- Title
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The RNA-binding protein Adad1 is necessary for germ cell maintenance and meiosis in zebrafish
- Authors
- Islam, K.N., Ajao, A., Venkataramani, K., Rivera, J., Pathania, S., Henke, K., Siegfried, K.R.
- Source
- Full text @ PLoS Genet.
Mutations disrupting the 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 PHENOTYPE:
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A-D: RT-PCR assaying |
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. EXPRESSION / LABELING:
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Histology of wild-type and 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. PHENOTYPE:
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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 PHENOTYPE:
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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 |
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 PHENOTYPE:
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Apoptosis and proliferation are not affected in A-F: Cell death assays in 60 dpf PHENOTYPE:
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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 ( |
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 |