Cap Analysis of Gene Expression (CAGE) in combination with single-molecule sequencing technology allows precision mapping of transcription start sites (TSSs) and genome-wide capture of promoter activities in differentiated and steady state cell populations. Much less is known about whether TSS profiling can characterize diverse and non-steady state cell populations, such as the approximately 400 transitory and heterogeneous cell types that arise during ontogeny of vertebrate animals. To gain such insight, we used the chick model and performed CAGE-based TSS analysis on embryonic samples covering the full 3-week developmental period. In total, 31,863 robust TSS peaks (>1 tag per million [TPM]) were mapped to the latest chicken genome assembly, of which 34% to 46% were active in any given developmental stage. ZENBU, a web-based, open-source platform, was used for interactive data exploration. TSSs of genes critical for lineage differentiation could be precisely mapped and their activities tracked throughout development, suggesting that non-steady state and heterogeneous cell populations are amenable to CAGE-based transcriptional analysis. Our study also uncovered a large set of extremely stable housekeeping TSSs and many novel stage-specific ones. We furthermore demonstrated that TSS mapping could expedite motif-based promoter analysis for regulatory modules associated with stage-specific and housekeeping genes. Finally, using Brachyury as an example, we provide evidence that precise TSS mapping in combination with Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-on technology enables us, for the first time, to efficiently target endogenous avian genes for transcriptional activation. Taken together, our results represent the first report of genome-wide TSS mapping in birds and the first systematic developmental TSS analysis in any amniote species (birds and mammals). By facilitating promoter-based molecular analysis and genetic manipulation, our work also underscores the value of avian models in unravelling the complex regulatory mechanism of cell lineage specification during amniote development.

基因表达的上限分析（CAGE）与单分子测序技术相结合，可以精确定位转录起始位点（TSS），并在全基因组范围内捕获分化和稳态细胞群中的启动子活性。关于TSS谱图是否可以表征多样化和非稳态细胞群体（例如在脊椎动物的个体发育过程中出现的约400种瞬时和异种细胞类型），人们所知甚少。为了获得这种洞察力，我们使用了雏鸡模型，并对涵盖整个3周发育期的胚胎样本进行了基于CAGE的TSS分析。总共将31,863个鲁棒的TSS峰（> 1个标签/百万个[TPM]）映射到最新的鸡基因组装配体，其中34％至46％在任何给定的发育阶段均具有活性。基于网络的ZENBU，开源平台，用于交互式数据探索。可以精确定位对谱系分化至关重要的基因的TSS，并在整个发育过程中跟踪其活动，这表明非稳态和异质细胞群体适合于基于CAGE的转录分析。我们的研究还发现了一大堆极其稳定的客房服务TSS和许多新颖的特定阶段的TSS。我们进一步证明，TSS定位可以加快与阶段特异性和管家基因相关的调控模块的基于基序的启动子分析。最后，使用 这表明非稳态和异质细胞群体适合基于CAGE的转录分析。我们的研究还发现了一大堆极其稳定的客房服务TSS和许多新颖的特定阶段的TSS。我们进一步证明，TSS定位可以加快基于基序的启动子分析，以分析与阶段特异性基因和管家基因相关的调控模块。最后，使用 这表明非稳态和异质细胞群体适合基于CAGE的转录分析。我们的研究还发现了一大堆极其稳定的客房服务TSS和许多新颖的特定阶段的TSS。我们进一步证明，TSS定位可以加快基于基序的启动子分析，以分析与阶段特异性基因和管家基因相关的调控模块。最后，使用以Brachyury为例，我们提供的证据表明，精确的TSS定位结合聚类的规则间隔的短回文重复（CRISPR）-on技术使我们第一次能够有效地靶向内源性禽类基因进行转录激活。综上所述，我们的结果代表了鸟类中全基因组TSS定位的第一个报告，以及任何羊膜物种（鸟类和哺乳动物）中第一个系统的发育性TSS分析。通过促进基于启动子的分子分析和遗传操作，我们的工作还强调了禽类模型在阐明羊膜发育过程中细胞谱系规格的复杂调控机制方面的价值。