Specification of primordial germ cells in medaka (Oryzias latipes)

Abstract

Background: Primordial germ cells (PGCs) give rise to gametes that are responsible for the development of a new organism in the next generation. Two modes of germ line specification have been described: the inheritance of asymmetrically-localized maternally provided cytoplasmic determinants and the induction of the PGC fate by other cell types. PGCs specification in zebrafish appears to depend on inheritance of germ plasm in which several RNA molecules such as vasa and nanos reside. Whether the specification mode of PGCs found in zebrafish is general for other fish species was brought into question upon analysis of olvas expression--the vasa homologue in another teleost, medaka (Oryzias latipes). Here, in contrast to the findings in zebrafish, the PGCs are found in a predictable position relative to a somatic structure, the embryonic shield. This finding, coupled with the fact that vasa mRNA, which is localized to the germ plasm of zebrafish but does not label a similar structure in medaka opened the possibility of fundamentally different mechanisms governing PGC specification in these two fish species.

Results: In this study we addressed the question concerning the mode of PGC specification in medaka using embryological experiments, analysis of RNA stability in the PGCs and electron microscopy observations. Dramatic alterations in the somatic environment, i.e. induction of a secondary axis or mesoderm formation alteration, did not affect the PGC number. Furthermore, the PGCs of medaka are capable of protecting specific RNA molecules from degradation and could therefore exhibit a specific mRNA expression pattern controlled by posttrancriptional mechanisms. Subsequent analysis of 4-cell stage medaka embryos using electron microscopy revealed germ plasm-like structures located at a region corresponding to that of zebrafish germ plasm.

Conclusion: Taken together, these results are consistent with the idea that in medaka the inheritance of maternally provided asymmetrically-localized cytoplasmic determinants directs cells to assume the germ line fate similar to zebrafish PGCs.

Figure 1

The early expression patterns of zebrafish-vasa, medaka-olvas and medaka-nanos1 mRNAs. In zebrafish, the vasa transcript is enriched at the marginal positions of the first two cleavage planes (A), while at a similar stage olvas mRNA is uniformly distributed throughout the cytoplasm of all blastomers (B). Throughout development in zebrafish, the vasa mRNA is expressed exclusively in the PGCs that can be found in random dorsoventral positions during blastula and gastrula stages (arrowheads in C). In contrast, at the first time point when medaka PGCs can be observed (stage 16), they are found on both sides of the embryonic shield on the dorsal side of the embryo (arrows in D). All images shown are whole-mount in situ hybridizations. The probes used are: vasa (A), vasa and chordin (C), olvas (B, D) and nanos1 (E to H). A, B, E and H are animal view, C is a lateral view with the dorsal aspect (labeled with chordin) to the left and D is a dorsal view, F and H are lateral views.

Figure 2

The effect of axis duplication and mesoderm induction modulation in medaka on PGC number. (A, B and C) Induction of a complete secondary embryonic axis was achieved by injection of β-catenin at the 8- and 16-cell stage. The injected embryos were fixed at stages 17 and 23 and their PGCs visualized by in situ hybridization using an olvas antisense RNA probe. A: a stage 23 control embryo injected with GFP-globin; B: a stage 23 embryo with a duplicated embryonic axis. In both stages analyzed, the average PGC number in embryos with a duplicated axis is similar to that of control embryos (C). (D to P) BMP-mediated mesoderm induction modulation was achieved by injection of either BMP2 (H to K) or dominant negative form of BMP2/4RI (DN(BMP2/4RI)) (L to O). To confirm mesoderm formation alteration, in situ hybridization was performed with either Goosecoid (Gsc) ventrolateral mesodermal marker (F, J and N) or HNF3-b axial mesodermal marker (G, K and O). One day post fertilization (dpf), the average PGC number in embryos with altered mesoderm is similar to that of control embryos (P). The number of embryos analyzed is provided within the corresponding bars.

Figure 3

Stabilization of zebrafish vasa and translational regulation of medaka olvas mRNA in PGCs of medaka. 100 pg of GFP tagged fusion RNAs were injected at the 2-cell stage. (A to D) The embryos were fixed at early somitogenesis and at 12-somite stage and in situ hybridization using a GFP antisense RNA probe was performed to reveal the spatial distribution of the injected RNAs. In the control embryos the GFP-globin RNA is degraded uniformly in all cells (A and C) whereas in vasa-GFP injected embryos (B and D), the PGCs retain the injected RNA (arrows in D) while it is degrading in somatic cells. (E to S) olvas-GFP (E to I) as well as GFP-zfvasa 3'UTR (J to N) constructs were injected in medaka and followed for GFP expression and compared to control GFP-β globin 3'UTR injected embryos (O to S). Interestingly, in medaka, olvas-GFP protein expression was rapidly restricted to PGCs (E to I), while zebrafish vasa 3'UTR did not induced selective GFP expression.

Figure 4

Sections of early 4-cell stage zebrafish and medaka embryos. Semi-thin section of zebrafish (A) and medaka (B) embryos visualized in a light microscope. Arrows indicate the areas shown in C-F representing the cleavage positions. The insets represent the orientation of the sections. Low magnification at the electron microscope showing the region of the germ plasm of zebrafish (C), and the corresponding position in medaka (D). At a higher magnification, the germ plasm of zebrafish is visible as distinct amorphous inclusions with a regular spherical shape and a filamentous fine structure lacking a surrounding membrane (E). At this magnification germ plasm resembling structure composed of irregularly shaped amorphous inclusions with a granular fine structure can be observed in medaka embryos. As described for zebrafish [28], the putative germ plasm of medaka can be followed in serial sections.