Zili is required for germ cell differentiation and meiosis in zebrafish

Abstract

Small RNAs exert an effect through diverse RNA interference pathways to transcriptionally or post-transcriptionally silence their targets. The Piwi-interacting RNAs (piRNAs) represent a germline-specific small RNA pathway where Piwi proteins themselves are thought to mediate piRNA biosynthesis. Here, we provide strong evidence for a piRNA amplification loop in zebrafish, in which Ziwi and Zili bind piRNAs of opposite polarity. Furthermore, we describe a function for Zili in transposon defense and germ cell differentiation, as well as a crucial function in meiosis, significantly extending the function of Piwi proteins beyond the control of transposable elements in vertebrates.

Figure 1

Zili is a nucleocytoplasmic protein. (A) Western blot showing specific expression of Zili in testis and ovary. The migration path of the Zili protein is distorted by highly abundant yolk proteins, resulting in a lower band for Zili in ovary than testis. (B) At 24 h post-fertilization (hpf), Zili protein cannot be detected in PGCs, whereas Ziwi protein is maternally provided and localizes to perinuclear granules. At 3 days post-fertilization (dpf), Zili protein is present in the nucleus of PGCs, as well as in the cytoplasm. From 7 dpf onwards, Zili protein starts localizing to the cytoplasm, whereas at this point, Ziwi protein exits the perinuclear granules and becomes diffuse in the cytoplasm. At 3 weeks post fertilization (wpf), Zili displays a granular distribution in the cytoplasm, whereas Ziwi is diffuse. Scale bar is 5 μm. (C) Zili (brown) in ovary is present in all stages of oogenesis in cytoplasm and/or nucleus (N). Stages of oogenesis are oogonia (I), stage I oocytes (7–140 μm; II), stage II oocytes (140–340 μm; III), stage III oocytes (340–690 μm; IV) and stage IV oocytes (0.69–0.73 mm; V) (Selman et al, 1993). Scale bar ia 50 μm. (D) Zili (red) in granules (white arrows) around the nucleus (N) of stage I oocyte. (E) Zili (brown) in testis is present predominantly in the cytoplasm of all stages of spermatogenesis, except the fully differentiated sperm. Stages of spermatogenesis are spermatogonia (SG), spermatocytes (SC), spermatids (ST) and sperm (S). Scale bar is 50 μm. (F) Zili (red) in granules around the nucleus of spermatogonia (upper panel) and spermatocytes (lower panel). Scale bar is 10 μm.

Figure 2

Amplification cycle for piRNAs. (A) Western blots for Ziwi and Zili protein, showing that Ziwi and Zili co-immunoprecipitate. (B) Ziwi piRNAs show a preference for uracil at their 5′-end, whereas Zili piRNAs have a preference for adenosine at position 10. (C) Sequence overlap of 10 nucleotides for Ziwi and Zili piRNAs of the opposite strand (upper panel), whereas piRNAs of the same strand show a 19–28 nucleotide overlap (lower panel). (D) Heat map showing a strand bias in Ziwi and Zili piRNAs mapping to several transposon species. Red depicts minus reads, whereas blue depicts plus reads. Calculated for loci that have 400+ total reads. Plotted ratio of plus/minus reads in log2 scale. (E) Size distribution of sequences cloned from zili^+/+ and zili^−/− mutant gonads at 5 weeks post fertilization (wpf), showing a clear reduction in the number of piRNAs in the zili^−/− mutant. (F) Nucleotide frequencies at positions 1 and 10. Position 10^* indicates all piRNAs without uracil at position 1. When comparing piRNAs that do not have a 5′ uracil preference (^*) to all piRNAs from zili^+/+, an increased preference for adenosine at position 10 can be detected. In zili^−/− piRNAs, the preference for adenosine at position 10 has disappeared in piRNAs without a 5′-uracil preference (^*).

Figure 3

Zili is required for germ cell differentiation. (A) Relative expression of transposon transcripts compared with vasa transcripts in zili^+/+ and zili^−/− gonads at 5 wpf. Several LTR (GypsyDR2, LTR2, DIRS1a), non-LTR (Ngaro, L1) and DNA elements (Polinton, EnSpmN1) show an increased transcript level in zili^−/− gonads compared with wild-type siblings. Error bars show average±s.d. for three replicates. (B) Whole mount in situ hybridization of wild type and zili^−/− (G51STOP) gonads at 6 wpf (upper panel) and 7 wpf (lower panel), using an antisense probe for vasa. Loss of Zili leads to a strong reduction in the amount of vasa-positive cells (arrow), which at 8 wpf are completely lost. Scale bar is 100 μM. (C) Ziwi (red) protein is still present in germ cells of zili^−/− gonads (6 wpf). Ziwi protein displays a granular distribution, reminiscent of earlier-stage germ cells. Scale bar is 100 μM. (D) zili^+/+ germ cells (5 wpf) (upper panel), as well as zili^−/− germ cell (5 wpf) (lower panel), are positive for active RNA polymerase II (red). Scale bar is 10 μM.

Figure 4

Zili has a function in meiosis. (A) Relative expression of transposon transcripts compared with vasa transcripts in zili^L590P/− ovaries. Several LTR (GypsyDR2, LTR2, DIRS1a), non-LTR (Ngaro, L1) and DNA (Polinton, EnSpmN1) elements were tested and show no difference in zili^L590P/− ovaries compared with wild-type siblings. Error bars show average±s.d. for three replicates. (B) DAPI staining on embryos before fertilization, at 10 min post-fertilization (mpf) and 30 mpf show a meiotic arrest in zili^L590P/−. In wild-type embryos, meiosis I is completed immediately after egg-laying, and meiosis II is completed after sperm entrance (arrow indicates male pronucleus), and both polar bodies are extruded (1 asterisk is first polar body, 2 asterisk is second polar body). The male and female pronuclei fuse and form a two-cell embryo at 30 mpf. When the embryo is unfertilized, meiosis II is completed, although the DNA eventually starts breaking down. In eggs laid by zili^L590P/− females, the egg is fertilized (arrow), but meiosis I is not completed, a first polar body is not formed and the cell fails to divide. Scale bar is 25 μm. (C) After 15 mpf, the male and female pronuclei have fused and the second polar body (2 asterisk) is surrounded by tubulin. In eggs laid by zili^L590P/− females, tubulin surrounds part of the arrested female pronucleus, but no polar body is formed. Scale bar is 25 μm. (D) vasa mRNA in zili^L590P/− localizes normally to Balbiani body (asterisk), whereas vitellogenic oocytes (IV) display a peripheral position of the nucleus (arrow). Diagram shows quantification of nuclear position by ratio of the longest distance and shortest distance from the nuclear membrane to the cell membrane. Error bars are s.e.m. (t-test P<0.01).