Figure 1.
MZ ewsa/ewsa mutant is a null mutant.
A. Schematic of the insertion site of the ewsa gene. After the 6th amino acid, a stop codon was introduced. Zebrafish ewsa gene (black), virus sequence (Red). B. Western blot of zebrafish cell lysates obtained from the wt/wt and MZ ewsa/ewsa embryos at 6dpf. Top panel: probed with the anti-Ewsa antibody (Ewsa, Ewsa protein; *: non-specific protein), bottom panel: probed with β-actin antibody.
Figure 2.
4 dpf MZ ewsa/ewsa mutants display an aberrant angle of Meckel’s cartilage and palatoquadrate.
A. Lateral views (anterior to the left) of wt/wt (left) and MZ ewsa/ewsa (right) and ventral views of adult zebrafish. The calcified bones were visualized by alizarin red staining. B. Lateral views (anterior to the left) of (a) wt/wt and (b) MZ ewsa/ewsa and ventral views of (c) wt/wt and (d) MZ ewsa/ewsa chondrocytes from 4 dpf zebrafish embryos visualized with alcian blue. (e and f) Angle formed by Meckel’s cartilage showing that the palatoquadrate is wider in the MZ ewsa/ewsa mutant than wt/wt at 4 dpf and 7 dpf. bh: basihyal, d: dentary, m: Meckel’s cartilage, pq: palatoquadrate, ch: ceratohyal, cb: ceratobranchial.
Figure 3.
Prehypertrophic chondrocytes of Meckel’s cartilage in MZ ewsa/ewsa fails to differentiate into hypertrophic chondrocytes.
A. Flat mounted Meckel’s cartilage of 4 dpf (left) wt/wt and (right) MZ ewsa/ewsa. B. Number of cells of Meckel’s cartilage of 4 dpf wt/wt and MZ ewsa/ewsa zebrafish (P = 0.0002). C. Lateral views (anterior to the left, dorsal to the top) of 4dpf (Left) wt/wt and (Right) MZ ewsa/ewsa visualized by in situ hybridization using probe for ihha (top panel) and colX (bottom panel). m: Meckel's cartilage. D. Ventral views (anterior to the left) of Meckel's cartilage of (Left) wt/wt and (Right) MZ ewsa/ewsa visualized by immunohistochemistry using (top) anti-IHH antibody (green), and anti-Collagen type II antibody (Red) (4dpf), and (bottom) anti-Collagen X antibody (green) and anti-Collagen type II antibody (Red) (5dpf).
Figure 4.
Sox9 interacts with Ewsa in zebrafish, and human SOX9 interacts with human EWS in HeLa cells.
A. Immunoprecipitation of the lysates obtained from 27 hpf zebrafish embryos using an IgG control and anti-SOX9 antibodies. Top panel: Blot with anti-Ewsa antibody (Input: 1/660). Bottom panel: Blot with anti-Sox9 antibody (Input: 1/70). B. Immunoprecipitation of lysates obtained from untransfected (untrans), empty vector-transfected (vector) and FLAG-SOX9-transfected HeLa cells using an IgG control and anti-FLAG M2 antibody. Bottom Panel: Blot with anti-SOX9 antibody. Top Panel: Blot with anti-EWS antibody (Input: 1/200).
Figure 5.
MZ ewsa/ewsa mutants display altered expression levels of Sox9 target genes.
The ratios of the Sox9 target mRNA expression levels between the wt/wt and MZ ewsa/ewsa mutants (27 hpf) are shown in a bar plot (* p<0.05 and ** p<0.01).
Figure 6.
MZ ewsa/ewsa mutants display altered expression domains of Sox9 target genes.
27hpf of (Left) wt/wt and (Right) MZ ewsa/ewsa visualized by in situ hybridization using antisense RNA probe for A. sox5, B. noggin1, C. noggin2, D. bmp4, E. ctgfa, F. ctgfb, G. col2a1a and H. col2a1b. Top panel: low magnification images, middle and bottom panel: high magnification images.
Figure 7.
Ewsa binds to ctgfa and ctgfb target genes.
A. Lateral views (anterior to the left, dorsal to the top) of 4dpf of (Left) wt/wt and (Right) MZ ewsa/ewsa visualized by in situ hybridization using probe for ctgfa (tom panel) and ctgfb (bottom panel). m: Meckel's cartilage. B. Ventral views (anterior to the left) of 4dpf Meckel's cartilage of (Left) wt/wt and (Right) MZ ewsa/ewsa visualized by immunohistochemistry using anti-Ctgf antibody (green), and anti-Collagen type II antibody (Red). C. (left) Schematics of ctgfa and ctgfb genes. Black circle: Sox9 binding site, black square: exon, arrows: PCR primer for ChIP assay. (right) ChIP assays were performed using 27 hpf zebrafish embryos. IgG: negative control of ChIP, anti-Ewsa: ChIP sample using EWSa antibody, input: 4.5% of DNA from total lysates was subjected to PCR, dw: negative control for PCR reaction.