Abstract
BackgroundIn 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.MethodsIn 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.ResultsSurprisingly, 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).ConclusionWhile 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.
Highlights
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 postembryonic formation from stem cells
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 [1,2,3,4,5,6,7]
The primordial PGCs in Platynereis arise early in development The four PGCs of Platynereis are first detectable at 96 hpf, when they leave the MPGZ and start to migrate anteriorly towards the primary gonad
Summary
Culture and breeding of Platynereis dumerilii Animals were cultured in artificial sea water (ASW) as described in [1]. 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) [22] was carried out in six well plates in ASW for different periods ranging from 1 hpf up to 7 days post fertilization (dpf). Specimens were fixed in 4% PFA/2xPBT and immunohistochemistry for Vasa protein was performed using affinity purified polyclonal antiVasa antibodies as described previously [1]. 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
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