RNA sequencing identifies trigger for ‘head-to-tail’ axis development in human embryo

Human embryo in the lab 9 days after fertilisation.

Scientists have identified key molecular events in the developing human embryo between days 7 and 14 – one of the most mysterious, yet critical, stages of our development.

The second week of gestation represents a critical stage of embryo development, or embryogenesis. Failure of development during this time is one of the major causes of early pregnancy loss. Understanding more about it will help scientists to understand how it can go wrong, and take steps towards being able to fix problems.

The pre-implantation period, before the developing embryo implants into the mother’s womb, has been studied extensively in human embryos in the lab. On the seventh day the embryo must implant into the womb to survive and develop. Very little is known about the development of the human embryo once it implants, because it becomes inaccessible for study.

Pioneering work by Professor Magdalena Zernicka-Goetz and her team developed a technique, reported in 2016, to culture human embryos outside the body of the mother beyond implantation. This enabled human embryos to be studied up to day 14 of development for the first time.

In a new study, the team collaborated with colleagues at the Wellcome Sanger Institute to reveal what happens at the molecular level during this early stage of embryogenesis. Their findings provide the first evidence that a group of cells outside the embryo, known as the hypoblast, send a message to the embryo that initiates the development of the head-to-tail body axis.

When the body axis begins to form, the symmetrical structure of the embryo starts to change. One end becomes committed to developing into the head end, and the other the ‘tail’.

The new results, published today in the journal Nature Communications, reveal that the molecular signals involved in the formation of the body axis show similarities to those in animals, despite significant differences in the positioning and organisation of the cells.

“We have revealed the patterns of gene expression in the developing embryo just after it implants in the womb, which reflect the multiple conversations going on between different cell types as the embryo develops through these early stages,” said Professor Magdalena Zernicka-Goetz in the University of Cambridge’s Department of Physiology, Development and Neuroscience, and senior author of the report.

She added: “We were looking for the gene conversation that will allow the head to start developing in the embryo, and found that it was initiated by cells in the hypoblast – a disc of cells outside the embryo. They send the message to adjoining embryo cells, which respond by saying ‘OK, now we’ll set ourselves aside to develop into the head end.’”

The study identified the gene conversations in the developing embryo by sequencing the code in the thousands of messenger RNA molecules made by individual cells. They captured the evolving molecular profile of the developing embryo after implantation in the womb, revealing the progressive loss of pluripotency (the ability of the embryonic cells to give rise to any cell type of the future organism) as the fates of different cells are determined.

Single cell RNA sequencing of post-implantation human embryos
and pluripotency transition profile

Fig. 1

a Identification of four major clusters coloured by lineage via UMAP: epiblast (red, 108 cells at 9 d.p.f., 58 cells at 11 d.p.f., total=166), hypoblast (purple, 82 cells at 9 d.p.f., 54 cells at 11 d.p.f., total = 136), cytotrophoblast (green, 1956 cells at 9 d.p.f, 226 cells at 11 d.p.f, total=2182), syncytiotrophoblast (cyan, 1058 cells 9 d.p.f., 1278 cells at 11 d.p.f., total = 2336). Number of embryos: n = 8 at 9 d.p.f., n = 8 at 11 d.p.f. Only embryos with all three lineages were analysed, refer to Supplementary Data 1b Heatmap displaying z-score values of the most differentially expressed genes between different lineages, alongside known lineage markers. In bold red the top most enriched genes expressed in a particular lineage as shown in Supplementary Data c Comparisons between pre-implantation single-cell sequenced epiblast and ICM cells previously published (cyan inner cell mass (ICM) at 5 d.p.f., green epiblast at 6–7 d.p.f.) versus our post-implantation datasets (purple epiblast at 9 d.p.f, red epiblast at 11 d.p.f.). Comparison of the expression levels of key markers of naïve pluripotency (d), primed pluripotency (e) and core pluripotency (f). Bars represent median. g Logistic regression analysis showing quantitative cell matching scores between human embryo datasets ICM at 5 d.p.f. (blue), pre-Epi at 6–7 d.p.f. (green) and post-Epi at 9 d.p.f. (purple) and at 11 d.p.f. (red) (y-axis) and human embryonic stem cells H9 and H9-Reset (x-axis). Comparison H9 vs H9-Reset by Sidak’s multiple comparisons ad-hoc test for 2 way ANOVA, H9 cells differ from the H9-Reset throughout stages: 5 d.p.f. (p = 0.0015**), 6–7 d.p.f. (p < 0.0001****), 9 d.p.f. (p = 0.0101*) and 11 d.p.f. (p < 0.0001****). This difference is highly significant at 6-7 d.p.f., with H9-Reset cells sharing similarities to 6–7 d.p.f. pre-implantion epiblast, and at 11 d.p.f. with H9 cells sharing similarities with 11 d.p.f. post-implantion epiblast.

“Our goal has always been to enable insights to very early human embryo development in a dish, to understand how our lives start. By combining our new technology with advanced sequencing methods we have delved deeper into the key changes that take place at this incredible stage of human development, when so many pregnancies unfortunately fail,” said Zernicka-Goetz.

SourceUniversity of Cambridge

Molè, MA, Coorens THH, Shahbaz, MN et al. (2021) A single cell characterisation of human embryogenesis identifies pluripotency transitions and putative anterior hypoblast centre. Nat Commun 12, 3679. [abstract]

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