A Chemical Screening System for Glucocorticoid Stress Hormone Signaling in an Intact Vertebrate

Glucocorticoids, steroid hormones of the adrenal gland, are an integral part of the stress response and regulate glucose metabolism. Natural and synthetic glucocorticoids are widely used in anti-inflammatory therapy but can have severe side effects. <i>In vivo</i> tests are needed to identify novel glucocorticoids and to screen compounds for unwanted effects on glucocorticoid signaling. We created the <b>G</b>lucocorticoid <b>R</b>esponsive <i><b>I</b>n vivo</i> <b>Z</b>ebrafish <b>L</b>uciferase activit<b>Y</b> assay to monitor glucocorticoid signaling <i>in vivo</i>. The GRIZLY assay detects stress-induced glucocorticoid production in single zebrafish larvae, measures disruption of glucocorticoid signaling by an organotin pollutant metabolite, and specifically identifies a compound stimulating endogenous glucocorticoid production in a chemical screen. Our assay has broad applications in stress research, environmental monitoring, and drug discovery

Rasl11b, an atypical cytoplasmic Ras small GTPase, is strongly conserved in Vertebrates.

<p>(A) Zebrafish <i>rasl11b</i> encodes a Ras-related small GTPase of 244 amino acids (accession number DQ983377) containing the 5 highly conserved domains (G1–G5, overlined in red) responsible for the guanine nucleotide-dependent molecular switches. Rasl11b has no obvious orthologues in <i>Drosophila melanogaster</i> or <i>Caenorhabditis elegans</i>, but is highly conserved among vertebrates. Note that, in contrast to most of the Ras small GTPases, Rasl11b lacks a COOH-terminal CAAX motif and any known recognition signal for C-terminal lipidation found in Ras proteins such as farnesylation or palmitoylation allowing membrane anchorage. The amino acid positions mutated to create the activated forms Rasl11b^S42V and Rasl11b^Q82L are indicated with stars. (B) Phylogenic analysis of zebrafish small GTPase proteins. The degree of relatedness is indicated by the length of the vertical lines. Numbers indicate bootstrap support for nodes. Red box: Rasl11b, Rasl11a, Rasl12 and Rerg constitute an uncharacterized branch of Ras proteins devoid of lipid modification signals. (C) Epifluorescent microscopy of zebrafish embryonic cells expressing myc-Rasl11b revealed by immunostaining.</p

Rasl11b inhibits endoderm and PP formation in an Oep deficient background.

<p>(A–D) Frontal views of 1 dpf wild-type embryo and Z<i>oep</i> embryos. Depending on the quantity of <i>oep</i> mRNA accumulated during oogenesis, Z<i>oep</i> embryos display different levels of anterior structure development: from absence of lens and forebrain (red class, B), via a cyclopia phenotype (green class, C) to two separate retinae (blue class, D, very rare). In each clutch, these categories can have slightly different proportions depending on the female, but a large majority of Z<i>oep</i> embryos do not even develop one retina. Whatever females (n>10) were used for the <i>rasl11b</i> knock down experiments, a drastic rescue (>70%) of the forebrain formation was always observed, and a large category of embryos with two retinae appeared. (E) Cumulated numbers for each class from 5 independent <i>rasl11b</i> MO-ATG knock down experiments. (F–H) <i>forkhead7</i> (<i>fkd7</i>) expression pattern in 1 dpf embryos. Gut defects (white arrowheads) are also rescued (black arrowheads) in Z<i>oep</i> embryos injected by <i>rasl11b</i> MO-ATG. (I–L) 1 dpf embryos lateral views. Rasl11b constitutively activated forms (Rasl11b*) prevent head formation and disturb the antero-posterior axis formation in Z<i>oep</i> and <i>oep</i>^+/− but not in wild-type embryos (not shown). <i>zrx2</i> is expressed in the retina, <i>krox20</i> in rhombomeres 3 and 5, <i>myod</i> is expressed in the somitic mesoderm. (M–V) dorsal view of late gastrulae. <i>rasl11b</i> knock down rescues expression of the prechordal plate marker <i>goosecoid</i> (<i>gsc</i>) and the endodermal marker <i>sox17</i> in Z<i>oep</i> whereas Rasl11b* reduces their expression in Z<i>oep</i> and <i>oep</i>^+/− embryos.</p

<i> rasl11b</i> expression pattern during zebrafish embryogenesis.

<p>(A) RT-PCR analysis showing that <i>rasl11b</i> has a maternal and a zygotic component. RNA extractions have been done before (1 cell stage) and after (gastrula) the midblastula transition, the time of activation of the zygotic transcription. The maternally and zygotically expressed <i>oep</i> gene and the strictly zygotic <i>sonic hedgehog</i> (<i>shh</i>) gene have been used as controls. (B) During the cleavage period (dome stage), <i>rasl11b</i> is ubiquitously expressed. (C) At the onset of gastrulation (shield stage), <i>rasl11b</i> is still detected at the animal pole (Black arrowhead) but is also expressed in a dorso-ventral gradient at the dorsal margin (white arrowhead). This marginal expression overlaps with the mesendodermal territory in zebrafish embryos. (D) Sagittal section. <i>rasl11b</i> transcript accumulates in both hypoblastic and epiblastic dorsal blastomeres. (E, F) <i>rasl11b</i> expression is maintained at the margin throughout gastrulation (white arrowheads). Gastrulae also expressed <i>rasl11b</i> mRNA in ectodermal precursors located at the lateral borders of the blastoderm (black arrowheads). (G–K) During somitogenesis and organogenesis, <i>rasl11b</i> is expressed in the tail tip and the spinal cord (sc), and in several head structures such as the hindbrain (hb), the isthmic organizer (i), the otic vesicle (ov), the pineal gland (p), the ventral hypothalamus (h) and the posterior boundary of the telencephalon (t). (J) lateral close up and (K) frontal view. (L) At 3 dpf, <i>rasl11b</i> is no longer expressed except in the otic vesicle (lateral close up).</p

<i> rasl11b</i> interacts specifically with <i>oep</i> but does not affect the Nodal/Smad2 transduction pathway.

<p>(A) The MZ<i>sqt</i>, MZ<i>oep</i>, Z<i>oep</i>, <i>bon</i> and <i>cas</i> mutants have a clear reduction of endodermal cells and so were used to quantify the putative impact of <i>rasl11b</i> knock down at different steps/levels of the nodal pathway. The Z<i>oep</i> mutant was the only one rescued by the <i>rasl11b</i> MO-ATG injection. This rescue was abolished by co-injection of <i>rasl11b</i> MO-resistant mRNA (<i>rasl11b^MOr</i>). Error bars indicate standard deviation. (B, C) It is impossible to generate clutches of 100% Z<i>oep</i> embryos, and because one embryo cannot give enough material for both immunoblot and genotyping experiments, 100% Z<i>oep-</i>like mutant clutches were produced by injecting clutches of 100% MZ<i>oep</i> eggs with low doses of wild-type <i>oep</i> mRNA. Half of them were then co-injected with <i>rasl11b</i> MO-ATG. Each batch was split in two, one used for phosphorylated Smad2 (Smad2-P) level analysis, the second for endodermal cell number count (assayed by sox17 in situ hybridization). Proteins were detected by western blotting using a Smad2-P antibody. Note that increasing doses of wild-type <i>oep</i> mRNA were correlated with an increase of Smad2-P and <i>sox17</i> endodermal cell number, whereas co-injection with <i>rasl11b</i> MO-ATG increased endodermal cell number without generating more Smad2-P (Error bars indicate standard deviation). The same experiment was done at three pregastrula stages: dome (B), sphere and 40% epiboly (C).</p

Opposite effects of Nodal and Oep on <i>rasl11b</i> expression.

<p>(A) Scheme of the Nodal cascade. The Oep coreceptor is necessary for the binding of the Nodal ligand Cyc and Sqt to the TGFβ receptors (containing the type I receptor Taram-A, the zebrafish Alk4 orthologue) that in turn phosphorylate Smad2. Smad2-P is translocated to the nucleus and triggers the transcription of a first set of genes encoding the transcription factors, Mixer, Gata5 and Mezzo. This set is required for the expression of the Sox factor Casanova that in turn initiates the transcription of the endodermal markers <i>sox17</i> and <i>axial</i>/<i>foxa2</i>. (B–M, Dorsal view of gastrulae save C, I, sagittal sections and G, lateral view) (B, C) In wild-type (WT) embryos <i>rasl11b</i> is expressed at the animal pole (arrows) and at the dorsal margin (black arrowheads). (D) This marginal expression is lost in MZ<i>oep</i> mutants (star), devoid of maternal and zygotic <i>oep</i>, consequently devoid of Nodal signaling, and so, unable to form most of the mesendoderm. (E, F) <i>rasl11b</i> dorso-marginal expression is normal in the <i>cyclops</i> Nodal mutant and largely reduced in the MZ<i>squint</i> Nodal mutant. (G) Activation of the Nodal signal by injection of a constitutively activated form of the Nodal type I receptor Taram-A (tar*) leads to a duplication of the <i>rasl11b</i> marginal expression domain (likely by inducing a second organizer, white arrowheads) (H, I) In Z<i>oep</i> mutants, devoid of zygotic <i>oep</i>, this mesendodermal expression domain is extended (white arrowheads). (J–M) A large series of embryos expressing different levels of Nodal signal was generated by injecting between blastula cells increasing doses of the recombinant Lefty protein, a Nodal pathway extracellular inhibitor. Here, only four representative doses are displayed. A progressive decrease of <i>rasl11b</i> marginal expression but no expansion was observed, even with concentrations of Lefty able to mimic a Z<i>oep</i>-like phenotype.</p

Rhythmic Clock Gene Expression under LD and Temperature Cycles

<div><p>Graphical summary of RPA assays are described:</p> <p>(A) <i>Per4</i> (solid line) and <i>cry3</i> mRNA expression (dashed line) in zebrafish larvae raised for 6 d either in a light (12 h) or dark (12 h) cycle at a constant temperature (25.3 °C).</p> <p>(B) <i>Per4</i> (solid line) and <i>cry3</i> mRNA expression (dashed line) in zebrafish larvae raised for 6 d in DD, under a temperature cycle of 4 °C (23.5 °C/11 h, 27.5 °C/11 h, plus 1 h for each heating and cooling phase). RNA samples were harvested during the seventh day (ZT0 is defined as the beginning of the heating and light periods).</p> <p>(C and D) Equivalent analysis of <i>clock1</i> (solid line) and <i>cry2a</i> (dashed line) expression in (C) LD, and (D) temperature cycle larvae.</p> <p>(E) <i>Per2</i> expression was assayed in LD (dashed line) or temperature cycle (ΔT) larvae (solid line). By linear regression analysis, the slope of the ΔT trace has no significant deviation from zero (R² = 0.033 and <i>p</i> = 0.66, F-test). The LD cycle curve fits to a 6th-order polynomial regression model (R² = 0.96 and Runs test for deviation from model <i>p</i> = 0.99).</p> <p>In each case, zeitgeber time is plotted on the <i>x</i>-axis while the relative expression levels (percentage) are plotted on the <i>y</i>-axis. <i>β-actin</i> levels were used to standardize the results. The highest band intensity in each experiment was arbitrarily defined as 100%, and then all other values were expressed as a percentage of this value. All experiments were performed in triplicate, and error bars denote the standard deviation.</p></div

Temperature Influences CLK Protein Expression and Function

<div><p>(A) In vitro luciferase assays of transiently transfected PAC2 cells. The combinations of CLK (Clk) and BMAL (Bml) expression vectors cotransfected with the 4x Ebox (−7) reporter plasmid are indicated for each assay result. Control cells were transfected with the reporter plasmid or with the pGL3 Control plasmid alone. Values represent the mean fold difference between luciferase activities measured in 30 °C and 20 °C, 60 h after transfection. All assays were standardized for transfection efficiency using a β-galactosidase assay. The results are based on four independent experiments, and error bars indicate the standard deviation.</p> <p>(B) Electrophoretic mobility shift assay of nuclear extracts from PAC-2 cells cultured at 20 °C or 30 °C on a LD cycle, and harvested at ZT3, 9, 15, and 21 (lanes 1 to 8). Three specific complexes are indicated by A, B, and an asterisk. Supershift assays of a ZT15, 30 °C extract (+Ab), used either a dopamine transporter antibody (Control) or a mouse clk antibody (Clock) (lanes 9 and 10). Complexes indicated by A, B, and an asterisk are all efficiently competed by a 25-, 50-, and 100-fold excess of cold E-box probe (lanes 12, 13, and 14, respectively, and compare with lane 11), but not with a 100-fold excess of a CRE probe (compare lane 15 with lane 11).</p> <p>(C) Western blotting assay using the anti-mouse CLK antibody of the same nuclear extracts tested in the electrophoretic mobility shift assay analysis of panel B. The migration of a 100-kDa marker band is shown. Below are shown western blotting results for the same extracts using an anti-mouse CREB antibody as a loading control.</p> <p>(D) Western blot assay of CLK protein in 30 °C extracts prepared at ZT9 or ZT21 (time points representing the trough and peak, respectively, of the CLK protein rhythm). Samples were prepared with (+) or without (−) treatment with alkaline phosphatase prior to electrophoresis and transfer. In panels B, C, and D, data are representative of at least three independent experiments.</p></div

Temperature Compensation and the Amplitude of E-box-Directed Rhythmic Expression

<div><p>(A) Bioluminescence profile of 4xE-box (−7) reporter cells held at 20 °C under a LD cycle and then transferred to DD conditions. Plates were counted once per hour and maintained in robotic stacking units between assays, where they were illuminated.</p> <p>(B) Equivalent experiment to panel A, with cells maintained at 30 °C.</p> <p>(C) Bioluminescence traces from 1.7-kb WT <i>per4</i> reporter cells maintained at 20 °C under LD cycle and DD conditions.</p> <p>(D) Bioluminescence traces from 1.7-kb WT <i>per4</i> reporter cells maintained at 30 °C under LD cycle and DD conditions.</p> <p>(E) RPA analysis of <i>per4</i> expression in WT PAC-2 cells held at 20 °C and 30 °C under an LD cycle for 3 d. RNA extracts were prepared on the fourth day at 3-h intervals during one 24-h cycle. Time 0 represents ZT 0: the onset of the light period. A white and black bar above the autoradiograph indicates the duration of the light and dark periods. RPA results with a β-actin loading control are also shown. “t” represents a tRNA control sample.</p> <p>(F) A bar graph shows quantification of the peak (ZT3) and trough (ZT15) <i>per4</i> expression values at 20 °C and 30 °C plotted as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030351#pbio-0030351-g001" target="_blank">Figure 1</a>, with error bars representing the standard deviation of three independent experiments.</p> <p>All bioluminescence traces represent the mean values of 16 independent wells. Each panel is representative of at least three independent experiments.</p></div

Temperature Steps Regulate Clock Gene Expression Levels

<div><p>(A) Larvae were raised in DD at 21 °C for 7 d and then shifted to 29 °C and harvested at the indicated times relative to the temperature shift (h). Controls remained at 21 °C and were harvested in parallel with the temperature shift larvae. RPA analysis of the indicated genes was then performed. “t” represents a tRNA control sample.</p> <p>(B) As in (A), except that 5-d-old larvae were shifted from 29 °C to 21 °C, and controls remained at 29 °C.</p> <p>All data are representative of at least three independent experiments.</p></div