CEREBELLAR PROTEOGLYCANS IN SONIC HEDGEHOG RESPONSES

这项建议研究了小脑发育过程中音速刺猬(Shh)反应的调节。Sonic Hedgehog在发育过程中会产生多种形态发生和有丝分裂效应，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信号转导机制知之甚少，远远落后于我们对其他几个生长因子的了解。在果蝇(Tout-velu)中，一种参与硫酸肝素蛋白多糖(HSPG)合成的糖基转移酶是HH反应所必需的。有许多HSPG调节生长因子信号的例子，以及几种HSPG合成疾病的显著发育异常和癌症风险增加。我们重点研究了HSPGs对发育中的小鼠小脑Shh受体结合和信号转导的影响。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，同样的处理会导致Shh结合增加。此外，我们还发现，脊椎动物Tout-velu同源基因ext2在小脑中的表达随着年龄的增长而增加，并且在有丝分裂后颗粒细胞中更为丰富。我们假设HSPG的合成在小脑发育过程中受到调节，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(EXT1-3，L1-3)在发育中的小脑中的表达模式来检验我们的假设。我们将在PTC-LacZ(正/负)小鼠的小脑原代培养中检测Shh途径的激活以及在遗传和生化操作HSPG合成后Shh受体的结合。直接HSPG-Shh相互作用的必要性将通过假定Shh肝素结合域的定点突变来确定。综上所述，这些实验将产生关于HSPG调节Shh结合和信号之间关系的新信息，并可能为Shh和HSPG功能障碍导致疾病的机制提供新的线索。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。罗莎琳德·西格尔博士在达纳-法伯癌症研究所儿科肿瘤科的实验室为我作为一名独立研究员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.