The Role of Nanog in Establishment and Patterning of Embryonic Pluripotency

Lead Research Organisation: University of Nottingham
Department Name: Sch of Biology

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description Research Grant
Amount £994,850 (GBP)
Funding ID MR/N020979/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 05/2016 
End 05/2019
 
Title Iterassemble 
Description A method to enable the iterative assembly of sequences surrounding coding sequence in a genome. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact Some users have employed these methods. 
URL https://github.com/LooseLab/iterassemble
 
Title Virtual Axolotl Genome Sequence 
Description We used virtual genome walking to construct a genome sequence for the axolotl, ambystoma mexicanum. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact It was considered a nature top 10 article in genetics an heredity https://www.nature.com/collections/pzcxmxnrhq 
URL http://pplgn962.nottingham.ac.uk/AxGen/trans/
 
Description Alexey Ruzov 
Organisation University of Nottingham
Department School of Clinical Sciences Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We are investigating epigenetic mechanisms that govern vertebrate development, and how they evolved. We have provided embryological material from a variety of model systems.
Collaborator Contribution Dr. Ruzov has investigated the epigenetic modification of DNA in embryos and adults.
Impact Alioui A, Wheldon L, Abakir A, Ferjentsik Z, Johnson AD, Ruzov A. (2012). 5-Carboxylcytosine is localized to euchromatic regions in the nuclei of follicular cells in axolotl ovary. Nucleus. Nov 1;3(6). Almeida RD, Loose M, Sottile V, Matsa E, Denning C, Young L, Johnson AD, Gering M, Ruzov A. (2012). 5-hydroxymethyl-cytosine enrichment of non-committed cells is not a universal feature of vertebrate development. Epigenetics. Apr;7(4):383-9. Almeida RD, Sottile V, Loose M, De Sousa PA, Johnson AD, Ruzov A. (2012). Semi-quantitative immunohistochemical detection of 5-hydroxymethyl-cytosine reveals conservation of its tissue distribution between amphibians and mammals. Epigenetics. Feb;7(2):137-40.
Start Year 2011
 
Description Cinzia Allegrucci 
Organisation University of Nottingham
Department School of Veterinary Medicine and Science Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We are identifying epigenetic mechanisms that lead to the reprogramming of breast cancer cells to acquire a normal cell division cycle. We provide extracts from axolotl oocytes to reprogram the cells. We also are investigating the mechanisms of primordial germ cell induction from human embryonic stem cells. We provide insights into PGC induction from our studies with axolotl embryos.
Collaborator Contribution Cinzia Allegrucci developed the method for epigeentic reprogramming of breast cancer cells using axolotl oocyte extracts. Her group also has expertise in the culture of human embryonic stem cells.
Impact Allegrucci C, Rushton MD, Dixon JE, Sottile V, Shah M, Kumari R, Watson S, Alberio R, Johnson AD. (2011). Reprogramming of breast cancer cells with oocyte extracts. Mol Cancer. Jan 13;10(1):7.
Start Year 2009
 
Description Martin Gering 
Organisation University of Nottingham
Department School of Biology Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We are investigating the mechanism of blood stem cell specification using the axolotl system.
Collaborator Contribution Dr. Gering is an expert on the development of haematopoietic stem cells in vertebrate embryos.
Impact Almeida RD, Loose M, Sottile V, Matsa E, Denning C, Young L, Johnson AD, Gering M, Ruzov A. (2012). 5-hydroxymethyl-cytosine enrichment of non-committed cells is not a universal feature of vertebrate development. Epigenetics. 7:383-9.
Start Year 2009
 
Description Matt Loose 
Organisation University of Nottingham
Department School of Biology Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution My group developed the axolotl embryo system as a tool to investigate the ancestral vertebrate mechanisms of development. Together with Dr. Loose's group we are investigating the mechanisms that control mesoderm induction. we are also investigating how the genetic regulatory mechanisms that govern vertebrate development evolved.
Collaborator Contribution Matt Loose is an expert in mesoderm induction and in bioinformatics. Together, we have identified the mechanisms that control mesoderm specification in axolotl embryos. We have also produced transcriptomes by Deep Sequencing to reveal the gene regulatory networks that govern axolotl development.
Impact Swiers G, Chen YH, Johnson AD, Loose M. (2010). A conserved mechanism for vertebrate mesoderm specification in urodele amphibians and mammals. Dev Biol. 343:138-52.
 
Description Ramiro Alberio 
Organisation University of Nottingham
Department School of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution My group developed the axolotl experimental system and we are using it to understand how pluripotency is regulated in early development, and how PGCs are specified from pluripotent cells.
Collaborator Contribution Dr. Alberio is isolating reprogramming factors from axolotl oocyte and embryo extracts and studying the protein complexes in which they exist. Together we are studying how these proteins remodel the chromatin of pluripotency associated genes such as Nanog.
Impact Allegrucci,C., Rushton, M., Sottile, V., Dixon,J.E., Alberio, R., and Johnson, A.D. (2011). Long term epigenetic reprogramming of breast cancer cells by amphibian oocytes extracts. Molecular Cancer 10(1), 7. Dixon, J.E., Allegrucci, C., Redwood, C. Kump, K., Bian, Y., Chatfield, J., Chen, Y., Sottile, V., Voss S. R., Alberio, R., and Johnson, A.D. (2010). Axolotl nanog activity in mouse embryonic stem cells demonstrates ground state pluripotency is conserved from urodele amphibians to mammals. Development:137, 2973-2980. Bian Y., Alberio, R., Allegrucci, C., Campbell, K.H., and Johnson, A.D.(2009). Epigenetic Marks In Somatic Chromatin Are Remodelled To Resemble Pluripotent Nuclei By Amphibian Oocyte Extracts. Epigenetics:4, 194-202. Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm. Alberio R, Johnson AD, Stick R, Campbell KH. Exp Cell Res. 2005 Jul 1;307(1):131-41
 
Description BBC Radio Nottingham 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact I was interviewed by the BBC on television and radio for work done on cancer cell reprogramming in my lab.

Extensive contact with the public.
Year(s) Of Engagement Activity 2011
 
Description Trent College 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Paper Presentation
Geographic Reach Local
Primary Audience Schools
Results and Impact I gave a talk to High School students about my research program.

I have had students from Trent College work in my lab.
Year(s) Of Engagement Activity 2009,2010,2011