Fig. 2

β-Arrestin1 Is Required for Zebrafish Embryonic Development

(A–R) Whole-mount in situ hybridization for embryos injected with 4 ng control morpholino (Ctrl MO), 4 ng β-arrestin1 morpholino (Arrb1 MO), or 4 ng β-arrestin2 morpholino (Arrb2 MO). Riboprobes to Hedgehog pathway target ptc1 (12 hpf; arrowheads denote aberrant expression in axial mesoderm [A–C]), cardiac marker nkx2.5 (14 hpf; arrowheads denote decreased expression in cardiac mesoderm [D–F]), hematopoietic markers lmo2 (12 hpf [G–I]) and fli-1 (12 hpf [J–L]) and ventral marker bmp4 (6 hpf; lateral views with ventral sides on the left [M–O]) were used. All embryos, except for bmp4, are dorsal views with anterior to the top. (P–R) Zebrafish microarray data analyses. 50 embryos injected with 4 ng control morpholino (Ctrl MO), 4 ng β-arrestin1 morpholino (Arrb1 MO), 4 ng β-arrestin2 morpholino (Arrb2 MO), or 4 ng β-arrestin1 morpholino plus 4 ng β-arrestin2 morpholino (Double MO) were lysed in Trizol reagent at 12 hpf. RNAs were extracted and microarray hybridization was performed on an Agilent platform. Venn diagram representation of differentially expressed genes was shown in (P). Upregulated and downregulated genes (at least 2 folds) were represented separately. Cluster analysis clearly distinguished β-arrestin1 from β-arrestin2 (red, upregulated genes; green, downregulated genes [Q]), with hematopoietic marker genes specifically downregulated in β-arrestin1 but not β-arrestin2 morphants (R). (S) Real-time quantitative PCR verification of hematopoietic marker changes. Data shown are means ± SEM of at least three independent experiments, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 versus the corresponding control.