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First published online 10 May 2006
doi: 10.1242/dev.02397

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Control of muscle regeneration in the Xenopus tadpole tail by Pax7

Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK.

View larger version (76K): [in a new window]   Fig. 1. Expression pattern of pax7 in Xenopus early development. Whole-mount in situ hybridization was performed with the pax7 antisense RNA probe. (A-C) Dorsal view of stage 13 (A), stage 16 (B) and stage 19 (C) embryos, anterior towards left. The inset in B is an anterior transverse section of a stage 16 embryo, showing that pax7 is expressed in the sensorial layer of neural ectoderm and in the lateral plate mesoderm. The inset in the bottom left-hand corner in C shows the segmented pattern of pax7 expression in a stage 19 embryo. The inset in the top right-hand corner is a view of an anterior transverse section. (D-F) Lateral view of stage 25, 33 and 35 embryos, anterior towards left. The arrowheads in D,E indicate the expression domain of pax7 in the pronephros. Arrows in D,E show the chevron pattern of pax7 expression in somites. The inset in F is an anterior view of stage 35 embryo. The lines in F indicate the relative position of cross-section planes in H-J,L. (G) Sagittal section of stage 35 embryos. The inset indicates the transcripts of pax7 in spinal cord concentrated on the dorsal side. (H) Transverse section through the midbrain of a stage 35 embryo. (I) Pax7 expression in the pituitary anlage is marked by an asterisk. (J,L) Transverse section through the trunk of a stage 35 embryo. (K) Parasagittal section of stage 35 embryo showing pax7 transcripts in posterior pronephric anlage. (M) Horizontal section of stage 35 embryo head. (N) Parasagittal section of stage 35 embryo. The black arrows in H,M,N indicate the mesenchyme cells with pax7 expression anterior to the eye. The white arrow in N indicates that pax7 transcripts locate in the edges of myotomes. (O) Parasagittal view of the tail of stage 35 embryo. Abbreviations: e, eye; mb, midbrain; n, notochord; pa, pituitary anlage; pr, pronephros; sc, spinal cord; so, somite.

View larger version (116K): [in a new window]   Fig. 2. Pax7 antibody detection in stage 46 tadpole. Immunostaining with anti-Pax7 monoclonal antibody was carried out on transverse sections of stage 46 tadpoles. (A-C) Detection of Pax7 with DAB staining (brown). The tissues were counterstained with 0.5% Methyl Green solution. (A) Midbrain; (B) hindbrain; (C) spinal cord. (D) Immunostaining of Pax7 (red) and DAPI (blue) on cross-section of tadpole head. The arrow in D indicates expression of Pax7 in eye muscle. (E) Co-immunostaining of Pax7 (red) and laminin (green) on cross-section of tadpole tail muscle. Abbreviations: hb, hindbrain; mb, midbrain; pg, pituitary gland; sc, spinal cord. Scale bars: 20 µm.

View larger version (142K): [in a new window]   Fig. 3. Immuno-electron microscopy study of Pax7 and BrdU labelling in tadpole tail. (A,B) Satellite cell with Pax7 antibody labelling in the nucleus. (C,D) High-power views of black box regions of A and B, respectively. The basement membrane is indicated by the arrows in C,D. (E) A myonucleus negative for Pax7 signal. (F) A Schwann cell with Pax7 antibody labelling in the nucleus. (G) A lymphocyte located between myofibres is positive for Pax7. (H) A fibroblast in the muscle connective tissues has a faint positive signal. (I) A satellite cell with BrdU labelling in the nucleus. (J) A myonucleus negative for BrdU.

View larger version (53K): [in a new window]   Fig. 4. Pax7 is upregulated in regenerating tadpole tails. (A) RT-PCR shows that pax7 messenger RNA is more abundant in regenerating tails at 3 dpa, compared with the tails without amputation. (B) PCNA antibody staining (red) on parasagittal section of regenerating tail. DAPI (blue) shows the nuclei. The dense region in the centre is the notochord tip, with the blastema above and below. (C) Pax7 antibody-labelled cells (brown) on parasagittal sections of regenerating tail. Dorsal side upwards and anterior leftwards. Scale bars: 100 µm.

View larger version (95K): [in a new window]   Fig. 5. Pax7EnR functions as a dominant-negative form of Pax7. (A,C,D,F) Embryos injected with 200 pg pax7EnR mRNA and 80 pg GFP into left side of dorsal animal hemisphere at four cell stage. (A) A stage16 embryo; the arrow indicates the anterior neural fold defect on left-hand side. (C) Left side of pax7EnR injected embryo, the arrow indicates the developing eye. The fluorescence in the inset shows a small lens underneath the epidermis. (D) Eye development on the un-injected side. (F) A tadpole with a smaller left eye (arrows). (B,E) Co-injection of 500 pg pax7 mRNA, 200 pg pax7EnR mRNA and 80 pg GFP is able to rescue the defective eyes to normal size. (G) Injection of 500 pg pax7 and 80 pg gfp mRNA into one side of the dorsal animal hemisphere results in an ectopic eye in the forebrain of stage 43 tadpoles. The GFP fluorescence in the insets indicates the injected region. (H-J) Transverse sections through ectopic eye of injected tadpole (showed in inset) were stained with Hematoxylin and Eosin. The arrows in G,H indicate the extra eyes. (I) A magnified view of the ectopic eye and one normal eye. The asterisk in J highlights the tube protruding from the ventral midbrain. Scale bars: 300 µm.

View larger version (45K): [in a new window]   Fig. 6. The number of muscle satellite cells is reduced in the heat shocked pax7EnR transgenic tails. (A) RT-PCR detection of pax7, pax6 and pax7EnR in wild-type and pax7EnR transgenic tadpoles with or without heat shock treatment. (B-E) The expression of Pax7 (red) in transverse sections of the tails. (B) Wild-type tail. (C) Wild-type regenerating tail with daily heat shock for 7 days after amputation. (D) pax7EnR transgenic tail. (E) pax7EnR transgenic regenerating tail with daily heat shock for seven days after amputation. (F) The histogram shows the number of satellite cells quantified by Pax7 antibody staining. Ten tails of similar size were examined in each case. **P<0.05. The d7 bars refer to 7-day regenerates. The d14 bars refer to 14-day regenerates amputated at a more distal level to provoke a second regeneration. Because of the more distal position, the absolute cell numbers per section are lower, but the wild type still has about twice the number of satellite cells compared with the heat-shocked pax7EnR. Scale bars: 20 µm.

View larger version (89K): [in a new window]   Fig. 7. Pax7EnR promotes satellite cell apoptosis during tail regeneration. (A-C) TUNEL assays were performed on parasagittal sections of regenerated tail at 3 dpa; the colour was developed with a Fast Red tablet and counterstained with Mayor's Hematoxylin. (A) Wild-type regenerated tail. (B) Heat-shocked pax7EnR transgenic regenerated tail. (C) Higher power view of B. TUNEL signals in the blastema region are arrowed. (A-C) Dorsal side upwards, anterior towards left. (D) Detection of Pax7 with DAB staining (left panel) and apoptosis with fluorescein labelling (middle panel), in a cluster of cells in pax7EnR transgenic tail. The right panel is the merged view. Red arrows: two TUNEL-positve/Pax7 low expression cells. (E) Quantification of the number of apoptotic cells in the two groups of tails. (F,G) Detection of MyoD (red) and cell death (green) on parasagittal sections of regenerated tails at 3 dpa. (F) Blastema region of wild-type tail. (G) Blastema region of heat-shocked pax7EnR tails. (H) Quantification of the percentage of apoptotic cells among the MyoD-positive cells in the two groups of tails. Ten tails of similar size were examined in each group. **P<0.01. Abbreviations: S.C., spinal cord; Not., notochord; Mes., mesenchyme; Blt., blastema. Scale bars: 100 µm.

View larger version (38K): [in a new window]   Fig. 8. 12/101 staining of regenerating tails in wild-type and pax7EnR transgenic tadpoles. (A) The flowchart in the experiment of repeated tail amputation. The first heat shock is given 3 hours before the first amputation and heat shocks are given daily thereafter. The distal 50% tail was cut in the first tail amputation. The second tail amputation was performed at 14 dpa at the site of distal 75% of the first regenerated tail. The purple and blue tails indicate the primary and secondary regenerated tail, respectively. (B-F) 12/101 antibody staining of the regenerated tails. (B) Wild-type tails 14 days post first amputation. (C) pax7EnR transgenic tails 14 days post first amputation. (D) Wild-type regenerated tails, 14 days after second amputation. (E,F) pax7EnR regenerated tails 14 days after second amputation. (G,H) Transverse section of second regenerated tail in wild type (G) and pax7EnR transgenic tadpoles (H). Section level is indicated by the black lines in D,E. Abbreviations: sc, spinal cord; not., notochord; mus, muscle. Scale bars: 500 µm in B-F; 50 µm in G,H.

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