How does signaling induce human primordial germ cells?

Primordial germ cells (PGCs) are the cells in an embryo that later become egg and sperm in adults, and they form in the early stages of an embryo's development. Understanding the molecular/genetic mechanism that controls PGC development is important for assisted reproduction, technologies, understanding the origins germ line cancers, and for regenerative medicine. However, to date little is known about how PGCs development in humans is controlled. Indeed, understanding how cells in an embryo decide to become PGCs, in vertebrates, generally, has posed a unique challenge to biologists for reasons that are only now becoming clear. At its heart is the process of evolution, and how evolutionary forces have affected the mechanisms for PGC development. Because, while many mechanisms that control development have been worked out in embryos of simpler animals, like those of frogs or fish, it has not been possible to use these species as "models" for human PGC development. This is because each of the model organisms typically studied in laboratories has evolved a unique mechanism for producing PGCs. Humans, in contrast, employ what is apparently the original mechanism that evolved in vertebrates to produce PGCs, the so-called conserved mechanism.
We recognized this problem several years ago, and to explain it we developed a novel theory of evolution concerning the relationship between PGCs and the other cells in an embryo, known as somatic cells. We hypothesized that human embryos retain the mechanism for PGC development that originally evolved in vertebrates, the so-called conserved mechanism. A major component of this theory is, also, that PGCs are derived from the same cells as somatic cells, not from specialized cells. To test this theory, the MRC funded development of an experimental system using embryos from axolotls, a salamander. Axolotls were chosen because they resemble the first vertebrates to move onto land, in other words, the amphibian ancestor to mammals. We predicted that axolotls and humans would share the same mechanism for PGC development, and axolotl embryos could therefore be used as an experimental model to unpick the mechanisms driving this process.
We determined the signals that govern PGC development in axolotls, and then considered a an established genetic pathway known to act downstream of these signals in other cell types. From this we identified a principle role for the transcription factor Elk-1, and its functional partner Med23, in PGC development. Elk-1 was discovered over 25 years ago, but its role in embryos has never been clearly determined because commonly studied animal models, including mice, evolved genetic circuits that circumvent its ancient role in embryos. We showed that the pathway discovered in axolotls also controls development of PGCs in pigs, whose embryos accurately model those of humans, strongly suggesting that the role for Elk-1/Med23 that we discovered also directs development of human PGCs.
Our proposal is designed to use axolotl embryos to define the biochemical and genetic mechanisms controlling the conserved pathway for vertebrate PGC development. We propose to reduce the activity of Elk-1 or Med23, and replace these proteins with mutant molecules lacking specific biochemical functions. We will also identify all of the genes either up or down-regulated by Elk-1/Med23 that control the distinction of PGCs from somatic cells. We will also test the function of Elk-1/Med23 using newly developed methods to induce PGC-like cells from human embryonic stem cells (hESC), and we will use this hESC system to identify the genetic elements responsible for switching-on genes that regulate human of PGC development. This will be a step towards defining the conserved network of gene required for vertebrate PGC development, enhancing our ability to understand and manipulate germ cells to address issues concerning human health.

We will use axolotl embryos and hESC to establish the link between FGF signalling and the molecular mechanisms of germ cell specification. Our goal is to understand how the Elk-1/Med23 axis represses mesoderm differentiation and activates the regulatory network for PGC development. This work will contribute to development of methods for in vitro production of human gametes. The project includes two work streams.
1.- Axolotl animal caps will be programmed to produce PGCs and used to identify direct targets of Elk-1 transcriptional repression or activation, and to identify the biochemical mechanisms through which Elk-1 represses somatic specification during PGC induction.
2- Induction of PGCLC from hESC will be used to test the role of Med23 in human PGCs. hPGCLC will be FACS sorted and gene expression will be assessed by qPCR or RNA-seq. ChIP-seq will be performed on hPGCLC using conventional methods.
Key informatics techniques will include:
3- RNA seq will be carried out following standard Illumina protocols with biological triplicates for all experiments. For transcriptomes, annotation will be derived from our axolotl transcriptome collection via reciprocal blast and/or other annotation pipelines such as blast2go. Differential expression analyses will be performed using DeSeq2 or TopHat/Bowtie/Cufflinks or equivalent pipelines to identify targets of Elk-1 and Med23. The intersection of these datasets should reveal PGC specific targets of the Elk-1/Med23 axis.
4- ChIP sequencing, to identify super enhancers for PGC specification, will be analysed using DiffBind or equivalent packages to identify Med23 targets which differ in the presence and absence of BMP in hPGCLC. Proximity of sequenced regions to specific genes will be determined by standard methods. Sequenced targets will be analyzed for Elk-1, Smad-1 and Nanog binding sites. Together, this will enable identification of candidate PGC specific super enhancers for further validation.

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 MRC of "Living Long and Healthy Life".
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 1- screening for effects of environmental pollutants in folliculogenesis, 2- screening for compounds that can modulate gamete development and for the treatment of infertility, 3- as a source oocytes with the capacity to reprogram somatic cells to pluripotency, with important applications in regenerative medicine. These developments will be of interest to regulators that will need to legislate for the use of in vitro gametes in humans. The pharmaceutical industry will be a primary beneficiary of these new developments. The applicants have established links with industrial partners (EvoCell, CellCentric, and Zoetis) 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 paediatric cancers will benefit from our research that will contribute to advancing the understanding of germ cell tumourigenesis. This new understanding will lead to a better diagnosis and treatment.
How will they benefit from this research?
The application of our research could impact reproductive health, by increasing reproductive potential of infertility patients, and paediatric germ cell tumours diagnosis and prognosis. Our research will establish the principles of gamete development in the growing embryo. The insight into these developmental mechanisms will contribute to 1- establish novel approaches for in vitro gamete production and their use in assisted reproduction, and 2- facilitate the development of novel diagnostic and treatment approaches for paediatric germ cell tumours.
What will be done to ensure that they benefit from this research?
The applicants have agreements in place with the University of Nottingham spin-off company EvoCell Ltd. to commercialize the research. During the course of the project regular meetings (twice a year) between the Business Development Executive of the Schools of Life Science and Biosciences, patent lawyers from the University and representatives from EvoCell will be organised to discuss patentable/commercial possibilities.