A single cell transcriptional roadmap for cardiopharyngeal fate diversification

Abstract

SummaryDynamic gene expression programs determine multipotent cell states and fate choices during development. Multipotent progenitors for cardiomyocytes and branchiomeric head muscles populate the pharyngeal mesoderm of vertebrate embryos, but the mechanisms underlying cardiopharyngeal multipotency and heart vs. head muscle fate choices remain elusive. The tunicate Ciona emerged as a simple chordate model to study cardiopharyngeal development with unprecedented spatio-temporal resolution. We analyzed the transcriptome of single cardiopharyngeal lineage cells isolated at successive time points encompassing the transitions from multipotent progenitors to distinct first and second heart, and pharyngeal muscle precursors. We reconstructed the three cardiopharyngeal developmental trajectories, and characterized gene expression dynamics and regulatory states underlying each fate choice.Experimental perturbations and bulk transcriptome analyses revealed that ongoing FGF/MAPK signaling maintains cardiopharyngeal multipotency and promotes the pharyngeal muscle fate, whereas signal termination permits the deployment of a full pan-cardiac program and heart fate specification. We identified the Dach1/2 homolog as a novel evolutionarily conserved second-heart-field-specific factor and demonstrate, through lineage tracing and CRISPR/Cas9 perturbations, that it operates downstream of Tbx1/10 to actively suppress the first heart lineage program. This data indicates that the regulatory state of multipotent cardiopharyngeal progenitors determines the first vs. second heart lineage choice, and that Tbx1/10 acts as a bona fide regulator of cardiopharyngeal multipotency.2 All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Combinatorial chromatin dynamics foster accurate cardiopharyngeal fate choices

“…To characterize the chromatin landscape underlying early cardiopharyngeal development, we used the assay for transposon-accessible chromatin (ATAC-seq 16 ) on lineage-specific samples isolated at successive time points, and following defined perturbations 15,17 ( Fig. 1A).…”

“…In Ciona , the transcripts of approximately half of the protein-coding genes undergo spliced-leader (SL) trans -splicing, causing the 5' end of mRNAs to differ from the transcription start site (TSS) 24,25 . Using annotated TSSs [26][27][28] , RNA-seq datasets 15 , and our ATAC-seq data ( Fig. S2), we determined that promoter regions and 5' untranslated regions (5'UTR) were over-represented in the accessome ( P <0.01, binomial test; Fig.…”

“…1A). Building on previous extensive transcription profiles 13,14 , including from single cells 15 , here we characterize the genome-wide chromatin accessibility dynamics underlying cardiopharyngeal fate specification. We identify regulatory inputs that govern cis -regulatory element accessibility and activity, as well as cell-type-specific enhancers for key cardiopharyngeal determinants, and propose models connecting combinatorial chromatin dynamics to cell-specific gene expression and fate choices.…”

Evolution of the embryonic cis-regulatory landscapes between divergent Phallusia and Ciona ascidians.

“…We conclude that the early expression patterns of a majority of our ten test genes are well conserved between Ciona and Phallusia, some of the observed differences possibly reflecting limitations of the colorimetric in situ hybridisation protocol we used. Future work based on single-cell RNA seq in ascidians (Horie et al, 2018;Sharma et al, 2018;Treen et al, 2018;Wang et al, 2017) may be required to discriminate technical issues from bona fide evolutionary changes. In the following sections, we used our extensive ATAC-seq dataset to identify the cis-regulatory sequences driving the conserved expression of these genes and to compare their activity in both species.…”

Neurogenin regulates effectors of migratory neuron cell behaviors in Ciona

“…cDNA synthesis was performed as described (Wang et al, 2017), with SMART-Seq v4 Ultra Low Input RNA kit (Takara). Sequencing libraries were prepared as described (Wang et al, 2017), with Ovation Ultralow System V2 (NuGen). Libraries were pooled and sequenced by Illumina NextSeq 500 Mid output 150 Cycle v2, to generate 75 bp paired-end reads, resulting in 192,396,840 single-end reads for the 6 samples.…”