Labelled cells do not leave the spinal cord
In our previous work [
3] we examined the fate of labelled spinal cord and showed that no cells left it during regeneration. However these experiments were conducted in such a way that only the ventral part of the cord was labelled and if new neural crest is being formed it is more likely to appear on the dorsal side. To create a more uniform label in the spinal cord we used two new methods: electroporation and grafting of labelled tissue at the tadpole stage. In the first method, a
GFP plasmid was injected into the tadpole spinal cord lumen and delivered to the lateral or dorsal wall by electroporation (Fig.
1A). 24 hours later, the tadpoles were amputated at the site of GFP expression, and the tail regenerate was examined. In all 29 specimens, the expression of GFP was observed solely within the regenerating spinal cord and no GFP-positive cells were observed outside it (Fig.
1B, C; Table
1).
| Figure 1Tail regeneration after spinal cord labelling or grafting. (A) Electroporation of GFP plasmid into the spinal cord lumen labels some spinal cord cells. (B) GFP+ cells were observed only within the regenerating spinal cord. (C) Enlarged view of the section (more ...) |
| Table 1 Non emigration of cells from the spinal cord |
Since electroporation labels only a small proportion of the cells in the spinal cord it is possible that export of a limited number of cells would not be observed in these experiments. For this reason we have also used spinal cord grafting. Here the donors were CMV-GFP transgenics which have previously been shown to express GFP ubiquitously and permanently [3]. This method has the advantage that all the donor cells are labelled, but the disadvantage that contamination from adhering non-neural tissues is impossible to exclude with certainty. To carry out the graft, a piece of spinal cord in non-transgenic tadpoles is replaced with an equivalent piece from a GFP transgenic (Fig. 1D). When these tails were amputated at a level such that at least 500 μm GFP spinal cord remained in the stump, the regenerated spinal cord is green along its entire length, showing that it is derived from the donor spinal cord (Fig. 1E,F). In 25 out of 27 cases the labelled cells were entirely confined to the spinal cord even though the tadpoles were allowed a long period of regeneration time and fixed just before metamorphosis (Fig. 1G). Only 2 grafts yielded any GFP-expressing cells outside the spinal cord. One showed some cells in the dorsal fin and the other near the ventral spinal root exit point (Fig. 1H). Because with this protocol it is not possible to guarantee that the grafts are free from contamination by cells outside the spinal cord, we do not regard these two cases as significant. The absence in the great majority of cases of any cells migrating from the spinal cord during regeneration argues against the reformation of a neural crest population.
Spinal ganglia are not regenerated
We also made a study to look for the presence of spinal (= dorsal root) ganglia in the regenerate. As shown in Fig.
2, in stage 49/50 tadpoles, the spinal ganglia in the trunk are large (at least 60 μm long) and are clearly distinguishable. Ganglion 9, the last of the lumbar ganglia, is found at the level where the spinal cord diminishes sharply in size with enlargement of the central canal (arrows in Fig.
2A). At more caudal levels, the spinal cord flattens and the ganglia are much smaller in size, only visible as cell clusters (arrow heads in Fig.
2A). In the middle of the tadpole tail (i.e. 50% postanal distance), a spinal ganglion typically consists of 10–20 cells in a cluster about 20 μm in diameter (Fig.
2B). The presence of these cell clusters continues to the distal part of the tail, where each cluster consists of only 3–5 cells (Fig.
2C). In regenerating tails collected one week after amputation, none contained structures resembling the spinal ganglia of the normal growing tails. When more advanced tail regenerates were examined only 2 out of 24 possessed any cell clusters comparable to those found in control tadpoles (Table
2). In most cases, we only found single cells occupying the position between the spinal cord and the notochord (Fig.
2D). These cells were previously observed by Filoni [
2] and referred to as "sporadic ganglion cells". This result shows that the
Xenopus tadpole does not reconstitute normal spinal ganglia after tail amputation, although occasional extramedullary sensory neurons may be present.
| Figure 2Morphology of the spinal ganglia viewed by haematoxylin-eosin staining. (A) A parasagittal section of the dorsal root ganglia in a stage 49 Xenopus tadpole is shown. The black arrow indicates ganglion 9. (B-D) Transverse sections of middle (B), distal (more ...) |
| Table 2 Presence of tail spinal ganglia in Xenopus tadpoles |
Neurons and axons are present in the regenerated tail
Despite the absence of typical sensory ganglia in the regenerate it is possible that sensory neurons are regenerated and remain within the spinal cord. To investigate this we examined the expression of genes encoding a neurotrophin receptor p75, and Brn3a, an important transcriptional regulator in sensory neurons [
32-
34] by
in situ hybridization. Both are expressed in the normal dorsal root ganglia and in a lateral position in the regenerating spinal cord (Fig.
3A–B').
| Figure 3Expression of neural markers in spinal cord of normal and regenerating tails. (A-B') In situ hybridization detection of mRNA expression of p75a (A, A') and Brn3a (B, B') on transverse sections. (A-B) Control tails. (A'-B') Tail regenerates. Arrows in (more ...) |
The tails were also immunostained for the neuron-specific beta III tubulin in order to reveal the overall arrangement of the fibre tracts and the neuronal cell bodies. In lateral view (Fig. 3C–D') it can be seen that the segmental pattern of axons seen in the original tail is not regenerated, and the axons innervating the regenerate are coming from more anterior levels. Transverse sections (Fig. 3D, D') show the presence of fairly similar fibre tracts in the original and regenerated spinal cord. Expression of Hu protein in the spinal cord of normal growing and regenerating tadpole tails confirms the presence of neurons (Fig. 3E, E')[35]. Some of these are in a dorsal location and so are probably sensory neurons. Others are in a lateral or ventral location and so are probably motor neurons. Some of these express the transcription factor islet1 (Fig. 3F, F') [36].
We noticed that a light touch to a regenerated tail provoked an escape response by the tadpole, suggesting the presence of sensory innervation in the regenerate. To further investigate this, we performed whole mount immunostaining on tail regenerates with an antibody recognizing 200 kda neurofilament (Fig. 4A–C). This shows that peripheral axons exist in the regenerated part of the tail (Fig. 4A,B). Retrograde labelling of nerve fibres was carried out by DiI injection just underneath the skin (Fig. 4C,D) and indicates that the regenerating tail is innervated with sensory fibres. As was apparent with the β III tubulin staining, these axons are mostly derived from the spinal cord more anterior to the amputation level, because the DiI can be found in cell bodies of the anterior spinal cord but is lacking in those of the regenerate (n = 7; Fig. 4E, F).
| Figure 4Peripheral nerve fibres in tail regenerates. (A) 1 month old tail regenerate. (B) Neurofilament 200 staining, enlarged view from the section in (A). Arrow indicates a neurofilament 200 positive nerve fibre. (C) Bright field image of a one week old tail (more ...) |
These results indicate that there is abundant sensory innervation in the regenerated tail, despite the great reduction of sensory ganglia. There are neurons, probably both motor and sensory, present in the regenerated part of the spinal cord but, at least for the stages examined, the axons of the regenerate originate from neurons in the stump.
Melanophores originate from committed precursors
A group of neural crest-derived cells whose presence is obvious in the tail regenerates is the pigment cells. This discussion will concern only the melanophores and not the xanthophores and iridophores, which are less abundant in the tail. The melanophores in a 7-day regenerate (Fig.
5C) are very numerous, similar in density to that of the normal growing tail (Fig.
5A). The cells are seen in the regenerating bud as early as one day after amputation (white arrow, Fig.
5B). We reasoned that there might be three possible cell sources for the melanophores in the tail regenerate.
| Figure 5Regeneration of pigmentation in Xenopus tadpoles. (A) Pigment pattern of a stage 49 tadpole. The inset is an enlarged view of the amputation level. (B) A regenerating tail 1 day post amputation (dpa). The white arrow indicates the melanophores near the (more ...) |
Firstly, as in early development, they may be derived from the spinal cord. Secondly, they might arise from some sort of pluripotent stem cell located in the dermis. Thirdly, as shown in the zebrafish, the regenerated melanophores might arise from pre-existing melanophore precursor cells present in the tail stump[30]. Experiments were carried out to test each of these three possibilities, and only the last is supported.
Melanophores arise from embryonic neural crest but not from tadpole spinal cord
When grafts are made of the neural fold region of neurula stage embryos (Fig.
6A) many melanophores become labelled, as well as fin mesenchyme and the spinal cord itself. This is shown in Fig.
6A–D and in the first line of Table
3. Grafts were taken from
CMV-GFP transgenic donor and were orthotopic, replacing a similar piece of tissue in the host embryo at stage 15–17. In the second line of Table
3 are shown a different series of grafts from the centre of the neural plate, similar to those previously described in [
3]. These do not label the melanophores because the graft populates just the ventral half of the neural tube and not the neural crest. This confirms, as expected, that the melanophores do arise from the neural crest in embryonic development, and that they do not arise from the ventral neural tube. It also confirms that labelled melanophores can readily be observed in the regenerates despite the presence of deep pigmentation (Fig.
6D). During fixation the pigment tends to contract towards the nuclei leaving the peripheral region of the cell unobscured so the GFP can be visualised.
| Figure 6Tail regeneration after embryonic grafting of neural crest or of posterior ventral epidermis+mesenchyme. (A-C) A piece of GFP transgenic posterior neural crest was grafted to the same positions in wild type hosts. The labelled embryos were grown to tadpoles (more ...) |
In contrast to the grafts of embryonic neural crest, the experiments presented above (Fig. 1 and Table 1), of labelling by electroporation or grafting of the tadpole spinal cord followed by amputation, did not normally result in the formation of labelled melanophores in the regenerate. In fact only one case out of the 27 spinal cord grafts did so and since this type of experiment cannot guarantee the absence of contamination by surrounding tissues this individual case is not considered significant.
Melanophores are regenerated from tadpole skin but not from dermal fibroblasts
If melanophores do not come from the spinal cord, then where do they come from? The next set of experiments involved skin grafts from
CMV-GFP transgenic donor tadpoles to wild type hosts. After healing for 2 days, the tails were amputated through the graft (Fig.
7A) and the GFP expression was followed for 14 days (Fig.
7B). Following this type of graft GFP-positive melanophores are regularly found (Fig.
7C, Table
3). It follows that some cell type present in the skin is the precursor of the melanophores, but skin contains a whole variety of cell types including the epidermis, the dermal fibroblasts, the blood vessels, the pre-existing melanophores, and perhaps also mesenchymal stem cells of some sort. So according to this experiment any of these might be the precursors.
| Figure 7Regeneration of melanophores after skin grafting in tadpoles. (A) A piece of GFP-labelled skin was grafted to the lateral region of the middle trunk of a non-GFP tadpole host, which was then amputated 3 days after. (B) A 7 day tail regenerate from a skin (more ...) |
To eliminate most of these possibilities, we performed early embryonic grafts of the posterior ventral ectomesenchyme (PVEM) of stage 15–17 CMV-GFP transgenics to wild type hosts (Fig. 6E). With such grafts a broad lateral region of the developing tail is labelled and the epidermis and dermis are both labelled, but not the melanophores (Fig. 6F). This type of graft sometimes also labelled a few myotomes, indicating contamination with muscle precursor cells. When amputated through the grafted area, the tail regenerate contains a great number of GFP-positive cells. As in the original tails, the epidermis and mesenchyme cells are labelled (Fig. 6G–H), but not the melanophores. Thus this experiment excludes an origin from epidermis or from any cell type derived from embryonic dermal mesenchyme, including any possible pluripotent stem cells, in the regeneration of the melanophores.
Melanophores are regenerated from pre-existing neural crest-derived precursors
The last possibility for the origin of the melanophores in the regenerate is the pre-existing melanophores or melanophore precursors near the amputation site. This seemed likely because the tadpole skin grafts do contain labelled melanophores, while the tails generated from embryonic grafts of PVEM do not. However, we could not be sure that there was not also some other difference in labelled cell composition between these two types of experiment.
To look for melanophore precursor cells in the tadpole tail we examined normal and regenerating tails by in situ hybridization for the expression of three melanoblast markers. They are the early melanoblast markers mitf (microphthalmia-associated transcription factor) [37-39] and kit [40,41], and the late melanoblast marker dct (dopachrome tautomerase) [22]. Cells expressing all these markers were found both in the normal and the regenerating tadpole tails (Fig. 8).
| Figure 8Detection of mitf, dct and kit expression. (A-D) Expression of mitf (A, B) and dct (C, D) transcripts in normal tadpole (A, C), and 3d tail regenerates (B, D) detected by in situ hybridization. Positive cells are present in the blastema region of the (more ...) |
To distinguish whether the regenerated melanophores come from pre-existing pigmented melanophores or from un-pigmented melanophore precursors, we used the tyrosinase inhibitor phenylthiourea (PTU) to block melanin synthesis [30]. The tails were allowed to regenerate in the presence of 0.1–0.2 mM PTU, which blocks the appearance of any melanophores in the regenerate. When the PTU is removed the tadpoles acquire pigmented cells in their regenerates over about a week (Fig. 9). This process is slower than in the zebrafish but the distribution of the cells is random across the regenerate, not appearing first at the proximal end as would be expected if melanophores were simply migrating from the stump region. These result suggest that the major source of the regenerating melanophore is the unpigmented melanophore precursor cells in the tail region.
| Figure 9Tail regeneration in Phenylthiourea (PTU) treated tadpoles. (A) Untreated 4 day tail regenerate. (B) Tail regenerate of a tadpole treated with PTU for 4 days, starting immediately after tail amputation. (C) same tadpole as in (B), 2 days after PTU withdrawal. (more ...) |
Previous work in our lab has shown that removal of the posterior neural fold of neurula stage embryos (inset in Fig. 10A) disrupts the development of the dorsal fin and creates a melanophore-free region in the tadpole tail [42]. Although it is not easy to make these tadpoles, a series of 16 was successfully prepared with complete absence of melanophores from the tail (Fig. 10A, white bracket). When these tadpoles were amputated through the melanophore-free region, at a distance more than about 500 μm away from the proximal visible melanophore (Fig. 10B), no melanophores appeared in the regenerated tail (Fig. 10C). In contrast, melanophore regeneration was observed in all control tadpoles 3 days after amputation (Fig. 10E). In situ hybridization of mitf and dct in these neural fold extirpated tadpoles showed a normal pattern in the anterior but an absence of positive cells in the region of the tail affected by the neural fold extirpation (fig. 10F,G). This experiment is the complement of the skin graft experiments, it shows that in the absence of neural-crest derived cells then no melanoblasts regenerate. Our conclusion therefore is that in embryonic development the melanophores arise from the neural crest, while in regeneration they arise from pre-existing melanophore precursors.
| Figure 10Tail regeneration in normal and neural crest-extirpated tadpoles. (A) A tadpole developed from a neural fold-extirpated embryo. The white bracket indicates the tail region with its pigment cells and dorsal fin depleted. The inset is a sketch of a stage (more ...) |