Fig. 5. Chase of cells of Ngn3⁺ and Ngn3^- lineages during the maturation of testes using Ngn3/Cre;CAG-CAT-Z double-transgenic mice. (A) Experimental design. In Ngn3-positive cells of Ngn3/Cre;CAG-CAT-Z mice, the reporter gene is irreversibly recombined between loxP sites by Cre recombinase driven by the Ngn3 regulatory sequence, thereby labeling their progenies with ß-gal (encoded by lacZ), while CAT expression is lost (Ngn3⁺ lineage). The reporter gene remains intact in cells that have never expressed Ngn3; these cells express CAT, but not lacZ (Ngn3^- lineage). (B-D) Serial sections of P3 testes probed for Ngn3, Cre and lacZ, representing the overlapping expression of these genes in a single seminiferous tubule (arrowheads). (E,F) A pair of adjacent sections of a P5 testis, which were hybridized for Kit and lacZ expression. The Kit-positive spermatogonia apparently outnumber the lacZ-positive ones. (G-I) P21 testis probed for CAT. H shows a higher magnification of a part of G; I shows a PI-stained fluorescence image of H. CAT signals are preferentially detected at the center of the seminiferous tubules (arrowheads). Arrows indicate CAT signals in the interstitial cells. (J-L) P28 testis probed for CAT and lacZ. (K) Higher magnification of a part of J; (L) the same location but in the next section. CAT-positive and lacZ-negative (Ngn3^- lineage) spermatogenic cells are preferentially found in the innermost layer (arrowheads). Seminiferous tubules marked 1-3 are examples of the different degrees of maturation (see Results). (M,N) P56 testis probed for CAT and lacZ. All germ cells are CAT negative and lacZ positive. Arrows indicate somatic cells positive for CAT (Sertoli and interstitial cells). Scale bars: 100 µm.