| dc.contributor.advisor | Dosch, Roland Dr. | |
| dc.contributor.author | Goloborodko, Alexander | |
| dc.date.accessioned | 2020-08-14T07:53:47Z | |
| dc.date.available | 2020-10-28T23:50:03Z | |
| dc.date.issued | 2020-08-14 | |
| dc.identifier.uri | http://hdl.handle.net/21.11130/00-1735-0000-0005-145A-0 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-8156 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-8156 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 572 | de |
| dc.title | Molecular Mechanisms of Germ Plasm Anchoring in the Early Zebrafish Embryo | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Dosch, Roland Dr. | |
| dc.date.examination | 2019-10-30 | |
| dc.description.abstracteng | Zebrafish 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 infertility. | de |
| dc.contributor.coReferee | Wouters, Fred Prof. Dr. | |
| dc.contributor.thirdReferee | Jäckle, Herbert Prof. Dr. | |
| dc.contributor.thirdReferee | Bucher, Gregor Prof. Dr. | |
| dc.contributor.thirdReferee | Günesdogan, Ufuk Dr. | |
| dc.contributor.thirdReferee | Vorbrüggen, Gerd Dr. | |
| dc.subject.eng | Germ plasm | de |
| dc.subject.eng | Bucky ball | de |
| dc.subject.eng | Zebrafish | de |
| dc.subject.eng | Non-muscle myosin | de |
| dc.subject.eng | zonula occludens | de |
| dc.subject.eng | ZO | de |
| dc.subject.eng | NMII | de |
| dc.subject.eng | Buc | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-21.11130/00-1735-0000-0005-145A-0-9 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.description.embargoed | 2020-10-28 | |
| dc.identifier.ppn | 1727092899 | |