Fig. 4

Gonads of fancl mutants have germ cells but fail to maintain a female gene expression profile.

Comparative expression analysis of the germ cell marker vasa, the early female somatic cell marker cyp19a1a, and the early male somatic cell marker amh in developing gonads of fancl homozygous mutants (fancl) and their wild-type sibling controls (WT), and in wild-type animals depleted of germ cells by dead end morpholino knockdown (dnd). To monitor the expression of vasa, cyp19a1a and amh, in situ hybridization (ISH) experiments were performed on adjacent cryo-sections of each animal at different stages of gonad development: undifferentiated gonads at 19 dpf (A–I), transitional juvenile gonads at 26 dpf (J–X) and post-transitional juvenile gonads at 33 dpf (Y-M′). Arrows point to examples of regions showing gene expression. ISH with vasa probe labeled germ cells in wild types (A,J,M,Y,B′) and fancl mutants (D,P,S,E′,H′), and confirmed the depletion of germ cells in dnd animals (G,V,K′). In undifferentiated gonads at 19 dpf, female and male markers were expressed in all genotypes: WT (B,C), fancl (E,F) and dnd knockdown animals (H,I), showing that the onset of cyp19a1a and amh expression does not depend on germ cells or on fancl function. At 26 dpf, controls had started to enter either the male pathway by down-regulating cyp19a1a and up-regulating amh (K,L) or conversely into the female pathway by up-regulating cyp19a1a and down-regulating amh (N,O), correlated with the presence of few or many oocytes, respectively. In contrast, most 26 dpf fancl mutants already showed a male expression profile by the absence of cyp19a1a and the up-regulation of amh (Q,R) and only one fancl mutant showed a low number of cyp19a1a-expressing cells while nevertheless maintaining high amh expression (T,U). Except for vasa, expression profiles of 26 dpf dnd knockdown gonads were similar to fancl mutants (W,X). At 33 dpf, wild-type controls showed either a male expression profile (no cyp19a1a and high amh expression, Z,A′) or a female expression profile (high cyp19a1a and no amh expression, C′,D′). Most 33 dpf fancl mutants showed a male expression profile (F′,G′), even if gonads maintained an ovary-like morphology (I′,J′). All 33 dpf dnd animals showed a male expression profile (L′,M′). Scale bar: 0.1 mm (A).