The Xenopus laevis tail-forming region

Transcription

1 Development 121, (1995) Printed in Great Britain The Company of Biologists Limited The Xenopus laevis tail-forming region Abigail S. Tucker and Jonathan M. W. Slack Imperial Cancer Research Fund Developmental Biology Unit, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK SUMMARY A fate map is produced for the Xenopus tail-forming region at the neurula stage by orthotopic grafting of tissue labelled with fluorescein-dextran amine. It is shown that the axial tissues of the tail are derived from a rectangle 700 µm wide by 600 µm long, while the epidermis of the tail is drawn from a much larger area. The fate map shows that much of the final tail is not formed from the tail bud itself, but by a displacement of trunk axial tissue relative to the proctodaeum. A specification map is also produced by culturing parts of the tail-forming region in vitro or as grafts on a neutral site on host embryos. For the axial tissues this map is identical to the fate map, showing that the tail-forming region is embryologically mosaic. The prospective tail epidermis can, however, regulate defects. It is shown that previous claims of regeneration of the Xenopus tail bud are misleading. Removal of the tailforming region totally prevents tail development. Removal of the tail bud leads to a partial tail, formed by the normal process of displacement of trunk tissue relative to the proctodaeum. Even when only part of the tail bud is removed the tail is still truncated. This shows that there is no terminal regeneration of the tail at embryonic stages. Key words: Xenopus, tail, tail bud, fate map, regeneration INTRODUCTION The tail is a major region of the body of most vertebrates and it has a special significance because its central core consists of notochord, neural tube and somites, hence representing a continuation of the structures of the main body axis posterior to the anus. An understanding of its mechanism of formation will necessarily tell us about the formation of the principal body axis itself. Surprisingly, in modern embryology textbooks the tail is not discussed (Gilbert, 1994; Browder et al., 1991), and in older ones there is only a brief statement to the effect that the tail develops from the tail bud (Balinsky, 1970). This lack of interest is presumably due to the lack of recent research activity on the subject, which is almost total, except for an important paper by Gont et al. (1993). In the present paper we have tried to lay the basis for a study of the mechanisms of tail development in Xenopus by establishing a fate map of the tailforming region, studying the development of isolated explants from the tail-forming region, and establishing the degree to which the tail bud can regulate following surgical defects. For many years there have existed two general concepts of the mode of tail development, which were summarised by Pasteels (1939). The first possibility is that the head, trunk and tail regions are created by a continuation of the morphogenetic processes of gastrulation with only quantitative differences in the intensity of growth. The second is that the morphogenesis of the tail represents a qualitatively different method of growth by a pluripotent group of cells in the tail bud. Pasteels himself favoured the first case. The concept of a pluripotential tail bud was supported by Holmdahl (1939), who proposed that the development of the body of vertebrate embryos occurred in two distinct phases, the first resulting in the establishment of the three classical germ layers at gastrulation, and the second phase commencing with the formation of an undifferentiated mass of mesenchymal cells, the tail bud, at the caudal limit of the embryo. In a recent review article, Griffith et al. (1992) described various experiments suggesting a pluripotential character to the tail bud mesenchyme in amniotes, and suggested that it does indeed consist of a blastema, thus favouring Holmdahl s theory. The amphibians, however, were specifically excluded from this conclusion. Evidence that the amphibian tail bud is not homogeneous was provided by Bijtel (1931, 1936) who showed by vital staining and by transplantation that the posterior part of the neural plate formed the tail mesoderm and that the tail bud developed at the junction of the neural and mesodermal regions. Further evidence was recently provided by Gont et al. (1993) who showed that the expression of two gene markers, Xbra and Xnot 2, could be followed from the blastopore lip into two distinct cell populations in the developing tail bud. These studies favour the view that the different tissues of the tail derive from distinct cell populations that arise during gastrulation, but leaves open the potency of these populations and the nature of any interactions between them. Our understanding of the mechanism of tail development thus remains very rudimentary, and given the level of molecular detail which we now expect, even a resolution of the dichotomy posed by Pasteels would only represent the beginnings of an explanation. This paper looks at the fate and specification maps of the tail in Xenopus embryos to provide some ground work on which

2 250 A. S. Tucker and J. M. W. Slack further experiments in this field can be based. A fate map is established by the labelling of tissues in situ, and shows what will become of each region in the course of normal development. A specification map shows how a region develops if cultured in isolation from the embryo and hence measures the commitment of tissues at the time of explantation. To construct a specification map, the medium into which the explants are isolated must be neutral with respect to the promotion of alternative developmental pathways. Some information about specification can also be gained by studying the development of the complementary defect embryo. An important result from both our fate and specification maps is that the tadpole tail, defined as the region posterior to the proctodaeum, does not originate only from the tail bud itself, but from a larger region of tissue extending more anteriorly. For this reason we distinguish between the tail bud, which is the region of apparently homogeneous cells at the posterior extreme of the embryo, visible as a bulge from stage 27 onwards; and the tail-forming region, which is the larger area that forms the complete tail of the stage 40 embryo. In this paper we have characterised the tail-forming region, assessing how it moves and changes shape as the tail develops. Our results show that the fate and specification maps are very similar. This indicates that the Xenopus tail is determined early on in development, by the end of gastrulation, and long before the onset of terminal differentiation. With respect to its axial components (neural, notochord and myotomes), the tail is embryologically mosaic. However, the tail epidermis does not become determined until fin induction take place during the tailbud stages. When the tail-forming region is extirpated, a tail-less embryo results. Since a partial tail is formed following removal of just the tail bud (Deuchar, 1975), it is generally believed that Xenopus tailbud stage embryos have some regenerative capacity. We show here that this is not the case, and that the supposed regenerate is formed from the part of the tail-forming region anterior to the tail bud, which is left behind in the operation. Using a molecular marker for the tail bud, we find that there is no regeneration when the bud is completely removed, and that terminally truncated tails arise even when part of the bud is left in place. Normal tail bud morphology The Xenopus tail bud is first visible as a bulge at stage 27, approximately 30 hours after fertilization at 24 C, and a well developed tail has formed by stage 40, after about 65 hours. In histological sections the tail bud appears to consist of a homogeneous mass of cells, but, as indicated above, it actually consists of several distinct regions. Sagittal and frontal sections of a stage 33 tail bud are shown in Fig. 1. The tail bud is directly descended from the cells of the dorsal lip, and expresses many genes that are associated with the blastopore and dorsal lip at gastrulation, e.g. Xbra, Xnot, XSna (Gont, 1993; von Dassow, 1993; Essex, 1993). As described Fig. 1. Structure of the Xenopus tail bud. (A,B) Photographs of stage 33 tail bud embryos, sagittal and frontal sections respectively. (A,B ) Diagrams of the respective sections showing key morphological structures.

3 The Xenopus laevis tail-forming region 251 by Gont et al. (1993), the bud develops from a chordoneural hinge which is the remanant of the dorsal lip at the end of gastrulation, together with the posterior neural plate, which closes over the blastopore to form a posterior wall. The closure results in the formation of a canal that maintains a continuous link between the neural tube and the gut, known as the neurenteric canal (Pasteels, 1939; Gont et al., 1993). In Xenopus this collapses at around stage 35. The posterior wall joins with the cells of the ventral lip to form the proctodaeum. The tail bud consists of the chordoneural hinge itself, together with tissue of the lateral blastopore lips and more lateral tissue from the posterior neural folds. MATERIALS AND METHODS Embryos Xenopus laevis embryos were obtained by standard procedures and staged according to Nieuwkoop and Faber (1967). Embryos were incubated in NAM/10 at 24 C after dejellying with 2% cysteine, ph 7.9, in Petri dishes coated with 1% noble agar. Operations were carried out in full strength NAM salts, after which embryos were cultured in NAM/2. Embryos were fixed with 10% formalin in PBSA overnight at 4 C, washed in PBSA and stored in the dark in methanol. Lineage tracing FDA Embryos were injected shortly after fertilisation with 9.6 nl of fluorescein dextran amine at 50 mg/ml in water. The operations were carried out in NAM plus 5% ficoll, which reduced blebbing due to injection damage. The label had been previously dialysed to remove any low molecular mass impurities. The label was visualised using a Zeiss fluorescence microscope. Embryos were cultured in NAM/2 plus 5% ficoll until needed. Grafted tissue was held in place for 30 minutes using small pieces of coverslip until the tissues had adhered, the host embryo being embedded in an agar hole to prevent squashing. Embryos were drawn using the camera lucida at stage 25, stage 33 and stage 40, and the position of the fluorescent tissue was recorded. Individual specimens were kept separate so the movement of particular grafts could be followed. Lineage tracing DiI DiI is a lipophilic dye, which intercalates in the cell membrane, marking small groups of cells. 0.3% DiI was made up in 99% alcohol and heated to 50 C to allow the dye to dissolve. This solution was then mixed with 0.2 M sucrose, which had been heated to 50 C, in a ratio of 9:1, sucrose to DiI respectively. The solution was vortexed and kept in the dark until use. The dye was loaded into a needle by suction, and injected onto the surface of a specimen. As DiI precipitates in salt solution, the embryos were injected in a sucrose solution in plates coated with agarose. A rectangle of spots was constructed around the blastopore of stage13 embryos. The live specimens were held posterior end up in agar and the movements of the spots were followed, as the neural folds formed, using a fluorescence microscope. Explant culture Extirpated tissue was incubated at 24 C in NAM/2 until control embryos had reached stage 40. The nutrient medium used to culture some of the explants consisted of 60 % L-15 with 10% foetal calf serum, 100 IU/ml penicillin/100 µg/ml streptomycin, and 70 µg/ml gentamycin (Laskey, 1970). After dejellying, the whole embryos were washed thoroughly in NAM/10 with 70 µg/ml gentamycin and 100 IU/ml penicillin/streptomycin, and incubated in this medium until they reached the neurula stage, at which point the explants were taken. Explants were cultured in 36- well plates, coated with agar. Plates were kept in a sterilised box at 20 C until controls reached stage 40. Whole-mount antibody staining After fixation, embryos were treated with 0.1 M potassium dichromate in 5% acetic acid for 30 minutes to inactivate phosphatases, and bleached with 5% (v/v of 100 vol.) hydrogen peroxide in PBSA for up to 90 minutes to make the embryos more permeable. Anti-muscle Fig. 2. Camera lucida drawings showing movement of FDA labelled grafts (hatched areas) from stages 18 to 40. Bottom specimen shows the development of the fluorescent donor embryo. (A) Graft of the whole tail-forming region. (B) Graft of the posterior half of the tail-forming region. (C) Graft of the anterior half of the tail-forming region. These diagrams show individual cases, but they are typical of the whole series. In all there were 28 cases of A, 29 of B and 28 of C.

4 252 A. S. Tucker and J. M. W. Slack Fig. 3. Stage 40 embryos showing fluorescent labelling from grafts carried out at stage 18. (A) Graft of whole tail-forming region, showing labelling of all the tail axial structures. (B) Graft of the posterior half of the tail-forming region. (C) Graft of the anterior half of the tail-forming region. (D) Graft of the tissue posterior to the tail-forming region, including the proctodaeum. Note the minimal contribution to tail formation. (E) Graft of the ventral epidermis from under the proctodaeum, showing its incorporation into the ventral fin.

5 The Xenopus laevis tail-forming region 253 Fig. 4. Contraction of width of the tail-forming region as the posterior neural folds close. Embryos embedded in agar, posterior upwards. (A and A ) Stage 13 embryo, prior to neural fold closure, showing a fluorescent rectangle of DiI spots (width 800 µm) which approximately demarcates the tail-forming region. (B and B ) The same embryo at stage 15. (C and C ) The same embryo at stage 18-19, neural folds having closed at the posterior. The lateral DiI spots have now moved inwards and the rectangle now has a width of 700 µm. Note that the centre spot has disappeared as it is now covered by the neural tube. All photographed at the same scale. Scale bar, 640 µm. A B Fig. 5. Fate maps of the tail. (A) Fate map of all three germ layers of the tail-forming region from stages 18 to stage 40, divided into anterior, posterior and proctodaeum sections, showing mapping of all axial structures in the tail and some fin. The 700 µm by 600 µm rectangle referred to in the text includes the anterior and posterior sections only. (B) Fate map of the epidermis only from stage 22 to stage 40. Note the larger area from which the epidermis is recruited into the tail in comparison with the axial structures. Map A is based on 85 grafts, map B 49 grafts.

6 254 A. S. Tucker and J. M. W. Slack was used as the first antibody to visualise the somite patterning (Kintner and Brockes, 1984). The antibody was obtained as a conditioned medium, and was used at a concentration of 1/10. The secondary antibody was anti-mouse IgG coupled to alkaline phosphatase, and was used at a concentration of 1/1000. Antibody binding was visualised by the BCIP/NBT reaction. In situ hybridisation To detect Xnot-2 transcripts in embryos that had had their tail-forming region, tailbud or tail tip extirpated the whole-mount in situ hybridisation protocol of Harland (1991) was used with modifications. Digoxigenin-labelled sense and antisense RNAs were generated by in vitro transcription of an Xnot-2 full-length cdna clone in Bluescript SK, kindly provided by Eddy De Robertis. To obtain the anti-sense probe the plasmid was linearised with EcoRI and transcribed with T7. To obtain the sense probe the plasmid was linearised with XhoI and transcribed with T3. Albino females were used to avoid endogenous pigment. Zygotic rescue of pigmentation occurred after stage 35 due to the use of normal sperm. Embryos were fixed in MEMFA for 1 hour and stored in ethanol at 20 C until required. Scoring of tails In order to improve the objectivity of the assessment of the extent of tail development, a tail-forming index was devised. This allows the scoring of partial and incomplete tail-like structures as follows: Presence of a neural tube 1 point Presence of a notochord 1 point Presence of segmented myotomes 1 point Elongation of the tissue 1 point Tapered end 1 point Complete fin formation 2 points One point is awarded for partial fin formation. A normal, fully formed, tail would score 7 points. The index is not perfect because the six characters are not completely independent. For example, a tapered shape is often linked with an elongated morphology. However no two characters are completely associated with each other and we regard the index as an improvement on an entirely subjective assessment of tail-like status. The tails can be scored by observing the morphology in whole mount without the use of tissue-specific antibodies. Some specimens were sectioned, and in all cases the scores given to the whole mounts matched those later given to the sections. Where a more precise quantitative measure was required, as in the studies of tail bud regeneration, the specimens were stained with the 12/101 antibody and the number of myotomes was counted. The somite number in normal Xenopus development may not be entirely determinate, given the diminishing size of the somites as the tail tapers out. The length of individual somites seems to be determined by the total length of tissue available, and the numbers of cells per somite (Cooke, 1975). However, an objective score can be produced by counting the number of somites formed in experimental situations and comparing these with controls. RESULTS Fate map for axial structures A fate map was produced using orthotopic grafting of tissue that had been labelled with the fluorescent lineage label FDA (fluorescein dextran amine). From a number of preliminary experiments, involving the removal and grafting of rectangles of tissue of various size from the area above the proctodaeum, the tail-forming region was identified. In mid-late neurulae it occupied a rectangle 700 µm wide by 600 µm long, 100 µm anterior to the base of the proctodaeum. This rectangle was extirpated from stage 18, mid-late neurulae, cutting through all the cell layers, and was replaced by similar rectangles from donor embryos of the same stage which had been labelled at the 1 cell stage by injection of FDA. The grafts were then followed through tail formation to show how the tail-forming region changes size and shape as it grows (Fig. 2A). This figure represents 28 similar grafts. It is clear from comparison of the labelled regions at stage 18 and stage 24 that the tail bud itself arises from just a part of the whole tail-forming region. In other words, quite a lot of the tail, defined as the region posterior to the proctodaeum, is not derived from the tail bud but from the posterior part of the trunk already laid down by the end of gastrulation. During the tailbud stages, this region moves posterior relative to the forming proctodaeum, as can clearly be seen by comparing the positions of the labelled domains at stage 33 and stage 40. In terms of tissue contributions, the standard rectangle can be seen to make up the whole of the axial portion of the tail, notochord, myotomes and neural tissue, but it is only responsible for the very tip section of fin epidermis, and some neural crest-derived fin mesenchyme, which has moved up out of the labelled neural tube (Fig. 3A). The axial portion of the tail remains highly fluorescent over the developmental period studied. The fin is naturally less fluorescent by stage 40 due to the cells having flattened out to cover a larger surface area, the presence of extracellular spaces, and the migration in of nonlabelled neural crest derived cells from the trunk (Collazo, 1993). The appearance of label just at the tip, however, implies that the bulk of epidermis and neural crest-derived mesenchyme responsible for fin formation must be located in areas outside the standard rectangle. Furthermore, as the fin is only labelled at the very posterior end, while the mesoderm is labelled throughout the tail, it follows that the posterior mesoderm must be extending at a greater rate than the posterior ectoderm, resulting in ectoderm and mesenchyme being pulled in from trunk regions to form the fin tissue. In order to determine the fate of subdivisions of this tailforming region, the standard rectangle was cut in half, either the anterior or the posterior half being grafted orthotopically. The grafts were followed from stage 24 to 40 (Fig. 2B,C). Fig 2B represents 29 similar grafts and Fig. 2C, 28 similar grafts. Once again the origin of the tail bud is clearly from just part of the tail-forming region, and there is obvious posterior displacement of the trunk relative to the proctodaeum. The posterior part of the rectangle, which would be predicted to include the chordoneural hinge, forms the mesoderm for most of the tail, as well as being a source for some of the ventral posterior fin (Fig. 3B). The anterior section of the rectangle contributes only to a small segment of more anterior mesoderm above the proctodaeum, as well as some of the dorsal posterior fin (Fig. 3C). The area posterior to the standard rectangle, lying directly above and including the proctodaeum at the neurula stage, did not contribute a great deal to tail tissue. The fluorescence was restricted to the area around the proctodaeum by stage 40, and to a small amount of ventral fin (Fig. 3D). The identification of the tail-forming region as a 700 µm by 600 µm rectangle applies to neurulae of stage 18 onwards, when the neural folds have closed at the posterior of the embryo. Before neural tube closure, the tail-forming region naturally occupies a wider rectangle. This may be shown by experiments such as that depicted in Fig. 4. Here, a rectangle

7 The Xenopus laevis tail-forming region µm wide by 600 µm long was marked out on a stage 13 embryo using small spots of DiI (Honig and Hume, 1989). The movements of the spots were followed, using a fluorescence microscope, as the neural folds closed. As predicted, the lateral spots moved inwards quite considerably, while the spots along the anterior-posterior axis retained a fairly constant position, despite the obvious A-P extension of the embryo as it grows. The 800 µm wide by 600 µm long rectangle in the stage 13 neurula was found to contract to the 700 µm wide by 600 µm rectangle, defined as the tail-forming region, in stage 18 neurulae. This change of width of the tail-forming region is due solely to neural tube closure. From the orthotopic grafting experiments a fate map of the tail-forming region has been constructed (Fig. 5A). It shows that as the tail expands it incorporates regions that were part of the trunk. This occurs because of the relative movement of the proctodaeum to a more anterior position. Much of the final tail, therefore, never passes through a period in the tail bud. Fate map for epidermis Although the defined tail-forming region produced all the axial tissues of the tail (neural, notochord and somites), the rectangle does not account for the complete origins of the fin. To look at fin development, small grafts of FDA-labelled epidermis were placed orthotopically onto unlabelled mid-late neurula hosts from which the epidermis had been removed (Fig. 3E). The grafts were followed to trace the movement of epidermis from out of the grafted region into the tail. Camera lucida drawings were made at stage 22, once the grafts had taken, and at stage 40 when the tail was fully formed. Labelled grafts behaved reproducibily, so an epidermal fate map could be constructed, complementary to the fate map of axial structures already produced (Fig. 5B). This figure is based on 49 grafts. The map shows how regions of epidermis contribute to the epidermis of the fin, but does not include the neural crest derived mesenchyme part of the fin. The neural crest pathways in the tail have recently been described by Collazo et al. (1993). Specification map The specification map was deduced by studying the development of isolated parts of the tail-forming region. Rectangles of tissue of various size were extirpated from the area above the proctodaeum in mid-late neurulae, stages The development of the isolated rectangles as well as that of the complementary embryos were followed over 2 days until controls reached stage 40. The explants were then scored using the tail index described in Materials and Methods. If the entire tailforming region, i.e. the 700 µm by 600 µm rectangle as identified by the fate map, was extirpated then a tail-less embryo resulted (Fig. 6A). The isolated standard rectangle developed as a tail (Fig. 6B). If a larger piece of tissue was extirpated, the isolates also included thoracic structures and included large blocks of yolky tissue, whereas if smaller regions were removed some part of a tail formed in the complementary Fig. 6. Specification of the tail-forming region. (A) Extirpation of the tail-forming region (the standard 700 µm by 600 µm rectangle) at stage 18. Control above, extirpation below. No tail is formed. (B) Development of tail-forming regions isolated at stage 18 and cultured to control stage 40. Grade 6 tails are formed by the explants. (C,D) Development of tail-forming regions isolated at stage 30 and cultured until control stage 40. Grade 7 tails are formed by the explants. Scale bars: (B) 800 µm; (C) 650 µm; (D) 2 mm.

8 256 A. S. Tucker and J. M. W. Slack Table 1. Scoring of explants cultured in isolation and when grafted onto a neutral site on a host embryo, grown up to stage 40 Sample no. Mean score Scoring range Explant type Explants in isolation Grafted explants Explants in isolation Grafted explants Explants in isolation Grafted explants st st st st st st st Scoring was based on the tail scoring index. The rectangles represent the tail-forming region at stage 18 and 13, anterior side uppermost. The shaded areas show the region extirpated from the embryo at these stages. For the stage 13 specimens the second rectangle represents the larger 800 µm by 600 µm rectangle which was shown to correspond to the 700 µm by 600 µm rectangle in later specimens. embryo. Extirpating the small area directly above and including the proctodaeum (green in Fig. 5) did not significantly affect tail formation (see Fig. 8C). In mid to late neurulae, the standard rectangle produces on average a grade 6 tail in culture, the deficit being due to lack of a complete, structured fin (Table 1). However, if the tailforming region is extirpated at stage 30, the cultured tails score the full grade 7 (Fig. 6C,D). This change is due to the fact that in the neurula much of the prospective fin lies outside the standard rectangle (Fig. 5), while by stage 30 the tail epidermal cells have been pulled posteriorly as the mesoderm starts to stretch out, and the tail mesoderm and epidermal layers have started to become aligned. Given the larger area of the tail-forming region at early neurula stages, as shown by the movements of the DiI spots, it would be predicted that tail-like explants could only be formed if a larger rectangle were extirpated at these stages. This is indeed shown by the fact that only grade 4 tails (lacking a fin and tapered morphology) are seen when rectangles of 700 µm by 600 µm are removed at stage 13, while a grade 6 is seen if more lateral non-neural tissue is included at this stage (Table 1). To define more precisely which cells can form which parts of the tail, the standard 700 µm by 600 µm rectangle from a stage 18 mid-late neurula was divided into smaller sections. The rectangle was first divided into a posterior half and an anterior half as for the fate map, and then these halves were divided into halves again along the midline. Stage 18 was chosen because earlier explants often disintegrated over the 2 day culture period. The explants were often difficult to score after culture owing to the fact that they only grew to approximately a quarter of the size of the corresponding regions in controls. This size reduction is due to the lack of any food reserve in the explants, which do not contain yolky tissue. Two methods were used to attempt to improve the situation. In the first, explants were placed in a nutrient medium of L-15 and FCS. However, no increase in size was seen, perhaps because they were no longer able to take up the nutrients once they had rounded up. In the second method, the explants were grafted to a putative neutral site on a host embryo. The host embryo was at the same stage to allow a comparison of size and shape of the explant with the host tail. The grafts were labelled with FDA so that the host and donor tissues could be distinguished. The site chosen was the ventral side of a mid-late neurula, halfway in between the anterior and posterior poles. There appeared to be little mixing of the fluorescent and non-fluorescent tissues, and the grafts did indeed grow larger than those explants left in isolation, to approximately half the size of controls. Although these grafts were larger and easier to score, the actual tail scores achieved were not greater than for explants cultured in NAM/2 (see comparison in Table 1). This supports the idea that the ventral side of the embryo really is a neutral site with respect to tail development. The isolated anterior halves formed non-elongated blocks of mesoderm, covered in a small amount of fin, and were graded on average as 3-4 (Fig. 7B,D). The posterior halves, by comparison, consisted of elongated, tapered structures, with some somite patterning and again a small amount of fin. These explants were graded on average as 5 (Fig. 7A,C; Table 1). The embryos which had had these areas removed developed tails of varying completeness, with missing morphologies complementary to those formed by the isolated pieces (Fig. 8A-C). For example, the embryos lacking the anterior section of the tail-forming region had a deficit of mesoderm from above the proctodaeum, visible as a thinning in the tail at this point, and were shorter than controls. They also had a reduction in dorsal fin. The embryos lacking the posterior section did not possess a tail, but were longer than those embryos missing the complete tail-forming region and contained a dorsal fin which curved around the cut surface. The quarters of the tail-forming region when isolated or grafted retained a similar grading and general morphology to that associated with the halves. These two sets of information were then pooled to create a specification map. This was identical to the fate map previously drawn (Fig. 5A) and so is not shown as an additional figure. The fact that cells develop in the same pathway whether in their normal environments or in isolation means that they are already specified to follow that pathway. The tail-forming region can thus be said to be mosaic, at least to the level of resolution provided by the subdivision into quarters.

9 The Xenopus laevis tail-forming region 257 Fig. 7. Explanted and grafted parts of the tail-forming region. Inserts show the explanted area, as indicated by the shaded region extirpated at the neurula stage. (A) Explants of the posterior half of the tail-forming region. (B) Explants of the anterior half of the tail-forming region. (C) Posterior half of the tail-forming region grafted to the ventral side of another embryo. (C ) Close up of same graft. (C ) Graft viewed under fluorescence. Note the minimal mixing of fluorescent and non-fluorescent tissue. (D) Anterior half of the tail-forming region grafted on the ventral side of another embryo. (D ) Close up of same graft. (D ) Graft viewed under fluorescence. Scale bars, 400 µm. The specification of the epidermis at stage 13 was also examined. If small areas of posterior epidermis are removed the cells around the cut can move in to form a completely normal fin. If larger stretches are removed the epidermis cannot grow over and a deficit of fin is seen. The removed pieces of epidermis round up to form balls of epidermis but do not carry

10 258 A. S. Tucker and J. M. W. Slack Table 2. Comparison of somite number in embryos and explants after extirpation of the tail bud and tail-forming region Tail bud Tail-forming extirpation region (as Deuchar) extirpation Controls Sample no embryos Mean somite 19.3± ± ±1.4 no. embryos Sample no explants Mean somite 22.7± ±1.9 no. explants Combined mean somite no. Somites were stained with the anti-muscle antibody and counted (± standard error of the mean). Differences in the number of embryos and explants is due to the fact that only those embryos with somites that could be accurately counted were included. Fig. 8. Defective embryos arising after removal of parts of the tailforming region. Inserts show area extirpated at the neurula stage, as indicated by the shaded region. (A) Embryo that has had the posterior half of its tail-forming region extirpated at stage 18, control below. (B) Embryo that has had the anterior half of the tail-forming region extirpated at stage 18, control below. (C) Embryo that has had the area under the tail-forming region, including the proctodaeum, extirpated at stage 18, control below. on to form fin. Thus, in contrast to the axial tissues, the epidermis in isolation does not seem to be committed to fin formation at this point. Regeneration The specification map shows the tail-forming region as a whole, and probably to some degree its constituent parts, to be specified by the early neurula stage. If the tail-forming region is removed from this stage onwards no tail will form, so there is no redirection of non-tail cells to a tail fate. We thus concluded that Xenopus laevis embryos should not have the ability to regenerate their tail-forming regions, or any large part thereof, at any stage up to stage 40. However, citations often suggest that Xenopus embryos can regenerate their tail buds (Deuchar, 1975; Gont, 1993). Deuchar (1975) was the first to look at regeneration in Xenopus. In her experiments, tail buds were extirpated from stages using a concave cut from above the proctodaeum. The resulting embryos did form tails, but they were only two thirds to a half the length of tails in control embryos and had a truncated tip. The tail-like structures were taken as evidence for tail bud regeneration, and Deuchar explained the tip defects as being due to damage during the operation. Our fate map data shows the movement and size of the tailforming region during stages (Fig. 2). If this region is compared to the material extirpated in the Deuchar experiments there is a discrepancy (Fig. 9). Her cuts remove the tail bud itself but not the whole tail-forming region. A large section of more dorsal tissue, which derives from the original tailforming rectangle, and is an essential region in tail formation, is left intact. It thus seemed likely that it was only the expansion of this remaining dorsal tail-forming material that was giving the illusion of regeneration. The tail bud may well not have regenerated at all. To re-investigate this problem, we compared the behaviour of embryos lacking the tail bud, as perceived by Deuchar, with those lacking the tail-forming region, as shown by our fate map. In these cases approximately the same amount of tissue was removed but the nature of the cuts is rather different (Fig. 9). Extirpating the tail-forming region removes more dorsal tissue than the cuts made by Deuchar, which remove more ventral tissue from above the proctodaeum. We also studied the possible re-formation of a tail bud by performing in situ hybridisations for a tail bud molecular marker, Xnot-2 (Gont et al. 1993), at stages 32, 34 and 36 as well as scoring the final morphology. The results showed that Deuchar s experiment was fully reproducible. The putative regenerated tails with their

11 The Xenopus laevis tail-forming region 259 Fig. 9. Diagram comparing the position of cuts to excise the tail bud (Deuchar) or the tail-forming region. Note the removal of more dorsalanterior tissue in the cuts associated with the excision of the whole tailforming region, in contrast to the more ventral tissue excised when just the tail bud is removed. truncated tips were produced by repeating Deuchar s cuts (Fig. 10A,B). By contrast, the removal of the entire tail-forming region resulted, as expected, in larvae without tails (Fig. 10B). However, the in situs showed that in neither case was there any re-formation of a tail bud at intermediate stages, as would be represented by re-expression of an Xnot-2 domain, making a claim for regeneration seem unlikely (Fig. 11A-C). A closer examination of the putative regenerates was made by counting the number of somites formed by the isolated bud and by the defective embryos (Fig. 10C). The control embryos at stage 41 had an average somite number of 42 (standard error 1.4; Table 2), of which 9 somites are associated with the trunk, the rest being found in the tail. The embryos produced by the Deuchar cut contained an average of 19 somites (s.e. 1.5). These specimens thus have less than half the number of somites present in controls. The isolated pieces produced from the extirpated tail buds formed tails with an average of 23 somites (s.e. 1.7). Together the somite number adds up to 42, the same as the mean for controls, showing that no extra somites have been formed. This provides clear evidence against regeneration, since we should obviously predict that regeneration would result in a higher somite number in the isolated explant plus the defective embryo as compared to controls. The defective embryos that had the whole of the tail-forming region removed formed an average of 13 somites (std.error 1.1), around 4 somites forming a stump posterior to the trunk. The isolated tail-forming regions produced an average of 29 somites (s.e.m. 1.9). So for this experiment the combined somite count again adds up to 42, the same as the controls. It can thus be concluded that the tail bud regeneration postulated by Deuchar does not occur. The tails formed arise entirely from the part of the tail originating from the trunk and not from the tail bud. However, Deuchar s experiment was quite a drastic one, involving as it did the entire removal of the tail bud. It might still be the case that tail bud regeneration occurs if a part of the tail bud were removed. We have carried out in situ hybridisation for Xnot-2 in embryos in which a small slice, half or most of the tail bud, was removed. The results at intermediate stages (stage 32-36) show clearly that the Xnot-2 expression does not recover if the entire domain is removed (Fig. 11C,F), but if some expression is left by removal of only half or less of the tail bud the expression is retained through intermediate stages (Fig. 11D,E). Whether the residual domain increases in size to indicate some regeneration cannot be decided by in situs. The number of somites were thus counted in similarly treated embryos at stage 41 (Table 3). The explants produced from extirpating the tail bud tips or halves were also grown up to stage 41 but did not produce definite somite patterns that could be counted once stained. This is probably due to their small size. Table 3 thus only shows the somite numbers of the defect embryos. The results show clearly that there is a deficit in somite number, and that this deficit is greater the larger the piece of tail bud removed. This deficiency in somite number was still clear at stage 43, 4 days after the operations were carried out. These results are compatible with a total mosaicism and a total absence of regenerative capacity by the tail bud. We cannot definitively prove that this is the case since we do not know the fate map to single somite resolution and so cannot exclude small adjustments of the fate map following Table 3. Comparison of somite number in embryos at st 41 after extirpation of regions of the tail bud at st 30 Complete tail Half tail bud Tail tip bud extirpation extirpation extirpation Controls Sample no Mean somite 19±1.4 27±1.7 34±1.5 42±1.4 no. embryos Somites were stained with the anti-muscle antibody and counted (± standard error of the mean).

12 260 A. S. Tucker and J. M. W. Slack as the fate map, at least to the resolution provided by a study of quarter size explants. These results show the mosaic nature of the tail, and show the formerly unappreciated fact that the tail does not just derive from the tissue located within the tail bud but also from more anterior tissue as well. The entire region that will later form the tail of a stage 40 embryo is here defined as the tail-forming region, while the term tail bud is reserved for the undifferentiated bud of the tailbud stages. Using the new fate map information we have shown that the tail of a tailbud stage embryo does not have the ability to regenerate, and we explain why previous experiments seemed to show that it did. Fig. 10. Extirpation of the tail bud and tail-forming region. (A) Comparison of the tail ends of a control embryo (top) and an embryo that has had its tail bud excised (bottom). Note truncated nature of the somite patterning, as stained with the anti-muscle antibody (B) Comparison of tail size when the tail-forming region (top) or tail bud (middle) is excised; control (bottom). (C) stained somite patterning of a tail bud, excised at stage 28 and cultured to stage 41. extirpation. However, our experiments provide no positive evidence for any regeneration at all. DISCUSSION The orthotopic grafting experiments presented here have enabled us to present fate maps of the prospective regions both for the axial tissues and for the epidermis of the Xenopus tail. The extirpation and isolation experiments allow us to conclude that the specification map of the tail-forming region is the same Fate mapping The fate map defines the tail-forming region at the mid-late neurula stage as a rectangle 700 µm wide by 600 µm long, 100 µm anterior to the base of the proctodaeum (Fig. 5A). Prior to neural tube closure the tail-forming region occupies an area of similar length but 800 µm wide (Fig. 4). This standard rectangle extends as the embryo develops and eventually can be shown to make up the whole of the axial component of the tail of a stage 40 embryo. It is, however, responsible only for the very tip section of fin (Fig. 3A). The epidermis over the tail bud is two layered. The layers have been characterised in Rana. The outer, pigmented layer is a substantial structure formed of a monolayer of cuboidal cells rising to columnar in the keel, the precursor of the tail fin. The inner, unpigmented, layer is a slight structure of flattened cells rising to cuboidal in the keel (Elsdale and Davidson, 1983). As the fin forms, the epidermis becomes elevated into a keel supported by a core of mesenchyme. The epidermal prospective region for the whole tail appears to be approximately two times the size of the area needed to provide the axial components (Fig. 5B). By the tailbud stage the tail epidermis moves posteriorly as the mesoderm stretches out and thus the tail mesoderm and epidermal layers eventually become aligned. By following the tail-forming region as it grows and develops it can be seen that the resulting stage 40 tail does not originate only from the cells in the prominent tail buds of stage embryos, but from a larger region. The anterior part of this is at first associated with the trunk, but as the embryo develops the proctodaeum moves anteriorly, displacing these trunk levels into the tail. Tail outgrowth has formerly been described as consisting of two processes: extension of presegmental tissue and expansion of somites already formed (Elsdale and Davidson, 1983). To these we can now add a third process: incorporation of segments from the trunk. Fate mapping experiments have previously been carried out on formation of the tail buds using localised vital staining, mainly in urodele embryos (Bijtel, 1931; Nakamura, 1942). But the availability of fluorescent lineage labels has allowed a much higher precision in our studies and also the labelling of deep tissue layers, not just those on the surface. Most of Bijtel s studies were terminated at the point of tail bud formation, however she did identify a region at the posterior of the neural plate as the tail primordium, and, although she did not draw attention to it, her Fig. 23 clearly shows the displacement of trunk somites into the tail in a Rana esculenta embryo. Specification mapping The specification map was constructed by looking at the devel-

13 The Xenopus laevis tail-forming region 261 Fig. 11. In situ hybridisation of Xnot-2. (A) Control stage 30 and 36. Arrowheads indicate Xnot-2 expression in the tail bud at stage 30 and at the tip of the tail at stage 36. (B) Extirpation of tail-forming region at stage 30 (above), showing removal of the Xnot-2 domain of expression. By stage 36 (below), no Xnot-2 expression is seen. (C) Extirpation ot tail bud region at stage 30 (above), showing removal of the Xnot-2 domain of expression. By stage 36 (below), no Xnot-2 expression is seen. (D) Extirpation of half the tail bud at stage 30(above), arrows indicate the presence of some Xnot-2 expression. At stage 36 (below), expression of Xnot-2 can be clearly seen at the tail tip. Note, however, that the tail is shorter than the control in A. (E) Close-up of the embryo in D, stage 30. Above, control showing Xnot-2 expression and below, embryo with half the tail bud extirpated showing the retention of some of the Xnot-2 expression domain. (F) Close-up of the embryo in C, stage 30. Above, control, and below, embryo with the whole tail bud removed, showing the removal of all the whole Xnot-2 expression domain. opment of divisions of the tail-forming region in isolation and when grafted onto a neutral site in a host embryo. Information was also gathered by studying the development of the complementary defect embryos. When the tail-forming region was extirpated, tail-less embryos were formed, which retained a completely normal head and trunk region (Fig. 6A). The explanted tail-forming region in culture in vitro, or after grafting to the ventral side of another embryo, continued to develop as a tail, with an elongated, tapered morphology, segmented muscle, and a fin of some description. Such tails were scored using our tail index, on average obtaining 6 points (Fig. 6B). A complete tail scores 7 using this system. In general, the explants scored less than control tails due to a deficit of fin. Explants would be expected to have a reduced fin compared with intact embryos given the larger size of the area from which the fin is derived compared to the mesoderm. By stage 30, the tail epidermis has been pulled posteriorly as the mesoderm starts to grow out, and if the tail-forming region

14 262 A. S. Tucker and J. M. W. Slack is extirpated at this stage the cultured tails score a grade 7 (Fig. 6C,D). The tail-forming region was divided into anterior and posterior halves and also into quarters by division along the midline, and these pieces were cultured and scored. The similarity of the structures formed by the quarters and the halves to the predictions from the fate map implies that the tissue is internally specified, at least to the level of resolution provided by this subdivision. Given the similarity of the fate and specification maps, the tail can be thought of as a mosaic structure. Regeneration The mosaic character of the tail-forming region leads to the strong prediction that the tail-forming region should not have the ability to regenerate at later stages. However, it is generally believed that Xenopus tailbud stage embryos do have the ability (Deuchar, 1975). To resolve this apparent contradiction the alleged regeneration was reinvestigated. When we repeated the extirpations using the same cut as in Deuchar s study, tails did form but these were two-thirds to a half the size of controls, with an average of 19 somites compared to the 43 found in controls, and were trucated at the tip (Fig. 10A). From our fate map it is clear that Deuchar s cut removes the tail bud but leaves the area of more anterior tissue which will later play a part in tail formation (Fig. 9). If the complete tail-forming region is extirpated then no tail forms (Fig. 10B). The structures produced after the removal of the tail bud is thus not due to a true regeneration of the bud but is due to the expansion of the tail-forming tissue that was left behind. This explains the shorter size of the tails and their truncated tips. In fact, although various references from the older literature are sometimes quoted to support the idea of tail bud regeneration, an examination of these papers suggests that the same arguments apply to other amphibian species, including urodeles. Studies of Schaxel (1922) and Vogt (1931) found that the tail bud could not be regenerated after complete removal. Münch (1938) published a more detailed study using three urodele species. These showed that extirpations corresponding roughly to our tail-forming region (ectoderm and mesoderm from the vorletzte part of the neural plate; or removal of bud with tail root from tailbud stage) both yielded tail-less larvae. Furthermore, all bilateral defects or unilateral defects of both tissue layers in the tail-forming region yielded defects in the final tail. Extirpations reported to give regeneration were those of the bud but not the root (similar to Deuchar); the region around the blastopore; or unilateral defects in the ectoderm alone. In these experiments somites were not counted and so the normalcy of a tail depended on subjective assessment. The illustrations in fact suggest that some of the tails actually do lack segments, and that in urodeles, like Xenopus, there is a contribution to the tail from trunk tissue outside the tail bud, and that the region around the blastopore is not part of the tail-forming region. Our results fail to show any regeneration: either recovery of an Xnot-2 domain following its removal, or the formation of complete tails from partial buds. In summary we believe that previous claims of regenerative ability at embryonic stages are mainly due to a subjective assessment of the results together with a misunderstanding of the normal fate map. The present paper concerns the normal fate map, specification map, and regenerative capacity of the tail-forming region. We hope that the results will serve as a resource on which future cellular and molecular work can be based. We are currently investigating the cell lineage of the tail tissues and the cell interactions involved in outgrowth, and hope thereby to clarify the roles of the many genes that play a key role in early axis formation and whose expression persists in the tail bud (e.g. Gont et al., 1993). This work was supported by the Imperial Cancer Research Fund. We are to E. de Robertis for the gift of the X-not 2 probe. REFERENCES Balinsky, B. I. (1975). An Introduction to Embryology, 5th edition. W. B. Saunders Company. Bijtel, J. H. (1931). Über die entwicklung des schwanzes bei Amphibien. Wilhelm Roux s Archiv. 125, Bijtel, J. H. (1936). Die mesodermbildungspotenzen der hinteren Medullarplattenbezirke bei Amblystoma mexicanum in Bezug auf die Schwantzbildung. Wilhelm Roux s Archiv. 134, Browder, L. W., Erickson, C. A. and Jeffery, W. R. (1991). Developmental Biology, 3rd edition. Saunders College Publishing. Collazo, A. Bronner-Fraser, M. Fraser, S. E. (1993). 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