Combinatorial action of transcription factors in open chromatin contributes to early cellular heterogeneity and organizer mesendoderm specification.

Abstract

During gastrulation, mesoderm is induced in pluripotent cells, concomitant with dorsal-ventral patterning and establishing of the dorsal axis. How transcription factors operate within the constraints of chromatin accessibility to mediate these processes is not well-understood. We applied chromatin accessibility and single cell transcriptome analyses to explore the emergence of heterogeneity and underlying gene-regulatory mechanisms during early gastrulation in Xenopus. ATAC-sequencing of pluripotent animal cap cells revealed a state of open chromatin of transcriptionally inactive lineage-restricted genes, whereas chromatin accessibility in dorsal marginal zone cells more closely reflected the transcriptional activity of genes. We characterized single cell trajectories in animal cap and dorsal marginal zone in early gastrula embryos, and inferred the activity of transcription factors in single cell clusters by integrating chromatin accessibility and single cell RNA-sequencing. We tested the activity of organizer-expressed transcription factors in mesoderm-competent animal cap cells and found combinatorial effects of these factors on organizer gene expression. In particular the combination of Foxb1 and Eomes induced a gene expression profile that mimicked those observed in head and trunk organizer single cell clusters. In addition, genes induced by Eomes, Otx2 or the Irx3-Otx2 combination, were enriched for promoters with maternally regulated H3K4me3 modifications, whereas promoters selectively induced by Lhx8 were marked more frequently by zygotically controlled H3K4me3. Our results show that combinatorial activity of zygotically expressed transcription factors acts on maternally-regulated accessible chromatin to induce organizer gene expression.