Show simple item record

Molecular Mechanisms of Germ Plasm Anchoring in the Early Zebrafish Embryo

dc.contributor.advisorDosch, Roland Dr.
dc.contributor.authorGoloborodko, Alexander
dc.titleMolecular Mechanisms of Germ Plasm Anchoring in the Early Zebrafish Embryode
dc.contributor.refereeDosch, Roland Dr.
dc.description.abstractengZebrafish germline is specified early during embryogenesis by inherited maternal RNAs and proteins called germ plasm. Only those cells containing germ plasm will become part of the germline, whereas other cells will commit to somatic cell fates. Therefore, proper localization of germ plasm is crucial for germ cell specification. In our lab we discovered the bucky ball (buc) gene. Buc is the first protein in vertebrates required for germ plasm aggregation and induction of primordial germ cells. Fascinatingly, Buc mirrors germ plasm localization during all stages of zebrafish embryogenesis and oogenesis. Hence, to investigate germ plasm localization, I used Buc as a molecular proxy. Previously, we mapped the localization signal of Buc and identified non-muscle myosin II (NMII) as one of the Buc interactors involved in germ plasm localization. However, the fundamental mechanisms responsible for germ plasm localization remain largely unexplored. In this study, we analyzed various NMII-associated cellular structures for their role in germ plasm localization, using immunohistochemistry. Moreover, we did a fine-mapping and mutagenesis of the localization domain of Buc to understand the requirement of predicted protein aggregation domains in germ plasm localization. After the refined mapping, we utilized the identified Buc localization sequence to isolate proteins involved in germ plasm localization. Finally, we investigated whether the function of Buc in germ plasm localization is conserved in vertebrates and invertebrates by a combination of protein overexpression in vivo and immunohistochemistry. We found that the cytoplasmic tight junction component Zonula occludens 1 isoform A (ZO1-A) colocalizes with Buc during oogenesis and embryogenesis. Furthermore, we demonstrated that ZO1 phosphorylation is required for Buc degradation. We also showed that Buc localization is mediated independently of the predicted aggregation domains. Additionally, we isolated 23 potential interactors with the Buc localization signal. In the end, we showed that the germ plasm organizer Xenopus Velo1 but not Drosophila short Oskar co-localizes with zebrafish germ plasm. Previously, it was shown that germ plasm interacts with actin. However, how exactly germ plasm is anchored to cleavage furrow remained unknown. Our data indicates for the first time that germ plasm is anchored by tight junction, as early as at the 8-cell stage. However, it remains to be addressed if mature tight junctions are present during the first embryonic cleavages in zebrafish. We demonstrated a role of ZO1 phosphorylation in Buc degradation. Our results indicate that Protein Kinase C (PKC)- mediated phosphorylation of ZO1 is required for the tight regulation of Buc levels during early embryogenesis. Our results also show that the predicted aggregation domains within the Buc localization signal are not required for germ plasm localization during early embryogenesis. This result is in line with a more liquid-like behavior of germ plasm during embryogenesis than during the oogenesis. Among the 23 potential interactors with the Buc localization signal, we identified Intracellular hyaluronan-binding protein 4 (Ihabp4) as the most promising candidate for anchoring germ plasm to cytoskeleton. Future study will focus on co-localization analysis of Buc and Ihabp4. In the end, our data shows that the molecular mechanism of germ plasm localization is conserved in vertebrates. We hypothesize, that Velo1 targets to zebrafish germ plasm also via its N-terminal localization signal. Further understanding of molecular mechanisms of germ plasm localization might lead to a better understanding of germ cell formation and to the establish new drug targets and therapies against
dc.contributor.coRefereeWouters, Fred Prof. Dr.
dc.contributor.thirdRefereeJäckle, Herbert Prof. Dr.
dc.contributor.thirdRefereeBucher, Gregor Prof. Dr.
dc.contributor.thirdRefereeGünesdogan, Ufuk Dr.
dc.contributor.thirdRefereeVorbrüggen, Gerd Dr.
dc.subject.engGerm plasmde
dc.subject.engBucky ballde
dc.subject.engNon-muscle myosinde
dc.subject.engzonula occludensde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

Files in this item


This item appears in the following Collection(s)

Show simple item record