CEREBELLAR PROTEOGLYCANS IN SONIC HEDGEHOG RESPONSES

本研究旨在探讨小脑发育过程中音刺猬反应的调控机制。 Sonic hedgehog在发育过程中产生多种形态发生和促有丝分裂效应，包括小脑颗粒细胞增殖。 Shh的失调具有深远的发育后果，并与几种癌症有关。 Shh信号传导的机制知之甚少，远远落后于我们对其他几种生长因子的了解。 在果蝇中，Tout-velu，一个假定的糖基转移酶参与硫酸肝素蛋白聚糖（HSPG）的合成是Hh反应所必需的。 有多个HSPG调节生长因子信号传导的实例和几种HSPG合成疾病，其具有显著的发育异常和增加的癌症风险。 我们集中在HSPGs对Shh受体结合和信号转导在发育中的小鼠小脑的影响。 在初步研究中，我们已经证明了Shh与肝素的直接相互作用，并表明细胞表面HSPG的减少导致Shh诱导的增殖减少，这与Tout-velu突变的效果一致。 令人惊讶的是，这些相同的处理导致增加的Shh结合。 此外，我们发现，小脑表达的EXT 2，脊椎动物Tout-velu同源物，随着年龄的增长，更丰富的有丝分裂后的颗粒细胞。 我们假设HSPGs的合成在小脑发育过程中受到调节，并且是Shh结合和Shh反应的决定因素。 我们将通过确定EXT家族成员（EXT 1 -3，L1-3）在小脑发育中的表达模式来验证我们的假设。 我们将测量Ptc-LacZ（加/减）小鼠小脑原代培养物中Shh通路的激活，以及HSPG合成的遗传和生化操作后Shh受体结合。 直接HSPG-Shh相互作用的必要性将通过推定的Shh肝素结合结构域的定点诱变来确定。 总之，这些实验将产生新的信息HSPG调制之间的关系Shh结合和信号传导，并可能揭示新的机制Shh和HSPG功能障碍产生的疾病。 此外，这些研究将为我提供学习现代细胞和分子生物学技术的机会，并建立研究神经发育的技能。 Rosalind Segal博士在Dana-Farber癌症研究所儿科肿瘤学系的实验室为我作为一名独立研究者的发展提供了一个极好的环境。

This proposal examines the regulation of sonic hedgehog (Shh) response in developing cerebellum. Sonic hedgehog produces multiple morphogenetic and mitogenic effects during development including cerebellar granule cell proliferation. Dysregulation of Shh has profound developmental consequences and is implicated in several cancers. The mechanism of Shh signaling is poorly understood and lags far behind our knowledge of several other growth factors. In Drosophila, Tout-velu, a putative glycosyltransferase involved in the synthesis of heparin sulfate proteoglycans (HSPG) is necessary for Hh response. There are multiple examples of HSPG modulation of growth factor signaling and several diseases of HSPG synthesis with significant developmental abnormalities and increased cancer risks. We have focused on the effects of HSPGs on Shh receptor binding and signaling in the developing mouse cerebellum. In preliminary studies we have demonstrated direct interaction of Shh with heparin, and shown that reduction of cell surface HSPGs results in decreased Shh induced proliferation consistent with the effect of the Tout-velu mutation. Surprisingly, these same treatments result in increased Shh binding. Further, we have found that cerebellar expression of EXT2, a vertebrate Tout-velu homolog, increases with age and is more abundant in post-mitotic granule cells. We hypothesize that the synthesis of HSPGs is regulated during cerebellar development and is a determinant of Shh binding and Shh responses. We will test our hypotheses by determining the pattern of expression of EXT family members (EXT1-3, L1-3) in developing cerebellum. We will measure Shh pathway activation in primary cultures of cerebellum from Ptc-LacZ (plus/minus) mouse and Shh receptor binding after genetic and biochemical manipulation of HSPG synthesis. The necessity for direct HSPG-Shh interactions will be determined by site-directed mutagenesis of the putative Shh heparin binding domain. Taken together these experiments will generate novel information regarding HSPG modulation of the relationship between Shh binding and signaling and may shed new light on the mechanisms by which dysfunction of Shh and HSPG produce disease. In addition, these studies will provide me with the opportunity to learn the techniques of modern cell and molecular biology and establish the skills with which to study neural developmental. Dr. Rosalind Segal's laboratory in the Department of Pediatric Oncology at the Dana-Farber Cancer Institute provides an excellent environment for me to develop as an independent investigator.