Romano et al., 2017 - G protein-coupled estrogen receptor regulates embryonic heart rate in zebrafish. PLoS Genetics   13:e1007069 Full text @ PLoS Genet.

Fig. 1 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Fig. 2 (A) Genomic DNA of gperuab102 zebrafish contains a 133 basepair deletion in the gper coding region between CRISPR guide RNA targets 1 and 2, resulting in a premature stop codon in the GPER protein. Red dashes indicate DNA deletions, mutated amino acids are shown in red. (B) Genomic DNA was harvested from individual embryos, gper was PCR amplified and separated on an agarose gel to identify deletion mutations. (C-D) 3 day post fertilization wildtype and maternal zygotic gperuab102 homozygous larvae (MZgper-/-) exhibit similar gross morphology. Images are lateral views, anterior to the left, dorsal to the top. Scale bar, 500 μm. (E) Neither estradiol (ER/GPER agonist, 3.67 μM), ICI182,780 (ER antagonist/GPER agonist, 10 μM) or G1 (GPER agonist, 1 μM) changed heart rate significantly compared to vehicle (0.1% DMSO) in MZgper-/-, two-way ANOVA, p = 0.27. (F) MZgper-/- exhibited lower basal heart rate than age-matched wildtype embryos. *, p<0.05 compared to wildtype, paired t test. Each black circle represents the mean heart rate from a single clutch of embryos (≥ 7 embryos per clutch). Horizontal blue lines are the mean of each treatment.

Fig. 3 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Fig. 4 gper expression in the brain and pituitary. (A-C) Whole mount colorimetric in situ hybridization was performed on wildtype embryos at 50 hours post fertilization (hpf). (A) amhc (alpha-myosin heavy chain) antisense RNA labels atrial myocardial cells in the heart (boxed). (B, C) gper antisense RNA labels a bilaterally symmetric cluster of cells in the olfactory area (white arrowheads) and preoptic area (black arrowhead) and a medial cluster of cells in the ventral hypothalamus (arrows). No label was detected in the heart. Lateral views with anterior to the left (A,B), ventral view with anterior to the top (C), scale bars = 100 μm. (D-F) Double fluorescent in situ hybridization performed on 48 hpf Tg(5xERE:GFP)c262 embryos following overnight exposure to 100 ng/ml estradiol. gfp marks cells with active nuclear estrogen receptors. Confocal images of selected Z-slices (0.975 μm) show that gper is expressed in the olfactory area (D) and preoptic area (E) in cells lacking gfp (D”, E”, scale bars = 50 μm). In the ventral hypothalamus (F), gper is expressed in a medial cluster of cells lacking gfp (arrows, F, F”), whereas gper is expressed together with gfp more laterally (arrowheads, F”, scale bar = 10 μm). (G-H) Double fluorescent in situ hybridization performed on 48 hpf wild-type embryos. Confocal images of selected Z-slices (0.975 μm) show that gper is expressed in the middle zone of the pituitary (G, arrows), together with tshb-positive thyrotropes (G”, arrowhead). gper is expressed in the anterior zone (H, arrows) together with pomca-positive corticotropes (H”, arrowhead). gper was not detected in the posterior zone of the pituitary, marked by a distinct population of pomca-positive cells (dotted arrows, H’, H”). Scale bars = 50 μm, dorsal views, anterior to the top. In merged images, gper is magenta, gfp is green and areas of colocalization are white.

Fig. 5 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Fig. S1 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Fig. S2 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Fig. S3 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Fig. S4 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Fig. S5 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Fig. S6 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Fig. S7 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Table 1 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Acknowledgments:
ZFIN wishes to thank the journal PLoS Genetics for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ PLoS Genet.