Fig. 5

Dmrt1 is required for normal testis development and gonadal sex-differentiation. A-N) H&E staining of dmrt1^E3ins heterozygous and homozygous mutant siblings throughout development. Specimens are progeny from a dmrt1 homozygous female and heterozygous male of from the Pair 1 family of Fig. 3B. These crosses consistently produced progeny with a strong female bias in mutants. A-B) At 3 wpf, mutant gonads typically had fewer apoptotic cells compared to heterozygous siblings (white arrowhead). Immunohistochemistry to detect Cleaved caspase 3 positive cells revealed half the quantity of apoptotic cells in mutant gonads compared to heterozygotes (2.03% vs. 4.04%, respectively, P value <0.0001; N=16 gonads). C-F) 5 wpf heterozygotes progressed to early ovary (C) and testes (D) stages, as indicated by developing oocytes in females (oc), and loss of oocytes combined with lumen formation (asterisk) in males. Mutant gonads were either apparently normal ovaries (E) or testes with no lumens that were typically reduced in size compared to heterozygotes (F). G-J) At 7 wpf heterozygotes were fully committed female (G) and male (H) gonads, whereas mutants that were not clearly female formed either intersex gonads (J) (marked by dying oocytes alongside clustering spermatogonia (arrowhead)), or presumptive testes (not shown). The latter were never observed to form tubules or lumens, and had severely reduced spermatocytes. K-N) By 10 wpf heterozygotes were maturing with ova (K) or spermatids (L). The mutant males (N) were marked by increased stroma (black arrows), and lack of spermatogenesis, although clusters of germ cells were apparent (white arrow). E, I, M) All mutant females developed normally with no detectable phenotype. O) Quantification of the number of gonads in each histological category shown in panels A-N. The number of individuals, “N”, analyzed for each age group is shown in the table. Categories of gonad development observed in each age group reveal delayed transition to testes fate in mutants. Ovary=comprised mainly of oocytes of various stages; transitioning=one or more of the following: apoptotic cells, lumen formation, loss of oocytes, gonial cell clustering; intersex=containing maturing oocytes in addition to testis-like gonial clusters and stroma; testis=containing clusters of spermatogonia (or spermatocytes), having no oocytes, lumen if heterozygous. Apoptotic cells=white arrowhead; spermatogonia=black arrowhead; lumen=black asterisk; oocyte=oc; stroma=black arrow; germ cell cluster=white arrow; liver=L. Scale bar=50 µm.