Zebrafish vasa is required for germ-cell differentiation and maintenance

Abstract

Vasa is a universal marker of the germ line in animals, yet mutations disrupting vasa cause sexually dimorphic infertility, with impaired development of the ovary in some animals and the testis in others. The basis for this sexually dimorphic requirement for Vasa is not clear; in most animals examined, both the male and female gonad express vasa throughout the life of the germ line. Here we characterized a loss-of-function mutation disrupting zebrafish vasa. We show that maternally provided Vasa is stable through the first ten days of development in zebrafish, and thus likely fulfills any early roles for Vasa during germ-line specification, migration, survival, and maintenance. Although zygotic Vasa is not essential for the development of juvenile gonads, vasa mutants develop exclusively as sterile males. Furthermore, phenotypes of vasa;p53 compound mutants are indistinguishable from those of vasa mutants, therefore the failure of vasa mutants to differentiate as females and to support germ-cell development in the testis is not due to p53-mediated apoptosis. Instead, we found that failure to progress beyond the pachytene stage of meiosis causes the loss of germ-line stem cells, leaving empty somatic tubules. Our studies provide insight into the function of zebrafish vasa during female meiosis, differentiation, and maintenance of germ-line stem cells.

Figure 1. The vasa^sa6158 allele results in a truncated protein lacking helicase domains

(A) Diagram of zebrafish Vasa protein structure, with the vasa^sa6158 allele resulting in a truncated protein lacking annotated Q-motif, ATP-binding motif, and helicase domains. (B) Seven annotated zebrafish vasa transcript variants are illustrated. Alternatively spliced exons are indicated in yellow, and the conserved mutated exon in the vasa^sa6158 allele is indicated in red. (C) Chromatography data from wild-type and vasa^sa6158-mutant genomic DNA. The arrow indicates the T-to-A vasa^sa6158 mutation, whereas the arrowhead points to the nucleotide mutation (G-to-C) inserted by the dCAPs primer to create a RsaI site. (D) The dCAPs PCR-based genotyping assay utilizes the RsaI restriction site in the vasa^sa6158 allele. Heterozygotes have both wild-type (upper) and mutant (lower) bands.

Figure 2. Maternal Vasa is stable throughout early germ-line and gonad development

(A, B) Lateral view (rostral to the left) of Vasa staining in germ cells in 10 d.p.f. larvae. The swim bladder (SB) is outlined in white. (C, D) Vasa staining in 12 d.p.f. larvae. Yellow outline marks the gut. (E, F) Vasa protein in 14 d.p.f. larvae. The blue arrowheads point to germ cells. (G) Table of embryonic and larval stages examined for Vasa staining in germ cells. (H, I) In situ hybridization for the germ-cell marker nanos3 expressed in germ cells of the shield-stage (H) and 30-hours-post-fertilization (I) embryos from vasa^sa6158/+ in-crosses. Dorsal is oriented toward the top in both panels. Scale bars, 100 μm (A–F, H–I).

Figure 3. vasa ^sa6158-mutant testes lack germ cells

(A–E) Lateral views (rostral to the left) show overall morphology of wild-type (A, D), vasa^sa6158 heterozygote (B, E), and mutant fish (C). (A′-E′) Lateral views (rostral to the left) of dissections reveal visible gonads in situ, outlined in white, in wild-type (A′, D′) and vasa^sa6158 heterozygote (B′, E′) fish, but not vasa^sa6158 mutants (C′). SB, swim bladder; L, liver. (A′′, B″) H&E-stained sections of wild-type and vasa^sa6158-heterozygous testes with numerous visible spermatocytes (SC) and spermatozoa (SZ) within tubules (outlined in yellow). (C″) vasa ^sa6158-mutant testis tubules have no detectable germ cells. (D″, E″) Oogenesis is normal in wild-type (D″) and vasa^sa6158-heterozygous females (E″). (F) Side-by-side comparison of dissected wild-type (top) and vasa^sa6158-mutant (bottom) testis. (G) RT-PCR of dissected gonads from adult wild-type, vasa^sa6158-heterozygote, and mutant fish. Scale bars, 0.1 cm (A′–E′); 10 μm (A″–E″, F).

Figure 4. Zygotic vasa is not required for the formation of the bipotential gonad

(A, B) Lateral view (rostral to the left) of d21 Tg[ziwi:GFP]-positive juveniles taken through the body wall. (C, D) Meiotic nuclei stained with DAPI. P, pachytene. (E) RT-PCR on samples from individual d21 juveniles. (F– F″, G–G″) Germ-cell (GC) clusters of d21 Tg[ziwi:GFP];vasa ^sa6158/+ (F–F″) and Tg[ziwi:GFP];vasa ^{sa6158/sa6158} (G–G″) gonads. (H) Quantification of germ-cell cluster size in d21 juveniles. Scale bars, 200 μm (A, B); 20 μm (C, D, F–F″ and G–G″).

Figure 5. Caspase-3 staining in d28 vasa^sa6158 mutants and siblings

(A, B) Lateral view (rostral to the left) of d28 Tg[ziwi:GFP]-positive juveniles taken through the body wall. (C–E) Tg[ziwi:GFP];vasa^sa6158/+ and Tg[ziwi:GFP];vasa^{sa6158/sa6158} gonads stained for GFP and active (cleaved) Caspase-3. (F) Intestinal tissue stained for GFP and active Caspase-3 served as a positive control for Caspase-3. Scale bars, 250 μm (A, B); 100 μm (C–F). Lateral views of organs (C–E) are oriented with the rostral-caudal axis aligned from top to bottom.

Figure 6. Zygotic vasa is required for meiotic progression, germ-cell differentiation, and germ-cell cluster development

(A– A″) Meiotic nuclei and of Tg[ziwi:GFP];vasa ^sa6158/+ d28 female. P, pachytene; D, diplotene. (B–B″) Meiotic nuclei and germ-cell (GC) clusters of a Tg[ziwi:GFP];vasa ^sa6158/+ d28 male. (C–C″) Meiotic nuclei and GC clusters of a Tg[ziwi:GFP];vasa ^{sa6158/sa6158} d28 male. Red arrowheads point to germ cells with a vacuolar phenotype. (D) Quantification of germ-cell cluster size in d28 juveniles. Error bars indicate the 95% confidence interval. (E) RT-PCR on samples from individual d28 juveniles. Scale bars, 20 μm (A–C).

Figure 7. Concurrent loss of tp53 does not rescue germ-cell loss in vasa^sa6158 mutants

(A–C) Transverse sections through the whole trunk of wild-type (A), vasa^{sa6158/sa6158} (B), and vasa^{sa6158/sa6158};tp53^M214K/M214K (C) fish identify the testis in situ (indicated by blue arrowheads). Dorsal is to the top. (A′, B′, C′) Higher magnification images of testes reveal that both vasa^{sa6158/sa6158} (B′) and vasa^{sa6158/sa6158};tp53^M214K/M214K (C′) contain no germ cells within the somatic tubules. (D, G) Wild-type differentiated gonads of d42 female (D) and male (G) fish. (E) d42 vasa^{sa6158/sa6158} gonad. (F) d42 vasa^{sa6158/sa6158};tp53^M214K/M214K gonad. (H, I) D42 vasa^{sa6158/sa6158} (H) and vasa^{sa6158/sa6158};tp53^M214K/M214K (I) gonads both show evidence of lymphocytic infiltration (indicated by black arrowheads). Scale bars, 0.1 cm (A–C); 10 μm (A′–I),

Figure 8. Proposed model for vasa function during gonad differentiation

(A) In normal zebrafish gonadogenesis, the bipotential gonad forms with numerous stage-Ia oocytes in meiotic prophase I (zygotene and pachytene) and rare stage-Ib oocytes (meiosis I arrested at the diplotene stage). As sexual differentiation takes place, oogenesis can either be sustained, resulting in female sexual development, or oocytes will undergo apoptosis and be replaced by spermatagonia, resulting in male sexual development. (B) We propose that in vasa^sa6158 mutants, stage-Ia oocytes are unable to progress to stage Ib of oogenesis. As differentiation proceeds, pachytene-stage oocytes display a vacuolated phenotype and are lost by p53-independent mechanisms. Although they are specified in the bipotential gonad, germ-line stem cells (GSCs) are not maintained in vasa mutants, ultimately resulting in an empty testis devoid of germ cells and sterility.