Hidden in the crowd: primordial germ cells and somatic stem cells in the mesodermal posterior growth zone of the polychaete Platynereis dumerillii are two distinct cell populations
© Rebscher et al; licensee BioMed Central Ltd. 2012
Received: 6 February 2012
Accepted: 18 April 2012
Published: 18 April 2012
In the polychaete Platynereis, the primordial germ cells (PGCs) emerge from the vasa, piwi, and PL10 expressing mesodermal posterior growth zone (MPGZ) at the end of larval development, suggesting a post-embryonic formation from stem cells.
In order to verify this hypothesis, embryos and larvae were pulse labeled with the proliferation marker 5-ethynyl-2'-deoxyuridine (EdU) at different stages of development. Subsequently, the PGCs were visualized in 7-day-old young worms using antibodies against the Vasa protein.
Surprisingly, the primordial germ cells of Platynereis incorporate EdU only shortly before gastrulation (6-8 hours post fertilization (hpf)), which coincides with the emergence of four small blastomeres from the mesoblast lineage. We conclude that these so-called 'secondary mesoblast cells' constitute the definitive PGCs in Platynereis. In contrast, the cells of the MPGZ incorporate EdU only from the pre-trochophore stage onward (14 hpf).
While PGCs and the cells of the MPGZ in Platynereis are indistinguishable in morphology and both express the germline markers vasa, nanos, and piwi, a distinct cluster of PGCs is detectable anterior of the MPGZ following EdU pulse-labeling. Indeed the PGCs form independently from the stem cells of the MPGZ prior to gastrulation. Our data suggest an early PGC formation in the polychaete by preformation rather than by epigenesis.
In many species, ranging from sponges, and cnidarians, to flatworms, annelids, tunicates, and sea urchins, both primordial germ cells (PGCs) and somatic stem cells are characterized by the expression of a similar set of genes, namely vasa, nanos, piwi, and PL 10 [1–7]. This fact led to the hypothesis that PGCs and stem cells are not only closely related, but also share a common gene regulatory module, the 'germline multipotency program' [8, 9]. Numerous examples show that pluripotent stem cells give rise to both PGCs and somatic derivates, such as choanocytes in sponges, large I-cells in cnidarians, neoblasts in planarians, or epiblast cells in mice [3, 7, 10, 11]. The generation of PGCs by undifferentiated cells occurs after gastrulation and sometimes is referred to as 'epigenesis' . It requires inductive signals by the surrounding tissue. In mice, for example, BMP 4 and 8 secreted by the extra-embryonic ecto-and endoderm induce the expression of germ cell specific genes in cells of the proximal epiblast . In other species, the PGCs form early in development self-autonomously by inheritance of cytoplasmic determinants contained in ribonucleotide particles of the germ plasm, which has been termed 'preformation' . Epigenesis is currently considered to constitute the ancestral mechanism, while preformation might have arisen in evolution several times independently as an adaptation to fast embryogenesis and/or extensive reorganization during gastrulation [14, 15].
A recent finding in the cephalochordate Branchiostoma, however, challenges the model of a stem cell origin of PGCs: in this species, the PGCs had for a long time been considered to arise post-embryonically from the stem cells of the posterior growth zone by epigenesis , but a closer examination now revealed that they are in fact specified during early cleavage stages by inheritance of a maternal vasa and nanos containing germ plasm, that is by preformation . Later, a second vasa and nanos expressing domain forms around the blastopore during gastrulation, which harbors the future somatic stem cells of the posterior growth zone. The PGCs then move from the ventral endoderm towards this region and blend into this cell mass, which they again leave as single cells in the late neurula stage . Thus, although the cells of the posterior growth zone in the early neurula stage are indistinguishable by morphology and both co-express vasa and nanos, they, in fact, constitute a patchwork of PGCs and somatic stem cells.
The fate of the putative PGCs in Platynerei s cannot be traced directly during gastrulation by immunohistochemistry, since maternal Vasa protein decreases to background levels after 10 hpf, while zygotic Vasa protein appears only in the MPGZ from the late trochophore larva stage onwards (> 24 hpf). The transcripts of vasa, nanos, and piwi are initially broadly distributed and only later get progressively restricted to the MPGZ during larval development, thus in situ hybridization is also not suitable for monitoring the PGCs at that stage . Finally, injection of lineage tracer into single blastomeres at later stages of development in Platynereis is extremely difficult due to small blastomere size and a tough vitelline envelope . Therefore, a different approach was employed to label the PGCs: Assuming that the four secondary mesoblasts constitute the definitive four PGCs these cells will form earlier than the cells of the MPGZ . Using EdU as a proliferation marker, the PGCs and the cells of the MPGZ were indeed shown to differ in their time of formation and proliferation. Furthermore, the PGCs form a distinct cluster lying at the anterior margin of the MPGZ. This challenges the assumption of PGC formation by epigenesis in the polychaete.
Culture and breeding of Platynereis dumerilii
Animals were cultured in artificial sea water (ASW) as described in .
Double-labeling for Vasa expression and cell proliferation
Embryos were dejellied by rinsing on a sieve of 15 μm mesh size with 0.5 l of ASW. Incubation with 0.5-2 μM 5-ethynyl-2'-deoxyuridine (Invitrogen)  was carried out in six well plates in ASW for different periods ranging from 1 hpf up to 7 days post fertilization (dpf). Subsequently, the specimens were washed thoroughly in ASW and allowed to develop further until the desired stage was reached. Specimens were then fixed in 4% PFA/2xPBT and immunohistochemistry for Vasa protein was performed using affinity purified polyclonal anti-Vasa antibodies as described previously . EdU incorporated during S-phase of mitosis was detected following immunohistochemistry using the click-it EdU Alexa Fluor 594 Imaging Kit (Invitrogen) according to the manufacturer's instructions. Specimens were mounted in DABCO glycerol and images were taken on a Nikon A1R confocal laser scanning microscope with a Plan Apo VC 20x NA 0.75 objective.
Results and discussion
The primordial PGCs in Platynereis arise early in development
The MPGZ forms during the pre-trochophore stage
The cluster of PGCs lies anterior of the MPGZ
Conclusion: the PGCs in Platynereis emerge early in development by preformation
days post fertilization
hours post fertilization
mesodermal posterior growth zone
primordial germ cell.
We thank Professor Dr Monika Hassel and Dr Jude Przyborski for critical reading of the manuscript. The valuable comments of three anonymous reviewers considerably improved the quality of this work. Robin Nostadt skillfully helped with the Platynereis culture and some of the drawings. This work was supported by funding from the Deutsche Forschungsgemeinschaft (DFG) to NR.
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