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.

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Mammalian digit tip regeneration is linked to the presence of nail tissue, but a nail–explicit model is missing. Here, we report that nail–less double–ventral digits of ΔLARM1/2 mutants that lack limb–specific Lmx1b enhancers fail to regenerate. To separate the nail′s effect from the lack of DV polarity, we also interrogate double–dorsal double–nail digits and show that they regenerate. Thus, DV polarity is not a prerequisite for regeneration and the nail requirement is supported. Transcriptomic comparison between wild–type and non-regenerative ΔLARM1/2 mutant blastemas reveals differential upregulation of vascularization and connective tissue functional signatures in wild–type versus upregulation of inflammation in the mutant. These results, together with the finding of uniform Lmx1b expression in the wild–type blastema and in the dorsal dermis underneath the nail, indicate that, in addition of the nail′s effect, a direct role for Lmx1b in driving the progression of digit tip regeneration is likely.