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First published online 30 November 2006
doi: 10.1242/dev.02697

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Functional redundancy among Nanos proteins and a distinct role of Nanos2 during male germ cell development

Atsushi Suzuki^1,*, Masayuki Tsuda^2,3,*, and Yumiko Saga^1,2,

¹ Department of Genetics, National Institute of Genetics, SOKENDAI, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
² Division of Mammalian Development, National Institute of Genetics, SOKENDAI, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
³ Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, 2-6-15, Shibakoen, Minato-ku, Tokyo 105-0011, Japan.

View larger version (33K): [in this window] [in a new window]   Fig. 1. Expression profile of Nanos proteins. (A) Characterization of Nanos2 and Nanos3 antibodies. Cell extracts were prepared from E14.5 and E12.5 male gonads, resolved by SDS-PAGE and subjected to western blot analysis with Nanos2 and Nanos3 antibodies, respectively. Arrows indicate the endogenous proteins. (B) Expression profile of Nanos proteins in embryonic gonads. Extracts were prepared from each single male embryonic gonad of wild-type and Nanos2-/- littermates. One-half of these extracts were resolved by SDS-PAGE and subjected to western blot analysis with Nanos2 and Nanos3 antibodies. Actin was used as a reference for protein amounts. (C) Analysis of Nanos mRNA and protein expression. Total RNA and protein extracts were prepared from E14.5 male gonads of wild-type and Nanos2-/- littermates. Expression of Nanos2 and Nanos3 mRNA was analyzed by RT-PCR. G3PDH was used as the internal standard for normalization. The expression levels of the Nanos2 and Nanos3 proteins were analyzed by western blotting as in B.

View larger version (40K): [in this window] [in a new window]   Fig. 2. Ectopic expression of a FLAG-tagged Nanos2 protein driven by the Oct4PE enhancer can rescue the defects in Nanos2-null testes. (A) Western blot analysis of Nanos2 from both wild-type and transgenic mouse lines expressing a FLAG-tagged Nanos2 under the control of the oct4PE enhancer. Extracts were prepared from E14.5 male gonads and actin was used as the loading control. The level of endogenous Nanos2 was decreased in the transgenic mice. The transgene construct is schematically depicted. (B) Comparison of testis size among littermates of Nanos2+/-, Nanos2-/- and Nanos2-/- mice expressing the FLAG-tagged Nanos2 transgene background (line #5). (C-E) Sections were prepared from the testes shown in B and stained with hematoxylin and Eosin. The genotypes are indicated in each panel. (E) Some empty (asterisk) or smaller tubules were observed in the rescued testis. (F) Comparison of testis weights among different genotypes at 8 weeks. The testis weight of Nanos2+/- was designated as 100%. Scale bar: 100 µm in D for C-E.

View larger version (146K): [in this window] [in a new window]   Fig. 3. Nanos2 can rescue the postnatel defects in Nanos3-/- testes and ovaries. Testis samples were prepared from littermates of Nanos3+/+, Nanos3+/- or Nanos3-/-, or from littermates of Nanos3-/- expressing the FLAG-tagged Nanos2 transgene, at 2 weeks (A-D) and 6 weeks (E-H) after birth. The testis weights of different genotypes were compared at 6 weeks (I). The testis weight of Nanos3+/- was designated as 100%. Ovary samples were prepared from Nanos3+/- (J), Nanos3-/- (K) and Nanos3-/- harboring the Nanos2 transgene (L) at 6-7 weeks after birth. The genotypes are indicated in each panel. Germ cells were detected by immunostaining with TRA104 (B-D) and TRA98 (F-H) antibodies. Scale bars: 100 µm in C for B-D; 100 µm in G for F-H; and 100 µm in K for J-L.

View larger version (102K): [in this window] [in a new window]   Fig. 4. Nanos2 can partially rescue the Nanos3-/- defects in the embryonic gonad. (A-D) Detection of migrating PGCs by ALP staining in E11.5 gonads from littermates of the Nanos3+/+ (A), Nanos3-/- (B) and Nanos3-/- expressing the Nanos2 transgene (C,D) backgrounds. (E-G) Sections were prepared from the male gonads of E14.5 Nanos3+/+ (E), Nanos3-/- (F) and Nanos3-/- expressing the Nanos2 transgene (G) backgrounds. Each section was then stained with TRA104 antibody. (H-J) Sections were prepared from the gonads of E16.5 females of Nanos3+/- (H), Nanos3-/- (I) and Nanos3-/- expressing the Nanos2 transgene (J) and stained with anti-TRA98 antibody. Signals are observed in the nuclei of germ cells. Blue, DAPI staining. Scale bars: 100 µm in E for E-G; and 100 µm in H for H-J.

View larger version (57K): [in this window] [in a new window]   Fig. 5. Nanos3 cannot rescue the Nanos2-/- defects. (A) Western blot analysis of Nanos3 from Nanos2+/- and from Nanos2+/- mouse lines with or without a FLAG-tagged Nanos3 transgene under the control of the Nanos2 enhancer. Extracts were prepared from E14.5 male gonads. The transgene construct is schematically depicted. (B-G) Immunostaining and detection of germ cells with TRA104 antibody. Sections were prepared from E16.5 gonads (B-D) and testes at 4 weeks after birth (E-G) from Nanos2+/- (B,E), Nanos2-/- (C,F) and Nanos2-/- expressing the FLAG-tagged Nanos3 transgene (D,G) male littermates. Scale bars: 100 µm in C for B-D; and 100 µm in F for E-G.