Gel mobility shift using increasing amounts of either in vitro translated Dmrt1bY or Dmrt1a proteins to shift a constant amount of radiolabelled dmrt1 probe. Binding reactions were resolved on a 5% polyacrylamide gel. (A) In vitro translated Dmrt1bY protein was incubated with radiolabelled Dmrt1bY target sequence as a probe, and non-radiolabelled probe was used as a competitor. (B) In vitro translated Dmrt1a protein was incubated with radiolabelled Dmrt1bY target sequence as a probe, and non-radiolabelled control (non-specific) probe was used as a competitor.

Chromatin immunoprecipitation using both Dmrt1bY::GFP and deltaDmrt1bY::GFP transgenic lines respectively expressing either Dmrt1bY or a control truncated Dmrt1bY (delta DM form lacking the DNA binding domain) fused to GFP revealed specific in vivo Dmrt1bY protein affinity to the Izanagi-nested dmrt1 target site, including the one described within dmrt1bY promoter. (A) Transgenic lines established for in vivo tissue–ChIP. (B) Dmrt1bY promoter primer sets map. (C) Specific enrichment of Izanagi-nested Dmrt1 binding sites subsequent to in vivo Dmrt1bY immunoprecipitation.

(A) Testis-specific GFP expression in 9KbDmrt1bYprom::GFP transgenic fish. (B) From testis formation (10 dph) up to adulthood, in 9KbDmrt1bYprom::GFP transgenic fish, robust testicular GFP expression is persistently noticeable in the epithelial cells of the intratesticular efferent duct. (C,D) Concomitantly, in agreement with the endogenous dmrt1bY expression, from 10 dph up to 30–35 dph strong specific GFP fluorescence is also detected in Sertoli cells. (E–G,I,J) By 35 dph, Sertoli cell-specific decline in fluorescence (mCherry) could be observed (E,I,H). In double transgenic fish (9KbDmrt1bYprom::mCherry and BACdmrt1a::GFP) this 9KbDmrt1bYprom-driven mCherry decline in expression is balanced by the rise of dmrt1a expression in Sertoli and germ cells (GFP in F,G,J). (H–J) In fully mature testes (after 45–50 dph), 9KbDmrt1bYprom-driven mCherry expression remains in scattered Sertoli cells around the germ cells (GFP in N) while Dmrt1a is now predominantly expressed in Sertoli cells (J).