Dysregulation of PTCH1-cyclin B1 pathway is involved in early arrest of GRK2 knockdown embryos. (A) Expression pattern of GRK2 during zebrafish developmental process by using whole-mount in situ hybridization (WISH) with a zebrafish GRK2-specific antisense riboprobe on embryos at the indicated stages. Areas with high intensity hybridization signal are indicated by arrows. (B) Expression levels of GRK2 during early developmental stages. Zebrafish embryos (15–20) were collected at each time point, and lysates were analyzed in Western blots by using rabbit anti-GRK2 antibody (Santa Cruz). Lysates from HEK293 cells transfected with beta-galactosidase (gal) or bovine GRK2 expression plasmids were taken as controls. GAPDH was used as a loading control (hpf, hours postfertilization; dpf, days postfertilization). (C) Effect of GRK2-MOs on GRK expression levels. Zebrafish embryos were injected at the 1- or 2-cell stage with 2- or 4-ng GRK2 splicing morpholino (GRK2-MO). GRK2 levels in embryo lysates were examined by Western blot analysis. (D) Somite stage arrest in GRK2 morphants, could be rescued by both zebrafish and bovine GRK2 catalytic domain. Each embryo was injected at the 1- or 2-cell stage with 2-ng con-MO, or 2-ng GRK2-MO alone, or with 0.15ng zebrafish or bovine GRK2 catalytic domain mRNA. Embryos were categorized, counted, and photographed 19 hpf. The percentages of arrest embryos are shown (200 embryos were counted for each bar). (E) GRK2, K220R, BP, or NLS-cyclin B1 rescues the arrest phenotype of GRK2 knockdown embryos. (F) GRK2, K220R, BP, or NLS-cyclin B1 rescues the deficiency in zebrafish eye and midbrain development. WISH was performed on 15–20 somite stage embryos injected with GRK2-MO and mRNA of indicated constructs, using pax6a, otx2 and gata1 riboprobes. Images of pax6a and otx2 were taken from a lateral view and those of gata1 from a dorsal view. (G) Effects of GRK2 on cell cycle progression in eye, midbrain, and hematopietic system were examined using p-H3 and BrdU assays. Twenty-four hpf injected embryos were collected and stained for p-H3 and BrdU. The images were taken from a lateral viewpoint, showing the eye, the midbrain, and the blood island.
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