The Role of Nanog in Establishment and Patterning of Embryonic Pluripotency

Embryonic stem (ES) cells can become any cell in the body, and so they have great potential as a tool for regenerative medicine to repair tissue damaged by injury or disease. A major goal of modern medicine, therefore, is to understand how to harvest the potential of embryonic stem cells for therapeutic purposes. The signature ability of ES cells to become other cells is called pluripotency, which is carefully regulated by protein factors, called pluripotency factors, which control the genes that regulate ES cell behaviour. We are working to identify how those genes are controlled, and we have focused on one of the most important pluripotency factors, called Nanog, a master regulator of stem cell behaviour.

ES cells resemble the cells that make up early embryos, and so by understanding how embryos develop it becomes it is possible to learn how to regulate ES cells to make specific types of adult cells. However, to understand how ES cells become pluripotent, it is necessary to consider the establishment of pluripotency in the embryo itself. However, the embryos of humans, and other mammals, are very small, and they develop inside the mother, so they are very difficult to access and to manipulate. A way around this problem is use the embryos of other species, whose cells resemble those of human embryos, but which are much easier to work with. This approach has been used for decades in biology to identify the function of genes in embryonic cells. However, we discovered that the embryos of frogs and fish, which most investigators use in the lab, do not contain pluripotent cells. For this reason we had to develop a novel experimental system using embryos from axolotls.

Mammals evolved from amphibians, of which there are two types, frogs and salamanders. These two types of amphibians last had a common relative 250 million years ago. Since then, frogs have evolved many traits are unique to them, however salamanders have remained relatively unchanged since they first walked the earth, and they evolved into reptiles and mammals. For this reason, the genes that control the development of embryos from salamanders and mammals are almost the same. In fact, axolotls are representative salamanders, and we have shown that they contain pluripotent cells that are basically the same as the ones that develop in human embryos. Also, importantly, they contain a Nanog gene, which for reasons that are not entirely clear, does not exist in frog embryos. For our purposes axolotl embryos are a perfect tool to understand how pluripotent cells respond to signals that tell them to become other cell types. We are using axolotl embryos to study how Nanog is regulated.

Axolotl embryos develop outside of the mother, so hundreds of embryos can be collected without harming the animals. Also, the embryos are enormous, about 10,000 times the size of human embryos, so it is very easy to dissect the cells you want to study in the lab. The axolotl experimental system that we developed is unique in the UK, and we are the only group in the world currently using it to understand pluripotency. When we isolated the Nanog gene from axolotls we showed that it works as well as human Nanog in controlling the behaviour of ES cells. But ES cells are not embryos, and we have the unique opportunity to understand how Nanog functions in a normal embryo, the embryo of an axolotl.

In this project we will take the pluripotent cells in axolotl embryos and induce them to become specific types of differentiated cells using solutions that contain the molecules that control development of normal embryos. We will then analyse how the loss of Nanog changes the response to these signals. By identifying how the cells respond we will understand the necessary first step in the establishment of pluripotency, and this will provide cues for how to produce human tissue for regenerating damaged body parts.

Pluripotency defines a cell's potential to give rise to progenitors of any somatic lineage, or to primordial germ cells which establish the germ line. Cells with this potential are not found in conventional animal models, including Xenopus and zebrafish, which have a predetermined germ line. Therefore we develop axolotl embryos as a model system. Axolotls represent the amphibian ancestor to vertebrates, and retain ancestral vertebrate developmental mechanisms that are conserved in mammals, including pluripotency. We recently isolated an axolotl Nanog ortholog, and showed it has conserved functional activity in mouse embryonic stem cells (ESC). Nanog is not conserved in Xenopus, but here we show it is a master regulator of axolotl development, required to initiate development, and then for specification to the ectodermal and mesodermal lineages. We identify Nanog interaction with Nodal signalling as a critical step in establishing pluripotency, as a prelude to further development.

We will establish transcriptomes from Nanog morphant embryos, identifying Nanog targets during the Nodal dependent priming that triggers somatic development. We will determine Nanog's role as a transcription factor in mesoderm development, using animal caps from wild-type and morphant embryos that are exposed to activin to identify Nanog targets. The mesoderm is subdivided into somatic (nodal dependent) and germ line (nodal independent) mesoderm. The loss of Nanog diverts both these tissue types to endoderm. Brachyury, itself not dependent on Nanog, is required for the induction of both mesoderm populations. We will use protein synthesis inhibition experiments to identify Nodal and Brachyury targets that are modulated by the presence of Nanog to establish the Nanog dependent regulatory networks for pluripotency and subsequent mesoderm specification. These studies will identify conserved mechanisms that govern development of differentiated tissues from pluripotent cells in mammals.

The prospects for regenerative medicine using patient matched stem cells looms on the horizon as a major contributor to the pharmaceutical/biotechnology sector of this country. Britain's strong position as a leader in the field of stem cell research offers the potential for a dominant position in this multi-billion pound industry. However, advanced technologies are ever changing and if the UK is to maintain its position it will need to adopt novel insights and technologies into the mainstream scientific-technological culture. A major problem in stem cell biology is to understand how to produce differentiated cells, from stem cells, and the work we propose brings a novel conceptual advance whose adoption could accelerate work in this field, yielding the attending economic benefits.

We have developed a novel experimental model based on a true representation of vertebrate evolution. Evolution underpins all aspects of biology, and its application to medicine derives from the presumed continuity of molecular processes among all species, enabling extrapolation of data from easy to use model system to biomedical issues concerning humans. However, we have discovered that accepted paradigms governing evolution are over-simplified and therefore some work with model systems is misleading with respect to its application to higher animals, like mammals. Major changes in evolution result from changes to the gene regulatory networks that govern the early stages of an animal's development, and therefore understanding how these networks change is critical to the application of data from basic biology to more applied problems, such as those confronted by regenerative medicine. Indeed, a major strength of the British pharmaceutical industry derives from its close links with university led research, whose world leading standing is recognized around the world as a source of innovation.

Our work has identified how the gene regulatory networks that govern stems cells, and their ability to differentiate into any cell type in the body, evolved. Previous assumptions about the evolution of stem cells were flawed by over-simplified presumptions about how vertebrates evolved. Our work has already identified major regulatory steps in how stem cells acquire differentiated fates that were impossible to identify with existing experimental systems, but which offer unique opportunities as a way to exploit stem cells for therapeutic potential. The work we propose here, with the axolotl experimental system, will impact the larger scientific community and achieve recognition within the biomedical sector.

The work proposed here is based on, and reinforces, an embryologically centred theory of evolution that we have proposed to explain how vertebrates evolved. Proof for this theory has only come in recent years with the advent of genomic technologies which permit genome analysis from a broad spectrum of previously understudied species. We have employed a systems based approach towards understanding the molecular genetic mechanisms that were conserved as mammals evolved from more primitive ancestors. Our work led us to identify pluripotency, the fundamental property of stem cells, as the unifying principle of vertebrate natural history. By analysing conserved mechanisms that govern pluripotency in species predating the evolution of mammals we have been able to identify the core processes around which stem cell behaviour is controlled. Novel mechanistic insights into the major evolutionary transitions of animal evolution are rare, and our findings will have a major impact on understanding the underpinning of how evolution is regulated at a genetic level. We anticipate that recognition of a novel evolutionary theory, involving stem cells, will find recognition in popular circles, and thus contribute to the cultural appreciation for the importance of evolution that has existed in Britain for over a century.