Establishing the GRN for the Germ Line Mesoderm of Axolotl Embryos

Lead Research Organisation: University of Nottingham
Department Name: School of Life Sciences

Abstract

Sperm cells and egg cells are produced from cells that form early in embryonic development called primordial germ cells, or PGCs. Understanding how PGCs are established during development of the embryos is important because it will lead to fertility treatments, and also because poorly formed sperm and eggs can give rise to embryos with developmental problems. Little is known about how PGCs are formed in humans because it is impossible to work with human embryos. In addition, mouse embryos, which share many characteristics with humans, also develop in a uterus, and are very small, and therefore are difficult to access. We developed an experimental system using embryos from a salamander called an axolotl. Unlike embryos from mammals, axolotl embryos are very large and they develop in water, and therefore hundreds of embryos are easy to acquire at a time. Also, because they do not develop in a uterus, the embryos can be acquired from natural fertilization, without harm to the adults. What makes axolotls unique for this study, however, is the fact that axolotl embryos produce PGCs using the same genes that humans do, and this makes the genes involved in making PGCs easy to identify. We developed a way to produce thousands of PGCs at a time using very specific conditions. Because the cells are so abundant we will be able to do DNA sequencing to identify the genes and do experiments to understand how the genes interact with each other. The results from our experiments will allow us to develop new ways to produce sperm cells and eggs from embryonic stem cells, which is a major goal of modern medicine.

Technical Summary

Understanding the genetic regulatory networks (GRN)s that govern the pattern of developing tissues is critical for understanding the underlying causes of genetic diseases, and for devising methods for the production of cells for cellular therapies. Embryos from lower vertebrate animal models are particularly useful for unpicking GRNs because they are readily accessible, so material is acquired with relative ease, and they facilitate the testing of predictions from the networks experimentally. An important problem in stem cell biology is how to derive gametes from pluripotent stem cells as a means of combating infertility, and as a vehicle for genetic manipulation. We developed axolotl embryos as an experimental model to understand how germ cells are specified because they share a conserved mode of primordial germ cell (PGC) specification with mammals, and this is not conserved in embryos from other models, like frogs and teleost fish. The axolotl system has the additional advantage that this species is representative of the ancestral amphibians from which all land-dwelling vertebrates evolved, and so can be used to understand how developmental mechanisms evolved in mammals.
In axolotl embryos PGCs emerge in the posterior lateral plate mesoderm, which we term germ line mesoderm. This tissue is ancestral to vertebrates, but was lost during the evolution of teleosts and frogs, so it has not been characterized before. In axolotl embryos, PGCs are closely related to haematopoietic stem cells (HSC)s, and these cells are induced with the PGCs in germ line mesoderm. The focus of this proposal is to understand how the germ line mesoderm is patterned to segregate these two closely related stem cell populations, and to establish the GRN that governs their production. We propose to use the inducible axolotl animal cap system to identify the mechanisms that pattern this novel mesodermal tissue, and we will use it to produce material for the GRN, which will be obtained under a variety of experimental conditions, and at several time points. We will validate predictions from the GRN in axolotl embryos, as well as in an in vitro system for the induction of PGCs from mouse embryonic stem cells. At the completion of this project we will deliver the conserved GRN that controls the production of PGCs from pluripotent precursors, an important step towards the efficient production of germ cells for therapeutic purposes.

Publications

10 25 50
 
Description Project Grant
Amount £632,588 (GBP)
Funding ID MR/L001047/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2014 
End 12/2016
 
Title Axolotl Colony 
Description We developed the axolotl embryo as a model system to understand the mechanisms that govern pluripotent cells in development. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2008 
Provided To Others? Yes  
Impact Evolution of electrosensory ampullary organs: conservation of Eya4 expression during lateral line development in jawed vertebrates. Modrell MS, Baker CV. Evol Dev. 2012 May;14(3):277-85. 
 
Title Conserved mechanism of germ cell induction 
Description We have identified the pathway for the induction of primordial germ cells and showed that it is conserved in vertebrates. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact The transcriptome of induced axoltol germ cells is underway and we will use this to identify the gene regulatory network that governs primordial germ cell specification leading to methods for in vitro production of germ cells from embryonic stem cells. 
 
Title Epigenetic Reprogramming using Axoltol oocytes 
Description We prepared extracts from the oocytes of axolotls and showed that it can reprogram the chromatin of somatic cells to a pluripotent configuration within a brief period of time. No other available material has simailr properties. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Understanding how chromatin is remodelled is a critical component of the process of reprogramming somatic clels to stem cells. We demonstrated axolotl oocyte extracts can remodel chromatin directly and we are using this to eluciate the reprogrammig process. 
 
Title Funding for complete transcriptome of axolotl development 
Description The Nanog and Mix dependent transcriptome of axolotl embryos is determined by Deep Sequencing. 
Type Of Material Database/Collection of Data/Biological Samples 
Provided To Others? No  
Impact Nanog and Mix are master regulators of development, yet their role in development is unknown. This project will determine the role of these genes in development which will have a direct impact on the directed differentiation of embryonic stem cells. 
 
Title Theory of Germ line soma relationship in vertebrate evolution. 
Description We developed a novel theory of evolution to explain how developmental mechanisms evolved in vertebrates. 
Type Of Material Data analysis technique 
Provided To Others? No  
Impact Bachvarova, R.F., Crother, B. I., and Johnson, A.D. (2009). Evolution of germ cell development in tetrapods: comparison of urodeles and amniotes. Evolution & Development:11, 603-609. Crother, B.I., White, M., and Johnson, A.D. (2007). Inferring developmental constraint and constraint release: Primordial germ cell determination mechanisms as examples. Journal of Theoretical Biology:248, 322-330. Johnson, A.D., Richardson, E., Bachvarova, R.F., and Crother, B.I. (2011). Evolution of the germ line-soma relationship in vertebrate embryos. Reproduction 141(3):291-300 
 
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
 
Title Biological materials and uses thereof. (WO2007010287) 
Description The patent protects the use of axoltol oocyte extracts as a tool to reprogram soamtic cells. 
IP Reference WO2007010287 
Protection Patent granted
Year Protection Granted 2007
Licensed Yes
Impact We have demonstrated the mechanisms that remodel the pluripotency gene network for activation during reprograming.
 
Company Name EvoCell Ltd. 
Description The company was started to commercialize the use of axolotl oocyte extracts as a reprogramming tool.; http://www.evocell.co.uk/ 
Year Established 2006 
Impact The compnay funded fundamental research into the process of cellualr reprogramming that is now under review for publication.
Website http://www.evocell.co.uk/
 
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