Beteiligte Signalwege an der Entstehung der Urkeimzellen in Platynereis dumerilii (Annelida, Polychaeta)

Primordial germ cells (PGCs) are important for the development of the next generation of an organism. Their development has to be controlled exactly; even minor defects may lead to a loss of fertility. Little is known about i the signaling pathways involved in the regulation of PGC development in invertebrates. Therefore, PGC development was investigated in the marine polychaete Platynereis dumerilii. Since PGC development is influenced by the steroid hormone 17βestradiol in vertebrates (Ge et al., 2012, La Sala et al., 2010), the effect of estradiol on PGC development in Platynereis was analysed. Upon exposure to estradiol from 2 to 24 h, larvae with supernumerary PGCs were found (Lidke et al., in press). Similiaryl, the xenoestrogen ethinylestradiol was found to increase PGC numbers. Incubation after 24 h with either estradiol and ethinylestradiol had no effect. In a next step, the sensitive phase for Estradiol could be narrowed down to 4 to 9 h, indicating thatthe PGCs can only be influenced with estradiol during their development. The estradiol antagonist ICI182 780 abolishes the effect of Estradiol. This suggests that the proliferative effect of estradiol is mediated by the estradiol receptor. In order to find out, when the estradiol receptor is expressed in Platynereis, several antibodies were tested in Westernblots, but none of them could detect a protein of the expected size. However, the ER transcript was detected by in situ hybridization in 6 h old embryos in the micromeres. Using of the proliferation marker EdU it could furthermore be shown, that the phase of PGC development is prolongated following incubation with estradiol.In order tTo elucidate the target genes of estradiol signaling, RT-PCR for putative target genes such as cyclin E was performed on estradiol treated and control embryos , but due to the small number of PGCs no difference could be observed. While it was initially assumed that the supernumerary PGCs arise from classical ‘genomic’ estradiol signaling, the fact that a proteinkinase B (AKT) inhibitor abolishes the estradiol effect suggests, that so called ‘rapid signaling’ (Moriarty et al., 2006) might be involved in PGC development. As a putativetarget of AKT, the role of GSK3β during PGC development was studied. The GSK3 β inhibitor Azakenpaullone (Azp)reduces PGC numbers after treatment from 4 to 5 h, while supernumerary PGCs occur upon treatment from 5,5 to 6,5 h. GSK3β and AKT seem to be indispensable for the development of the correct number of PGCs. Since the protein β-catenin is regulated by GSK3β, it was assumed, that β-catenin levels are altered by estradiol or azakenpaullone treatment. Unfortunately, the antibodies at hand failed yield reproducible results. Since FGFR is a possible activator of AKT (Kalff and Spencer, 2012), the FGFR inhibitor SU5402 was tested on Platynereis embryos. Indeed it produced larvae with less than four PGCs, which suggests a connection between FGFR and AKT signalling might exist. In the 3’ UTR region of the FGFR a putative Nanos response element was detected. A down-regulation of FGFR via Nanos could be possible in Platynereis. Besides AKT, MAP Kinase and Notch/Delta signaling can be influenced by FGFR (Espinosa et al., 2003, Kalff and Spencer, 2012, Kim and Snider, 2011). Treatments with specific inhibitors lead to no differences in PGC numbers. However, since only a single concentration was tested with both inhibitors, the involvement of this pathways has to be investigated more intensely. Furthermore, te complete sequence of the PduVasa insert in the plasmid ‘Vasa ClonA’ was obtained. Additionally, fragments of the genes PduActin and PduCyclinB1 were cloned and sequenced. Antibody staining against α-Tubulin could be shown to be a good counter-staining for staining with β-Catenin antibody. As a possible and fast staining of the PGCs in Platynereis embryos, DNA staining with DAPI and Propidium Iodid was tested. This method however did not allow the vizualisation of PGC formation because of the difficulty of finding the PGCs in the embryo. In my thesis I was able to show, that the formation of supernumery PGCs in Platynereis can be induced by estradiol when applied during a sensitive phase. A model explaining the involvement of FGFR, AKT and GSK3β within the development of PGCs was developed.