The impact of experience-dependent and independent factors on gene expression in songbird brain [e03]. The impact of experience-dependent and independent factors on gene expression in songbird brain [e03]

Songbirds provide rich natural models for studying the relationships of brain anatomy, behavioral function, environmental signals and gene expression. Under the Songbird Neurogenomics Initiative, investigators from more than a dozen laboratories collected brain samples from six species of songbird under a range of experimental conditions, and 488 of these samples were analyzed systematically for gene expression by microarray. ANOVA was used to test 32 planned contrasts in the data, revealing the relative impact of different factors. The brain region from which tissue was taken had the greatest influence on gene expression profile, affecting the majority of signals measured by the 18,848 non-redundant cDNAs spotted on the microarray. Social and environmental manipulations had highly variable impact, ranging from nil in some cases to robust in others. Several specific genes were identified as points of interest in the evolution of mechanisms by which environmental signals influence behavior. The data were also analyzed using Weighted Gene Co-expression Network Analysis (WGCNA) followed by Gene Ontology (GO) analysis. This revealed modules of co-regulated genes that are also enriched for specific functional annotations such as “ribosome” (expressed more highly in juvenile brain) and “dopamine metabolic process” (expressed more highly in striatal song control nucleus Area X). These results underscore the complexity of influences on neural gene expression and provide a resource for studying how these influences are integrated during natural experience. Overall design: RNA samples were collected from six different songbird species (phylogeny in SI Appendix, Figure S1) and representing 80 different “treatments”, i.e., combinations of species, brain region, sex, age, and behavioral state. The tissue samples were organized around 15 standalone experiments (Table 1 and SI Appendix, Table S1), contributed by investigators in a dozen different laboratories but analyzed under uniform conditions in a single laboratory as originally planned under the Songbird Neurogenomics Initiative. Each sample was hybridized to a zebra finch cDNA array along with a universal reference (a pool of zebra finch telencephalic RNA). e03: Diencephalons were collected under short days (8:16 light:dark) from birds on either standard or enriched diets, in 2 species that differ in effects of diet on reproductive behavior (responsive Red crossbills, non-responsive House finches). Red crossbills on a standard diet and long days (20:4 light:dark) were also compared to the short day group to assess effects of photostimulation in this species. All samples were hybridized against the SoNG universal RNA reference pool.

鸣禽是研究大脑解剖结构、行为功能、环境信号与基因表达之间关联的优质自然模型。在鸣禽神经基因组学计划（Songbird Neurogenomics Initiative）框架下，来自十余家实验室的研究人员在多种实验条件下，采集了6种鸣禽的脑组织样本，并通过微阵列（microarray）技术对其中488份样本进行了系统性的基因表达分析。研究采用方差分析（ANOVA）对数据中的32项预设对照进行检验，以此明确不同因素对基因表达的相对影响程度。结果显示，组织取材的脑区对基因表达谱的影响最为显著，该因素会影响微阵列上点样的18848个非冗余互补DNA（cDNA）所检测到的绝大多数信号。社会与环境干预的影响则具有高度异质性，部分情境下无显著效应，而在另一些情境下则效应显著。研究还鉴定出若干关键基因，可作为探究环境信号调控行为的进化机制的研究靶点。此外，团队还采用加权基因共表达网络分析（Weighted Gene Co-expression Network Analysis, WGCNA），随后开展基因本体（Gene Ontology, GO）分析对数据进行二次解析，该分析揭示了共调控基因模块，这些模块同时富集了特定功能注释，例如"核糖体"（在幼年大脑中表达量更高）以及"多巴胺代谢过程"（在纹状体鸣唱控制核团X区（Area X）中表达量更高）。上述结果凸显了神经基因表达调控影响因素的复杂性，同时也为探究自然体验过程中这些影响因素如何协同整合提供了重要的数据资源。

实验整体设计：研究人员从6种不同鸣禽（系统发育信息详见补充材料附录图S1）中采集了RNA样本，涵盖80种不同的"treatments"，即物种、脑区、性别、年龄与行为状态的不同组合。这些组织样本围绕15项独立实验进行整理（详见表1与补充材料附录表S1），由十余家实验室的研究人员共同提供，但按照鸣禽神经基因组学计划的初始规划，统一在单个实验室中完成标准化分析。每份样本均与通用参考样本（斑胸草雀端脑RNA混合池）一同，在斑胸草雀cDNA微阵列上进行杂交反应。

e03实验：研究人员在短光照条件（光照:黑暗=8:16）下，从2个对日粮调控生殖行为效应存在差异的物种中采集间脑组织——分别为具有响应性的红交嘴雀（Red crossbills）与无响应性的家朱雀（House finches），受试鸟类均饲喂标准日粮或富集日粮。此外，研究还将饲喂标准日粮且处于长光照条件（光照:黑暗=20:4）的红交嘴雀，与短光照组的红交嘴雀进行对比，以此评估该物种的光刺激效应。所有样本均与SoNG通用RNA参考池进行杂交反应。