Skeletal muscle abnormalities in ddx27 mutant zebrafish.

(A) Microscopic visualization of control and mutant larval zebrafish (osoi) at 5 days post fertilization (dpf). Mutant fish display leaner muscles (left panel) and exhibit highly reduced birefringence in comparison to control (right panel). Mutant fish also exhibit pericardial edema (arrow) (B) Genetic mapping of osoi mutant by initial bulk segregant analysis identified linkage on chromosome 6. Fine mapping of chromosome 6 resolved flanking markers z41548 and z14467, with a candidate genome region containing six candidate genes that were sequenced by Sanger sequencing (C) Overexpression of human DDX27 mRNA results in a significant decrease in mutant zebrafish phenotype (D) Whole-mount Immunofluorescence was performed on control and ddx27 mutant larvae (Z-stack confocal image, 4dpf) (scale bar: 50μm) (E) Immunofluorescence on newly isolated (Day 0) and cultured (Day1 and 3) EDL myofibers from wild-type mice (scale bar: 10μm). (F) Western blot showing relative expression of Ddx27 and myogenic markers (MyoD, MyoG and MF20) in proliferating C2C12 myoblasts in growth media (50% confluence) or in differentiation media for 3 days (D0-3). GAPDH was used as the control. (G) Schematic diagram of nucleolus depicting nucleolar domains. Eukaryotic nucleolus has tripartite architecture: Fibrillar center (FC); Dense fibrillar component (DFC) and granular compartment (GC). Immunofluorescence of human myoblasts with DDX27 and nucleolar markers labeling each compartment of nucleolus (scale bar: 2μm).

Skeletal muscle hypotrophy and precocious differentiation in Ddx27 deficiency.

(A-B) Histology of longitudinal skeletal muscle sections in control and ddx27 mutant stained with toluidine blue exhibiting enlarged nucleoli (arrowhead) and areas lacking sarcomeres (arrow) at 5 dpf. High magnification view (boxed area) (C-G) Transmission electron micrographs of skeletal muscles (longitudinal view: C-E, and cross-section view: F-G) in control and ddx27 mutant (5 dpf). Arrows indicating disorganized sarcomere (H) Quantification of myofiber size in control and ddx27 mutant fish (5 dpf) (n = 10) (I) qRT-PCR of control and ddx27 mutant fish showed a reduction in the expression of muscle stem cell markers (pax3, pax7) and an increase in expression of myogenic commitment genes (myod1 and myf5). The expression of late differentiation markers was reduced in ddx27 fish suggesting that pre-mature expression of early myogenic genes results in abnormal disorganization of skeletal muscles.

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Decrease in muscle precursor cells (MPC) proliferation and skeletal muscle regeneration in Ddx27 deficiency.

(A) Whole mount immunofluorescence of zebrafish at different time intervals (2 dpf and 4 dpf) with MPC marker (Pax7) and late differentiation marker (Mef2) demonstrating a decrease in of MPC in ddx27 mutant during post-embryonic skeletal muscle growth (4 dpf) (scale bar: 50μm) (B) Control and ddx27 mutant zebrafish were pulse-labeled with EdU for 2hr and immunostained with Pax7. Fish were analyzed for EdU and Pax7 labeling (4 dpf) by whole mount immunofluorescence. The proportion of proliferative Pax7 population was estimated by quantifying Pax7⁺/Edu⁺ double-positive nuclei out of total Pax7⁺ nuclei in control and mutant fish (scale bar: 50μm) (C) Trunk muscles in control and ddx27 zebrafish were injected with cardiotoxin (3 dpf). Skeletal muscles were analyzed at 5 dpf by whole mount immunofluorescence with Pax7 and phalloidin. Control muscles show an accumulation of Pax7 expressing cells at the site of injury (arrow) that was lacking in ddx27 muscles (arrow) (scale bar: 50μm).

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).

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Impaired differentiation in Ddx27 deficiency.

(A) Hematoxylin and eosin staining of embryonic skeletal muscle (2dpf) in control and ddx27 zebrafish.

(B) Skeletal muscle growth during embryogenesis (2 dpf) and larval stages (4 dpf) was assessed by measuring cross-section area of myofibers in control and mutant fish.

(C) Myonuclear content in control and ddx27 mutant fish was evaluated by quantifying the number of nuclei/myofiber in muscle cross-sections. 5 different areas in myotome were analyzed (n = 4).

(D) Control and ddx27 zebrafish myofibers (4 dpf) were cultured and immunofluorescence analysis was performed. Expression of sarcomeric α-actinin labeling Z-line was reduced in mutant myofibers. Expression of sarcoplasmic reticulum marker, Ryr1 showed a disorganized pattern in comparison to control myofibers (scale bar: 100μm).

(E) Three different guide RNAs (sgRNA) were designed targeting mouse Ddx27 gene. sgRNA targeted to exon3 of all three Ddx27 transcripts resulted in a 166 base pair homozygous insertion and generation of several stop codons. PCR analysis of genomic DNA revealed an insertion in exon3 of Ddx27 gene.

(F) Control and Ddx27 mutant C2C12 were plated at equal concentration and grown in the proliferation media. Cells were pulsed treated with Edu (FITC signal) and counterstained with DAPI.

(G) Control and Ddx27 mutant C2C12 cells were plated at equal cell density and grown in the differentiation media for 7 days. Control cells differentiated in to well differentiated myotubes/myofibers whereas Ddx27 cells exhibited a severe differentiation defects (scale bar: 50μm).

Cell death analysis of zebrafish skeletal muscle.

(A) Whole mount TUNEL labeling was performed in WT and ddx27 mutant zebrafish (3 and 4 dpf, n = 20) and myotome was analyzed.

(B) protein extracts were prepared from control and ddx27 mutant fish (3 and 4 dpf) and western blot analysis was performed with caspapse 3 antibody. Quantification of western blot revealed no significant differences in cell death in control and mutant zebrafish.