20β-hydroxycortisone is the most abundant glucocorticoid in extracts from adult zebrafish holding water.

<p>The measurement of four glucocorticoids in extracts from adult zebrafish holding water was performed by a separate LC-MS/MS analysis of each fraction: (A) unconjugated steroids; (B) glucuronidated steroids; (C) sulfated steroids. Each bar represents the mean value of three independent samples with standard deviations added. Sample abbreviations: X-mixed gender group; F-all female group; M-all male group; X-E-mixed gender group, without addition of digestive enzymes; N-water control without fish.</p

Challenge of 5 dpf zebrafish larvae with a physical stressor up-regulates <i>hsd20b2</i> and <i>hsd11b2</i> mRNA.

<p>The fold changes of target genes from six biological replicates were calculated after normalization to the geometric mean of three reference genes and compared with unstressed fish. The time courses for <i>crh</i>, <i>mc2r</i>, <i>star, cyp11c1, hsd11b2,</i> and <i>hsd20b2</i> are shown as mean values with standard deviations and significant changes to unstressed fish are indicated as follows: * p<0.05, ** p<0.01.</p

Primer sequences for quantitative real-time PCR.

<p>Primer sequences for quantitative real-time PCR.</p

3–24 hpf exposure to cortisol up-regulates <i>hsd20b2</i> and <i>hsd11b2</i> mRNA in 24 and 48 hpf larvae.

<p>Zebrafish embryos were treated with cortisol and its vehicle DMF until 24 hpf and were then placed in cortisol-free embryo water. The fold changes of <i>hsd20b2</i> (A) and <i>hsd11b2</i> (B) expression in three replicates were calculated after normalization to β-actin and compared with untreated fish embryos. Mean values with standard deviations are shown. White bars-0.1% DMF; grey bars-50 mg/L cortisol (137.9 µM). Significance levels are indicated as follows: * p<0.05, ** p<0.01.</p

20β-HSD type 2 morphants display developmental abnormalities upon cortisol treatment.

<p>Animals are presented from a lateral view, with the anterior end at left. Representative specimens of the ‘mild’ and the ‘severe’ phenotype of the cortisol-treated 20β-HSD type 2 morphants are shown. (A) Embryos at 48 hpf and (B) at 72 hpf. Black arrows point to yolk deformations, white arrows point to altered somitogenesis resulting in kinked or truncated tails, and the asterisk denotes pericardial edema. WT-wild-type; MO-morpholino; C-control morpholino. Magnification 5x.</p

20β-hydroxycortisone is not a physiological ligand for zebrafish GRα or MR.

<p>An analysis of reporter gene activation in COS-1 cells mediated by zebrafish GRα (A) and zebrafish MR (B) using four different steroidal ligands. Firefly luciferase values were normalized to renilla luciferase and corrected for background. Mean values with standard deviations are shown.</p

Distribution of phenotypes in <i>hsd20b2</i> morphant zebrafish larvae.

<p>Abbreviations: hpf-hours post fertilization; MO-morpholino-injected fish; C-control morpholino-injected fish; n-number of larvae. The significant appearance of the phenotype as determined by Fisher’s Exact Test is indicated by a p-value below 0.001. Data were pooled from four experiments and expressed as means ± SD.</p

mRNA expression of <i>hsd20b2</i> and <i>hsd11b2</i> is increased by cortisol treatment.

<p>The effects of cortisol and its vehicle DMF were examined in embryos at 24 hpf and 48 hpf and in larvae at 72 hpf. Final cortisol concentrations of 10, 25, 50, 75, and 100 mg/L are equivalent to 27.5, 69.0, 137.9, 206.9, and 275.9 µM, respectively. The fold changes of three replicates were calculated after normalization to β-actin and compared with untreated fish embryos. White bars-the gene of interest in DMF treated fish; grey bars-the gene of interest in cortisol treated fish. Mean values with standard deviations are shown. Significance levels are indicated as follows: * p<0.05, ** p<0.01.</p

Analyses of knock down efficiency of an <i>hsd20b2</i> splicing morpholino reveal reduction of enzymatic activity.

<p>(A) In the genomic structure of zebrafish <i>hsd20b2</i>, exons are indicated by boxes and numbered. Below, the important short-chain dehydrogenase/reductase motifs (single letter amino acid code) are denoted. ‘x’ denotes any amino acid residue, and when present, the subsequent number indicates the number of×residues. The splice site targeted by the morpholino is indicated by a dash. Morpholino-induced mis-splicing is illustrated by the triangle above the genomic structure. Exons are to scale, whereas the space between the exons does not reflect the respective intron size. (B) The analyses of morpholino efficiency at the mRNA level by RT-PCR using primers that prime within the first and fourth exons demonstrate mis-splicing in morpholino-injected fish (MO). The smaller PCR product is absent from samples of the control morpholino-injected fish embryos (C). The respective morpholino concentration used is denoted. β-actin controls were included for normalization. (C) The knock down efficiency was analyzed by assaying the enzymatic activity that converts cortisone to 20β-hydroxycortisone in morpholino (MO)- and control morpholino (C)-injected fish. The respective morpholino concentration used is denoted and mean values with standard deviations from four biological replicates are presented. Significant levels are indicated: ** p<0.01.</p

Reduced visual acuity in the <i>Fgf9^Y162C</i> mutants.

<p>Spatial frequency thresholds indicated a reduced vision in the <i>Fgf9^Y162C</i> mutants. Values represent means ± standard deviation of measurements from twelve C57BL/6J controls (WT), ten heterozygous <i>Fgf9^Y162C</i> mutants (A/+), and ten homozygous <i>Fgf9^Y162C</i> carriers (A/A). *<i>p</i><0.001.</p