Zhao Qing, Kho Alvin, Kenney Anna Marie, Yuk Di Dong-in, Kohane Isaac, Rowitch David H
Department of Pediatric Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5704-9. doi: 10.1073/pnas.082092399.
Hedgehog pathway activation is required for proliferation of cerebellar granule cell neuron precursors during development and is etiologic in certain cerebellar tumors. To identify genes expressed specifically in granule cell neuron precursors, we used oligonucleotide microarrays to analyze regulation of 13,179 genes/expressed sequence tags in heterogeneous primary cultures of neonatal mouse cerebellum that respond to the mitogen Sonic hedgehog. In conjunction, we applied experiment-specific noise models to render a gene-by-gene robust indication of up-regulation in Sonic hedgehog-treated cultures. Twelve genes so identified were tested, and 10 (83%) showed appropriate expression in the external granular layer (EGL) of the postnatal day (PN) 7 cerebellum and down-regulation by PN 15, as verified by in situ hybridization. Whole-organ profiling of the developing cerebellum was carried out from PN 1 to 30 to generate a database of temporal gene regulation profiles (TRPs). From the database an algorithm was developed to capture the TRP typical of EGL-specific genes. The "TRP-EGL" accurately predicted expression in vivo of an additional 18 genes/expressed sequence tags with a sensitivity of 80% and a specificity of 88%. We then compared the positive predictive value of our analytical procedure with other widely used methods, as verified by the TRP-EGL in silico. These findings suggest that replicate experiments and incorporation of noise models increase analytical specificity. They further show that genome-wide methods are an effective means to identify stage-specific gene expression in the developing granule cell lineage.
在发育过程中，刺猬信号通路的激活对于小脑颗粒细胞神经元前体的增殖是必需的，并且在某些小脑肿瘤中具有病因学意义。为了鉴定在颗粒细胞神经元前体中特异性表达的基因，我们使用寡核苷酸微阵列分析了新生小鼠小脑异质原代培养物中13,179个基因/表达序列标签对有丝分裂原音猬因子（Sonic hedgehog）的反应调控情况。同时，我们应用特定实验的噪声模型来对音猬因子处理的培养物中基因上调情况进行逐个基因的稳健指示。对如此鉴定出的12个基因进行了测试，其中10个（83%）在出生后第7天（PN7）小脑的外颗粒层（EGL）中表现出适当的表达，并且到PN15时表达下调，这通过原位杂交得到了验证。从PN1到30对发育中的小脑进行全器官分析，以生成一个时间基因调控图谱（TRP）数据库。从该数据库中开发了一种算法来捕捉EGL特异性基因典型的TRP。“TRP-EGL”准确预测了另外18个基因/表达序列标签在体内的表达，灵敏度为80%，特异性为88%。然后我们通过TRP-EGL在计算机模拟中的验证，将我们分析程序的阳性预测值与其他广泛使用的方法进行了比较。这些发现表明重复实验和纳入噪声模型可提高分析特异性。它们还进一步表明全基因组方法是识别发育中的颗粒细胞谱系中阶段特异性基因表达的有效手段。
