The cis-regulatory dynamics of embryonic development at single-cell resolution

Abstract

Understanding how gene regulatory networks control the progressive restriction of cell fates is a long-standing challenge. Recent advances in measuring single cell gene expression are providing new insights into lineage commitment. However, the regulatory events underlying these changes remain elusive. Here we investigate the dynamics of chromatin regulatory landscapes during embryogenesis at single cell resolution. Using single cell combinatorial indexing assay for transposase accessible chromatin (sci-ATAC-seq)1, we profiled chromatin accessibility in over 20,000 single nuclei from fixed Drosophila embryos spanning three landmark embryonic stages: 2-4 hours (hrs) after egg laying (predominantly stage 5 blastoderm nuclei), when each embryo comprises ~6,000 multipotent cells; 6-8hrs (predominantly stage 10-11), to capture a midpoint in embryonic development when major lineages in the mesoderm and ectoderm are specified; and 10-12hrs (predominantly stage 13), when each of the embryo’s >20,000 cells are undergoing terminal differentiation. Our results reveal spatial heterogeneity in the usage of the regulatory genome prior to gastrulation, a feature that aligns with future cell fate, and nuclei can be temporally ordered along developmental trajectories. During mid-embryogenesis, tissue granularity emerges such that individual cell types can be inferred by their chromatin accessibility, while maintaining a signature of their germ layer of origin. The data reveal overlapping usage of regulatory elements between cells of the endoderm and non-myogenic mesoderm, suggesting a common developmental program reminiscent of the mesendoderm lineage in other species2–4. Altogether, we identify over 30,000 distal regulatory elements exhibiting tissue-specific accessibility. We validated the germ layer specificity of a subset of these predicted enhancers in transgenic embryos, achieving 90% accuracy. Overall, our results demonstrate the power of shotgun single cell profiling of embryos to resolve dynamic changes in the chromatin landscape during development, and to uncover the cis-regulatory programs of metazoan germ layers and cell types.

MOFA+: a probabilistic framework for comprehensive integration of structured single-cell data

“…Little coordinated variation is observed in promoters ( Figure 4b ), even for genes that show strong differential expression between germ layers ( Figure S16 ). These results are in agreement with other studies that pinpointed distal regulatory elements as a major target of epigenetic modifications during embryogenesis [45][46][47] .…”

Single-cell reconstruction of developmental trajectories during zebrafish embryogenesis

“…Moreover, combining URD with Seurat enabled the anchoring of developmental trajectories to their spatial origins. We anticipate that similar augmentation of URD with information about lineage relationships (30, 31), chromatin dynamics (32), and signaling will further deepen insights into developmental processes. Additionally, some of URD’s limitations could be addressed by future enhancements.…”

Developmental Transcriptional Enhancers: A Subtle Interplay between Accessibility and Activity

“…[ 46 ] Using ATAC‐seq, a handful of recent publications focusing on the early embryogenesis in D. melanogaster have explored in great detail how enhancer accessibility varies both along the temporal and spatial axes of development. [ 15,45,47,48 ] Additionally, by considering quantitative accessibility differences, they provided valuable insights into chromatin organization of different enhancer states.…”

“…Second, Cusanovich et al. [ 48 ] performed single‐cell ATAC‐seq on broader embryonic collections and computationally ordered individual nuclei into temporal trajectories based on their accessibility profiles. As a result, the authors recreated rapid transitions of cell identities during the earliest embryogenesis and identified enhancers characterized by dynamic accessibility changes.…”