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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - TOTIPOTENCY2014 (Dissecting the epigenetic control of totipotency.)

Teaser

Summary

Mammalian development is a remarkable process. Following fertilisation of the egg by the sperm, the embryo must grow from a single cell capable of generating every cell type, to a group of cells each with their own identity. This requires tight regulation and control, which is carried out by factors that turn the correct genes on and off at the right developmental stage. However, due to the small number of cells, technical and ethical limitations, it has been traditionally extremely difficult to study this important developmental time.

Using the mouse as a model, this project uses exciting cutting-edge technologies to investigate how genes are regulated through early development. It explores the similarities and differences between single cells within and between embryos, and how these are regulated. Additionally, these findings will be applied to a new technology termed â€˜reprogrammingâ€™, which induces adult cells into a different cell type, for example turning a skin cell into a liver cell. The future medical promise of this technology for regenerative medicine and organ replacement is enormous. However before its clinical use becomes reality, it must be better understood and its pitfalls amended, which this project aims to help achieve.

The work plan consists of four specific objectives:
1. Characterisation and validation of early-embryonic like (EEL) cells as a model of totipotency.
2. Identification of EEL regulators through a novel candidate-based screen.
3. Mechanistic studies on selected EEL regulators, including 2 preliminary candidates.
4. Assessing the role of EEL regulators in the efficiency and fidelity of somatic reprogramming.

Work performed

\"For objective 1, the researcher developed a new in vitro model system for studying early embryonic development. These \"\"2C-like\"\" or \"\"early-embryonic like\"\" cells exist within embryonic stem cell cultures as a rare subpopulation of cells, yet had been poorly characterised. To this, the researcher has performed extensive molecular characterisation of these cells, describing the unique open chromatin structure as well as loss of global DNA methylation that temporarily takes place in these cells. This has resulted in a first author and co-corresponding publication for the researcher.

Objective 2 set out to perform a candidate-based screen to identify novel potential regulators of zygotic genome activation. This has successfully been carried out and has resulted in the identification of 10 new factors. Two of these were selected to study further in objective 3 and extensive mechanistic insight into their mode of action has been identified. A manuscript is currently in preparation to describe the results in objective 2 and 3. Given the large number of factors identified and the interesting mechanistic work, objective 4 was not pursued, instead additional experiments on the identified factors and addition leads into their roles in later stages of development have been the main focus of the second half of the project period.

During the project, the results have been disseminated in several ways, including oral presentations at 4 international scientific conferences and symposiums (Epigenetics in Development, IMB Conference, Mainz, Germany; Epigenetics and Chromatin, Keystone Symposium, Whistler, Canada; Stem Cell Institute Postdoc Symposium, The University of Cambridge; International Postdoc Retreat, Lisbon, Portugal). The project has already given rise to one scientific publication (Eckersley-Maslin et al. 2016 Cell Reports), with an additional publication currently being prepared.\"

Final results

The proposal utilises many new and innovative technologies, including single-cell RNA-sequencing and single-cell bisulfite sequencing technologies being pioneered in the host lab. The work is highly original in that it provided the first in vitro system for studying totipotency, which has been traditionally difficult to study. As such the experimental system developed in this proposal is of wide interest to others studying the many facets of early embryonic development. The proposed methods using existing yet innovative techniques, build on the combined experience of the host and the researcher. The important new insights are not only of scientific importance, but have potential medical relevance, benefiting the European Research Area. The researcher has benefited the European Research Area through a valuable exchange of ideas, knowledge and expertise, and will promote to increase international cooperation, in line with Horizon 2020 objectives, to develop the European knowledge based economy and society.