Buc is localized to the germ plasm during early embryogenesis

So far, the localization of germ plasm during early embryogenesis (0-4 hpf) has been examined in detail only by analyzing localization of mRNA germ plasm components, such as vasa mRNA (Braat et al., 1999; Yoon et al., 1997). However, very little is known about the localization of protein germ plasm components during cleavage and blastula stages.

3.1.2.1 Transgenic Buc-GFP marks germ plasm in vivo

The localization of zebrafish Buc during early embryogenesis is of special interest as Buc is able to induce the formation of primordial germ cells in the embryo (Bontems et al., 2009). As buc mRNA was not localized and only expressed during the first four hours of early embryogenesis, it was unclear how Buc protein is localized or if it is expressed at all.

The transgenic buc-gfp line was used to investigate the localization of Buc in different stages

Results of living embryos by stereo or confocal fluorescence microscopy. In 1-cell stage embryos, Buc-GFP was localized in small granules at the cytokinetic ring by the vegetal part of the embryo, leaving the animal pole free of granules (Figure 11). During cytokinesis, these granules were recruited to the distal ends of the first and second cleavage furrows, thus forming four aggregates at the 4-cell stage (Figure 11). At this stage, a considerable amount of small granules was still present at the cortex of the embryo. The four aggregates were stable during early cleavage stages resulting in a 256-cell stage embryo with four aggregates in the cortical region of the embryo (Figure 11). Until this stage, Buc-GFP granules and other smaller aggregates had disappeared, suggesting their degradation or fusion with the four main aggregates.

Buc-GFP localization in living buc-gfp embryos mimics the localization pattern of previously described germ plasm components such as vasa mRNA (Yoon et al., 1997). Hence, the newly generated transgenic buc-gfp line is the first transgenic line that marks the germ plasm in vivo throughout cleavage and blastula stages.

Figure 11: Buc-GFP localizes to the germ plasm during early embryogenesis. Transgenic buc-gfp embryos at indicated stages imaged by fluorescence microscopy. Embryos are depicted in animal view. A Confocal z-stack projection of a 1-cell stage embryo is additionally depicted in a lateral view, animal pole to the top. The Blastomere is outlined by a yellow dashed line.

The image of the 256-cell stage embryo is likewise a z-stack projection. Scale bars represent 100 µm.

3.1.2.2 Buc-GFP is maintained only in the first and second cleavage furrow

It has been described previously that, in addition to the four aggregates at the cleavage furrows, small granules of vasa mRNA are localized to the distal cortex of all four cells in 4-cell stage embryos (Wolke et al., 2002). However, due to the lack of in vivo reporters, the role of these granules at the cortex is not known.

To study the dynamics of these additional granules in detail, living transgenic buc-gfp embryos were imaged in high resolution time-lapse movies (digital Appendix, Chapter 7.4).

Similar to vasa transcripts, Buc-GFP protein localized in these granules at the cortex in early

Results cleavage stages (Figure 12). At the onset of 8-cell stage, small Buc-GFP granules seemed to fuse with the closely adjacent aggregates in the first and second cleavage furrow. Additionally, granules accumulated at the distal ends of the forming third cleavage furrows (Figure 12).

However, the aggregations in the third cleavage furrows were unstable and disassembled at the beginning of the 16-cell stage. The disassembled granules as well as the smaller granules in the cortical region were strongly reduced in number during further development of the embryo (Figure 12). In contrast to the small granules, the four aggregates originating from the first two cleavage furrows were stabilized in a rod-like structure.

Hence, smaller cortical Buc-GFP granules are only temporary stable whereas the first four aggregates at the distal ends of the first and second cleavage furrow are stable. This observation suggests that proper localization of germ plasm is crucial for its stabilization and the inheritance of the germ cell fate. This four cells with inherited Buc-GFP labeled germ plasm are in line with the identification of four founding primordial germ cells at late blastula stage (Wolke et al., 2002; Yoon et al., 1997).

Figure 12: Buc-GFP is stable only in four condensed rod-like aggregates during early embryogenesis. Still pictures of a time lapse movie showing a transgenic buc-gfp embryo starting at onset of 8-cell stage and ending at onset of 128-cell stage imaged by confocal fluorescence microscopy. Note that the signal at the cortex disappears over time. Embryo is shown in animal view. Numbers at eight-cell stage indicate first, second and third cleavage furrows. Images are z-stack projections. Scale bars represent 100 µm.

3.1.2.3 Transgenic Buc-GFP reflects endogenous Buc during early embryogenesis The transgenic buc-egfp line marks the germ plasm in vivo. However, the GFP-tag might alter the localization and thus Buc-GFP does not reflect endogenous Buc.

To investigate, whether Buc-GFP localization resembles the so far unknown localization of endogenous Buc during early zebrafish embryogenesis, wild type embryos were analyzed by Buc immunostaining and subsequent confocal fluorescence microscopy. For comparison, living transgenic buc-gfp embryos were examined by stereo fluorescence microscopy at comparable stages. Endogenous Buc was localized in two aggregates at the distal ends of the first cleavage furrow in 2-cell stage embryos (Figure 13A). In addition, smaller granules were localized at the cortex of the embryo, similar to Buc-GFP in living transgenic embryos (Figure 13B). Identical localization of endogenous Buc and transgenic Buc-GFP persisted throughout early embryogenesis, leading to four aggregates and some smaller clusters at early blastula stages (2.5-3 hpf, Figure 13A, B).

These results show that endogenous Buc is localized to the germ plasm till blastula stage as previously described for the germ plasm component vasa mRNA (Yoon et al., 1997). The

Results endogenous Buc localization during early embryogenesis is reflected by transgenic Buc-GFP in living embryos.

Figure 13: Endogenous Buc and transgenic Buc-GFP are localized to the germ plasm in early embryos. (A) Wild type embryos immunostained for Buc at 2-cell respectively 256-cell stage imaged by confocal fluorescence microscopy. Embryos are shown animal and in lateral view, animal pole to the top (images in animal view are z-stack projections). (B) Living transgenic buc-gfp embryos at 2-cell and high stage imaged by stereo fluorescence microscopy. Embryos are shown in animal view.

Embryos are outlined by a yellow dashed line. Scale bars represent 50 µm (A) and 100 µm (B).

In summary, these results show that throughout cleavage and blastula stages endogenous Buc is localized to the germ plasm and this localization is reflected by Buc-GFP in living embryos of the transgenic buc-gfp line. Therefore, the transgenic line can be used as an in vivo marker line, representing the endogenous Buc localization throughout oogenesis and early zebrafish development.