular basis for this control, however, is largely unknown. Our goal is to understand the roles of specific HS fine structures in morphogenesis using a genetically tractable model organism, Drosophila melanogaster. We recently identified Drosophila HS 6-O-sutfotransferase (dHS6ST) as a positive regulator of FGF signaling during tracheal system development. This is consistent with recent biochemical data, which suggested that 6-O-sulfation is responsible for the activation of FGF signaling in vertebrate systems. However, the molecular mechanism by which growth factor signaling is controlled by this specific sulfation of HS remains to be elucidated. We have also investigated sulfation at the 3-O-position of HS, which is known to be critical for blood coagulation and viral infection in mammals. However, the role of 3-O-sulfation in development is poorly understood. In our preliminary studies, Drosophila HS 3-O-sulfotransferase-b (dHS3ST-b) is involved in Notch signaling and the assembly of many tissues, implicating 3-O-sulfation in development. In the proposed research, we will study the in vivo functions of three genes for Drosophila HSSTs, dHS6ST, dHS3ST-a, and -b, during development. Specific aims are: Aim 1. Explore the molecular functions of dHS6ST in growth factor signaling during Drosophila development. Aim 2. Determine the molecular functions of the dHS3ST-b gene in the Notch signaling pathway. Aim 3. Localize the ligand-specific HS in Drosophila tissues. Two recently developed techniques, transgenic RNA interference (Aim 2) and an in situ ligand binding assay (Aim 3), will be used in this study.