Figure 3.

Figure 3.

ROME is expressed in zebrafish and mice and its knockdown in zebrafish leads to severe defects. A, ISH staining for rome in zebrafish at 24 to 36 hpf. The black arrows indicate Rohan beard neurons, lateral line ganglia, and the olfactory placode. B, Representative images of developmental defects after rome knockdown: (1) normal embryo, (2) cardiac edema (mild defect), (3) bent tail (mild defect), (4) tail necrosis (severe defect), (5) cranial cell necrosis, cyst, and axis extension defect (severe defects), (6) group of embryos with defects including necrosis, missing head, and missing tail, and (7) mass of cells (monster phenotype). C, Developmental defects in zebrafish embryos injected with rome MO at 24 hpf. Embryos were injected with 10–50 ng of MO at the two- four-cell stage. The quantification used the definitions of the mild and severe phenotypes from B (n = 10 for each dose). D, Expression of rome mRNA (400 ng/embryo) rescued survival in rome MO (20 ng/embryo)–injected zebrafish embryos (P = 0.02, χ² test). E, Developmental defects in zebrafish embryos with rome KO by CRISPR/Cas9 system at 24 hpf. Embryos were injected with the RNP complex [Cas9 + rome cr:tracrRNA or Cas9 + tracrRNA (Ctl)] at the one-cell stage. F, Rome mRNA expression in the liver of mouse embryos from E13.5 was analyzed via RNAscope. Ppib and dapB probes were used as positive and negative controls, respectively. G, ROME protein expression was detected via immunoprecipitation followed by Western blotting with an ICD-6 antibody in all the mouse organs profiled (the red arrows indicate ROME protein).