GSE126691
Genome-wide maps of chromatin accessibility governing the transition from founder cells to distinct fate-restricted cardiopharyngeal precursors
We used the assay for transposon-accessible chromatin using sequencing (ATAC-seq) on FACS-purified cells to profile chromatin accessibility during early cardiopharyngeal fate choices in Ciona.  We obtained ~500 million unique reads from samples comprising cardiopharyngeal mesoderm and mesenchymal cells, as well as whole Ciona embryos and genomic DNA control. We combined ATAC-seq peaks from all the replicates to generate an atlas of 56,090 unique and non-overlapping accessible regions (accessome) covering 9.25% of the C. robusta genome. We used the accessome to analyze differential accessibility and integrated expression data to compare the chromatin and gene expression dynamics underlying cardiopharyngeal fate specification. In summary, we revealed that most changes in accessibility occur upon induction of multipotent cardiopharyngeal progenitors from the founder cells. Comparing differential expression to differential accessibility shows that genes activated in the multipotent progenitors tend to have regions that specifically open nearby them. We found that the elements accessible specifically in multipotent cardiopharyngeal progenitors were enriched in Fox, GATA and nuclear receptor binding motifs. CRISPR-mediated loss of Foxf function followed by FACS, ATAC-seq and RNA-seq showed that Foxf is required to open ~22% of the cardiopharyngeal-specific elements. Notably, elements associated with de novo expressed genes, which turn on either in heart or pharyngeal muscle progenitors, were also opening in multipotent progenitors, whereas only ~10% of differentially expressed genes had differentially accessible elements. Finally, we propose a general model for chromatin dynamics whereby most lineage-specific elements open in multipotent progenitors, and control both early pan-cardiopharyngeal and late cell-type-specific expression. Overall design: Examination of 52 ATAC-seq samples: we collected ~4,000 cells per independent replicate for samples from embryos dissociated at five time points encompassing cardiopharyngeal development: naive Mesp+ mesoderm (aka founder cells) at 6 hours post-fertilization, hpf), anterior tail muscle (aka ATMs), first and second multipotent cardiopharyngeal progenitors (aka trunk ventral cells, TVCs) at 10hpf,  fate-restricted progenitors obtained from larvae dissociated at three time points (15, 18 and 20hpf) encompassing second multipotent cardiopharyngeal progenitors (and aka STVCs), as well as fate-restricted first and second heart precursors (FHPs and SHPs), and pharyngeal muscle precursors (aka atrial siphon muscle cells -ASMs). From the same dissociated embryos embryonic cell populations, we isolated B-line mesenchymal cells at 10, 15, 18, and 20hpf. We also collected samples from whole embryo at 10hpf and genomic DNA as input control. We obtained FACS-purified cells at 10hpf after inducing loss of Foxf function using CRISPR/Cas9-mediated mutagenesis (FoxfCRISPR – 10hpf). We examined 6 RNA-seq samples from FoxfCRISPR and ControlCRISPR at 10hpf. B7.5 = cardiopharyngeal mesoderm precursors The "new" ATAC-seq method includes addition of EDTA quenching to block the tagmentation reaction (i.e. 1.1  l of 500mM EDTA (final concentration approx. 50mM, incubating at 50  C for 30mins). No EDTA quenching step was used in the "old" method.