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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.