Developmental roadmap of primordial germ cells in humans and pigs

Studying the development of the gametes, the cells that make egg and sperm, in mammals is important for understanding the basis of animal development and evolution. It also has important applications in biotechnology, assisted reproduction, species conservation and regenerative medicine.

We and others have shown that germ cell precursors, the primordial germ cells (PGCs), emerge in the posterior region of early embryos in large mammals (e.g. pigs and non-human primates), and that key genes that control development of germ cell precursors is conserved in these species. Although the period when PGC form is known, the features of the cells that respond to inducing signals is unknown. In this project we will test the hypothesis that cells with a unique molecular signature localized in the posterior end of the embryo can respond to specific signals to form germ cell precursors.

We will carry out experiments to establish the genetic and epigenetic profile of PGC precursors in an embryo, and functional experiments using pig epiblast stem cells and human embryonic stem cells to identify key master regulators of germ cell development. The results of the project will help us gain a detailed understanding of how PGCs are induced from a pool of progenitor cells. This will be achieved by mapping the genetic and epigenetic signatures of pig PGCs and their precursors to build an atlas of their developmental progression, and by performing experiments using stem cells to identify role of a key master gene (SOX17) regulating this process. Intriguingly, the master regulator SOX17 is involved in the program of germ cells and gut progenitors. Here we will determine how this protein can elicit a differential response to inducing signals produced in the surrounding tissues to initiate two different programs of differentiation in an embryo.

These comprehensive investigations will provide detailed molecular understanding of the formation of the PGCs in a large mammal for the first time. This new knowledge will be important for establishing improved methodologies for the study of human PGC development from stem cells in humans and livestock species. Development of new technologies that allow the generation of gametes in the laboratory will have broad applications for understanding the causes of infertility in humans through modelling of gametogenesis, for gaining better understanding of the origin of germ cell tumours, and for assisted reproduction in livestock.

This project addresses questions of strategic relevance in the BBSRC remit, such as lifelong health and wellbeing. We anticipate that the outcomes of the current project will contribute to the academic and clinical advance in the areas of regenerative medicine and global food security.

Detailed understanding of primordial germ cell (PGC) development in humans is lacking, hindering progress in the development of robust in vitro systems for creating gametes in the laboratory. The pig is an excellent model that shares key developmental principles with humans that serves as a suitable, ethically acceptable, alternative for studying germline development that may be relevant to humans.

Based on our previous findings that pig PGCs are induced in the posterior primitive streak, we propose that precursors with a unique transcriptional and epigenetic signature are able to respond to inducing signals to produce PGCs. We will carry out the following experiments to gain detailed molecular understanding of the features of these precursor cells and early PGCs:

1- Establish the gene expression profile of the posterior end of early gastrulating pig embryos to build an atlas of PGC development using scRNASeq.
2- Identification of SOX17 targets during in vivo differentiation of PGCs and definitive endoderm using genome wide profiling methods. We will validate the function of putative candidate regulators (such as enhancers or alternative promoters) during differentiation using in vitro differentiation of human embryonic stem cells and pig epiblast stem cells.
3- Delineate the epigenetic landscape of in vivo derived pig PGCs using genome wide profiling approaches, and compare the findings with available datasets from human in vivo and in vitro produced PGCs. We will also investigate the role of macroH2A during PGC specification.

These experiments will enable the identification of critical molecular determinants of pig PGC development in vivo, and for testing these putative regulators in human in vitro systems. These findings will serve as the basis for the improving the differentiation procedures to generate in vitro gametes from domestic animals and humans.

The insight into these developmental mechanisms will contribute to 1- establish improved approaches for in vitro gamete production and their use in assisted reproduction in livestock and humans, and 2- facilitate the development of novel diagnostic and treatment approaches for germ cell tumours.

Who will benefit from this research?
Advances in the field of gamete and stem cell biology will benefit the areas of health science, regenerative medicine, assisted reproduction, pharmaceutical industry and biotechnology. These areas are within the strategic priorities of the BBSRC in Lifelong Health and Wellbeing and Global Food Security.

The key impact areas of this research are:

1. Advance in methodologies for in vitro production of gametes which are required by the pharmaceutical and medical industries as tools for drug screening and toxicological studies. These in vitro produced gametes will be useful for i) screening for effects of environmental pollutants and toxic compounds on folliculogenesis, ii) screening for compounds that can modulate gamete development and for the treatment of infertility. The applicants have established links with industrial partners and will seek to develop these technologies further in partnership with them.

2. Clinicians working in the area of cancer, in particular those related to germ cell cancers will benefit from our research that will contribute to advancing the understanding of tumourigenesis. This new understanding will lead to a better diagnosis and treatment.

3. Advances in methodologies for generating gametes in vitro will be instrumental for developing in vitro breeding procedures in farm animals that will greatly impact genetic selection and livestock productivity. We anticipate that these approaches could be developed rapidly within the course of this project for experimental testing, before they are taken up by livestock selection companies (within 5-10 years).

4. The training potential for the PDRAs working in this project is outstanding, as it offer highly specialized and cutting edge techniques in molecular genetics, bioinformatics and bioengineering that are relevant in the regenerative medicine (Biotech companies) and to livestock genetic selection (animal genetics companies).

5. Development of in vitro gametes will be of interest to regulators and advisory bodies (HFEA, Academy of Medical Sciences) that will need to issue recommendations on the use of the new sources of human gametes.