Compartmentalised expression of in epithelial somites is required for the formation of intervertebral joints-5

<p><b>Copyright information:</b></p><p>Taken from "Compartmentalised expression of in epithelial somites is required for the formation of intervertebral joints"</p><p>http://www.biomedcentral.com/1471-213X/7/68</p><p>BMC Developmental Biology 2007;7():68-68.</p><p>Published online 17 Jun 2007</p><p>PMCID:PMC1924847.</p><p></p>). () The number of chondrocytes undergoing hypertrophic differentiation is reduced in transgenic embryos (arrow). At E17.5 () chondrocytes of wild-type embryos lose the cartilage matrix and vascularisation and invasion of osteocytes starts (arrow). The notochord is extruded from the vertebrae. () In transgenic vertebrae of the same age fewer cells are hypertrophic and are dorsally displaced (arrow). Ossification is delayed and notochord extrusion is incomplete. Scale bar: 200 μm. Asterisks: nucleus pulposus

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-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

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

Compartmentalised expression of in epithelial somites is required for the formation of intervertebral joints-3

<p><b>Copyright information:</b></p><p>Taken from "Compartmentalised expression of in epithelial somites is required for the formation of intervertebral joints"</p><p>http://www.biomedcentral.com/1471-213X/7/68</p><p>BMC Developmental Biology 2007;7():68-68.</p><p>Published online 17 Jun 2007</p><p>PMCID:PMC1924847.</p><p></p>ed vertebrae (arrow) and reduced costal heads of ribs (asterisks) compared to () wild-type animals are found. () Dorsal examinations exhibit reduced spinous processes, open neural arches (white arrowheads) and reduced (black arrowheads) or malformed (black and white arrows) intervertebral joints. () Reduced intervertebral joints occur in () transgenic mice compared to () wild-types but no fused adjacent neural arches (dashed lines) are observed. iap, inferior articular process; ivd, inter-vertebral disc; la, lamina; p, pedicle; pr, proximal rib; sp, spinous process; sap, superior articular process; TG, transgenic; tp, transverse process; vb, vertebral body; WT, wild-type

Compartmentalised expression of in epithelial somites is required for the formation of intervertebral joints-4

<p><b>Copyright information:</b></p><p>Taken from "Compartmentalised expression of in epithelial somites is required for the formation of intervertebral joints"</p><p>http://www.biomedcentral.com/1471-213X/7/68</p><p>BMC Developmental Biology 2007;7():68-68.</p><p>Published online 17 Jun 2007</p><p>PMCID:PMC1924847.</p><p></p>eals no differences between wild-type and transgenic embryos. () At E13.5 presumptive IVDs stain with alcian blue in transgenic embryos (arrowhead) but not in () wild-types. At E14.5 () vertebrae of wild-type embryos are separated by weaker stained IVDs (arrowhead). The notochord is extruded to the nucleus pulposus. () Transgenic vertebrae are shortened, stain uniformly with alcian blue, IVDs are missing and the notochord remains as a rod like structure. Newborn transgenic mice () exhibit two lateral centres of ossification compared to one centre in wild-type mice (). ivd, intervertebral disc; L, lumbar vertebra; nc, notochord; np, nucleus pulposus; s, somite; T, thoracic vertebra; TG, transgenic mouse; vb, vertebral body; WT, wild-type

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

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