Germplasm stability in zebrafish requires maternal Tdrd6a and Tdrd6c

Alessandro Consorte, Yasmin El Sherif, Fridolin Kielisch, Nadine Wittkopp, René F. Ketting

Posted on: 13 December 2024

Preprint posted on 4 October 2024

Maternally deposited proteins Tdrd6a and Tdrd6c act redundantly to regulate Bucky ball activity and ensure the retention of germplasm in zebrafish embryos

Selected by Justin Gutkowski

Categories: cell biology, developmental biology, molecular biology

Background:

Early in development, the cells of an embryo begin to differentiate to assume different functions in the adult organism. Several factors influence this specification, but maternal deposition, in which the mother leaves specific molecular signals in the embryo, is arguably the most important in the earliest stages of development.

The zebrafish germline, or the lineage of cells essential for sexual reproduction, including spermatozoa and oocytes, goes through multiple phases of specification. An important checkpoint in this process is the formation of germplasm, or the mass in an early embryo containing cells that will later develop into the gametes. Before fertilization, the protein

Bucky ball (Buc) coordinates Balbiani body formation on the vegetal pole of the oocyte. After fertilization, this organelle migrates to the animal pole to coordinate Primordial Germ Cell (PGC) specification there. Although this process is characterized [1-4], its regulation is yet to be fully understood.

One factor known to regulate Buc mobility is the maternally deposited protein Tdrd6a. Zebrafish that do not receive this signal have reduced PGC counts. Tdrd6a has one paralog known to be expressed in zebrafish germ cells, Tdrd6c. In this preprint, the authors set out to uncover the mechanism of interaction between these proteins and Buc, as well as the effects of these proteins on the development and specification of the zebrafish germplasm.

Key Findings:

Structural elements of Tdrd6a and Tdrd6c:

The authors used AlphaFold to predict the structures of Tdrd6a and Tdrd6c. Both exhibit seven extended Tudor domains (eTDs), which are typically involved in substrate-specific interactions. These motifs may be involved in the interactions between Tdrd6a and Buc. Tdrd6c exhibits one prion-like domain (PrLD), a motif typically exhibited by regulators of phase-separated processes, such as germplasm development.

Localization of Tdrd6a and Tdrd6c:

The authors used antibody staining and a Tdrd6c fluorescent reporter line to determine the localization of Tdrd6a and Tdrd6c in zebrafish embryos. Both proteins were localized in the germplasm by the 4-cell stage and were enriched in the PGCs from 24 hours post-fertilization (hpf). Tdrd6a localized with the Balbiani body while Tdrd6c localized around the nucleus.

Paralog-associated Phenotypes:

The authors created three transgenic knock-out lines: 1) Tdrd6a mutant (Tdrd6a-/-), 2) Tdrd6c mutant (Tdrd6c-/-), and 3) Tdrd6a/Tdrd6c double-mutant (Tdrd6a-/- Tdrd6c-/-). Mutants of all three lines exhibited normal development, behavior, and fertility. However, zebrafish born to Tdrd6a-/- mothers (6a mmut) and zebrafish born to Tdrd6c-/- mothers (6c mmut) exhibited reduced PGC counts at 24hpf. Zebrafish born to double-mutant mothers (6a6c mmut) exhibited no PGCs at 24hpf, were 100% male, and were infertile as adults.

Using a Buc-eGFP transgenic line to visualize the germplasm, the authors observed normal development in the offspring of the 6a mmut and 6c mmut populations. However, in 6a6c mmut fish, the germplasm formed normally until 2hpf, but then disappeared before 3hpf. This means neither Tdrd6a nor Tdrd6c are necessary for germplasm formation. However, at least one is required to stabilize and maintain this structure.

Figure 1: the images and graphs that the preprint used as Figure 4. A) the localization of germplasm, visualized using BucEGFP, in control, 6a mmut, 6c mmut, and 6a6c mmut embryos at 1hpf, 2hpf, and 3hpf. B) Quantification of the volume of germplasm (μm³) based on BucEGFP signal.

Interactions with Buc:

Finally, to determine the mechanism of interaction between Tdrd6c and Buc, the researchers co-expressed Buc with each protein as well as different combinations of the domains of each protein. Buc did not interact with Tdrd6c or its eTDs in vitro, although the first three eTDs bound each other. However, Buc would co-localize with the PrLD of Tdrd6c. This means the PrLD of Tdrd6c interacts with Buc, while the eTDs do not. However, they are likely important for the interactions of Tdrd6c with itself.

Significance:

I highlighted this preprint because I am interested in germ cell specification and development. This preprint details an aspect of these processes that I was unaware of: the loss of germplasm in an embryo due to the lack of maintenance signals, even after it is specified. I also found it interesting that the genetic signals involved in this process exhibit redundancy, which is typically characteristic of critical processes in vertebrate development.

Questions for the Authors:

Would overexpressing Tdrd6a or Tdrd6c affect zebrafish germplasm development? The PrLD of Tdrd6c was found to bind to Buc in vitro, but these proteins did not interact in vivo. How would increasing the expression of Tdrd6c to make this interaction more favorable affect germplasm development in the embryos?
Figure 4B shows no significant difference in germplasm volume between mmut and wild-type zebrafish, even though 6a and 6c mmut fish have reduced PGC counts. Does this mean the density/composition of the germplasm was different between these groups? Could this also affect the signaling between PGCs during development?
Are there any other proteins expressed in the zebrafish germline known to exhibit the same functional domains (eTDs and PrLDs) as Tdrd6a and Tdrd6c? If so, what role do they play in germplasm regulation?
Why does the germplasm disappear between 2 and 3hpf in 6a6c mmut zebrafish? Did you observe any possible mechanism, or have other research groups identified one?

References:

1) Boke, E., Ruer, M., Wühr, M., Coughlin, M., Lemaitre, R., Gygi, S. P., Alberti, S., Drechsel, D., Hyman, A. A., & Mitchison, T. J. (2016). Amyloid-like Self-Assembly of a Cellular Compartment. Cell, 166(3), 637–650. https://doi.org/10.1016/j.cell.2016.06.051

2) Bontems, F., Stein, A., Marlow, F., Lyautey, J., Gupta, T., Mullins, M. C., & Dosch, R. (2009). Bucky Ball Organizes Germplasm Assembly in Zebrafish. Current Biology, 19(5), 414–422. https://doi.org/10.1016/j.cub.2009.01.038

3) Marlow, F. L., & Mullins, M. C. (2008). Bucky ball functions in Balbiani body assembly and animal–vegetal polarity in the oocyte and follicle cell layer in zebrafish. Developmental Biology, 321(1), 40–50. https://doi.org/10.1016/j.ydbio.2008.05.557

4) Raz, E. (2003). Primordial germ-cell development: The zebrafish perspective. Nature Reviews Genetics, 4(9), 690–700. https://doi.org/10.1038/nrg1154

Tags: danio, gametogenesis, germline, germplasm, oogenesis

doi: https://doi.org/10.1242/prelights.39185

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Author's response

René F. Ketting shared

1) Would overexpressing Tdrd6a or Tdrd6c affect zebrafish germplasm development? The PrLD of Tdrd6c was found to bind to Buc in vitro, but these proteins did not interact in vivo. How would increasing the expression of Tdrd6c to make this interaction more favorable affect germplasm development in the embryos?

In fact, also in vivo Buc is found to coIP with both Tdrd6a and Tdrd6c. How overexpression would act in the fish is unclear, and I find that hard to predict.

2) Figure 4B shows no significant difference in germplasm volume between mmut and wild-type zebrafish, even though 6a and 6c mmut fish have reduced PGC counts. Does this mean the density/composition of the germplasm was different between these groups? Could this also affect the signaling between PGCs during development?

Using RNA ISH we previously showed that in 6a mmut embryos there is indeed a slight dispersion of some mRNAs from the germplasm. But indeed, the effect is much weaker than in the double mutant. It may be that in the single mutants the effects are simply to subtle at the level of germ plasm volume to be recorded by our experiments. However, I tend to think that perhaps some transcripts are more prone to be lost from germ plasm in these single mutant scenarios and that that relates to the PGC defect in these single mutants.

3) Are there any other proteins expressed in the zebrafish germline known to exhibit the same functional domains (eTDs and PrLDs) as Tdrd6a and Tdrd6c? If so, what role do they play in germplasm regulation?

There are quite some other eTD containing proteins in zebrafish (and other animals). For instance there is Tdrd1, which we previously showed plays a role in the formation/maintenance of perinuclear granules in the adult germ cells. Tdrd1 helps to drive the piRNA pathway. Besdies that, there are, for instance, Tdrd5 and Tdrd7, and even more. They have not been studied in much detail, but Tdrd7 was shown to affect PGCs by the Raz lab. For PrLDs: I am sure there will be more, but we have not specifically looked.

4) Why does the germplasm disappear between 2 and 3hpf in 6a6c mmut zebrafish? Did you observe any possible mechanism, or have other research groups identified one?

Possibly a more extensive loss of mRNA from the germ granules leads to the slow dissolution of them, as mRNA is known to be a strong driver of germ any type of granule formation. However, this has not yet been tested. We are, however, testing that very idea using smFISH.

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