The impact of experience-dependent and independent factors on gene expression in songbird brain [e07]

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. 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). e07: HVC of p55 and adult male zebra finches were collected after 20 days of food ad libitum or Timed Access to Food (TAF) to assess singing-driven differences in gene expression.

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天然模型生物。在鸣禽神经基因组学计划（Songbird Neurogenomics Initiative）框架下，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，并通过基因芯片（microarray）对其中488份样本开展了系统性的基因表达分析。研究人员采用方差分析（ANOVA）对数据中的32项预设对照进行检验，以此揭示不同因素对基因表达的相对影响程度。脑组织采集区域对基因表达谱的影响最为显著，该因素可影响基因芯片上点样的18848条非冗余互补DNA（non-redundant cDNAs）所检测的绝大多数信号。社会与环境干预的影响则呈现高度异质性，部分干预无显著效应，部分则可产生强烈调控作用。多项特定基因被鉴定为环境信号调控行为的进化机制研究中的关键靶点。研究团队还先后采用加权基因共表达网络分析（Weighted Gene Co-expression Network Analysis, WGCNA）与基因本体论（Gene Ontology, GO）分析对数据集进行了深入挖掘，鉴定出了多组共调控基因模块，这些模块同时富集了特定功能注释条目，例如"核糖体（ribosome）"（在幼鸟脑组织中表达量更高）与"多巴胺代谢过程（dopamine metabolic process）"（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宝贵的数据资源。本次研究采集的RNA样本来自6种不同鸣禽（物种系统发育信息详见补充材料附录图S1），涵盖80种不同的"处理组"，即物种、脑区、性别、年龄与行为状态的各类组合。这些组织样本围绕15项独立实验进行归类整理（详见表1与补充材料附录表S1），尽管样本由十余家实验室的研究人员分别提供，但均按照鸣禽神经基因组学计划的原始设计，在单一实验室中采用统一标准完成分析。每份样本均与通用参考RNA（即斑胸草雀端脑组织RNA混合池）共同与斑胸草雀cDNA芯片进行杂交反应。实验编号e07：在自由采食或定时进食（Timed Access to Food, TAF）处理20天后，采集55日龄与成年雄性斑胸草雀的HVC核团脑组织，以此评估鸣唱行为诱导的基因表达差异。