Mutations disrupting the adad1 gene cause germ cell loss in adult zebrafish.

A: Graphical representation of Slc34a1a and Adad1 wild-type and mutant proteins. Missense mutations are shown in red below wild-type protein schematics in the approximate position they occur in the protein. Representative images of truncated proteins resulting from nonsense mutations are shown. The frameshifted region of slc34a1a^umb10 is colored orange. Numbers denote the number of amino acids in each protein. Abbreviations: Na-Pi cotrans (sodium dependent phosphate co-transporter domain), dsRBM (double-stranded RNA binding motif), WT (wild type). B-E: Hematoxylin-eosin staining of testes from adult wild type (B), t30103 homozygous mutant (C), slc34a1a^umb10 trans-heterozygous to t30103 (D), and adad1^Y67X trans-heterozygous to t30103 (E). In the wild-type testis spermatogonia, spermatocytes, and mature sperm are marked by red, blue, and yellow outlines, respectively. The t30103 homozygous and adad1^Y67X/t30103 trans-heterozygous testes are small and lack germ cells (red dotted outlines in C and E). Conversely, there is no visible difference between wild-type and slc34a1a^umb10/t30103 trans-heterozygous testes. Scale bar: 50 μm.

Adad1 mRNA is expressed in both sexes and is germline specific.

A-D: RT-PCR assaying adad1 expression in wild-type tissues. RT-PCR on adult tissues demonstrates that adad1 is expressed only in ovary and testis (A), whereas the control gene rpl13a is expressed in all organs tested (B). During embryonic and larval development adad1 is expressed in all stages tested (C), similar to the germ cell marker piwil1 (D). E: RT-PCR on testes lacking germ cells revealed that adad1 is not expressed in somatic cells of the testis. Piwil1 expression was not detected confirming that germ cells are not present while the Leydig cell marker gene cyp11c1 confirmed the presence of testes tissue. F-K: In situ hybridization (ISH) and single cell RNA sequencing data showing adad1 is expressed in germ cells of adult ovaries and testes. (F-H) In testes adad1 expression was highest in spermatogonia, spermatocytes, and less expressed in early spermatids. Testes were counterstained with periodic acid-Schiff. (I-K) In ovaries adad1 expression was found in oogonia through early follicle stages. Scale bars: 20 μm for testes and 50 μm for ovaries. Abbreviations: SPG1 = spermatogonia cluster 1; SPG2 = spermatogonia cluster 2; SPC = spermatocytes; E_Round = early round spermatids; M_Round = middle round spermatids; L_Round = late round spermatids; Elongated = elongated spermatids; GSC_Pro = germline stem cell and progenitor cells; E_Meio = early meiosis, Meio = Meiosis, L_Meiosis = Late meiosis; E_Oo1 = early follicle stage oocyte cluster 1; E_Oo2 = early follicle stage oocyte cluster 2; E_Oo3 = early follicle stage oocyte cluster 3; Follicle_Lhx9 = Lx9-expressing follicle cells; Follicle1 = follicle cells cluster 1; Follicle2 = Follicle cells cluster 2; Theca = Theca cells.

Adad1 protein expression in adult ovaries and testes.

A-H: In the ovary, Adad1 is expressed in the cytoplasm of prefollicle stages through stage II oocytes (A and E) similar to the Ddx4/Vasa protein (B and G). Similar levels of Adad1 were seen in nuclei and cytoplasm of pre-follicle stage germ cells (E-H, arrows). Images in panels A-D and E-H are from different samples I-L: In the testis, Adad1is expressed in the cytoplasm of spermatogonia and spermatocytes but not in mature sperm (I), overlapping with Ddx4/Vasa expression (J). Magnification of the insets shows that Adad1 is both nuclear and cytoplasmic in single spermatogonia and spermatogonia in cysts of 2–4 cells (M-P, arrows). Scale bars: 200 μm for A-D and I-L, 20 μm for E-H.

Histology of wild-type and adad1^Y67X mutant gonads at 45, 60, and 90 dpf.

A-B: At 45 dpf, germ cells are present in both the wild-type (N = 8) (A) and mutant (N = 8) (B) testes. C-D: At 60 dpf, spermatogonia (Sg, red outline) and spermatocytes (Sc, blue outline) are present in both wild-type (N = 10) (C) and mutant (D), but the mutant lacks mature sperm (N = 10) (Sp, yellow outline). E-F: At 90 dpf, the wild-type testis is larger with abundant sperm (N = 6) (E) whereas the mutant testis is small and devoid of germ cells (N = 6) (F). Dotted red lines outline the gonads in A, B and F. Scale bar: 50 μm.

Hematoxylin-eosin staining on 28, 35, and 42 dpf wild-type and mutant gonads.

A-B: At 28 dpf, gonads are undifferentiated in wild-type (A) and adad1^Y67X mutant (B) fish. C-H: At 35 and 42 dpf, wild-type fish were developing as either female (C, F) or male (D, G), whereas mutant fish were developing only as males (E, H). Gonads are marked by dotted red outlines in all images. Scale bar: 50 μm.

Immunostaining with meiotic marker Sycp3.

A-F: Sycp3 immunofluorescence (green) and DAPI nuclear stain (white in B, E and blue in C, F) on 60 dpf wild-type (A-C) and adad^Y67X testis (D-F) showed the presence of leptotene (red dotted outline), early zygotene or bouquet (white dotted outline), and late zygotene/pachytene (yellow dotted outline) stage cells in both wild-type (A-C) and adad1 mutant (D-F) testes. N = 9 for each genotype. Arrows point to sperm. Scale bar: 20 μm.

Telomere and synaptonemal complex protein labelling on meiotic chromosome spreads.

A-T: Wild-type chromosome spreads have leptotene (A-E), zygotene (F-O), and pachytene (P-T) stage cells. A’-J’: Chromosome spreads from adad1^Y67X testes lack pachytene stage cells. Telo-FISH (white), IF for Sycp1 (magenta) and Sycp3 (green), and DAPI nuclear staining (blue). Scale bar: 10 μm.

Apoptosis and proliferation are not affected in adad1 mutants.

A-F: Cell death assays in 60 dpf adad1^Y67X wild-type and mutant testes. Neither TUNEL assays (A,B,E) nor cleaved-Caspase 3 immunofluorescence (B,D,F) showed a significant difference between wild-type and mutant testes. Arrowheads indicate examples of TUNEL and cleaved-Caspase3 positive cells. G-P: Cell proliferation assay in 45 dpf testes using BrdU labeling. Fish were treated overnight with BrdU then waited for 6 hours without BrdU prior to fixation (G). Immunostaining was done with BrdU, Ddx4/Vasa, and Sycp3 antibodies to detect proliferating spermatogonia. Spermatogonia were identified as Ddx4/Vasa positive and Sycp3 negative (arrowheads, H-O). Quantification of the percentage of BrdU positive spermatogonia showed no significant difference between wild-types and mutants (P). SG = spermatogonia. Scale bar: 20 μm.

Label retaining cell assays (LRC) to identify spermatogonial stem cells.

A: 32 dpf fish were treated with BrdU three times for about 16 hours per treatment (overnight) followed by a 25 day chase where fish were in water with no BrdU. At the end of the chase period, fish were fixed and processed for BrdU detection. B-F: Wild-type testes all had LRCs (B-C, arrows) with median percentage of 9.34% out of the total spermatogonia (N = 8 fish) (F). Four out of eight mutant (adad1^Y67X) testes had no LRCs (D-E) and the median percentage of LRCs of all mutant samples was less than the wild-types (F). G-I: Fluorescent in situ hybridization for nanos2 in wild-type and adad2^Y67X mutant testis. Wild-type testes had cells with bright (solid arrowhead) and faint (outlined arrowhead) nanos2 label (N = 6) (G and I). Mutant testes had either no visible nanos2 labeled cells (N = 3/6) (H and I) or had only faint nanos2 labeled cells (N = 3/6) (I). Scale bar: 20 μm.

Bulk RNAseq from wild-type and mutant testes.

A: Venn diagrams showing numbers of differentially expressed genes in each mutant line compared to their wild-type siblings using fold change >1.5 and false discovery rate (FDR) <0.05 as the cutoff. B: Gene ontogeny pathway analysis performed on genes that were dysregulated in both adad1 alleles showing down- and up-regulated pathways in the mutant testes. C-D: Dot plot analysis of adult testes scRNAseq data shows expression pattern of genes listed in Tables 2(C) and 3(D).

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