Figure 1.

The Nanos2^TAG mouse allele is functional

(A) Schematic representation of the NANOS2 protein and the TAG-NANOS2 fusion protein, with corresponding lengths of amino acid sequences (aa).

(B) Number of litters per animal from Nanos2^CTL(Nanos2^+/+) and Nanos2^TAG (Nanos2^TAG/TAG) mice. Data are mean and s.d., n = 6 for both genotypes. n.s. indicates not significant, p value > 0.05 using two-tailed Student's t-test.

(C) Testicular weight of Nanos2^CTL and Nanos2^TAG from six-months-old mice. Data are mean and s.d., n = 5 for both genotypes. Statistical evaluation as in panel b.

(D) Representative testis cross-sections stained with hematoxylin and eosin (H&E) from six-months-old Nanos2^CTL and Nanos2^TAG mice. Scale bar, 100 μm.

(E) Representative immunofluorescent images of Nanos2^TAG seminiferous tubules stained with anti-GFP (green) and anti-GFRα1 (red) antibodies. Representative examples of GFRα1^Pos, NANOS2^Pos cells (A_s and A_pr) are highlighted (white boxes). Scale bar, 50 μm.

(F) Enumeration of GFRα1^Pos NANOS2^Pos and GFRα1^Neg NANOS2^Pos testicular populations. The number of cells (N) present in each cluster is normalized to 1,000 Sertoli cells (y axis). Error bars represent SEM (standard error of mean).

(G) Representative immunofluorescent images of Nanos2^TAG seminiferous tubules stained with anti-GFP (green) and anti-c-KIT (red) antibodies. Representative example of NANOS2^Pos, c-KIT^Neg cell (A_s) is highlighted (white box). Scale bar, 50 μm.

(H) Representative immunofluorescent image of an A_s cell from Nanos2^TAG seminiferous tubules stained with with DAPI (blue), anti-GFP antibody (green) and anti-DCP1a (red). Scale bar, 5 μm.

(I) Representative bright field images of Nanos2^CTL and Nanos2^TAG SSC lines cultured on mouse embryonic fibroblasts (MEF) feeders.

(J) Western blot using anti-GFP and anti-Actin antibodies on Nanos2^CTL and Nanos2^TAG SSC lines lysates.

(K) Flow cytometry analysis of Nanos2^CTL and Nanos2^TAG SSC lines. The GFP intensity of the Nanos2^TAG population is represented in a single-parameter histogram, with the overlay of the Nanos2^CTL control.