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Single-Cell RNA Sequencing Provides Insight Into Mouse Embryo Development

NEW YORK (GenomeWeb) – Researchers from the UK have used single-cell RNA sequencing to gain insight into how a mouse embryo first begins to transform from a ball of unfocused cells into a small, structured entity.

"If we want to better understand the natural world around us, one of the fundamental questions is, how do animals develop?" Bertie Göttgens, senior author and research group leader at the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, said in a statement.

Until now the molecular mechanisms underlying this process have been difficult to parse out since the numbers of gastrulating cells are very limited. However, the rise of single-cell sequencing has given researchers the opportunity to "make direct observations to see what's going on during the earliest stages of development," John Maironi, another senior author and research group leader at EMBL-EBI, the Wellcome Trust Sanger Institute and the University of Cambridge, said in a statement.

"We can look at individual cells and see the whole set of genes that are active at stages of development, which until now have been very difficult to access," Maironi added.

The research, led by the European Bioinformatics Institute (EMBL-EBI) and the Wellcome Trust-MRC Cambridge Stem Cell Institute, was published yesterday in the journal Nature.

The researchers analyzed 1,205 single cells collected from early gastrulation at embryonic day (E) 6.5 to the generation of primitive blood cells at E7.75. They collected mouse embryo samples from CD1 mice, prepped the samples for sequencing using an Illumina DNA prep kit, and performed single-cell RNA sequencing using an Illumina HiSeq 2000.

Maironi, Göttgens, and their colleagues confirmed that their data were of high quality using previously published methods and obtained 501 single-cell transcriptomes from cells taken from distal halves of E6.5 embryos. Using the transcriptomes, the researchers identified 2,085 genes that had significantly more heterogeneous expression across the 1,205 cells than they could expect to find by chance.  

Then the researchers used knockout mice to study the function of Tal1, a key hematopoietic transcription factor. Contrary to previous studies performed in retrospective assays, they found that Tal1 knockout does not immediately bias precursor cells towards a cardiac fate.

"It wasn’t what we expected at all," Maironi said. "We found that cells which in healthy embryos would commit to becoming blood cells would actually become confused in the embryos lacking the key gene, effectively getting stuck. What is so exciting about this is that it demonstrates how we can now look at the very small number of cells that are actually making the decision at the precise time point when the decision is being made. It gives us a completely different perspective on development."

The researchers also analyzed the T gene, which plays a role in the nascent primitive streak. They noted that T expression correlated with other gastrulation-associated genes, including Mixl1 and Mesp1. They added that "genes negatively correlated with [the T gene] were consistently expressed across the majority of epiblast cells, suggesting that cells outside the primitive streak have not yet committed to a particular fate," which is consistent with observations of epiblast cell plasticity in transplant experiments.

Essentially the researchers have created an atlas of gene expression during very early, healthy mammalian developments. "Many of the things that go wrong, like birth defects, are caused by problems in early development," Göttgens said. "We need to have an atlas of normal development for comparison when things go wrong."

The research team believes that as technologies advance that "resolve epigenetic processes at single-cell resolution and match single-cell expression profiles with spatial resolution," there will be a great deal more progress in the field of single-cell genomics that will allow greater insight into early mammalian development. "This is just the beginning of how single-cell genomics will transform our understanding of early development," Göttgens said.