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First published online August 23, 2004
Journal of Experimental Biology 207, 3329-3338 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01146

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Characterization of Fxr1 in Danio rerio; a simple vertebrate model to study costamere development

Bart Engels, Sandra van 't Padje, Lau Blonden, Lies-anne Severijnen, Ben A. Oostra and Rob Willemsen^*

Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands

View larger version (98K): [in a new window]   Fig. 1. Comparison of human FXR1P and the zebrafish Fxr-protein family. Identical residues are shaded in black, and conserved substitutions in grey. The following functional domains are indicated: the nuclear localization signal (NLS), the two KH1 RNA-binding domains (KH1), the nuclear export signal (NES), the conserved casein kinase II phosphorylation site found in Drosophila Fmrp (CKII), the region containing the RNA interaction RGG box (RGG) and two nucleolar targeting signals (NoS1 and NoS2). The boxed region of mouse and zebrafish Fxr1p that contains the second NoS indicates the alternatively spliced exon 15 of Fxr1p. The epitope of the affinity-purified anti-Fxr1p antibody Affi5 is depicted with the synthetic peptide.

View larger version (86K): [in a new window]   Fig. 2. Specificity of affinity-purified antibody Affi5. Cos-1 cells were transiently transfected with EGFP-Fxr1 (A,B) or with EGFP-Fxr2 (C,D), and stained with Affi5. The EGFP signal (A,C, green) is present in both transfected Cos-1 cells, while Affi5 labelling is only present in EGFP-Fxr1 transfected Cos-1 cells (B, red). Cos-1 cells transfected with EGFP-Fxr2 show absence of labelling after Affi5 incubation (D). Lysates of EGFP-Fxr1 (lanes 1) and EGFP-Fxr2 (lanes 2) transfected Cos-1 cells were immunoblotted using antibodies against EGFP (E, lanes 1 and 2) and Affi5 (F, lanes 1 and 2). Note the absence of cross reactive material in the lane with EGFP-Fxr2 transfected Cos-1 cells using Affi5 antibodies (F, lane 2). Bars, 10 µm.

View larger version (133K): [in a new window]   Fig. 3. Immunohistochemical analysis of Fxr1p expression in adult zebrafish tissues. Cryostat sections from adult zebrafish were immunoincubated using Affi5 antibodies. Significant Fxr1p expression was present in the brain (A), the testis (B) and in skeletal muscle tissue (C). Note the nuclear labelling in Purkinje cells of the cerebellum (A). All immature spermatogenic cells of the testis showed Fxr1p expression (B). A transversal section of skeletal muscle shows that Fxr1p expression is mainly present at the sarcolemma and at the myoseptum (C). A higher magnification of the myoseptum is shown in (D). P, Purkinje cells; I, immature spermatogenic cells; S, sarcolemma; M, myoseptum. Scale bars, 3.5 µm (A); 30 µm (B); 80 µm (C); 320 µm (D).

View larger version (96K): [in a new window]   Fig. 4. Immunohistochemical analysis of Fxr1p expression in zebrafish embryos during embryonic development. (A) Longitudinal cryostat sections of 6 h.p.f. embryos, (B,C) head (B) and tail (C) of 1-day-old embryos, and (D,E) head (D) and tail (E) of 3 d.p.f. embryos stained for zebrafish Fxr1p using Affi5. Inset in D shows a higher magnification of the boxed region in the hindbrain from D. Note the nuclear staining in neurons from 3 d.p.f. embryos (D) and the very intense staining of myoblasts in muscle tissue within the somites (E). Y, yolk sac; M, myoseptum; N, neural tube. Arrows in C point to Fxr1p immunoreactive myoblasts in the developing somite. Scale bars, 15 µm (C); 30 µm (B); 35 µm (E); 70 µm (A); 100 µm (D).

View larger version (55K): [in a new window]   Fig. 5. Analysis of Fxr1p isoforms in adult zebrafish tissues, Homogenates of adult zebrafish brain, muscle and testis were immunoblotted using Affi5 antibodies to detect the presence of molecular forms from Fxr1p. In brain a prominent band of approximately 73 kDa and a low-intensity band of approximately 70 kDa are present. In skeletal muscle high molecular mass isoforms of Fxr1p are present of approximately 86-88 kDa. The detection of Fxr1p isoforms in testis results in a prominent band of 73 kDa and weaker bands of 77 kDa and 88 kDa.

View larger version (78K): [in a new window]   Fig. 6. Colocalization of Fxr1p with components of the translational machinery. Transverse cryostat sections of skeletal muscle tissue from adult zebrafish were stained for Fxr1p (B, red; D, green), Staufen (A, green) and P0 (E, red). (C) and (F) The merged images of A,B and D,E, respectively. Colocalization is shown by yellow staining. M, myoseptum; S, sarcolemma. Bars, 40 µm.

View larger version (78K): [in a new window]   Fig. 7. Fxr1p is localized next to vinculin and dystrophin. Longitudinal cryosections of skeletal muscle tissue from adult zebrafish were stained for Fxr1p (A and D, green), vinculin (B, red) and dystrophin (E, red) and examined using confocal microscopy. Images were taken in one confocal plane. (C) and (F) show overlays of Fxr1p with vinculin (bar, 5 µm) and dystrophin (bar, 50 µm), respectively. Note that only partial overlap is observed. M, myosepta.