nanos1 is required to maintain oocyte production in adult zebrafish

Abstract

Development of the germline requires the specification and survival of primordial germ cells (PGCs) in the embryo as well as the maintenance of gamete production during the reproductive life of the adult. These processes appear to be fundamental to all Metazoans, and some components of the genetic pathway regulating germ cell development and function are evolutionarily conserved. In both vertebrates and invertebrates, nanos-related genes, which encode RNA-binding zinc finger proteins, have been shown to play essential and conserved roles during germ cell formation. In Drosophila, maternally supplied nanos is required for survival of PGCs in the embryo, while in adults, nanos is required for the continued production of oocytes by maintaining germline stem cells self-renewal. In mice and zebrafish, nanos orthologs are required for PGC survival during embryogenesis, but a role in adults has not been explored. We show here that nanos1 in zebrafish is expressed in early stage oocytes in the adult female germline. We have identified a mutation in nanos1 using a reverse genetics method and show that young female nanos mutants contain oocytes, but fail to maintain oocyte production. This progressive loss of fertility in homozygous females is not a phenotype that has been described previously in the zebrafish and underlines the value of a reverse genetics approach in this model system.

Fig. 1

nanos1 is expressed in adult female germ cells. RNA in situ hybridization reveals that at 21 dpf (A and B) and 35 dpf (C and D). nos1 expression (A and C) appears to be restricted to perinuclear stage oocytes (arrows) as compared to vasa (B and D) that appears to be expressed in both perinuclear stage oocytes (p.o.) as well as germ cells that are less than 20μm in diameter (arrow in D). (E) RNA in situ hybridization of a transverse section through a 2 month old female zebrafish reveals that nos1 is expressed at high levels in early stage oocytes (oriented dorsal up). In stage Ib oocytes, nos1 transcripts are enriched in a spherical cytoplasmic structure that resembles the Balbiani body (arrow). (F) In adult ovaries, vasa expression reveals that early stage oocytes localize to a ventral zone on the ovarian surface (arrow; ovary is oriented with anterior to the left and dorsal up). (H) Higher magnification of region boxed in (F) reveals an overall organization of early stage oocytes within this ventral zone, with stage Ia oocytes located dorsal to stage Ib oocytes. Similar to nos1, vasa RNA is also enriched in spherical cytoplasmic structures within stage Ib oocytes (arrow). (G) nos1 is highly expressed in stage Ib oocytes, but is either not expressed in clusters of stage Ia oocytes that localize to the ventral zone, or is expressed at levels below our ability to detect (Compare G and H). nos1 expression can not be detected in testes by in situ hybridization (I) while vasa expression can be readily detected in developing spermatocytes (J). (K) RT-PCR analysis confirms that nos1 expression in adults is female specific. ornithine decarboxylase (odc) expression analysis is included as a positive control. (L) A confocal image of an ovary double-labeled for Vasa protein (green) and nos1 RNA (red) show that nos1 and Vasa co-localize in early stage oocytes (Ib) but not in the smallest (<20μm) Vasa-positive germ cells (arrow). Scale bars: 50 μm in A-D, L; 200 μm in E, I and J; 1 mm in F; 100 μm in E and F.

Fig. 2

The nos1(fh49) mutant allele encodes a truncated form of the protein. (A) Sequence traces of nos1 from wild-type (top) and nos1(fh49) heterozygotes revels that nos1(fh49) is a point mutation that converts the Leu(110) codon into a stop codon. The mutation disrupts an MseI restriction enzyme site present in wild-type. (B) Schematic of the predicted wild-type and mutant Nos1 protein structures. The CCHC zinc finger RNA binding domains are shown in yellow. (C) PCR-based genotyping assay utilizing the MseI restriction site polymorphism. nos1(fh49) heterozygotes have both wild-type (lower) and mutant (upper) bands.

Fig. 3

Maternal nos1 is required for primordial germ cell survival. (A) Numbers of germ cells in embryos at the stages identified on the X axis as assayed by in situ hybridization using a vasa probe. Shield and 14-somite stage embryos were derived from a cross between nos1^−/− females and nos1^+/+ males, while 17-somite stage embryos were derived from a cross between nos1^−/− parents. Standard deviations and numbers of embryos analyzed for each class are indicated. In legend, Mnos1⁻ indicates embryos derived from nos1^−/− females. PGC localization in representative shield-stage embryos derived from nos1^+/+ females (B) or nos1^−/− females (C) as reveled by vasa in situ hybridization (blue). PGC localization in representative 17-somite stage embryos stained for myod and egr2b in red, and vasa in blue, derived from nos1^+/+ females (D),nos1^−/− females (E) or nos1^−/− females injected with 200 pg synthetic nos1 mRNA (F). Arrows point to correctly localized vasa expressing PGCs, while arrowheads indicate ectopically localized PGC’s. Germ cell development in Mnos1 embryos can be rescued by injection of nos1(wt) RNA(G; n=12/12) but not nos1(fh49) mutant RNA (H; n=0/23) at the one cell-stage, as assayed in a 24 hpf embryo. Arrow in G points to EGFP expressing germ cells (see materials and methods). Scale bars: 100μm.

Fig. 4

Zygotic nos1 is required for adult germline maintenance. Histology of 6 month old females (A and B) and male (C and D) gonads. (A) Wild-type ovaries contain oocytes (stages indicated). (B) nos1 mutant females do not contain oocytes. The testes of wild-type (C) and nos1 mutants (D) are indistinguishable. 3 month-old wild-type (E, G and I), and nos1 mutant (F, H and J) ovaries freshly isolated (E and F), or stained for vasa (G and H) or ziwi (I and J) RNA. Wild-type ovaries contain numerous stage I oocytes, visualized as small (7−140μm diameter) clear cells on the surface of the ovary (E) or as small vasa RNA⁺ (G) and ziwi RNA⁺ (I) cells (arrows). In contrast, nos1 mutant ovaries contain very few stage I oocytes (F, H, J; arrows in H and J). RNA in situ hybridization to detect vasa RNA reveals that 35 dpf nos1 mutant ovaries (L) contain fewer <20 μm germ cells (arrow) than wild-type ovaries (K). sg, spermatogonia; sp, sperm; l, liver. Scale bars: 100 μm in A-D, K, L; 200μm in E-J.