Nucleus of a 8-cell stage early embryo before (up) and at the time of the bleaching (down). The area targeted by the laser is shown within the white frame.
Genes Dev May 15, 2014
May 15, 2014
What gives a cell the possibility to be totipotent, i.e. to be able to differentiate into any cell type? The group of Maria Elena Torres Padilla from IGBMC publishes new results to answer this question in the journal Genes & Development on May, 15th. The chromatin and its histone proteins are involved in this property.
The zygote is the result of the fusion between the sperm and the oocyte. This unique cell is totipotent, which means that it can generate all cell types. Upon division, it generates the 2-cell stage embryo, which divides then further, giving the 4-cell stage, the 8-cell stage and so on. The more the cells divide, the more they differentiate and lose their totipotency. How? By modifying the plasticity of the chromatin, according to the team of Maria-Elena Torres Padilla.
The chromatin is the combination of DNA and specific “packaging” proteins called histones. Upon fertilization, embryonic chromatin undergoes major changes. Ana Boskovic and her collaborators found that these changes could be responsible for the loss of totipotency. They mainly noticed that the mobility of the histones within the cell nucleus decreases as development proceeds.
A technical feat
It is generally assumed that the more the cell is differentiated into a specific type, the less its chromatin is plastic. However, this question was not yet addressed in vivo, during development. To answer this question, the researchers developed for the first time a technique to address protein mobility, Fluorescence Recovery After Photobleaching (FRAP) in live cells of the early mouse embryo.
To achieve this aim, they first collect early embryos just after fertilization and culture them so that they can develop, just like in the mothers’womb. To analyze the mobility of the histone proteins, they “micro-inject” zygotes with fluorescent histones that can therefore be monitored in the microscope by FRAP. They next bleach a pool of these fluorescent histones by directing a laser to a small part of the nucleus at different developmental stages (2-cell, 8-cell…) (see illustration). In the place where the bleaching occurs, the fluorescence of the histones disappears. If the fluorescence signal comes back fast, this means that some new fluorescent histones come rapidly in this place and that they are really mobile, while if the reappearance of fluorescence is slow, it indicts that histones are less mobile.
Using this live cell imaging approach, they showed that histones are much more mobile at the 2-cell stage than at the 8-cell stage when cells lose their totipotency, highlighting a link between the plasticity of chromatin and the extremely high cellular plasticity of totipotent cells.
A major breakthrough
To confirm this link, they also analyzed other specific cells called “totipotent 2-cell like” cells. These cells appear stochastically by culturing embryonic stem (ES) cells, but they are less “differentiated” than ES cells: they somehow went backward in the developmental process and became totipotent again. Ana Boskovic and her collaborators showed that also on these specific cells, histones display higher mobility than in normal ES cells. This is a clear evidence of a link between totipotency and chromatin plasticity.
This important breakthrough contributes to a better understanding of the cell differentiation machinery, but most importantly, on the molecular features that help to keep a cell “plastic” and with the potential to generate multiple cell types. Deciphering these mechanisms is essential in the field of regenerative medicine where scientists try to get new living functional tissues from differentiated cells. Being able to understand how cells can become totipotent would allow them to make any differentiated cell totipotent and then differentiate them again in any tissue.