Summary Cell lineage tracing during Xenopus tail regeneration

The tail is a major region of the body of most vertebrates, constituting that part of the body posterior to the caudal opening of the digestive tract. It consists of a continuation of the axial structures of the body, with segmented muscle flanking the spinal cord and the vertebral column. Even humans exhibit a tail in embryonic life, vestiges of which may be observed on any human skeleton as the lowest three or four caudal (coccygeal) vertebrae. During embryonic stages, a notochord is present instead of the vertebral column. In the larvae of anuran amphibians, the tail is a transitional structure that becomes resorbed during metamorphosis mediated by thyroid hormones. The tail of Xenopus laevistadpoles retains this core organization common to the other vertebrates: with a spinal cord and notochord flanked by paired myotomes, in continuation with the axial trunk. Some capacity for regeneration of missing structures is found in several classes of vertebrates, especially the urodele amphibians, but complete tail regeneration can also occur in anuran tadpoles. Since the 18th century (Spallanzani, 1768) and more recently (Morgan and Davis, 1902; Locatelli, 1924; Stefanelli, 1947; Baita, 1951; Holtzer, 1956; Roguski, 1957; Niazi, 1966; Hauser, 1972), the tail regeneration of urodele and anuran amphibians has attracted interest, but the mechanism of new tissue formation from the stump is still not yet clearly understood. Elsewhere, we have addressed the molecular signalling processes involved in regeneration (Beck et al., 2003), and in this paper we address the issue of cell lineage. Does the regenerate originates from mature differentiated cells or from undifferentiated ‘reserve’ cells? If it is the former, then does each tissue give rise to just its own cell type in the regenerate, or can it switch to different cell types (metaplasia)? These questions are still being answered. In the axolotl, a urodele amphibian, Echeverri and Tanaka (Echeverri and Tanaka, 2002) have demonstrated neural cell plasticity during tail regeneration. During regeneration, the ependymal or radial glial cells of the axolotl spinal cord are able to give rise to neurons, melanocytes, muscle and chondrocytes, cell types of both ectodermal and mesodermal origin. In previous experiments on limb regeneration in newts it has been shown that the multi-nucleate muscle fibres can de-differentiate. The nuclei re-enter S phase, and the multinucleate cells break up into mononuclear cells. These enter the undifferentiated blastema of the limb and some can later differentiate into other tissue types, such as cartilage (Lo et al., 1993; Kumar et al., 2000). These experiments clearly show that de-differentiation of mature differentiated cells, and metaplasia of one cell type to another, can take place during urodele regeneration. Recently the term ‘transdifferentiation’ has become popular as an alternative to ‘metaplasia’, particularly in the context of the ability of bone marrow stem cells to populate various other tissue types after grafting. In our own publications we have used the term ‘transdifferentiation’ in its original sense to refer to direct transformation of one differentiated cell type to another with or without cell division, and the term ‘metaplasia’ to refer to any conversion of one differentiated tissue type to another regardless of pathway or mechanism (Tosh and Slack, 2002). The tail of the Xenopustadpole will regenerate following amputation, and all three of the main axial structures – the spinal cord, the notochord and the segmented myotomes – are found in the regenerated tail. We have investigated the cellular origin of each of these three tissue types during regeneration. We produced Xenopus laevis embryos transgenic for the CMV (Simian Cytomegalovirus) promoter driving GFP (Green Fluorescent Protein) ubiquitously throughout the embryo. Single tissues were then specifically labelled by making grafts at the neurula stage from transgenic donors to unlabelled hosts. When the hosts have developed to tadpoles, they carry a region of the appropriate tissue labelled with GFP. These tails were amputated through the labelled region and the distribution of labelled cells in the regenerate was followed. We also labelled myofibres using the Cre-lox method. The results show that the spinal cord and the notochord regenerate from the same tissue type in the stump, with no labelling of other tissues. In the case of the muscle, we show that the myofibres of the regenerate arise from satellite cells and not from the pre-existing myofibres. This shows that metaplasia between differentiated cell types does not occur, and that the process of Xenopus tail regeneration is more akin to tissue renewal in mammals than to urodele tail regeneration.