DNA Copying Hits Snag In Early Embryos

At the 8-cell stage of mouse embryo development, the coordination between DNA unwinding and replication appears to improve. © MENG ZHU, ZERNICKA-GOETZ LABORATORY, CAMBRIDGE UNIVERSITY / SCIENCE PHOTO LIBRARY

In the first days of human embryonic growth, cells divide rapidly, copying their DNA as they go. About 24 hours after fertilization, the first DNA copy and cleavage occurs, resulting in a 2-cell embryo. Roughly 12-18 hours later, this copy and cleavage process happens again, resulting in a 4-cell embryo, and so on.

This process was long thought to be smooth and stable, but a new RIKEN study in mice has revealed one potential bumpy transition¹.

The researchers behind the work found that at the 1- and 2-cell stage, relatively uniform copying of DNA occurs in embryos. But for a brief window around the 4-cell stage, mouse embryos hit a chaotic period where DNA replication suddenly becomes messy and prone to mistakes.

At this point, the cells begin to copy their genetic material in stages, but without the coordination between DNA unwinding and replication seen at later stages. This can lead to increased stress and errors in the copying process.

The researchers found that chromosomes at this stage can fail to sort properly, sometimes causing breakages and other anomalies that could impact the embryo's development.

"These kinds of peculiar replication patterns have never been observed before," says lead author Ichiro Hiratani, a developmental biologist at the RIKEN Center for Biosystems Dynamics Research.

He hopes the finding could inform practices in reproductive medicine clinics, potentially leading to improved outcomes in fertility treatments. Applying these insights to human embryos could, for instance, help identify why early-stage development goes wrong-both naturally, and during in vitro fertilization (IVF) procedures.

"There's a lot of interesting work to do," Hiratani says.

The discovery was made possible by a cutting-edge genomics technique known as single-cell DNA replication sequencing, or scRepli-seq.

Developed by Hiratani and members of his Laboratory for Developmental Epigenetics at RIKEN, in collaboration with colleagues from Japan's Mie University in the city of Tsu, the method works by precisely measuring the amount of DNA that has been copied in a single cell, like taking snapshots of a book being photocopied page by page. The scRepli-seq method allows scientists to see which 'pages' of the genome are complete, and which are still blank, capturing a moment-by-moment view of the replication process.

We immediately thought of applying [the technique] to early mouse embryos," Hiratani says. But it wasn't easy. "We did have some technical hurdles to overcome," he says, especially concerning embryos preservation and the separation of individual cells for analysis.

With some troubleshooting, however, the team-a coalition of researchers from Hiratani's lab, members of RIKEN's Laboratory for Chromosome Segregation led by Tomoya Kitajima, and collaborators from nearby Japanese universities-finally got the method to work in the first cycles of DNA replication during embryo formation.

Briefly, around the 4-cell stage in mouse embryos, DNA replication becomes prone to mistakes. © 2024 RIKEN

From the 8-cell stage onward, they found that the timing of replication followed a predictable pattern similar to that seen in adult cells, much like a photocopier that prioritizes certain chapters first, duplicating some sections of DNA early and others later, to ensure that the entire genome is copied quickly and efficiently.

In contrast, in 1- and 2-cell embryos, the process of DNA replication is different: it proceeds slowly and uniformly, resembling a photocopier that methodically works through every page at the same pace, without prioritizing any particular sections.

However, things seemed to break down at the 4-cell stage. These early embryos showed a staggered, adult-like program of DNA replication, with certain segments of the genome predictably copied before others. But the replication 'fork'-the Y-shaped region of an actively replicating genome, where DNA strands are unwound and copied-still moved as slow as did in the 1- and 2-cell stages, creating a mismatch between the timing and speed of DNA duplication.

This leads to stress and errors, the researchers found, so much so that around half of all early embryos would be expected to have some form of chromosomal abnormality in some of their cells. Yet, such abnormalities are rarely seen in fully developed embryos, nor is such a high fraction of embryos lost before they reach maturity. This suggests, says Hiratani, that "early embryogenesis has some trick to cope with these kinds of errors."

Exploring the nature of such a trick is an obvious future direction, Hiratani says. One possibility is error-filled cells are directed away from the part of the embryo that will form the fetus and toward the section destined to become the placenta. Such a process would minimize the inheritance of genetic defects.

Yet, even if the embryo succeeds in this redirection, it likely wouldn't be 100% accurate: some of the copying errors introduced at the 4-cell stage might still make their way into cells destined for fetal development. So, Hiratani's group is investigating other factors that may minimize this risk.

In another study, they demonstrated that supplementing the growth media of embryos with extra DNA building blocks, known as nucleosides, helped speed up the movement of the replication fork, reducing the likelihood of chromosome abnormalities-but only when those nucleosides were added at precisely the right developmental moment.

Such an approach, if validated in humans, might one day be applied to enhance genetic stability during embryo cultivation for IVF, Hiratani suggests.

There are also implications for IVF and pre-implantation genetic profiling, a technique commonly used to screen embryos for genetic deficiencies before they are implanted into mothers.

Such tests analyze just a few cells from early embryos. However, this method is likely to miss any abnormalities introduced at the 4-cell stage, unless the specific cells containing these errors happen to be sampled by chance.

Of course, these possibilities are all based on the assumption that the bumpy transition in DNA replication regulation is also conserved in humans and not just mice, Hiratani points out.

Nevertheless, with this issue now identified, he and his RIKEN colleagues are keen to gain a deeper understanding of the biology of early embryos.

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