Epigenetics and cell diversity in the embryo.

A research team at the Max Planck Institute for Molecular Genetics in Berlin has explored the role of factors in embryonic development that do not alter DNA sequence, but only epigenetically modify its “packaging.” In the scientific journal Nature, describe how regulatory mechanisms contribute to the formation of different tissues and organs in the embryos of early mice.

A fertilized egg becomes a complete organism with a multitude of different tissues and organs, although the genetic information is exactly the same in each cell. A complex mechanism of molecules regulates which cell in the body performs each task and determines the right time and place to activate each gene.

Epigenetic regulatory factors are part of this molecular mechanism and act to modify the “packaging” of the DNA molecule without altering the underlying genetic information. Specifically, they act to mark DNA and control what parts can be accessed in each cell.

Most of these regulators are essential, and embryos that lack them tend to die during development time when organs begin to emerge. However, these regulators may have specific functions that differ in each cell, making it difficult to study. This has also been a major obstacle to the study of these proteins, which are not only relevant to embryo development, but are also involved in cancer formation.

Detailed examination of embryos.

“The same regulator is present in all cells, but it can have very different tasks, depending on cell type and development time,” says Stefanie Grosswendt, one of the first authors of a new study in the scientific journal. Nature.

Grosswendt and his colleague Helene Kretzmer from Alexander Meissner’s laboratory at the Max Planck Institute for Molecular Genetics (MPIMG) in Berlin together with Zachary Smith of Harvard University, MA, have now managed to elucidate the importance of epigenetic regulators for embryonic development. with unprecedented precision.

The researchers analyzed ten of the most important epigenetic regulators. Using the CRISPR-Cas9 system, they first specifically removed genes encoding regulatory factors in fertilized oocytes, and then observed the effects on embryo development days later.

After the embryos developed for approximately six to nine days, the team examined the anatomical and molecular changes that resulted from the absence of the respective regulator. They found that the cellular composition of many of the embryos was substantially altered. Cells of certain types existed in excessive numbers, while others were not produced at all.

Analyzing thousands of individual cells

In order to make sense of these changes at the molecular level, the researchers examined hundreds of thousands of individual embryo cells, from which individual epigenetic regulators had been systematically removed. They sequenced the RNA molecules of nearly 280,000 individual cells to investigate the consequences of loss of function. RNA transmits information encoded in DNA, allowing researchers to understand the identity and behavior of cells using sequencing technologies.

In their analysis, the scientists focused on a development phase, in which epigenetic regulators are particularly important. When they compared data from altered and unaltered embryos, they identified genes that were dysregulated and cell types that are abnormally overproduced or underproduced. From this general picture, they deduced previously unknown functions of many epigenetic regulators.

Complex effects during development.

An eight-day-old mouse embryo looks a bit like a seahorse and still doesn’t have any organs. “From the outward appearance of an early embryo, you can often only guess which structures and organs will form and which will not,” say bioinformatics scientist Helene Kretzmer and biologist Zachary Smith, who are also the publication’s first two authors. “Our sequence allows for a much more accurate and high-resolution view.”

Single cell analysis gave them a very detailed view of the first nine days of mouse development. Often turning off a single regulator caused ripple effects across the entire network of interacting genes, with many genes differentially switched on or off throughout development.

Extraction of the epigenetic regulator Polycomb (PRC2) had a particularly surprising impact. “Without PRC2, the embryo looks egg-shaped and very small after eight and a half days, which is very unusual,” says Kretzmer. “We see huge changes in the way DNA is packaged that occurs long before, long before the embryo develops morphological abnormalities.”

The researchers found that PRC2 is responsible for limiting the number of germline progenitor cells, the cells that later develop into sperm and eggs. Without PRC2, the embryo develops an excessive number of these cells, loses its shape, and dies after a short time.

Starting point for future analysis

“With the combination of new technologies we address the problems that have been in the air for 25 years,” says Alexander Meissner, who led the study. “We now better understand how epigenetic regulators organize different types of cells in the body.”

The work is only the first step for even more detailed investigations, says Meissner. “Our method allows us to investigate other factors such as transcription or growth factors or even a combination of these. Now we can see very early stages of development at a level of detail that was previously unthinkable.”