Establishing the GRN for the Germ Line Mesoderm of Axolotl Embryos

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.

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.