Mechanisms of hedge-hog induced neuroproliferation

描述（由申请人提供）： Sonic hedgehog（SHH）通路激活是发育过程中小脑颗粒神经元前体细胞（CGNP）扩增所必需的，并且是人类小脑肿瘤髓母细胞瘤的病因。然而，刺猬调控细胞周期装置的分子机制知之甚少。初步数据和最近发表的工作表明，在CGNP增殖过程中，原癌基因N-myc在SHH信号下游通过调节D型细胞周期蛋白促进细胞周期进程中起重要作用。此外，翼形螺旋转录因子FoxM1是B型细胞周期蛋白表达的调节因子，与HH相关的皮肤癌有关。我们的关键假设，新的见解，发展和肿瘤发生将出现从一个详细的了解刺猬调节作用的细胞周期机制内中枢神经系统前体。为了验证这一假设，我们提出了以下三个具体目标：具体目标1是确定在CGNP谱系发育过程中对N-myc的遗传要求。这将使用条件性（"floxed"）等位基因来建立，该等位基因特异性地（a）在培养物中用SHH处理的增殖的出生后CGNP中，和（B）通过与颗粒细胞特异性（Math 1）cre转基因系的杂交来评估体内小脑原基。具体目标2的目的是在转基因小鼠中使用经典的缺失分析来鉴定N-myc的组织特异性、Hedgehog响应性顺式作用DNA调控序列。同时，我们建议开发和测试新的全基因组工具，以获得对CGNP中刺猬信号传导的其他直接靶点和最初激活N-myc表达的上游事件的看法。具体目标3是确定CGNP谱系发育期间FoxM1的遗传需求。由于在FoxM1-/-新生儿中发现的新生儿致死性，我们将采用Aim 1中使用的相同组织特异性敲除策略，使用Math 1-cre组合FoxM1的floxed等位基因靶向颗粒细胞谱系。这项工作的目的是建立一个"增殖途径"的刺猬信号和其对细胞周期机制的神经前体。高水平的N-MYC表达是Hedgehog相关髓母细胞瘤（一种主要影响儿童的肿瘤）病例的保守特征。因此，阐明中枢神经系统发育过程中Hedgehog信号的细胞内和细胞间的新相互作用可以为人类肿瘤的治疗干预提供线索。

DESCRIPTION (provided by applicant): Sonic hedgehog (SHH) pathway activation is required for expansion of cerebellar granule neuron precursors (CGNP) during development and is etiologic in the human cerebellar tumor, medulloblastoma. However, the molecular mechanisms underlying Hedgehog regulatory effects on the cell cycle apparatus are poorly understood. Preliminary data and recently published work suggests an important role for proto-oncogene N-myc during CGNP proliferation downstream of SHH signaling in promoting cell cycle progression via regulation of D-type cyclins. Additionally, the winged-helix transcription factor, FoxM1, a regulator of B-type cyclin expression, has been implicated in HH-associated skin cancer. Our key hypothesis that new insights into development and tumorigenesis will emerge from a detailed understanding of Hedgehog regulatory effects on the cell cycle machinery within CNS precursors. To test this hypothesis, we propose the following three Specific Aims: Specific Aim 1 is to determine genetic requirements for N-myc during development of the CGNP lineage. This will be established using a conditional ("floxed") allele to inactivate N-myc specifically (a) in proliferating post-natal CGNP treated with SHH in culture, and (b) through intercrosses with a granule cell specific (Math 1) cre transgenic line to assess the cerebellar anlagen in vivo. The objective of Specific Aim 2 is to identify tissue-specific, Hedgehog-responsive cis-acting DNA regulatory sequences for N-myc using a classic deletion analysis in transgenic mice. In parallel, we propose to develop and test novel genome-wide tools to gain perspective on other direct targets of hedgehog signaling in CGNP and the upstream events that initially activate N-myc expression. Specific Aim 3 is to determine genetic requirements for FoxM1 during development of the CGNP lineage. Because of neonatal lethality found in FoxM1-/- neonates, we will employ the identical tissue-specific knockout strategy used in Aim 1 using Math 1-cre combined a floxed allele of FoxM1 to target the granule cell lineage. The proposed work is intended to establish a "proliferative pathway" for Hedgehog signaling and its effects on the cell cycle machinery within neural precursors. High levels of N-MYC expression are a conserved feature of Hedgehog-associated cases of medulloblastoma, a tumor primarily affecting children. Thus, elucidation of novel intra- and intercellular interactions of Hedgehog signaling during CNS development could provide clues for therapeutic intervention in human tumors.