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Development
2022 Aug 01;14915:. doi: 10.1242/dev.200465.
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The germ plasm is anchored at the cleavage furrows through interaction with tight junctions in the early zebrafish embryo.
Rostam N
,
Goloborodko A
,
Riemer S
,
Hertel A
,
Riedel D
,
Vorbrüggen G
,
Dosch R
.
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The zebrafish germline is specified during early embryogenesis by inherited maternal RNAs and proteins collectively called germ plasm. Only the cells containing germ plasm will become part of the germline, whereas the other cells will commit to somatic cell fates. Therefore, proper localization of germ plasm is key for germ cell specification and its removal is crucial for the development of the soma. The molecular mechanism underlying this process in vertebrates is largely unknown. Here, we show that germ plasm localization in zebrafish is similar to that in Xenopus but distinct from Drosophila. We identified non muscle myosin II (NMII) and tight junction (TJ) components, such as ZO2 and claudin-d (Cldn-d) as interaction candidates of Bucky ball (Buc), which is the germ plasm organizer in zebrafish. Remarkably, we also found that TJ protein ZO1 colocalizes with germ plasm, and electron microscopy of zebrafish embryos uncovered TJ-like structures at the cleavage furrows where the germ plasm is anchored. In addition, injection of the TJ receptor Cldn-d produced extra germ plasm aggregates, whereas expression of a dominant-negative version inhibited germ plasm aggregate formation. Our findings support for the first time a role for TJs in germ plasm localization.
DO 740/2-3 Deutsche Forschungsgemeinschaft, Gattinger Graduiertenschule fur Neurowissenschaften, Biophysik und Molekulare Biowissenschaften, Deutscher Akademischer Austauschdienst, Göttinger Zentrum für Molekulare Biowissenschaften, Göttinger Graduiertenzentrum für Neurowissenschaften, Biophysik und Molekulare Biowissenschaften, University Medical Center Göttingen
Figure 3. Localization of BucLoc is not dependent on the aromatic amino acids in the PLD essential for Balbiani Body aggregation. (A) Alignment of Buc11-88 with the N-terminus of Xenopus Velo1 (aa7-88). Red letters highlight the regions enriched in aromatic amino acids in the PLD previously discovered in Velo1 (Boke et al., 2016) to be essential for Balbiani Body aggregation and their corresponding amino acids in Buc. (B-C″) BucLoc (11-88)-m-cherry colocalizes to the germ plasm with endogenous Buc-GFP in transgenic embryos. (B-B″) Living sphere stage transgenic Buc-GFP embryo injected at the one-cell stage with RNA encoding BucLoc-m-Cherry, showing colocalization. Embryo is shown in lateral view with the animal pole towards the top, outlined by the white dashed line. (C-C″) Magnification of the localized spot of germ plasm shown in B-B″. (D) Summary of BucLoc mapping showing that the domains enriched in aromatic amino acids (red boxes) are not important for the localization of Buc. PLDs are indicated with red boxes. (E) Quantification of BucLoc mapping and 5aa deletions in D. Buc31-88 (60.1±7.9%) and Buc31-71 (21.1±6.4) show significantly less localization compared with Buc11-88 (P=0.01 and 0.0004). There was no significant difference between the localization of Buc11-88 and Buc 31-78 (P=0.41), excluding the role of the first domain enriched in aromatic amino acids in localization. 5aa deletions of Buc31-78 showed that residues other than the second domain with aromatic amino acids are important in the localization of Buc. Buc31-78Δ31-35 (30.0±10) showed significantly less localization compared with Buc31-78 (P=0.009). Colocalization of constructs in D is shown in Fig. S7. n=30 for Buc11-88; n=30 for Buc31-88, Buc31-78, Buc35-78, Buc31-78Δ62-66, Buc31-78Δ67-71 and Buc31-71. Data are mean±s.d. *P≤0.05, **P≤0.01, ***P≤0.001. Scale bars: 50 µm in B; 2 µm in C.
