Systematic analysis of transcription start sites in avian development

Abstract

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.

Characterisation of subpopulations of chicken mononuclear phagocytes that express TIM4 and the macrophage colony-stimulating factor receptor (CSF1R)

“…An earlier study comparing mouse and chick, examined TIMD4 mRNA in the chick semi-quantitatively at embryonic days (ED) 4-7, and inferred that TIMD4 mRNA was likely expressed by yolk sac-derived macrophages. As noted in the introduction, this conclusion was supported by expression profiling of the chick embryo (19, 20). To confirm the location of TIM4 protein, we first examined yolk sac and embryos from embryonic day 3 (HH19) by whole mount immunohistochemistry (IHC).…”

“…Nevertheless, the first evidence that macrophages are produced by the yolk sac derived from studies of chicken development and these cells are involved extensively in the clearance of apoptotic cells (reviewed in (18)). A recent study of the time course of chicken embryonic development based upon cap analysis of gene expression (CAGE) detected expression of both TIMD4 and HAVCR1 (encoding the related TIM1 protein) around day 2 of development, when the first CSF1R-dependent macrophages are also detected (19, 20). In the current study, we utilise anti-TIM4 antibodies in combination with a CSF1R- mApple reporter to locate and characterise the gene expression profiles of cells that express this receptor.…”

Combination of novel and public RNA-seq datasets to generate an mRNA expression atlas for the domestic chicken

“…Relatively few RNA-seq datasets were available for chicken embryonic development. Lizio et al [53] have recently analysed the time course of chicken development using Cap Analysis of Gene Expression (CAGE). Their dataset complements a CAGE-based analysis of gene expression in multiple tissues of the mouse during embryonic development [92].…”

“…The X-axis shows the different samples (blue – embryo developmental time course from 1.5 hours to day 20 after fertilisation (HH45); green – extraembryonic tissues; yellow – limb buds; orange – hepatocytes; red – bone marrow derived mesenchymal stem cells; dark red – aortic smooth muscle cells. Full detail of the samples can be found in Lizio, et al [53]. Y axis shows average TPM for TSS in the cluster for each sample.…”

“…Table S17. Clusters of co-expressed CAGE tags, obtained via network analysis of the Lizio, et al dataset [53].…”