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Fig. 5
Disruption in nucleolar architecture, rRNA synthesis and ribosomes in Ddx27 deficiency.
(A) Immunofluorescence of control and ddx27 mutant fish with antibodies labeling different nucleolar compartments at 5 dpf (scale bar: 10μm) (B) rRNA transcription was measured in MPCs (labeled with Pax7) in myotome (labeled with myosin) or myonuclei (labeled with Actn2/3) at 5 dpf by quantifying the incorporation of 5-ethynl uridine (5-EU). Zebrafish or myofibers were treated with Actinomycin D for two hours to block background transcription and subsequently, were incubated with or without Actinomycin D and freshly synthesized rRNA was quantified by incorporation of 5-EU by fluorescent detection. Representative single Z-section images are shown. (scale bar: 5μm) (C) Northern blot analysis of total RNAs extracted from skeletal muscles of control and mutant ddx27 zebrafish larvae (5 dpf). 5’ETS, 5’ITS1 and ITS2 probes were used to identify pre-rRNA and intermediate species targeted different steps of the processing pathways. The pre-rRNA intermediates are described in zebrafish. The corresponding human precursors are indicated into brackets. (D) Quantification of the pre-rRNA intermediates in zebrafish skeletal muscles. (E) Polysomal profiles of skeletal muscle in control and ddx27 mutant larvae (5 dpf).