Histological evidence of disrupted testis cords in Dax1-deficient testis. Histological comparison of Dax1-deficient (B,D,F,H,J) and wild-type (A,C,E,G,I) testes taken at 11.5 dpc (A,B), 12.5 dpc (C,D), 13.5 dpc (E,F), 14.5 dpc (G,H) and 17.5 dpc (I,J). Although development of the testis cords is readily discerned in wild-type testis, little organization is present in Dax1-deficient testis. Asterisks identify testis cords; arrows indicate peritubular myoid cells.

The basal lamina is disrupted in Dax1 deficient testis at 12.5, 13.5 and 14.5 dpc. Immunohistochemistry with a laminin antibody reveals that laminin deposition clearly separates the testis cords from the peritubular space in wild-type testis (A,C,E); the basal lamina is disrupted or absent in Dax1-deficient testes (B,D,F).

Disorganization of Sertoli cells in Dax1-deficient testis. In situ hybridization for Dhh (A,B), Amh (C,D) or Sox9 (E-H) reveals robust expression confined to the cords in wild-type testis, whereas expression is decreased and not clearly organized in Dax1-deficient testis. In situ hybridization for Dhh and Amh was performed at 12.5 dpc, and Sox9 was analyzed at 12.5 and 14.5 dpc. (I,J) Immunohistochemistry for Gata4 (green) and laminin (red) reveals decreased organization and differentiation of Sertoli cells at 13.5 dpc in Dax1-deficient testis (J) compared with wild-type males (I). Left panel, wild-type testis (A,C,E,G,I); right panel, Dax1-deficient testis (B,D,F,H,J).

Leydig cells in Dax1-deficient are restricted to the ventral surface. In situ hybridization for Scc reveals that while Leydig cells are present on the ventral surface of Dax1-deficient gonads (B,D) similar to wild-type males (A,C), but decreased in number and organization. Lateral examination (D) reveals that Leydig cells do not extend throughout the full ventral to dorsal thickness of the Dax1-deficient testis (arrow). Immunohistochemistry for Sf1 demonstrates that Sf1-positive Leydig cells are restricted to the coelomic surface in the Dax1-deficient testis (above the broken line, F) compared with the wild-type testis (E). Arrows identify the columns of Leydig cells in wild-type testis (E).

Dividing peritubular myoid cells are reduced in the Dax1-deficient testis. TUNEL staining of the wild-type (A) and Dax1-deficient (B) testis at 13.5 dpc. (C) Cell counts taken from gonads at 13.5 dpc. (D,E) BrdU labeling of dividing cells (brown precipitate) in wild-type (D) and Dax1-deficient (E) testis at 13.5 dpc. (F) Cell counts included flat, curved epithelial cells (arrows, inset in D) in eight wild-type and ten Dax1 knockout gonads (^*P≤0.001).