SEARCH

SEARCH BY CITATION

Keywords:

  • gene expression;
  • larva;
  • regeneration;
  • tail;
  • Xenopus laevis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The regeneration of the amputated tail of Xenopus laevis larvae is an excellent model system for regeneration research. The wound left by the amputated tail is covered with epidermis within 24 h. Then, the cell number increases near the amputation plane at the notochord, spinal cord and muscle regions. An apparently complete tail with notochord, muscle and spinal cord is regenerated within two weeks. To reveal whether the molecular mechanism underlying the tail regeneration is the same as that in embryonic tail development, the gene expression patterns of the embryonic tail bud and the regenerating tail were compared by in situ hybridization and reverse transcription-polymerase chain reaction. Most genes analyzed were expressed at similar levels in both tissues, whereas two bone morphogenetic protein (BMP)-antagonists, chordin and noggin, were detected only in the embryonic tail bud. The regenerating tail also lacked expression of Xshh in the floor plate and expression of Xdelta-1 in the spinal cord and presomitic mesoderm. These results show that there are some differences in gene expression between the two processes. Furthermore, when the tail of Xenopus larvae is amputated, the regenerating tail has a gene expression pattern similar to the distal portion of the larval tail rather than the embryonic tail bud, suggesting that the cut larval tail does not make a new embryonic tail bud, but rather a new larval tail tip for regeneration.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Urodeles and larval anurans have been widely used for regeneration research because of their ability to regenerate lost body parts at organ level (Goss 1969; Stocum 1995; Tanaka 2003). The Xenopus laevis larva has a powerful regeneration capacity in limb and tail, although the regeneration capacity is gradually limited as development proceeds (Stocum 1995). Intensive studies using X. laevis for early embryogenesis have resulted in the isolation of numerous marker genes to describe many developmental events and, recently, many more collections of cDNA as expressed sequence tags (EST). Establishment of an efficient transgenic method for X. laevis made it possible to manipulate gene expression in tadpoles and frogs (Kroll & Amaya 1996). In fact, the transgenic method was recently used for regeneration research of Xenopus (Ryffel et al. 2003; Beck et al. 2003).

Studies in urodeles and anurans have shown that many aspects of their morphology and gene expression are common between normal development and regeneration (Stocum 1995). It has been reported that several genes expressed in the embryonic tail bud are re-expressed in the regenerating tail. For example, some Hox genes and bone morphogenetic protein (BMP) signal- and Notch signal-related genes are expressed both in the embryo and the regenerating tail (Beck et al. 2003; Christen et al. 2003). But it is not clear to what extent the molecular mechanism of the tail regeneration resembles that of normal tail development. To elucidate fundamental events in the tail regeneration of Xenopus larvae, we performed a gene expression analysis to compare the processes of normal tail development and tail regeneration.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Animals and surgery

All experiments were carried out on embryos and larvae of X. laevis maintained at 22°C. Developmental stages were determined according to Nieuwkoop & Faber (1967). Tadpoles were mounted on wet paper and the tail was amputated vertically at its midpoint using a razor. The amputated tadpoles were maintained in water containing penicillin (50 U/mL) and streptomycin (50 µg/mL) for 4 days without feeding, and for an additional 6 days with feeding.

Histological and immunological analysis

Isolated tail regions were mounted on slides after fixation with Buin solution and observed using Nomarski optics. For histology, specimens fixed with Buin solution were embedded in paraffin, sectioned, then stained with hematoxylin and eosin by a standard method.

For immunological staining, the isolated tails were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 2 h and treated with 10% H2O2 in ethanol for bleaching. The bleached tails were successively treated with PBS containing triton X-100 (0.1%, PBST), PBST containing goat serum (10%), and a monoclonal antibody diluted in PBST containing goat serum. After washing with PBST, the tails were treated with horse radish peroxidase (HRP)-labeled antimouse IgG (Bio-Rad, Hercules, CA, USA). Detection was performed by incubation with 0.05% diaminobenzidine (DAB) and 0.003% H2O2. Monoclonal antibodies, 12/101 for muscle cells (Kinter & Brockes 1984) and Xen1 for neural cells (Ruiz i Altaba 1993), were obtained from the Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, IA, USA).

In situ hybridization

Bluescript plasmids encoding Xsox-2 (xl014h03), chordin (xl046c22), Xshh (xl09620), Xnot (xl049a03), and XmyoD (xl078m12) were characterized by reference to the results of the NIBB/NIG X. laevis EST Project (http://xenopus.nibb.ac.jp/). The plasmids noggin-pGEM5Z (Smith & Harland 1992) and Xwnt5a-pBS (Moon et al. 1993) were gifts from Drs R. M. Harland (University of California, Berkeley, CA, USA) and R. T. Moon (University of Washington, WA, USA), respectively. Complementary DNA for Xdelta-1 (Chitnis et al. 1995), Xbra (Smith et al. 1991) and Xbra-3 (Hayata et al. 1999) were amplified from gastrula cDNA using specific primers and cloned into pBluescript vector. The primers used for the cloning were as follows: Xdelta-1 (US: 5′-TGAATAAGAAGGGACTGCTGGG-3′; DS: 5′-TTCTGCACTCTAACCCTCACAC-3′), Xbra (US: 5′-CCTGGATCCCA-ATGCAATGT-3′; DS: 5′-GTGCTAAGCTTCTATATCCA-3′), and Xbra-3 (US: 5′-ATCTTGCCTTTGGGACTGGT-3′; DS: 5′-AGGATCTACTGGTGGACAAAAC-3′). DIG-labeled RNA probes were synthesized with T7 or T3 RNA polymerase (Invitrogen, Carlsbad, CA, USA) and DIG nucleotide mixture (Roche, Basel, Switzerland).

Whole mount in situ hybridization was performed as described by Harland (1991), with some modifications. Embryos or larval tails were fixed with methanol–formalin solution (50% methanol, 30% formalin) at 4°C overnight. RNase treatment was omitted. Specific signals were detected with BM purple reagent (Roche). After color development, embryo samples were bleached in a solution containing 10% H2O2 and 70% methanol.

For in situ hybridization of sections, the methanol–formalin-fixed embryos and 4% paraformaldehyde-fixed regenerating tail were embedded in paraffin and sectioned as described previously (Kobayashi et al. 1998). The sectioned specimens were incubated with a prehybridization solution containing formamide (50%), standard saline of citrate (SSC; 2 x), and sodium dodecylsulfate (SDS; 1%) at 60°C for 30 min, and then hybridized with DIG-labeled RNA probes in a solution containing formamide (50%), SSC (5 x), SDS (0.5%), and torula yeast RNA (100 µg/mL; Sigma, St. Louis, MO, USA) at 60°C for 17 h. After washing with the prehybridization solution at 60°C, specific signals were detected with alkaline phosphatase-labeled anti-DIG antibody and BM purple reagent according to the manufacturer's manual (Roche).

Reverse transcription-polymerase chain reaction analysis

Total RNA was extracted from the embryo or tail sample with TRIzol reagent according to the manufacturer's manual (Invitrogen). The regenerating tail was cut off vertically at a site about 0.5 mm proximal to the amputation plane. Complementary DNA was synthesized from total RNA (0.5 µg) with Superscript II reverse transcriptase (RT; Invitrogen) and random hexamers. The primers, annealing temperature, and cycle number for each polymerase chain reaction (PCR) were as described in Table 1. The reaction conditions were empirically determined in each case within the linear range of amplification.

Table 1.  Primer pairs and cycling conditions of reverse transcription–polymerase chain reaction
 OriginSequenceAnnealing temp. (°C)Cycle no.
nogginXenopus molecular markerU: 5′-AGTTGCAGATGTGGCTCT-3′5520
resourceD: 5′-AGTCCAAGAGTCTGAGCA-3′  
chordinXenopus molecular markerU: 5′-AACTGCCAGGACTGGATGGT-3′5525
resourceD: 5′-GGCAGGATTTAGAGTTGCTTC-3′  
XnotXenopus molecular markerU: 5′-ATACATGGTTGGCACTGA-3′5023
resourceD: 5′-CTCCTACAGTTCCACATC-3′  
XbraXenopus molecular markerU: 5′-GGATCGTTATCACCTCTG-3′5526
resourceD: 5′-CTGTAGTCTGTAGCAGCA-3′  
Xbra-3Strong et al. (2000)U: 5′-CAAACCCTGTTGGAGTTG-3′5027
resourceD: 5′-CCTTCTCACTTCCAACTTGC-3′  
XshhEkker et al. (1995)U: 5′-CTTCGCTCGGACGAGATGCTGG-3′5524
D: 5′-CCTTCGTATCTGCCGCTGGCC-3′  
XmyoDXenopus molecular markerU: 5′-AACTGCTCCGATGGCATGATGGATTA-3′5520
D: 5′-GATGCTGGGAGAAGGGATGGTGATTA-3′  
Xsox-2NewU: 5′-CCACACGCCGCCTCGATGT-3′5524
D: 5′-TCAGCCCCCAGCCTCTTGC-3′  
Xdelta-1Wittenberger et al. (1999)U: 5′-AATGAATAACCTGGCCAACTG-3′5524
D: 5′-GTGTCTTTTGACGTTGAGTAG-3′  
Xwnt-5aNewU: 5′-GATCCTACAGCTCCTCCT-3′5524
D: 5′-CTAACGACCACCAGGAGCT-3′  

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Morphological and immunological observations of the tail regeneration

The posterior half of the tails of stage 48 tadpoles were amputated, and then the tadpoles were allowed to recover for 0.5–10 days. The remaining tails were observed with and without antibody staining (Fig. 1). In a normal tail, the notochord is located centrally, in contact with a spinal cord dorsally, with bilateral muscle masses covering both the notochord and the spinal cord (Fig. 1A). After amputation, a transparent mass of the notochord protruded posteriorly from the amputation plane (Fig. 1B). Within 24 h after the operation, the protruded notochord was covered with an epidermis (Fig. 1C). The epidermis became thick in the most distal region and formed a wound epidermis of multicell layers (Fig. 1F). Immediately after the wound was covered, round blood cells appeared near the amputation plane both in the fin and the notochord regions (Fig. 1C,G and data not shown). At the same time, mesenchymal cells appeared in the notochord and muscle regions near the amputation plane, and their numbers increased in the later stages (Fig. 1G,H,I). Between 1.5 and 2 days after amputation, a compact cell mass of the notochord precursor was formed within a notochord sheath near the amputation plane (Fig. 1G). Then, the compact cells formed a new notochord, growing posteriorly (Fig. 1E,H). In the forming notochord, cells were arranged along the anterior–posterior axis, and more differentiated cells were located in the more anterior regions. The arranged cells became flat and then vacuolated to differentiate terminally into mature notochord cells (Fig. 1H). A dorsal and ventral fin grew posteriorly as the notochord elongated. Between 1.5 and 2 days after amputation, a posterior portion of the spinal cord formed a terminal vesicle located dorsally to the forming notochord (Fig. 1D,G,L). The spinal cord elongated as the notochord grew until 10 days after amputation, but the terminal vesicle had disappeared by day 4 (Fig. 1M). In the muscle region just anterior to the amputation plane, the number of mesenchymal cells increased after day 1. Muscle cells positive for the monoclonal antibody 12/101 began to appear on day 4 at the proximal portion of the regenerated area and increased their number as the regenerated tail grew (Fig. 1J,K). The newly formed myotubes were arranged along the anterior–posterior axis, but did not form the hook-shaped segments observed in a normal tail.

image

Figure 1. Morphological and immunological analysis of the tail regeneration. Transverse section of the normal tail of the stage 48 larva was stained with hematoxylin and eosin (A). The amputated tail was observed with Nomarski optics at days 0 (B), 1 (C), 2 (D), and 3 (E) after amputation. Sagittal sections of the regenerating tail at days 1 (F), 2 (G), and 3 (H) after amputation, and frontal section at day 3 (I), were stained with hematoxylin and eosin. Muscle cells were detected on days 4 (J) and 5 (K) by staining with the monoclonal antibody 12/101, and neural cells on day 2 (L) and day 4 (M) with the monoclonal antibody Xen1. The amputation plane is indicated by two arrowheads. NC, notochord; SC, spinal cord; M, muscle mass; NS, notochord sheath; TV, terminal vesicle of the regenerating spinal cord. Bar, 50 µm.

Download figure to PowerPoint

Expression of organizer-specific genes

The tail region of the larva is formed from the trunk region and the tail bud region of the embryo (Tucker & Slack 1995). Gene expression analyses in the tail-forming region of embryos revealed that some organizer-specific genes are expressed in the tail bud, suggesting that differentiation of neural and mesodermal cells is continuously induced in this region in the same fashion as in the dorsal-blastopore region at the gastrula stage (Gont et al. 1993; Beck & Slack 1998). If the tail regeneration of the larva proceeds in a similar fashion as the embryo, some organizer-specific genes may be expressed in the regenerating tail and induce neural and mesodermal cells. To verify this hypothesis, we performed a gene expression analysis using whole mount in situ hybridization and RT–PCR.

The expression of three organizer-specific genes, chordin, noggin and Xnot, was analyzed in embryos (stages 30 and 36) and regenerating larval tails (days 2 and 4 after amputation) by whole mount in situ hybridization. Chordin and Xnot were expressed in the tail bud and notochord of stage 30 embryos, as reported previously (Sasai et al. 1994; Gont et al. 1993), but noggin was expressed in the dorsal region of the tail bud in addition to the notochord (Fig. 2A,B,C). These expressions were also detected in stage 36 embryos. Whole mount in situ hybridization using the regenerating tails did not detect expression of chordin or noggin either on day 2 or 4, while Xnot was expressed faintly on day 2 and clearly on day 4 in the regenerating notochord (Fig. 2C). To confirm the above result, the RT–PCR method was employed and revealed no expression of chordin or noggin in the regenerating tail (Fig. 3).

image

Figure 2. Whole mount in situ hybridization of embryos and regenerating tails. Expression of the indicated genes was detected in stage 30 and stage 36 embryos and in regenerating tails on days 2 and 4 of regeneration. Arrowheads indicate positive signals with relatively weak intensity. Magnification common to each stage. Bar, 100 µm.

Download figure to PowerPoint

image

Figure 3. Reverse transcription–polymerase chain reaction (RT–PCR) analysis of gene expression in developing and regenerating tails. Gene expression was analyzed by the RT-PCR method in regenerating tails on days 0, 2 and 4 of regeneration, in the tail buds of stage 30 embryos, and in the tail tip regions of stage 48 larvae. Specific primer pairs and PCR conditions are indicated in Table 1.

Download figure to PowerPoint

Expression of notochord-specific genes

The expression of genes reported to be expressed in the embryonic notochord was analyzed to reveal whether the notochord is formed in the regenerating tail by a gene expression mechanism similar to that of embryogenesis. Like Xnot, the genes Xbra, Xbra3 and Xshh were expressed in the regenerating notochord at day 4 after amputation (Fig. 2D,E,F). Signals for Xbra-3 and Xshh were also detected on day 2. This result was confirmed by RT–PCR analysis showing that the expression of Xnot, Xbra, Xbra3 and Xshh were upregulated during the tail regeneration process (Fig. 3).

Gene expression in the spinal cord

The expression of genes known to be expressed in the embryonic neural tube and spinal cord was analyzed in the regenerating tail by whole mount in situ hybridization. Xsox2 was expressed in both the embryonic and the regenerating spinal cords, suggesting the expression of pan neural genes in the regenerating spinal cord (Fig. 2G). Xdelta-1 was not detected in the regenerating spinal cord, but was expressed in the neural tube at stages 30 and 36 (Fig. 2H). In embryos, Xshh is known to be expressed in the floor plate of the neural tube in addition to the notochord, and it plays a role in ventral specification of the spinal cord (Ruiz i Altaba 1995). Because whole mount analysis can hardly distinguish between the hybridization signals for the floor plate and notochord, we examined Xshh expression by in situ hybridization of transverse sections. No Xshh expression was observed in the ventral spinal cord of the regenerating tail, while Xshh expression was clear in the stage 30 neural tube and stage 42 spinal cords (Fig. 4A,B,C).

image

Figure 4. In situ hybridization of sections. The expression of Xshh, Xbra-3, and Xwnt-5a was analyzed by in situ hybridization using sectioned samples. Transverse sections of stage 30 (A) and stage 42 (B) tail regions, and of the regenerating larval tail on day 4 (C), were stained with Xshh probe. Sagittal sections of stage 30 (D) and stage 36 (E) tail regions, and of the regenerating tail on day 2 (F), were stained with Xbra-3 probe. Sagittal section of stage 30 (G) and stage 36 (H) tail regions and regenerating tail on day 4 (I) were stained with Xwnt-5a probe. Broken line indicates neural tube. Arrowheads in (A) and (B) show Xshh expression in the floor plate. Arrowheads in (D) and (E) show the posterior expression of Xbra-3. Bars, 20 µm (C), 50 µm (A,B,H,F), 100 µm (D,G,E,I).

Download figure to PowerPoint

Genes expressed in other mesodermal tissues

Xbra-3 and Xbra were expressed in the posterior wall of the tail bud in addition to the chordoneural hinge (Fig. 2D,E), as reported previously, indicating their roles in dorsal, lateral and ventral mesoderm specifications (Gont et al. 1993; Hayata et al. 1999). Whole mount in situ hybridization of the regenerating tail shows no obvious signals corresponding to the posterior expression in the embryonic tail bud (Fig. 2D,E). In situ hybridization of sections was carried out to examine the expression pattern in detail. The posterior expression of Xbra-3 was clearly identified in stage 30 and 36 tail-bud sections (Fig. 4D,E), but no signals other than those for the notochord were detected in the regenerating tail, indicating that there were no Xbra-3 expressing cells corresponding to the lateral and the ventral mesoderm during regeneration (Fig. 4F).

To identify the region where muscle cells are formed, the expression of XMyoD was examined by whole mount in situ hybridization. As expected from the results of staining with 12/101 monoclonal antibody (see Fig. 1), the signal for XmyoD was not detected in the distal region of the regenerating tail where the notochord is formed, but was detected in the proximal region (Fig. 2I). This result shows that muscle cells are differentiated in or near the remaining muscle tissue during the regeneration process.

Xdelta-1 is expressed in broad tail bud and presomitic mesoderm regions of embryos (Fig. 2H). Whole mount in situ hybridization revealed that it was also expressed in a broad pattern in the regenerating tail, as reported previously (Beck et al. 2003). But an expression domain corresponding to the presomitic mesoderm was not found in the regenerating tail.

Xwnt-5a was found to be expressed both in the embryonic tail bud and regenerating tail by whole mount in situ hybridization (Fig. 2J). We attempted to determine the precise expression domain by sectioning these tail samples and performing whole mount in situ hybridization. Xwnt-5a was expressed in a broad region of the tail bud of a stage 30 embryo, including the chordoneural hinge, spinal cord, posterior wall, dorsal region and ventral regions (Fig. 4G). This expression persisted at stage 36 (Fig. 4H). A section of the regenerating tail at day 4 after amputation showed that Xwnt-5a was expressed in a broad region of the regenerating tail, including the posterior notochord, posterior spinal cord, dorsal and ventral mesenchymes, and epidermis.

Comparison between the regenerating tail and a larval tail tip

Xenopus tail grows continuously during the larval period. The notochord, spinal cord and posterior fin grow, and new myotubes are formed near the tail tip region. It may be possible that the regenerating tail is functionally homologous to the larval tail tip. To clarify this point, the gene expression patterns of the normal tail tip and the regenerating tail were compared. Because in situ analysis with the larval tail tip failed to detect gene expression (data not shown), RT–PCR was employed. The levels of expression of Xnot, Xbra, Xbra-3, Xshh, XmyoD, Xsox-2, Xdelta-1 in the larval tail tip were similar to those in the regenerating tail, and chordin and noggin showed low expression in the tissue of both (Fig. 3). These results show that the gene expression profile of the regenerating tail is more similar to that of the larval tail tip than that of the embryonic tail bud.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In the present study, the regenerating Xenopus tail did not show the typical blastema found in the urodele limb and tail, which is composed of uniform undifferentiated cells (Stocum 1995). The blastema cells proliferate extensively and differentiate into several types of cells. Our morphological observations showed that the notochord, spinal cord and muscle tissue seem to grow by increasing their own cells during the tail regeneration process. Further analysis using a lineage tracing method will be needed to clarify this point.

We did not detect the expression of chordin or noggin in the regenerating tail, which were expressed in the tail bud of the embryo. Chordin and Noggin are well-known BMP antagonists expressed in the organizer region, and both play roles in neural induction. Our results suggest that neural tissue is not formed by BMP antagonists through the induction mechanism in the regenerating tail, although they do not exclude the possibility that other BMP antagonists are expressed in the regenerating tail. The absence of BMP antagonists is consistent with the morphological observation that the spinal cord appeared to grow by increasing its own cells, as mentioned above. Xnot was expressed both in the organizer region and notochord in embryos. The fact that Xnot was expressed in both the embryonic tail bud and the regenerating tail suggests that it plays a role in notochord formation rather than induction. Whole mount in situ hybridization and RT–PCR analysis showed that the expression of the notochord specific genes, Xnot, Xbra, Xbra3 and Xshh, were upregulated during the tail regeneration process, suggesting the presence of a common mechanism underlying notochord formation in both embryonic development and regeneration.

Expression of Xdela-1 and Xshh were not detected in the regenerated spinal cord, although they were expressed in the normal spinal cord. The lack of Xshh expression suggests an abnormal patterning of the spinal cord because Xshh is specifically expressed in a floor plate and has a critical role in ventral specification. Furthermore, we were unable to identify the floor plate or dorso-ventral patterning by the morphology performed in the present study. These results are consistent with a previous observation that the anuran larva does not regenerate a complete spinal cord but rather produces an immature ependymal tube (Goss 1969; Feretti 2001). The regenerating spinal cord of Xenopus was separated from the notochord by mesenchymal cells (see Fig. 4C), while the stage 42 spinal cord was in contact with the notochord, which is thought to induce formation of the floor plate in embryos (Yamada et al. 1991). Thus, the induction signal from the notochord to make a floor plate may be physically interrupted by the mesenchymal cells located between the spinal cord and the notochord. Alternatively, the floor plate was not formed in the regenerated tail because the larval tail may contain no floor-plate precursor cells. Floor-plate cells are suggested to have the same origin as notochord cells but a different origin from cells in other parts of the neural tube (Gont et al. 1993).

Xdelta-1 was expressed in both the tail bud of the embryo and the distal portion of the regenerating tail, suggesting a critical role in tail formation. Xdelta-1, however, was not expressed in the proximal region of the regenerating tail where the muscle cells were formed, but it was expressed in the presomitic mesoderm of the embryos. Delta proteins are known to be the ligands in the Notch-signaling pathway, which plays a critical role in segmentation of the presomitic mesoderm (Jiang et al. 1998). The expression pattern of Xdelta-1 shows that the regenerating tail lacks all or part of the segmentation mechanism of the mesoderm. This is consistent with our morphological observation that regenerated muscle tissue did not form the typical segments (See Fig. 1K).

Xwnt-5a was expressed in the broad regions of both the embryonic tail bud and the regenerating tail, suggesting that it has a similar function in development and regeneration. It was reported that Xwnt-5a expression was correlated with the normal tail growth in mice (Gofflot et al. 1998). Xwnt-5a may play a critical role in tail formation in both development and regeneration.

A comparison of the gene expressions of the embryonic tail bud and the regenerating tail revealed that many genes are expressed similarly in both tissues, but some are not. For example, chordin and noggin were not expressed in the regenerating tail and Xbra3, Xshh and Xdelta-1 showed different expression domains. These results suggest that the mechanism underlying larval tail regeneration is somehow different from that underlying embryonic tail growth. Xenopus tails grow continuously during the larval period, especially in the posterior region. It may be possible that the regenerating tail is functionally homologous to the larval tail tip. To clarify whether the regenerating tail is similar to the distal portion of the normal larval tail in gene expression, the gene expression patterns of the both tissues were compared. RT–PCR analysis revealed that the gene expression profile of the regenerating tail is more similar to that of the larval tail tip than that of the embryonic tail bud, suggesting the homologous function of the normal tail tip and the regenerating tail in larvae. Of course, the larval tail tip is not the same as the regenerating tail; for example, the growth rate of the regenerating tail is much higher than that of the larval tail tip. In conclusion, Xenopus larvae may regenerate the amputated tail by making a new distal portion of the larval tail rather than by making a new embryonic tail bud.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by the Sasakawa Scientific Research Grant from The Japan Science Society to T. S., the ‘Research for the Future Program’ of the Japanese Society for the Promotion of Science to N. U., a Grant-in-Aid from the Ministry of Education, Science, Sports, and Culture, Japan and a grant from the Sumitomo Foundation both to M. M.. We thank Dr Orii and other members of our laboratory for helpful discussion and maintenance of animals.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References