Single-cell and single-embryo RNA sequencing reveals gene activity during early development

Studying the earliest stages of human development is difficult because embryos contain very few cells and donated human embryos are limited. Traditional gene expression methods often require much more biological material, making it challenging to examine what is happening inside individual cells or embryos during these critical stages.

Advances in RNA sequencing have made it possible to analyze extremely small samples, including individual cells and entire early embryos. Researchers from the Université Paris Cité and INSERM 1016 in Paris, France describe techniques for measuring which genes are active at different stages of development and compare how gene expression changes as cells begin to take on specialized roles.

Embryonic RNA-sequencing: at the single-embryo (A), single-cell (B), or single-embryo compartment (C) scale

Measuring RNA from very small samples

RNA sequencing works by measuring RNA molecules produced when genes are active. Because different cell types activate different sets of genes, RNA sequencing can provide a detailed view of cellular identity and biological activity.

Single-cell RNA sequencing applies this approach to individual cells. Instead of averaging gene expression across many cells, researchers can examine differences between cells that may appear similar but are following different developmental paths.

Single-embryo RNA sequencing applies similar principles to an entire embryo. This is particularly useful during preimplantation development, the period between fertilization and implantation in the uterus, when an embryo progresses through several stages while undergoing major changes in gene activity.

The method described by Ducreux and colleagues provides a workflow for studying gene expression in single embryos and individual embryonic cells, from collecting the biological material through sequencing and bioinformatic analysis.

Following early embryonic development

During the first days after fertilization, cells within an embryo initially look similar but gradually acquire different identities.

By the blastocyst stage, two important populations have emerged. The inner cell mass contains cells that will contribute to the developing embryo, while the trophectoderm forms much of the tissue that will later contribute to the placenta.

RNA sequencing can help researchers identify the gene expression programs associated with these developmental decisions.

The approach can also be adapted to separately analyze embryonic and abembryonic regions of the embryo. Examining these regions individually can provide a more detailed picture of how gene expression differs across an embryo as its structure becomes increasingly organized.

From sample collection to sequencing

Working with single cells and embryos requires careful laboratory procedures because the amount of RNA available is extremely small.

After an embryo or individual cell is collected, its RNA must be converted into complementary DNA, or cDNA. The cDNA can then be amplified so that enough material is available to prepare a sequencing library.

Sequencing generates millions of short sequence reads representing RNA molecules that were present in the original sample. Computational analysis then maps these reads to the genome and estimates the expression levels of individual genes.

Because small-input RNA sequencing is particularly sensitive to technical variation, careful sample preparation and quality control are essential for distinguishing real biological differences from experimental noise.

Applications in reproductive biology

Single-cell and single-embryo RNA sequencing can help researchers investigate many questions about reproduction and early development.

For example, researchers can examine how gene expression changes from fertilization through the blastocyst stage, identify genes associated with specific embryonic cell populations, and investigate how environmental conditions may influence early development.

The technology can also be used to study questions relevant to assisted reproductive technologies. Previous work involving Ducreux and colleagues has used single-embryo transcriptomics to investigate whether different embryo culture conditions are associated with changes in gene expression during preimplantation development.

A detailed view of early gene regulation

Early embryonic development involves rapid and carefully coordinated changes in gene activity. Studying those changes has historically been difficult because each embryo contains so little biological material.

Single-cell and single-embryo RNA sequencing provide researchers with ways to examine gene expression at a resolution that was previously difficult to achieve.

By combining careful sample handling, low-input RNA sequencing, and bioinformatic analysis, these approaches can help researchers better understand how cells establish their identities during early development and how gene regulation contributes to successful embryo development.