Analysis of subcellular localization of zebrafish 20beta-HSD type 2 showed that the enzyme is localized in the endoplasmic reticulum. Pearson’s correlation coefficient for colocalization of 20beta-HSD type 2 and the endoplasmic reticulum marker is 0.84. The panels depict detection of recombinant 20beta-HSD type 2 with anti-myc FITC-labeled antibody (A), counterstaining of endoplasmic reticulum with DsRed (B), and nuclei with Hoechst 33342 dye (C), respectively, and the overlay of fluorescence patterns (D). Magnification: 40x.

Expression pattern of 20beta-HSD type 2 appears comparable to that of 11beta-HSD type 2 but different to ubiquitously expressed 11beta-HSD type 3. RT-PCR was performed to analyze the expression pattern of all three enzymes during zebrafish embryogenesis (A), in adult female tissues (B), and in adult male tissues (C). Gel photographs show the observed distribution of RT-PCR signals for all genes analyzed. Beta-actin controls were included for normalization. hpf, hours post fertilization.

Whole mount in situ hybridization shows distinct expression of 20beta-HSD type 2 during zebrafish development. In 30 hpf embryos (B and C), expression is observed in brain, eye, ear, intestine, liver, and pronephric ducts. In 48 hpf (E) and 3 dpf larvae (G), signals are detected in all parts of brain, eye, intestine, liver, pronephric ducts, and branchial arches. 4 dpf larvae (I) show expression in brain, eye, ear, pharyngeal arches, intestine, liver, pronephric ducts, and neuromasts. Specific hybridization is indicated by blue color. Control hybridizations with sense probe are shown for all stages (30 hpf (A), 48 hpf (D), 3 dpf (F) and 4 dpf (H)). hpf, hours post fertilization; dpf, days post fertilization; b, brain; el, eye and lens; e, ear; l, liver; i, intestine; pd, pronephric ducts; pa, pharyngeal arches; ba, branchial arches; n, neuromasts. Magnification: 5x for A, B, and D–I, 10x for C.