Stochastic specification of primordial germ cells from mesoderm precursors in axolotl embryos.

A common feature of development in most vertebrate models is the early segregation of the germ line from the soma. For example, in Xenopus and zebrafish embryos primordial germ cells (PGCs) are specified by germ plasm that is inherited from the egg; in mice, Blimp1 expression in the epiblast mediates the commitment of cells to the germ line. How these disparate mechanisms of PGC specification evolved is unknown. Here, in order to identify the ancestral mechanism of PGC specification in vertebrates, we studied PGC specification in embryos from the axolotl (Mexican salamander), a model for the tetrapod ancestor. In the axolotl, PGCs develop within mesoderm, and classic studies have reported their induction from primitive ectoderm (animal cap). We used an axolotl animal cap system to demonstrate that signalling through FGF and BMP4 induces PGCs. The role of FGF was then confirmed in vivo. We also showed PGC induction by Brachyury, in the presence of BMP4. These conditions induced pluripotent mesodermal precursors that give rise to a variety of somatic cell types, in addition to PGCs. Irreversible restriction of the germ line did not occur until the mid-tailbud stage, days after the somatic germ layers are established. Before this, germline potential was maintained by MAP kinase signalling. We propose that this stochastic mechanism of PGC specification, from mesodermal precursors, is conserved in vertebrates.

Evolution of the germ line-soma relationship in vertebrate embryos.

The germ line and soma together maintain genetic lineages from generation to generation: the germ line passes genetic information between generations; the soma is the vehicle for germ line transmission, and is shaped by natural selection. The germ line and somatic lineages arise simultaneously in early embryos, but how their development is related depends on how primordial germ cells (PGC) are specified. PGCs are specified by one of two means. Epigenesis describes the induction of PGCs from pluripotent cells by signals from surrounding somatic tissues. In contrast, PGCs in many species are specified cell-autonomously by maternally derived molecules, known as germ plasm, and this is called preformation. Germ plasm inhibits signaling to PGCs; thus, they are specified cell-autonomously. Germ plasm evolved independently in many animal lineages, suggesting convergent evolution, and therefore it would be expected to convey a selective advantage. But, what this is remains unknown. We propose that the selective advantage that drives the emergence of germ plasm in vertebrates is the disengagement of germ line specification from somatic influences. This liberates the evolution of gene regulatory networks (GRNs) that govern somatic development, and thereby enhances species evolvability, a well-recognized selective advantage. We cite recent evidence showing that frog embryos, which contain germ plasm, have modified GRNs that are not conserved in axolotls, which represent more basal amphibians and employ epigenesis. We also present the correlation of preformation with enhanced species radiations, and we discuss the mutually exclusive trajectories influenced by germ plasm or pluripotency, which shaped chordate evolution.

Expression of Dazl and Vasa in turtle embryos and ovaries: evidence for inductive specification of germ cells.

In bilaterian animals, germ cells are specified by the inductive/regulative mode or the predetermined (germ plasm) mode. Among tetrapods, mammals and urodeles use the inductive mode, whereas birds and anurans use the predetermined mode. From histological data it has been predicted that some reptiles including turtles use the inductive mode. Examining turtle oocytes, we find that Dazl RNA, Vasa RNA, and Vasa protein are not localized, suggesting that germ plasm is not present. In turtle embryos at somite stages, primordial germ cells (PGCs) expressing Dazl lie on a path from the lateral posterior extraembryonic endoderm through the gut to the gonad as previously described. In gastrulating embryos, cells expressing Dazl are found in the blastoporal plate and subsequently below the blastoporal plate, indicating that PGCs are generated at the equivalent of the early posterior primitive streak of mammals. Vasa RNA is expressed in somatic cells of gastrula to early somite stages, and Vasa RNA and protein are expressed in PGCs of later embryos. Taken together the evidence strongly suggests that turtles, and other reptiles (lacertoid lizards) with the same location of PGCs in embryos, use the inductive mode of germ cell specification. Phylogenetic analysis of the available evidence supports the following hypotheses: (1) the inductive mode is basal among reptiles, indicating that this mode was maintained as basal tetrapods evolved to amniotes, (2) the predetermined mode arose twice within reptiles, and (3) the induced mode may be used in several lepidosaurs whose PGCs are located in an unusual pattern distributed around the embryo.

Evolution of germ cell development in tetrapods: comparison of urodeles and amniotes.

The embryonic development of germ cells in tetrapods is described, focusing on groups with the inductive mode of germ cell specification. In mammals PGCs are induced early in the gastrulation process, they are internalized with future extraembryonic mesoderm in the early posterior primitive streak, and specified soon thereafter. Strong evidence indicates that a similar process occurs in turtles and some other reptiles. In amniotes, the PGCs appear well before formation of the gonad in the posterior trunk, resulting in a period in which they are located outside the embryo before their migration to the gonad. In contrast, in urodeles the PGCs appear relatively late, and throughout development maintain a position close to precursors of the somatic cells of the gonad so that migration is not required. In lampreys early development of germ cells is strikingly similar to that in urodeles, suggesting this is the primitive process. As amniotes evolved large yolky eggs and better access to nutrition, development of the posterior half of the trunk became more dependent on cell proliferation; this was followed or accompanied by a shift of early germ cell development to the equivalent of the early primitive streak. A similar process may have occurred as some basal vertebrates developed large yolky eggs.

Gene expression in the axolotl germ line: Axdazl, Axvh, Axoct-4, and Axkit.

Primordial germ cells (PGCs) in embryos of mammals and urodele amphibians are formed by induction in the absence of germ plasm. We describe expression of four germ cell-related genes through the germ cell cycle of the axolotl. The orthologs of vasa and daz-like are up-regulated in PGCs of tail bud embryos before the gonad forms and are expressed throughout the female germ cell cycle. Mammalian Oct-4 is a marker of pluripotency in embryonic cells. Axolotl Oct-4 has higher homology to Oct-4 than that found in other vertebrates. It is expressed in the equivalent of the mouse epiblast, in the posterior mesoderm of late gastrulae that gives rise to PGCs, and in diplotene growing oocytes, but not in presumptive PGCs after gastrulation. Finally, a c-kit homolog is expressed in gonadal oogonia and growing oocytes as in mice but is also not found in PGCs. The expression pattern in urodele gonadal germ cells is similar to that of other vertebrates, although the pattern in pregonadal PGCs is distinctly different from that of mice. We conclude that PGCs are restricted to the germ line later in urodeles than in mice or lack migration and proliferation programs.

Regulative germ cell specification in axolotl embryos: a primitive trait conserved in the mammalian lineage.

How germ cells are specified in the embryos of animals has been a mystery for decades. Unlike most developmental processes, which are highly conserved, embryos specify germ cells in very different ways. Curiously, in mouse embryos germ cells are specified by extracellular signals; they are not autonomously specified by maternal germ cell determinants (germ plasm), as are the germ cells in most animal model systems. We have developed the axolotl (Ambystoma mexicanum), a salamander, as an experimental system, because classic experiments have shown that the germ cells in this species are induced by extracellular signals in the absence of germ plasm. Here, we provide evidence that the germ cells in axolotls arise from naive mesoderm in response to simple inducing agents. In addition, by analysing the sequences of axolotl germ-cell-specific genes, we provide evidence that mice and urodele amphibians share a common mechanism of germ cell development that is ancestral to tetrapods. Our results imply that germ plasm, as found in species such as frogs and teleosts, is the result of convergent evolution. We discuss the evolutionary implications of our findings.

Evolution of predetermined germ cells in vertebrate embryos: implications for macroevolution.

The germ line is established in animal embryos with the formation of primordial germ cells (PGCs), which give rise to gametes. Therefore, the need to form PGCs can act as a developmental constraint by inhibiting the evolution of embryonic patterning mechanisms that compromise their development. Conversely, events that stabilize the PGCs may liberate these constraints. Two modes of germ cell determination exist in animal embryos: (a) either PGCs are predetermined by the inheritance of germ cell determinants (germ plasm) or (b) PGCs are formed by inducing signals secreted by embryonic tissues (i.e., regulative determination). Surprisingly, among the major extant amphibian lineages, one mechanism is found in urodeles and the other in anurans. In anuran amphibians PGCs are predetermined by germ plasm; in urodele amphibians PGCs are formed by inducing signals. To determine which mechanism is ancestral to the tetrapod lineage and to understand the pattern of inheritance in higher vertebrates, we used a phylogenetic approach to analyze basic morphological processes in both groups and correlated these with mechanisms of germ cell determination. Our results indicate that regulative germ cell determination is a property of embryos retaining ancestral embryological processes, whereas predetermined germ cells are found in embryos with derived morphological traits. These correlations suggest that regulative germ cell formation is an important developmental constraint in vertebrate embryos, acting before the highly conserved pharyngula stage. Moreover, our analysis suggests that germ plasm has evolved independently in several lineages of vertebrate embryos.