Molecular Control of Germ Plasm Localization in Zebrafish
by Nadia Rostam
Date of Examination:2021-08-18
Date of issue:2022-05-24
Advisor:Prof. Dr. Ernst A. Wimmer
Referee:Dr. Roland Dosch
Referee:Dr. Melina Schuh
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Name:Nadia Rostam Doctoral thesis.pdf
EnglishGerm plasm (GP) consists of a maternally inherited ribonucleo-protein (RNP) condensate, which controls in many animals the formation of germline. Only the cells which contain GP commit to germ cell destinies, whereas all other cells will commit to somatic cell fates. Therefore, correct GP localization is important for germ cell specification, and its elimination is important for the development of soma. In this thesis, I show that zebrafish (Danio rerio) GP localization is similar to that of Xenopus but different from Drosophila. Bucky ball (Buc), the GP organizer in zebrafish, was used in this research as a molecular proxy to follow GP localization. The identification of Buc localization domain (BucLoc) is presented here as a tool to identify the protein interactome of Buc recruitment, which led to the identification of non- muscle myosin II (NMII) and tight junction (TJ) components such as ZO2 and Claudin-d (Cldn- d) as interacting candidates of Buc. The data here show that Buc colocalizes with TJ proteins ZO1 and Cldn-d. Moreover, TJ- like structures, at the cleavage furrows where the GP is anchored, was revealed under electron microscopy (EM). Furthermore, the detailed protein machinery anchoring GP at TJs is presented here. I am showing that multiple PxxP motifs in Buc interact with the SH3 domains of the TJ proteins ZO2a and ZO2b. I am showing a detailed mutation analysis of PxxP motifs in Buc, leading to the identification of the key motifs for interaction with SH3 domain and ultimate localization of GP at the TJs. Finally, overexpression of TJ-receptor Cldn-d created additional GP aggregates, whereas expression of a dominant negative version prevents the production of GP aggregates. Taken together, the data presented in this thesis discovered a comprehensive molecular mechanism for GP localization in zebrafish.
Keywords: Germ plasm localization, zebrafish, tight junctions, Bucky ball, ZO proteins, Claudin-d, SH3 domain, PxxP motif.