The role of transcription factors and the signaling environment in commitment of cells into the germline

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Molecular. Genetics & Pop Health

Abstract

Background
In mammals, specification of the germline occurs shortly after implantation. Understanding how germ cells become specified is important both to inform the treatment of human infertility, for example through in-vitro spermatogenesis, and for the wider field of regenerative medicine. The proposed work is a follow-on project from our studies showing that the transcription factor OTX2, restricts entry of pluripotent cells into the germline (Zhang et al. 2018). In cultures of wild type cells, expression of primordial germ cell (PGC) transcription factors (TFs) does not increase for over 24 hours after changing the culture to PGC differentiation media containing BMP4. This increase in PGC TF expression is preceded by a decrease in the level of Otx2, suggesting that BMP4 may suppress Otx2 expression, thereby stimulating entry to the germline. Consistent with this, Otx2 null cells enter the germline at enhanced efficiencies of >80% and Otx2 null cells can enter the germline even in the absence of BMP4. However, the efficiency of the latter process is only 30% indicating that cytokines provide additional functions in addition to Otx2 repression. Our published studies also show that, in the absence of Otx2, the PGC TF, Blimp1 is not required for the initial stages of germline entry but is required for cells to develop a mature PGC transcriptome. This project will use single cell transcriptomic analyses to investigate the changes occurring at early time points during germline differentiation of wild-type cells, Otx2-/- cells and cells carrying compound mutations in Otx2-/- and selected PGC TF genes. These studies will reveal the important genomic changes required to enable subsequent efficient germline differentiation.
Aims
1 to determine whether Otx2+/+ and Otx2-/- cells express the same genes when they initiate PGC-like differentiation
2 to determine at what stage PGC-like differentiation goes awry in Otx2-/-;Blimp1-/- cells. Analyses will be extended to Otx2-/- cells deleted for the additional PGC TFs Prdm14 and AP2g.
3 to determine the chromatin structural changes occuring during progression of differentiation from ESC EpiLC PGC-like cells using single cell ATAC-seq
Training outcomes
The student will be trained in a combination of wet and dry lab techniques. CRISPR/Cas9-mediated gene disruption in embryonic stem cells will be used to generate new lines for study (Otx2-/-;Prdm14-/- and Otx2-/-;AP2g-/-). Cell culture approaches to differentiating ESCs to germline competent EpiLCs and further differentiation into initial PGC-like cells will be provided. In addition, single cell biochemical techniques will be employed to generate high throughput libraries. Training will also be provided in statistical techniques required to analyse and interpret single cell sequencing data.
References
1. Zhang, J., Zhang, M., Acampora, D., Vojtek, M., Yuan, D., Simeone, A. and Chambers, I. Otx2 restricts entry to the mouse germline. Nature, 562, 595-599.
2. Zhang, M. and Chambers, I. Segregation of the mouse germline and soma. Cell Cycle, accepted.

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