Characterization of the GRE:Luc reporter
line. (a) Schematic showing
the structure of the reporter construct. Tol2, Tol2 transposase sites
to facilitate integration into the genome; (GRE)4, four
concatemerized Glucocorticoid Response Elements (GRE); Pmin, minimal promoter; pA, polyadenylation site. (b–d) Luciferase
expression is ubiquitously upregulated upon GC treatment. (b, c) Immunohistochemistry
with an anti-luciferase antibody (anti-Luc) and an Alexa Fluor 488
labeled secondary antibody. Fluorescence intensity is shown color
coded. Representative examples of 5 dpf larvae treated with solvent
control (0.3% DMSO (b)) or 40 μM betamethasone (c). Scale bar
0.5 mm. (d) Quantification of fluorescence intensity shows a significant
(p = 0.0115, n = 10) increase in
betamethasone treated larvae. (e–h) The bioluminescence response
of GRE:Luc larvae (n = 48) to GC signaling is specific.
Graphs show the relative reporter activity in response to treatment
with the indicated concentrations of a GR agonist (dexamethasone (e)),
the natural GC cortisol (hydrocortisone (f)), the mineralocorticoid
aldosterone (g), and a GR antagonist (mifepristone, in presence of
5 μM dexamethasone (h)). Red, control treatments. (i) GRE:Luc
larvae increase bioluminescence in response to a rise in endogenous
cortisol levels under osmotic stress. Upon salt stress, endogenous
cortisol levels (gray, left y-axis) rise to a peak
within 20 min (p ≤ 0.01, n = 5) and a significant increase in bioluminescence (black, right y-axis) is observed after 4 h of treatment (p ≤ 0.01, n = 22). (j) Developmental time
course of GC signaling activation by DEX. Treatment with DEX leads
to a significant rise in bioluminescence starting at 2 dpf (black
bars, p ≤ 0.001, n ≥
48). Error bars represent mean values ± SEM. (k) Developmental
time course of osmotic stress response. A trend for increased bioluminescence
is observed at 4 dpf, which becomes significant at 5 dpf (black bars, p ≤ 0.001, n = 288).