Dzip1 is dynamically expressed in the vertebrate germline and regulates the development of Xenopus primordial germ cells

Abstract

Primordial germ cells (PGCs) are the precursors of sperms and oocytes. Proper development of PGCs is crucial for the survival of the species. In many organisms, factors responsible for PGC development are synthesized during early oogenesis and assembled into the germ plasm. During early embryonic development, germ plasm is inherited by a few cells, leading to the formation of PGCs. While germline development has been extensively studied, how components of the germ plasm regulate PGC development is not fully understood. Here, we report that Dzip1 is dynamically expressed in vertebrate germline and is a novel component of the germ plasm in Xenopus and zebrafish. Knockdown of Dzip1 impairs PGC development in Xenopus embryos. At the molecular level, Dzip1 physically interacts with Dazl, an evolutionarily conserved RNA-binding protein that plays a multifaced role during germline development. We further showed that the sequence between amino acid residues 282 and 550 of Dzip1 is responsible for binding to Dazl. Disruption of the binding between Dzip1 and Dazl leads to defective PGC development. Taken together, our results presented here demonstrate that Dzip1 is dynamically expressed in the vertebrate germline and plays a novel function during Xenopus PGC development.

Keywords: Dazl; Dzip1; Germ plasm; Germline development; Primordial germ cell; Xenopus.

Fig 1.. Specificity of the anti-Dzip1 antibody.

A. Lysates from control and a combination of DMO1 and DMO2 (10 ng total) injected embryos (stage 20) were immunoprecipitated using the affinity-purified anti-Dzip1 antibody. IP samples were subjected to western blot. This experiment was performed three times. B. Whole mount immunostaining of the control and a combination of DMO1 and DMO2 (10 ng total) injected embryos (stage 25) using the affinity-purified anti-Dzip1 antibody. Arrows point to two multi-ciliated cells in the epidermis.

Fig 2.. Expression of Dzip1 protein in Xenopus and zebrafish germline.

A - H are Xenopus oocytes (A - E) and embryos (F - H). I - K are zebrafish oocytes (I) and embryos (J and K). F - H are 16-cell stage (F), stage 10 (G), and stage 13 (H) embryos from the Dria transgenic frog, which carries a mitochondria-specific GFP transgene. I - K are zebrafish oocyte (I), 4-cell stage (J), and 8-cell stage (K) embryos. Arrows in A and I point to the Bb. Arrowheads point to germ plasm.

Fig 3.. Expression of Dzip1 protein in the mouse ovary.

A shows a primary follicle. B shows a secondary follicle. C shows an antral follicle. The arrowhead in B points to the nucleolus-like structure in the GV. DAPI stains nuclei on each section. The scale bar represents 20 μm.

Fig 4.. Expression of Dzip1 in mouse testis.

A - C are immunofluorescence, showing the expression of Dzip1 in mouse testis tissue. A. Dzip1 (green) and PLZF (red) double staining. B. and C. Dzip1 (green) and Sycp3 (red) double staining. C is the high magnification view, which shows the spermatocyte nuclei in B. D - L are immunostained spreads of spermatocyte chromosomes from adult mouse testes. D, D’, D”, and D’” are Dzip1 (green) and γH2AX (red) double staining. E - L are Dzip1 (red) and Sycp3 (green) double staining.

Fig 5.. Dzip1 is required for PGC development in Xenopus.

A. knockdown of Dzip1 impaired Xenopus PGC development. Whole-mount in situ hybridization shows the expression of pgat in control and Dzip1 morpholino (DMO1 and DMO2, 10 ng per embryo) injected embryos. B. Quantification of the results shown in A. The number of pgat-positive PGCs in each embryo was counted and plotted on the graph. C. The specificity of Dzip1 knockdown. Whole-mount in situ hybridization shows the expression of pgat in control, DMO2 (10 ng) injected, and DMO2 (10 ng) + myc-Dzip1 (500 pg) injected embryos. D. Quantification of the number of pgat-positive PGCs shown in C. E. Knockdown of Gli1 and IFT88, which impaired Hh signaling and ciliogenesis respectively, had no detectable effect on Xenopus PGC development. Quantification of the number of pgat-positive PGCs in control, Gli1 morpholino (10 ng) injected, and IFT88 morpholino (10 ng) injected embryos at stage 33. F. Knockdown of Dzip1 does not affect Xenopus PGC specification. Quantification of the number of pgat-positive PGCs in control and DMO2 (10 ng) injected embryos at stages 16, 22, and 28. Statistical analysis in B, D, E, and F was done by two-tailed t-tests. *P<0.05; **P<0.01; ****P<0.0001. Data are mean±s.d.

Fig 6.. The interaction between Dzip1 and xDazl is required for Xenopus PGC development.

A. Dzip1 physically interacts with xDazl. FLAG-xDazl and Myc-Dzip1 were expressed in HEK293T cells for CoIP analysis. The CoIP experiment was done four times. B. Schematic drawing to show the structure of Dzip1 protein and Dzip1 deletion constructs. Whether Dzip1 constructs bind xDazl was indicated by “+” (bind) or “-” (not bind). C. Co-IP results show that xDazl interacts with the full-length Dzip1, 1–550, and 282–550, but not 1–221 and 550–850. This CoIP experiment was performed twice. D. Co-IP results show overexpression of Dzip1^282−550 interfered with the complex formation between the full-length Dzip1 and xDazl. This CoIP was performed three times. E. Interfering with the complex formation between Dzip1 and xDazl impaired PGC development in Xenopus embryos. Control and Dzip1^282−550 (1 ng) injected embryos were harvested at stage 33 for pgat in situ hybridization. The number of pgat-positive PGCs in control and Dzip1^282−550 injected embryos were counted and plotted on the graph. F. Knockdown of Dzip1 and xDazl impaired Xenopus PGC development synergistically. Embryos were injected with DMO2 (2.5 ng) and xDazl morpholino (xDazl-MO, 3 ng) alone, or in combination. Control and injected embryos were harvested at stage 33 for pgat in situ hybridization. The number of pgat-positive PGCs was counted and plotted on the graph. Statistical analysis in E and F was done by two-tailed t-tests. *P<0.05; ***P<0.001; ****P<0.0001. ns – not significant. Data are mean±s.d.