Taniguchi Y et al. (2008), Spinal cord is required for proper regeneration...

XB-ART-37177

Dev Growth Differ 2008 Feb 01;502:109-20. doi: 10.1111/j.1440-169X.2007.00981.x.

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Spinal cord is required for proper regeneration of the tail in Xenopus tadpoles.

Taniguchi Y , Sugiura T , Tazaki A , Watanabe K , Mochii M .

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Tail regeneration in urodeles is dependent on the spinal cord (SC), but it is believed that anuran larvae regenerate normal tails without the SC. To evaluate the precise role of the SC in anuran tail regeneration, we developed a simple operation method to ablate the SC completely and minimize the damage to the tadpole using Xenopus laevis. The SC-ablated tadpole regenerated a twisted and smaller tail. These morphological abnormalities were attributed to defects in the notochord (NC), as the regenerated NC in the SC-ablated tail was short, slim and twisted. The SC ablation never affected the early steps of the regeneration, including closure of the amputated surface with epidermis and accumulation of the NC precursor cells. The proliferation rate of the NC precursor cells, however, was reduced, and NC cell maturation was retarded in the SC-ablated tail. These results show that the SC has an essential role in the normal tail regeneration of Xenopus larvae, especially in the proliferation and differentiation of the NC cells. Gene expression analysis and implantation of a bead soaked with growth factor showed that fibroblast growth factor-2 and -10 were involved in the signaling molecules, which were expressed in the SC and stimulated growth of the NC cells.

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Species referenced: Xenopus laevis
Genes referenced: dnai1 fgfr1 ncam1 tbxt.2
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	Fig. 1. Ablation of the spinal cord (SC) in the amputated tail. (A) Schematic diagram of surgery. The red line shows the SC. The SC-ablated and control tails were whole-mount stained with a monoclonal antibody against N-CAM (B, C), or sectioned transversally and stained with hematoxylin and eosin (D, E). The inset in (D) shows a section of the removed spinal cord. The arrow in (C) indicates the signal for the SC. The asterisk in (D) shows the meningeal cavity where the SC was located before the surgery. The arrowheads indicate sensory ganglion cells. NC, notochord.
	Fig. 2. Regeneration of the spinal cord (SC)-ablated tail. Regenerations of the SC-ablated tail and control tail were compared under a stereoscopic microscope (A, B) and Nomarski differential interference contrast (DIC) optics (C, D). A pair of arrowheads marks the amputation plane. The white dashed line in (C, D) marks the shape of the notochord (NC). Days after the operation are indicated in each panel. The length and diameter of the regenerated NC were quantified (E, F). The data points represent the average length. The error bars are standard deviations. The diameter was measured at the midpoint of the regenerated NC.
	Fig. 3. Histological analysis of regeneration in the spinal cord (SC)-ablated tail. The SC-ablated (A) and control (B) tails were sagittally sectioned and stained with hematoxylin and eosin. The notochord precursor cells accumulated at the edge of the notochord sheath (2 d, arrow), formed a compact cell mass (2.5 d), aligned along the proximo-distal axis (3 d), elongated and finally vacuolated (5 d). The SC ablation resulted in a reduced size of the compact cell mass (2.5 d, 3 d) and delayed vacuolation (5 d). A pair of arrowheads marks the amputation plane. Days after the operation are indicated in each panel. The magnification is the same in all images except for the image taken at 5 days.
	Fig. 4. Proliferation of the notochord (NC) cells in the spinal cord (SC)-ablated tail. Proliferative cells in the SC-ablated (A–C) and control (D–F) tails were detected by BrdU labeling (magenta; B, E) at 2.5 days after amputation. (A, B) All nuclei were stained with Hoechst 33342 (green). (C, F) show merged images of (A, B) and (D, E), respectively. The white dashed line in (C, F) marks the shape of the NC precursor. All sections are horizontal. (G) The proliferation rate of the NC precursor cells was determined by counting the BrdU-positive cells in five tadpoles for each experiment. The error bars indicate standard deviations. The P-value is 0.019.
	Fig. 5. Immunohistochemical detection of nerve cells and myofibers in the spinal cord (SC)- ablated tail. The SC-ablated (A) and control tail (B) were wholemount stained with monoclonal antibody Xen-1 to detect nerve cells. The arrows show dorsal and ventral peripheral nerves in the regenerated tail. The white arrow shows the regenerated SC. (C, D, E, F) Immunological staining of the regenerating tails with monoclonal antibody 12/101 to detect myofibers. A pair of arrowheads mark the amputation plane. Days after the operation are indicated in each panel. (F′) Higher magnification view of (F). Bar, 500 μm.
	Fig. 6. Gene expression analysis in the spinal cord (SC)-ablated and control tails. Semi-quantitative gene expression analysis by reverse transcription–polymerase chain reaction (RT–PCR) was carried out (A–C). The expression of marker genes for the tail regeneration was analyzed in regenerating tails of the SC-ablated and control tadpoles (A). The expression of fibroblast growth factor (FGF) and FGF receptor (FGFR) genes was analyzed (B). Isolated SC was also analyzed by RT–PCR (C). Specific primer pairs and PCR conditions are indicated in Table 1. (D–N) Section in situ hybridization of the regenerating tails at day 2.5. The spatial expression of Xfgf-2, -8, and -10 (D–F) and Xfgfr-1, -2, -3, and -4 (K–N) was analyzed on the regenerating tail. Expression of Xbra3 was analyzed to mark the regenerating notochord (G). The expression of Xfgf-2, -8 and -10 was analyzed on the SC-ablated regenerating tail as well (H–J). A pair of arrowheads marks the amputation plane. Black and white dashed lines indicate the shapes of the regenerating SC and NC, respectively. NC, notochord.
	Fig. 7. Effect of fibroblast growth factor (FGF)-2 and -10 on regeneration in the spinal cord (SC)-ablated tail. A bead soaked with FGF or phosphate-buffered saline (PBS) was implanted in the SC-ablated tail on day 1. The regenerated notochord (NC) (marked with a line) with the FGF- 10-soaked bead is larger than that of the control with the PBSsoaked bead 4 days after amputation (A, B; see Table 2). The implanted bead was located by its blue color. (C) The proliferation rate of the NC precursor cells was determined at day 3 by counting the BrdU-positive cells (see Table 3). The control in (C) shows the proliferation rate in the regenerating tail with SC. The error bars indicate standard deviations.