Developing single cell multiomics technologies to understand mammalian development
A single human cell contains about 6 picograms of DNA. Nonetheless, recent advances in DNA sequencing technologies have made it possible to sequence the genome of thousands of individual single cells. In addition to the DNA bases present, DNA sequencing libraries can contain information on RNA transcripts as well as epigenetic features central to cellular identity like DNA methylation (5mC), DNA hydroxymethylation (5hmC), and DNA accessibilty. Connecting these epigenetic features to gene expression is challenging, particularly in complex heterogeneous systems. To resolve this, I have developed techniques to capture the mentioned features simultaneously from a single cell and then applied them to 4 key areas of human development: 1. pre-implantation development, 2. gastrulation and primordial germ cell (PGC) specification, 3. primordial germ cell maturation, and 4. stem cell pluripotency. This thesis defense will discuss two of these works.
Gastrulation and primordial germ cell specification: By developing a sequencing technique to investigate 5mC, DNA accessibilty, and the transcriptome from the same cell (scMAT-seq), we have characterized the epigenetic landscape of major cell types in 3D human gastruloids corresponding to the germ layers and primordial germ cell-like cells (hPGCLC). Here we find hPGCLCs are specified from progenitors which emerge from epiblast cells and show transient characteristics of both amniotic- and mesoderm-like cells. Finally, we find that during gastrulation DNA accessibility is tightly regulated while 5mC regulation is more lineage specific.
Stem cell pluripotency: Genome wide erasure of 5mC is associated with the acquisition of pluripotency. To investigate this process, we have developed scDyad&T-seq, which allows for the detection of 5mC dynamics along with RNA expression from the same single cell. By applying scDyad&T-seq to different time points of mouse embryonic stem cells transitioning from a primed to a naïve state of pluripotency, we observe extreme demethylation dominated by passive processes and discover this process is highly heterogenous and delayed in some cells. By connecting RNA expression from the same cells, we detect a set of genes directly linked to 5mC levels during this transition.