Mechanisms of hedge-hog induced neuroproliferation

描述(由申请人提供)： Sonic hedgehog(SHH)通路激活是小脑颗粒神经元前体(CGNP)在发育过程中扩张所必需的，是人类小脑肿瘤-髓母细胞瘤的病因。然而，Hedgehog对细胞周期装置的调节作用的分子机制却知之甚少。初步数据和最近发表的工作表明，原癌基因N-myc在SHH信号下游CGNP增殖过程中通过调节D型周期蛋白促进细胞周期进展。此外，翼状螺旋转录因子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。我们的关键假设是，通过详细了解Hedgehog对中枢神经系统前体细胞周期机制的调控作用，将出现对发育和肿瘤发生的新见解。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CGNP谱系发育过程中对N-myc的遗传需求。这一点将通过使用条件性(“开花”)等位基因来特异性地灭活N-myc，(A)在用SHH处理的出生后CGNP的增殖中，以及(B)通过与颗粒细胞特异性(Math1)cre转基因系的交叉杂交来评估体内的小脑神经原。具体目标2的目的是利用转基因小鼠中经典的缺失分析来识别组织特异性的刺猬反应顺式作用DNA调控序列。同时，我们建议开发和测试新的全基因组工具，以获得关于CGNP中Hedgehog信号的其他直接靶点的观点，以及最初激活N-myc表达的上游事件。具体目标3是确定在CGNP谱系发育过程中FOXM1的遗传需求。由于在FOXM1-/-新生儿中发现了新生儿致死性，我们将使用在目标1中使用的相同的组织特异性基因敲除策略，使用数学1-cre结合FOXM1的开花等位基因来靶向颗粒细胞谱系。这项拟议的工作旨在为Hedgehog信号及其对神经前体细胞周期机制的影响建立一条“增殖途径”。高水平的N-MYC表达是Hedgehog相关的髓母细胞瘤的保守特征，这是一种主要影响儿童的肿瘤。因此，阐明在中枢神经系统发育过程中Hedgehog信号在细胞内和细胞间的新的相互作用可能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.