Manipulation of glucocorticoid activity induces changes in cardiac structure Confocal images of embryonic (120 h post fertilization (hpf)) of Tg (CMLC2: GFP) zebrafish hearts after 120 h glucocorticoid receptor (GR) manipulation with either GR agonist dexamethasone, (Dex) [100 µM] or targeted GR knockdown using morpholino (GR Mo). Whole mounted GFP embryonic hearts (green) were stained with DAPI (blue) and imaged at high power with a confocal microscope. Gross cardiac morphology can be observed at ×20 magnification, higher power images (×40 magnification) demonstrate advanced maturity of the Dex treated hearts in contrast to the GR Mo hearts which show considerable immaturity of myofibrillar and trabecular patterning. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Effects of glucocorticoid receptor morpholino and rescue on the embryonic heart Western blot analysis was carried out to determine whether glucocorticoid receptor protein (Gr) was reduced in isolated hearts from zebrafish embryos (5 days post fertilization (5 dpf)) or adults (120 dpf) which had been treated with either glucocorticoid receptor (GR) translational blocking morpholino (GR Mo) or with GR agonist dexamethasone (Dex) [100 µM] for 120 h (5 dpf). A) Densitometry of Western blots of Gr abundance in isolated hearts. Data are n = 3 (150 embryo heart or 10 adult hearts per n), given as a mean percentage of their respective controls (mism Mo for GR Mo and vehicle for Dex) ± SEM analysed by two-way ANOVA and Bonferroni post hoc test. *p ≤ 0.05. B) Example of Western blot of embryonic (5 dfp) and adult (120 dpf) heart tissue lysates probed with antibodies against Gr with alpha-tubulin (loading control), C) Success of GR Mo was further determined following co-injection of rescue mRNA with GR Mo and calculation of ventricle length at 120 h post fertilization (hpf) D) Calculation of cardiomyocyte number at 120 hpf. C and D) n = 3 (6 embryos per group). Data are mean ± SEM analysed by two-way ANOVA and Bonferroni post hoc test vs each individual group. *p ≤ 0.05 and **p ≤ 0.01. E) Densitometry of Western blots of Gr abundance in 120 hpf whole embryo homogenate. Data are n = 3 (10 embryo per n), data given as relative protein abundance for control (mism Mo), GR Mo, and Gr mRNA rescue (rescue) n = 3 mean ± SEM analysed by two-way ANOVA and Bonferroni post hoc test, *p ≤ 0.05, **p ≤ 0.01. F) Confocal images of isolated 120 hpf Tg (CMLC2: GFP) zebrafish hearts (green) co-stained with DAPI nuclear stain (blue), images are isolated from embryos which were treated with control morpholino (mism Mo), GR Mo and capped GR mRNA (rescue) co-injected with GR Mo. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Effects of embryonic glucocorticoid manipulation on adult heart structure Long-term impact of embryonic glucocorticoid receptor (GR) manipulation with either GR agonist dexamethasone (Dex) [100 µM], or targeted translational GR knockdown using morpholino (GR Mo) was assessed in the heart of 120 day post fertilisation (dpf) adult zebrafish. Structural analyses of A) ventricle length, B) weight and C) cavity area were carried out. Data are n = 8 hearts per group, displayed as mean ± SEM analysed by one-way ANOVA with Dunnett′s post hoc comparison (*p < 0.05, **p < 0.01, ***p < 0.001). D–F) Histology (haematoxylin and eosin staining) in adult fish hearts D) Controls (Vehicle only control shown here), E) Dex, F) GR Mo.