Pluripotency transcription factor function during primordial germ cell development

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

The aim of the proposed work is to study the factors regulating formation of functional germ cells, oocytes in females and sperm in males. We will focus on the role of Nanog, an important molecule involved at distinct stages during embryonic development.
We will determine what parts of Nanog are required to mediate function in developing primordial germ cells (PGCs for short), the embryonic cells that will give rise to the adult germ cells. We will compare Nanog proteins from different species for their ability to function in mouse cells, since the mouse is the mammalian species where these processes are best understood. We will investigate whether Nanog function is unique, or whether other factors, such as Esrrb, can play a similar role in PGCs. We will determine how Nanog exerts its function in driving germ cell development by identifying the genes regulated by Nanog in this process.
This study will delineate the mechanisms through which Nanog functions. We will learn if Nanog function has been conserved throughout evolution and which parts of the Nanog molecule are important. In a broader sense, since primordial germ cells and the cells of the early embryo that retain unrestricted differentiation potential are similar, we will understand better how the factors required for the formation of these distinct cells work. Understanding how functional germ cells are formed is of great importance for the progress of reproductive medicine. Learning how pluripotent cells are regulated has high impact on regenerative medicine strategies.
The continued existence of a sexually reproducing species requires effective formation of germ cells that possess a haploid DNA complement and that can join with another germ cell of the opposite sex during fertilisation to form a new individual. PGC development from cells set aside early in development involves changes in gene expression and modifications of the chromatin, the complex of DNA and protein in which the genome is packed, as the PGCs migrate to the site where the genitals form, known as the genital ridges. Once the PGCs enter the genital ridges they undergo further changes to their chromatin that erase memories of their parental origin. This sets a blank slate onto which instructions for gene expression in the next generation can be written.
These changes are directed by a number of important factors able to influence the expression of other genes, amongst which is Nanog. We have shown that Nanog is required for continued PGC development during the period of chromatin remodelling. Interestingly, Nanog is also required at the other developmental period in which profound chromatin remodelling occurs; the formation of cells with unrestricted differentiation potential (pluripotency) in the pre-implantation embryo.
Pluripotency can be conveniently studied in cells grown in culture and derived from pre-implantation embryos: embryonic stem (ES) cells. Nanog controls the ability of ES cells to indefinitely duplicate. Our recent work has characterized the genes acting downstream of Nanog in ES cells. We consistently found that the gene most up-regulated by Nanog induction was Esrrb. We further showed that Esrrb could recapitulate many of the functional properties previously ascribed to Nanog and, importantly could do this in the absence of Nanog. A further key finding is that, in ES cells lacking Esrrb, the ability of Nanog to sustain pluripotency is compromised. This demonstrates unequivocally that Esrrb is a critical downstream mediator of Nanog function in ES cells.
We now propose to investigate in greater detail the importance of Nanog function in germ cell development and determine whether a similar functional relationship exists between Nanog and Esrrb in PGCs. Our study has the potential to highlight new similarities between the pluripotent cells of the pre-implantation embryo and PGCs, ultimately improving our understanding of the principles governing both cell populations.

Technical Summary

Nanog is required for PGC development beyond the time of epigenetic reprogramming at E11.5, but little is known about how Nanog exerts this function. Here we propose to investigate the role of Nanog and its target gene Esrrb at this time in order to provide a more detailed understanding of the molecular changes occurring at this important biological transition.
In this work we aim to determine
1 what structural features are required for Nanog protein to function in PGC development?
2 to what extent can Nanog function in PGC development be rescued by its target gene, Esrrb?
3 what are the target genes that function downstream of Nanog and Esrrb to facilitate PGC development?
In aim 1, we will determine which domains of Nanog mediate function in PGCs. Nanog mutants will be knocked-in to the Nanog locus in Nanog-/- ES cells and in vitro differentiation used to test their ability to sustain PGC development.
We have shown that Esrrb is the most upregulated Nanog target gene in ES cells and that Esrrb can compensate for Nanog in driving maintenance and acquisition of pluripotency. In aim 2 we will investigate the existence of a similar functional overlap during PGC development by crossing mice with an Esrrb knock-in at Nanog and mice with a conditional Nanog knock-out allele that is specifically deleted in PGCs to establish whether complete functional rescue by Esrrb occurs.
To address aim 3, we will take advantage of the fact that deletion of the Nanog conditional knock-out allele leads to expression of GFP. Therefore, the transcriptional profiles of FACS purified GFP+ PGCs from Nanog+/CKO, Nanog-/CKO or NanogEsrrbKI/CKO embryos will be used to characterise the overlap in Nanog and Esrrb target genes during PGC development.

Planned Impact

The social beneficiaries of the proposed research are to be found within the wider public.
The general public will benefit from the results of the proposed work mainly in three ways:
1) The proposed research has potential medical implications in two fields of broad interest: reproductive medicine and regenerative medicine. Reproductive medicine: the potential contribution of the outlined experiments to the understanding of the molecular control of germ cell specification can not be directly translated in advances of clinical relevance. Nonetheless, delineating the steps and mechanisms of germ cell specification is of pivotal importance in understanding the process of gamete production, and correcting potential defects observed in this lineage. Regenerative Medicine: The proposed work has the potential to enhance the current understanding of the principles governing the specification of pluripotent cell populations. Since the ability to promote reprogramming to pluripotency of differentiated cells from patients is a key step in most cellular replacement therapies, the direct investigation of the principles and factors governing this process is of immediate relevance to the field.
2) The biological research carried out during the proposed project will contribute towards maintaining the high standard of academic excellence currently enjoyed by the Centre for Regenerative Medicine. This will be reflected in the ability of the Centre for Regenerative Medicine and the University of Edinburgh being able to offer educational opportunities for undergraduate and post-graduate students training in our group.
3) The conceptual advances and tangible material, such as pictures, diagrams and illustrations generated to present the results of the proposed experiments will add to the resources used by our science communication staff during outreach activities aimed at raising awareness of the latest advances in the field of stem cell biology and regenerative medicine.
 
Description Targeted expression of the Nanog target gene Esrrb from the Nanog locus can rescue defect in PGC development caused by Nanog deletion in vivo.

The absence of Nanog expression during PGC-like development in vitro prevents PGC-like cell differentiation.
This defect can be rescued by inducible expression of a Nanog transgene.
This defect can also be rescued partially by inducible expression of an Esrrb transgene

We have found that the NANOG target gene OTX2 plays a key role in determining the efficiency of germline differentiation in vitro and development in vivo.

Deletion of the OTX2 gene dramatically increases the efficeincy of PGC differentiation in the presence of cytokines, including BMP4
Deletion of OTX2 enables PGC differentiation even without BMP4
Deletion of OTX2 enables entry of cells into the early stages of PGC differentiation even in the absence of the key PGC transcription factor BLIMP1
Deletion of OTX2 increases the number of cells entering the germline in vivo both in Otx2-null embryos and in chimaeras of Otx2-null and wildtype cells
Exploitation Route by publication
Sectors Pharmaceuticals and Medical Biotechnology

URL http://www.crm.ed.ac.uk/research/group/embryonic-stem-cell-biology
 
Description BBSRC International Partnering Award
Amount £47,065 (GBP)
Funding ID BB/N022599/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2016 
End 04/2020