Pluripotent stem cells have the capacity to form any other type of cell in the body. How pluripotency is maintained in cells is not well understood, and this is the major focus of the lab. We use several approaches to this question.
Specific Areas of Research
Area 1. Embryology of PGCs. We developed embryos from the axolotl, a salamander, as a model to understand how pluripotency is maintained in stem cells. We have identified a population of pluripotent stem cells in axolotl embryos that later become the primordial germ cells (PGCs), the cells that give rise to gametes (sperm and eggs) later in development. Our focus is then on two questions: what prevents pluripotent stem cells from differentiating into somatic cells? Also, how do pluripotent stem cells decide to become germ cells?
Area 2. Nuclear Reprogramming. As a second approach towards understanding how pluripotency is controlled we are using extracts from the oocytes and eggs of axolotls, and the frog Xenopus laevis, to reprogram mammalian adult cells, i.e. somatic cells, to pluripotency. This is accompanied by parallel studies with embryonic stem (ES) cells.
Area 3. Evolution of PGC Specification (Evo-Devo). Embryos use different mechanisms to specify the production of PGCs. The embryos of some animals produce PGCs from pluripotent stem cells, in the embryos of other animals PGCs are predetermined by germ cell determinants laid down in the egg. The evolution of PGC specification strategies has had an enormous impact on the evolution of developmental mechanism. We are trying to understand how these different mechanisms of PGC specification evolved, what the ramifications of the different mechanisms are, and what selective pressures drive the evolution of these mechanisms.
Area 1. Embryology of PGCs.
Pluripotent cells have the ability to differentiate into any of the thousands of different cell types in the body, and their potential as therapeutic agents is widely recognized. However, how pluripotent stem cells form during the normal development of embryos is unknown. To address this question we have developed embryos from the axolotl, a salamander, as a model system. Axolotl embryos have a major advantage over other model systems in that they produce pluripotent stem cells through a mechanism that has been conserved in mammals, yet they are very accessible and easy to manipulate.
We study the development of Primordial Germ Cells, or PGCs. In axolotls and mammals PGCs are derived from pluripotent stem cells that can also give rise to many other cell types of the body, or soma. We are using the embryos to address two specific questions: Firstly, how do pluripotent cells form, and how do they avoid the signals that could cause them to differentiate into somatic cells; Secondly, what are the signals that trigger pluripotent stem cells to enter the germ line?
We can harvest hundreds of axolotl embryos from natural matings. The embryos can then be injected with synthetic messenger RNAs that encode either specific growth factors or transcription factors. These RNAs are translated into proteins that will affect the development of the stem cells. We then test the effects of these treatments on the stem cells in developing embryos. In other experiments we dissect the injected regions of the embryo to test the effects of injected molecules in isolated cells. The injected cells are located with green fluorescent protein (GFP), which is co-injected with other RNAs (See the adjacent figure.). This approach exploits the ease with which axolotl embryos can be cut and pasted in classic embryological manipulations. Recently we have begun a major effort to clone the genes that regulate the development of pluripotent stem cells, as well as the genes that trigger stem cells to differentiate into PGCs. When these are isolated, modified forms of these genes will be injected into embryos to understand how they affect stem cell development. In parallel studies we will transfect these genes into into embryonic stem (ES) cells from mice or humans to test their activities in mammalian systems.
Isolated Animal Caps at Blastula Stage
Animal cap at larval stage stained for expression of the Axdazl gene, a marker for PGCs
Embryos co-injected with RNA encoding fibroblast growth factor (FGF) and Green Flourescent protein GFP
PGCs in Axolotl embryos
A) Section through an embryo stained for Axdazl. Arrow shows PGCs Blowup of B) PGC region in Figure A C) Section stained for control gene
Members of the lab and collaborators from other labs involved in this project: Prof. Rosemary Bachvarova (Cornell University Medical College, New York, USA), Dr. Maz O'Reilly, Dr. James Folwell, Catherine Jackson, Jodie Edgeson
Area 2. Nuclear Reprograming.
Early mammalian embryos are composed of pluripotent cells that have the ability to become any cell type in the body. As development proceeds, however, the cells make decisions to differentiate into specific cell types, like liver, nerve, muscle, etc., and they sacrifice pluripotency. Our goal is to re-programme differentiated cells back to a pluripotent state.
The cloning of adult mammals using nuclei from differentiated adult cells demonstrates that nuclei can be re-programmed to pluripotency by egg cytoplasm. However mammalian eggs are very small and very difficult to obtain. For this reason we have focused on the eggs of amphibians. Amphibian eggs are up to ten thousand times the size of a typical mammalian egg, and they are available by the thousands. In addition, amphibian eggs contain most, if not all, of the same factors that are found in mammalian eggs. Therefore, we are using amphibian eggs as a starting material for the re-programming of adult mammalian cells to pluripotency.
Epigenetic marks are removed from Nuclei after treatments in extracts.
Histone H3 Lysine 9 (K9) methylation staining of mouse foetal fibroblasts incubated in extracts for 3 hs. Green: Fitc-anti H3H9 antibody, Blue: DAPI.
Two central issues underlie the problem of re-programming. Firstly, the genes that regulate the pluripotent state of cells must be activated. Typically these genes are inactive in differentiated cells. Secondly, the epigenetic marks that regulate gene activity must be erased so that adult nuclei more closely resemble those of pluripotent cells. Epigenetic marks are chemical modifications to the chromatin that are applied during normal development to stabilize fate decisions. In one aspect of this project we inject differentiated cells into the nuclei of amphibian oocytes (oocytes are eggs before they are ovulated). Using this approach we have demonstrated the robust activation of specific pluripotency genes. Also, we have shown genome wide chromatin alterations that result in a nucleus with characteristics expected of pluripotent cells. In a parallel approach, we prepare extracts from oocytes or eggs, and incubate somatic cells in these. We have shown that the major epigenetic marks that restrict gene expression in somatic cells, DNA methylation and histone methylation, are erased by components within the extracts, and extracts trigger the activation of pluripotency genes. We are working towards culturing treated cells to achieve and stabilize a pluripotent state, with the ultimate goal of expanding these cells for future therapeutic applications.
Members of the lab and collaborators from other labs involved in this project: Prof. Keith Campbell (School of Biosciences, Univ. of Nottingham), Dr. Ramiro Alberio (School of Biosciences, Univ. of Nottingham), Sebastian Lazar (School of Biosciences, Univ. of Nottingham), Dr. Maz O'Reilly, Yuhong Bian
Area 3. Evolution of Vertebrate Embryos (Evo-Devo).
The germ line and soma make different contributions to the maintenance of an organism's lineage. The soma manifests the effects of natural selection, which stabilizes traits that enhance competitiveness within a niche. The germ line is responsible for the passage of the genetic information between generations. Importantly, an organism's lineage is terminated if the germ line is interrupted, and within this context we have considered the evolutionary consequences of the mode of PGC specification employed by developing embryos.
In many organisms, such frogs, the germ line is established early in embryogenesis by germ cell determinants that are deposited in the egg. These determinants, known as germ plasm, are distributed only to the cells that will give rise to PGCs. Thus, in these organisms the germ line is established during the earliest stages of development, and the PGCs develop without any influence from the surrounding somatic cells. In contrast, other organisms produce PGCs from pluripotent stem cells, as described above for axolotls and mammals. In these cases the PGCs are produced in response to signals from the surrounding somatic cells, so the development of germ line and soma are intricately linked.
Frogs and salamanders diverged from a common ancestor over 250 million years ago. And salamander embryos have retained more ancestral traits than those of frogs, which are highly derived. Moreover, salamanders, such as axolotls, produce PGCs from pluripotent stem cells, while frog embryos contain germ plasm and have "predetermined" germ cells. Because frogs form PGCs one way and salamanders form PGCs the other way, we can exploit these experimental model systems to understand how the evolution of germ cell determining mechanisms affects the evolution of embryological strategies and the signaling pathways that control early development.
We are using computer modeling to predict how the gene regulatory networks (GRNs) that control the early development of vertebrate embryos might evolve in animals with either form of PGC specification. We believe that induced PGCs act as a constraint on the evolution of GRNs, and that the evolution of predetermined PGCs relieves that constraint. We test predictions based on this theory directly, using the axolotl and Xenopus models that we maintain in the laboratory. Our work is consistent with a model in which the evolution of germ plasm is favoured during natural selection since it can enhance the evolvability of species within a specific lineage. In contrast, the production of PGCs from stem cells is a mechanism that has been conserved during vertebrate evolution, and is largely responsible for the retention of the highly conservative embryology whose existence underpins the marvelous conservation of the vertebrate body plan through evolutionary history.
Members of the lab and collaborators from other labs involved in this project: Prof. Rosemary Bachvarova (Cornell University Medical College, New York, USA), Prof. Brian Crother (Southeastern Louisiana University, USA), Prof. Mary White (Southeastern Louisiana University, USA), Dr. Matt Loose (Institute of Genetics, Univ. of Nottingham), Emma Richardson, Jodie Edgeson
Research in the laboratory is funded by the Wellcome Trust, The Medical Research Council, and EvoCell Ltd., a private stem cell technology company founded by Dr. Johnson.