The induction of PGCs from pluripotent cells in axolotl embryos

During normal animal development the cells that are egg and sperm come from cells called primordial germ cells, or PGCs. Little is known about how PGCs form in humans, but this is very important, because it can help with fertility problems, and with understanding the origins of ovarian and testicular tumors. Also, PGCs come from precursor cells that are pluripotent, which means they can become any other cell in the body. In this way PGCs are very similar to embryonic stem cells, or ES cells. Understanding how ES cells form, and retain their capacity for becoming other cells, is a major goal of modern biomedical science.
A fundamental approach to modern developmental biology is to study how cells behave in the embryos of less advanced animals, then transfer knowledge from these studies to understand how similar processes are controlled in mammals, and ultimately humans. This approach is useful because the embryos of lower animals are usually much larger and easy to manipulate, and so experiments are less expensive and easier to perform. Also, this approach minimizes the distress that more advanced animals might encounter. Many scientists use embryos from frogs or from fish, but the species usually used form PGCs through a mechanism that is not relevant to humans. We developed axolotl embryos as model experimental system because it produces PGCs in a manner very similar to humans. As proof of this we have previously cloned genes from this species that are known to be required in human pluripotent cells. Using embryos from axolotls has allowed us to understand how pluripotent cells are produced in normal embryos, and how they are governed during development.
In this work we prove for the first time that PGCs can be induced in vast numbers, in a highly enriched form, using very simple methods that we developed. Here we are working to further validate and refine our methods, and to understand the actual genes and mechanisms that are involved in PGCs formation. This system will make PGCs useful as novel tools to understand how pluripotency is controlled. Also, this work will be very important in aiding our understanding of how stem cells develop in embryos, and how germ cells are made distinct from other types of cells in the body.

Pluripotent cells can differentiate into any somatic cell type, or into germ cells. Cells fulfilling this definition are only known to exist in mammals and include early epiblast cells and cultured lineages like ES cells. In other animal models, primordial germ cells (PGCs), the germ cell precursors, can only be derived from cells that contain germ plasm.
Here we demonstrate ectopic PGC induction from animal caps of axolotl embryos. Since these cells can also give rise to any somatic cell, they are therefore pluripotent. In accordance with this, we recently showed that axolotl animal caps express all genes associated with the mammalian pluripotency network, suggesting pluripotency is conserved between axolotls and mammals.
Here we propose to define the signal transduction pathways that induce PGCs from pluripotent cells in caps and in embryos. Axolotl PGCs develop in vivo from the ventral marginal zone (VMZ), adjacent to the blood. Our preliminary experiments show that ectopic PGCs are induced in animal caps at high efficiency by a combination of FGF-4 and BMP-4 RNA, in a dose-dependent manner. This combination of factors does not produce blood. In contrast, blood, but not PGCs, is induced by low activin with BMP-4. We propose to determine the signal transduction pathways downstream of FGF4 and activin to understand the different tissues induced by these regimes. We will also investigate natural patterning of the VMZ to determine if FGF and activin function in vivo as they do during the induction of ectopic PGCs. We will test the effects of blocking either FGF or nodal signalling in VMZ explants on PGC and blood development. Next we will examine the endogenous role of FGF4 in whole embryos by using an anti-sense morpholino to block AxFGF4 function. Conversely, we will also determine whether we can increase the population of PGCs in intact embryos by increasing FGF4 levels ventrally, to see if somatic cells can be diverted to the germ line.
Finally, we propose to test the functional properties of ectopic PGCs using tissue transplantation and einsteck chimera assays.
Axolotl PGCs develop from cells that retain pluripotency until late in development. The work proposed here will provide important experimental data that will aid in understanding how pluripotency is controlled and how the germ line is established during axolotl development, This in turn may provide important clues to understanding how these processes are governed in mammals.