Fig. 3. Distribution of the newly-born Ngn3- and Kit-positive spermatogonia, and the seminiferous epithelial cycle prepattern. (A-H) Double-staining ISH of testes sections at the indicated ages with the indicated genes. Overlaid images of Kit or Ngn3 (purple, bright field) and galectin 1 (red fluorescence) are shown in A,C,E,G; galectin 1 signals in the same field are shown in B,D,F,H. Arrows in A and C indicate the Kit- and Ngn3-positive spermatogonia, respectively. E,F and G,H are adjacent sections. Note the variable intensities of galectin 1 signals within the seminiferous tubule cross-sections and their relationship with Kit- or Ngn3-positive spermatogonia (see Results). Asterisks (E-H) represent typical segments with a high galectin 1 mRNA signal; Kit-positive, but not Ngn3-positive, spermatogonia preferentially localize to these segments. Segments marked by hearts show lower galectin 1 signals and exhibit a preference for Ngn3-positive spermatogonia. Scale bars: 100 µm, in D for A-D, in H for E-H. (I) Quantification of Kit- or Ngn3-positive spermatogonia localized in tubule segments categorized by different levels of galectin 1 mRNA (high, medium or low). Tubule cross-sections marked with H, M or L in B and D are examples of segments with high, medium and low levels of galectin 1 mRNA, respectively. Data are represented as `preference' to each category of tubule segment. a and b represent significant deviation with P-values of <0.0001 and <0.05, respectively. See Materials and methods and Tables S2-S7 in the supplementary material. The number of Ngn3-positive spermatogonia at P3 and P4 was too low for statistical analyses. (J) Model for the generation of spermatogonia subpopulations. One area of seminiferous tubules is represented to align tubules with increasing age. The galectin 1 mRNA level is shown by the gradient: black, highest; white, lowest. As galectin 1 expression increases and reduces in cycles (black to gray to white to black, etc.), its expression domain shifts leftward in a wave-like manner. Kit-positive spermatogonia (red ovals) appear specifically at the galectin 1-high segments (red bands). By contrast, Ngn3-positive spermatogonia (green ovals) are generated separately from Kit-positive spermatogonia around the segments of medium level of galectin 1 expression (green bands).