Patterning of Germ Line and Soma from Pluripotent Cells 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. This will allow us to identify the genes involved in this process. We have identified a previously unknown way for cells to talk to each other that is essential for producing PGCs. 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.

How germ cells form during metazoan development is a classic question of developmental biology. In many organisms primordial germ cells (PGCs) are specified cell-autonomously by maternal molecules, known as germ plasm. In other species, including mammals, PGCs are formed from pluripotent cells in response to extracellular inducing signals. We propose to investigate how germ cells are segregated from somatic cells in embryos from axolotls, a urodele amphibian (salamander). Axolotl embryos do not contain germ plasm, and, similar to mammals, PGCs are formed by induction. Moreover, axolotls are of special interest because they retain basal vertebrate traits, and in this way are instructive for understanding how the mechanisms governing vertebrate development evolved. Here we investigate how the PGCs are segregated from the somatic lineages during axolotl development.
We developed a highly efficient in vitro system for inducing PGCs from isolated explants (animal caps) of axolotl embryos using fibroblast growth factor-4 (FGF) in combination with BMP-4, and we show these signals are required for PGC specification in intact embryos. In response to the induction of PGCs animal caps become refractory to TGFbeta signals which direct the production of somatic mesoderm during normal development. The induction of PGCs, and the block to TGFbeta signalling, requires expression of the axolotl ortholog of Nanog (AxNanog), but how these mechanisms function are unknown. Intriguingly, studies with mouse embryos show that PGCs are specified by bone morphogenetic proteins (BMPs), which induce the transcription factor Blimp1. A critical role of Blimp1 is to prevent the PGC precursors from specification to a somatic fate by the local signalling environment, and this is accomplished by the establishment of repressive epigenetic marks. However, we have been unable to detect Blimp1 in natural or artificially induced axolotl PGCs, suggesting that a different mechanism is involved in preventing somatic specification of the germ cells.
We propose to identify the transcriptome of axolotl animal caps that are induced to form PGCs, and we will focus on identifying genes whose activation is involved in PGC specification, and dependent on AxNanog expression. The products of these genes will be tested in functional assays. In addition, we will investigate the mechanisms that prevent TGF?O signalling from affecting induced cells. These studies will reveal novel mechanisms that regulate cell signalling during vertebrate development, and will uncover basal mechanisms that govern PGC specification in vertebrates.