Regeneration from half lower arms in the axolotl

Limb regeneration in amphibians provides a system for the study of the formation of a complex pattern of skeletal and soft tissue elements. Information for this pattern is believed to lie in the cells close to the amputation plane and to be present in both the dermis and underlying muscles (see review by Tank & Holder, 1981). Several techniques for assaying regenerative ability have shown that this information is not evenly distributed within the upper arm (symmetrical limbs: Stocum, 1978; Tank & Holder, 1978; Holder, Tank & Bryant, 1980; irradiation: Maden, 1979; isolation of half limbs using head skin: Wigmore & Holder, 1985). These techniques have shown that normal 4-digit limbs can only form if the stump contains posterior or dorsal tissues. Stumps lacking these tissues produce hypomorphic regenerates, anterior halves in particular only regenerate single digits.

The dorsoventral axis, assayed using the pattern of muscles, appears to be controlled independently of the anteroposterior axis, allowing the formation of limbs with a normal skeletal pattern but abnormal muscle distribution. The dorsoventral axis also tolerates the presence of discontinuities in the pattern while discontinuities present in the anteroposterior axis of the stump result in the formation of additional structures in the regenerate (Maden, 1980; Holder & Weekes, 1984,1985; Burton, Holder & Jesani, 1986).

The lower arm has been less-intensively studied but there is some evidence that the amount of structure regenerating from different halves is different from the upper arm. Surgical removal of the posterior half of the lower arm of the newt (Goss, 1957a; Dinsmore, 1983) produced regenerates with between 1 and 4 digits but with a mean digit number of 2·4. Irradiation of half the limb (Goss, 1957b; Maden, 1979) showed that both posterior and anterior halves could produce on average slightly more than 2 digits but that posterior halves tended to produce more than anterior halves. The implantation of unirradiated half limbs into irradiated stumps (Stinson, 1964) or the construction of symmetrical lower legs (Stocum, 1978) produced a greater difference between anterior and posterior halves; the posterior half producing approximately three quarters of the normal limb and the anterior one quarter. Construction of symmetrical lower arms in the axolotl (Krasner & Bryant, 1980) produced regenerates with on average less than 2 digits from either double anterior or double posterior stumps while construction of limbs with symmetrical skin produced double posterior regenerates from double posterior stumps and normal or hypomorphic regenerates from stumps with double anterior skin (Slack, 1980). These results indicate that in contrast to the upper arm no part of the lower arm is capable of producing a complete regenerated limb. Despite the apparent inability of lower arm regenerates to compensate for deficiencies in the stump this region of the limb shows strong powers of regulation by the production of supernumeraries when anterior and posterior tissues are opposed (Tank, 1978). No study of the dorso ventral axis has been done after manipulations on the lower arm and part of this study was designed to ascertain whether this axis behaves in the same way in the lower arm as in the upper.

The present work is the application of a new technique which has been able to demonstrate differences in regenerative ability in different regions of the upper arm (Wigmore & Holder, 1985). This technique uses the fact that head skin contains no positional information affecting limb regeneration (Wigmore & Holder, 1985) and rapidly heals to a wound made in the limb. These properties are . used to isolate halves of the arm by grafting head skin onto the medial surface of amputated stumps from which half of the limb has been removed. The position of the head skin prevents any contribution from proximal parts of the limb corresponding to the half of the stump that has been removed and restricts regeneration to that produced by the isolated half. The controls consisted of an identical operation but without any grafting of head skin. This allows all parts of the limb to interact around the amputation plane and contribute to the regenerate.

All experiments were carried out using axolotls (Ambystoma mexicanum), 15–20 cm in length spawned in the colony at King’s College. Animals were kept in tap water in individual containers and fed on raw heart.

During surgery animals were anaesthetized in MS222 (Sigma), after recovery they were kept at 10°C in the dark for two days in order to facilitate healing. All operations were performed on the zeugopodium with the arm at right angles to the body and the elbow flexed using the axes of the body to determine the position of different halves of the limb (see Burton et al. 1986). Experimental operations comprised the removal of half the lower arm (either the dorsal, ventral, anterior or posterior half). Posterior and anterior half stumps included either the ulna or radius respectively while both forearm bones were included in both dorsal and ventral halves. The medial wound surface formed by the removal of half the forearm was covered with a 3–4 mm wide strip of skin removed from the forehead which was sutured into place (see Wigmore & Holder, 1985). The limb was then amputated through the graft and trimmed so that the amputation plane passed through the middle third of the forearm. Thus the stumps consisted of either a single forearm bone in the case of anterior and posterior halves or both radius and ulna in dorsal and ventral half stumps together with approximately 50 % of the circumference of limb skin and soft tissues, the remainder of the circumference being covered by head skin. Controls consisted of carrying out the same procedure as described above but without grafting any head skin onto the limb.

All experimental animals were checked for retention of the graft every few days for the first few weeks after the operation. Operated animals were left for 60 days after which time the limbs were amputated, fixed in Bouin’s solution, dehydrated and stained with Victoria Blue (Bryant & Iten, 1974) to show the pattern of cartilages. Fig. 1 shows a control regenerate with a normal skeletal pattern. Camera-lucida drawings were made of all regenerates. Limbs were then wax embedded and serial transverse sections were cut at 10 μm and stained with haematoxylin and eosin. These sections were used to check the position of the graft and to analyse the dorsoventral muscle pattern. Limbs where the graft was not in the expected location were discarded.

The muscle pattern was examined at two levels, the proximal metacarpals and the midforearm. In normal limbs at the level of the proximal metacarpals, ventral muscle is continuous across all digits while dorsal muscle occurs as discrete crescents over each metacarpal and a large vascular sinus is present only on the ventral side (Maden, 1980,1982; Maden & Mustafa, 1982; Burton et al. 1986). At the midforearm level, the dorsoventral axis can be determined from the overall muscle pattern and the presence of the pronator quadratus (pq) muscle which runs from a spine on the ventral side of the ulna towards the radius (Holder & Weekes, 1984; Burton et al. 1986). Head-skin-derived tissue was normally easily recognizable as a fold of tissue on the side of the limb overlying an extensive region of loose connective tissue. Serial sections of limbs showed this fold to be continuous with the original head-skin graft. The anatomical nomenclature used was obtained from Francis (1934) and Grim & Carlson (1974).

Twenty posterior half stumps were constructed all of which produced a regenerate (see Table 1). Regenerates had between 1 and 4 digits, the most frequent type (40 % of limbs) having 2 digits. Of the 4-digit limbs all except for one was normal; the exception was a limb that had a proximally incomplete radius and lacked phalanges from digits 1 and 2. One 3-digit limb possessed the distal part of the radius (Fig. 2A) while the remaining 3-digit limbs had only an ulna (Fig. 2B). All 3-digit limbs appeared to consist of digits 2, 3 and 4. These limbs were asymmetrical and although lacking anterior structures, had apparently normal posterior halves. All 2-digit limbs appeared to be digits 3 and 4 (Fig. 2C) but unlike the 3-digit regenerates six out of eight had a complete radius although this did not always articulate with the carpals (Fig. 2D). Two regenerates were distally incomplete, lacking phalanges and having only the proximal ends of metacarpals. Both single-digit regenerates (Fig. 2E) possessed only an ulna and one was distally incomplete.

Sections of regenerates from posterior half stumps showed a fold of loose connective tissue that was normally located on the anterior side. This feature was continuous with the head skin graft and is assumed to be tissue derived from the graft. The muscle pattern of all limbs was normal at the forearm level, however, graft-derived tissue was found to lie in a dorsoanterior location in some limbs and this may have led to the absence of dorsal muscle in the metacarpal region of two of the 2-digit limbs. One limb of 3 digits possessed a duplicate radius distally and the graft-derived tissue in this limb was located anteriorly and ventrally. The remaining limbs had a normal muscle pattern at the metacarpal level.

Thirteen anterior half limbs were made using the contralateral limbs of the animals used to make posterior half stumps (see Table 1). Anterior half stumps regenerated considerably less structure than those from posterior halves with a mean of only 1·3 digits compared to 2·7. The majority of anterior half stumps produced only a single digit with a single forearm bone, the radius. Of the three regenerates with more than 1 digit, the 3-digit limb had both radius and ulna but the anterior digit appeared unconnected to the posterior pair (Fig. 3A). One 2-digit limb had both radius and ulna (Fig. 3B) while the other possessed only a radius and consisted of digits 1 and 2 (Fig. 3C). All the single-digit regenerates had only a radius with two or three carpals, a metacarpal and two phalanges. Occasionally spikes of cartilage were found lying posterior to the radius in the position expected of the ulna (Fig. 3D).

It was not normally possible to recognize dorsoventral muscle patterns in singledigit limbs but the distribution of muscle was often asymmetric. Both 2-digit limbs had dorsal and ventral muscles but the pattern in the 3-digit limb was unrecognizable. All limbs had graft-derived tissue present on their posterior side.

Eight dorsal half stumps were constructed and all but one produced a regenerate (see Table 1), consisting of between 2 and 4 digits with a mean digit number of 2-8. All regenerates had both a radius and ulna. One 4-digit regenerate was normal while the remaining two lacked posterior carpals and in one case the phalanges of digit 4. The 2- and 3-digit limbs appeared symmetrical about their anteroposterior axis (Fig. 4A,B) but unlike regenerates from posterior half stumps the digits could not be identified with any certainty.

Sections of regenerates from dorsal half stumps showed graft-derived tissue on the ventral side of the limb at proximal levels but this tissue tended to be localized more anteriorly at more distal positions. All of the 4-digit and one 3-digit limb had normal muscle patterns at both metacarpal and forearm levels although in one regenerate the hand was folded to enclose the ventral surface. The three remaining 2- and 3-digit limbs were either double dorsal (Figs 6, 7) or had dorsal muscle but no muscle on the ventral side. This was true both at the metacarpal and forearm levels. One 3-digit limb had three bones in the distal forearm (Fig. 7).

Ten ventral half stumps were completed and all produced a regenerate varying between 1 and 4 digits (see Table 1) with 50 % of limbs producing 2 digits. Both 4-digits limbs had a normal pattern of bones and all other regenerates had both radius and ulna. The digits of the 2- and 3-digit limbs were not identifiable (Fig. 5A,B); digits were often not in the same plane as the rest of the limb and were sometimes fused at the metacarpal level. Single-digit limbs also had both a radius and an ulna but although having five or six carpals only a single metacarpal with two phalanges was present distally. One of the 2-digit limbs had three bones in the distal forearm.

As with the 4-digit dorsal half stump regenerates, 4-digit regenerates from ventral stumps had normal muscle patterns at both metacarpal and forearm levels. The remaining limbs had either ventral muscle on both sides of the limb (five limbs) or ventral muscle on one side and an absence of muscle on the dorsal side (three limbs). Double ventral limbs had continuous muscle between the digits on both sides of the metacarpals and two pronator quadratus muscles in the forearm (Figs 8, 9). One limb, which was double ventral in the forearm lacked any muscle distally.

Control consisted of ten anterior, ten posterior, five dorsal and five ventral half limbs. All but one of these limbs regenerated 4 digits, the exception being a 5-digit limb produced from an anterior half stump. All regenerates possessed both a radius and an ulna. Some anterior half stumps were seen to have medium bud blastemas with two apices and this type of stump had the highest proportion of abnormalities in the final regenerate. The commonest abnormality that was seen on three posterior half and six anterior half controls was the appearance of an additional basal carpal between the fused basal carpals one and two and basal carpal three (Fig. 10). Five anterior limbs and one posterior and one dorsal limb also had three phalanges on two of their digits. The dorsoventral muscle pattern was normal at both metacarpal and forearm levels for all control regenerates.

This work was carried out to investigate the regenerative potential of different parts of the lower arm and, in particular, to compare these results with those from the upper arm (Wigmore & Holder, 1985). Previous authors (see Introduction) have produced results indicating that the lower part of the limb is not able to compensate for defects in the stump. This contrasts with the upper arm, where normal regenerates can be produced from dorsal or posterior half stumps. In the present work all halves of the lower arm produced a high proportion of hypo-morphic regenerates and the ability of posterior and dorsal tissue to consistently regulate to produce normal regenerates was not found.

In the upper arm the greatest difference in the amount of skeletal tissue regenerating was found between anterior and posterior half stumps. The mean number of digits regenerating from posterior upper arm stumps was 3·8 but only 1·2 regenerated from anterior half stumps. In the lower arm the posterior half still regenerated more digits than the anterior half but the difference was less marked (2·7 digits from posterior halves, 1·3 from anterior). Compared with the upper arm there was little change in the regenerative ability of the lower arm anterior halves with the commonest regenerate still being a single digit. This was always associated with a radius although parts of the ulna were occasionally present (Fig. 3D) and a complete ulna was present in two of the multidigit limbs (Fig. 3A,B).

Posterior half stumps produced the greatest proportion of normal limbs but the majority of regenerates (70 %) were hypomorphic due to the absence of anterior digits and carpals. This contrasts with the upper arm where 85 % of limbs were normal. The radius was also absent in all 3-digit regenerates but surprisingly was present in many 2-digit limbs. The posterior parts of hypomorphic limbs from posterior half stumps appeared normal but the amount of the normal skeletal pattern present varied, often ending abruptly and sometimes even splitting individual carpals (Fig. 2B). Similar limbs were made from anterior half stumps (Fig. 3C) but these consisted of anterior structures and occurred at a much lower frequency. The two single-digit limbs regenerating from posterior half stumps are difficult to identify but since they were associated with only an ulna they can tentatively be identified as digit 4. Single-digit limbs were never produced from upper arm posterior half stumps. The dorsoventral muscle pattern could be recognized in multidigit limbs and in these limbs the pattern was normal irrespective of whether the limb was from an anterior or posterior half stump.

Regenerates from dorsal and ventral half stumps were similar in their anteroposterior axis; although a proportion of both types of stump produced normal limbs, the majority of regenerates, while possessing both radius and ulna, had only 2 or 3 digits. Some digits could be tentatively identified from their articulation with their carpals, for example in Fig. 4B the digits may be 3 and 4 while Fig. 5A may show digits 1 and 2. In general however it was not possible to be sure what part of the limb had regenerated. These limbs therefore differ from the asymmetric regenerates produced from posterior or anterior half stumps which clearly contained part of the normal anterior–posterior pattern.

The major difference between regenerates from dorsal and ventral half stumps was in their muscle patterns. Limbs with 4 digits had a normal muscle pattern but nearly all hypomorphic regenerates had only dorsal or ventral muscle depending on whether they were from dorsal or ventral half stumps. The muscle present was either on both sides of the limb making them double dorsal or double ventral or only occupied one side leaving head-skin-derived tissue on the other (half-ventral or half-dorsal limbs). Double or half distributions of muscle were present at both forearm and metacarpal levels. Where double ventral muscle occurred at the forearm the symmetry of the soft tissues was also reflected in the bones as a normally ventral spine on the ulna was present on both sides of the bone. In the upper arm, ventral half stumps regenerated a high proportion of limbs with very similar muscle patterns to those found here. Upper arm dorsal half stumps however, nearly always regenerated limbs with a normal dorsoventral muscle pattern; the exception was one limb that was half dorsal (Wigmore & Holder, 1985). A possible explanation for the normality of muscle patterns from upper arm dorsal regenerates was that the boundary between dorsal and ventral tissue (see Meinhardt, 1983) lies dorsal to the humerus in the upper arm. In the experimental protocol used, this could have led to the inclusion of ventral tissue in dorsal half upper arm stumps. The higher proportion of regenerates from lower arm dorsal half stumps with only dorsal muscle present may indicate that this boundary is located more ventrally in the forearm. Despite the above differences, the production of limbs with only dorsal muscle from dorsal half stumps and only ventral muscle from ventral half stumps in both upper and lower arms is evidence that these two tissue types act mosaically during regeneration. Results from limbs symmetrical in the dorsoventral axis (Burton et al. 1986) and the surgical construction of discontinuities in this axis within the stump (Holder & Weekes, 1984) have provided further evidence for this.

Control limbs had normal dorsoventral muscle patterns for all four types of stump which was not surprising, as, despite the uneven shape of the stump after removal of half of the limb, there was no barrier to the interaction and incorporation of all parts of the stump. The anteroposterior skeletal pattern was also normal for most posterior, dorsal and ventral half stumps but a frequent abnormality in anterior halves was the presence of an extra basal carpal between basal carpals two and three (Fig. 10). This bone did not appear in any experimental regenerates but its occurrence may be linked to the presence of ‘double’ blastemas seen on anterior half control stumps. Without the head skin that was present in experimental stumps, control stumps had two transverse amputation plançs at slightly different proximodistal levels. It seems likely that both planes start to produce blastemas which subsequently fuse to form a single regenerate. The presence of the extra basal carpal maybe evidence that the integration between the two blastemas was not perfect.

The technique described here enables the isolation of half stumps with head skin. This tissue, despite contributing cells to the regenerate, appears to have no morphogenic effects (Wigmore & Holder, 1985) but by preventing any contribution from cells on the side of the limb that was removed we were able to show differences in the regenerative ability of different parts of the limb. The major difference between upper and lower arm results was the inability of any part of the lower arm to produce a high proportion of normal regenerates. In the lower arm posterior half stumps can produce a variable amount of the anterior–posterior axis from 1 to 4 digits while the anterior half can produce between 1 and 3 digits but with a high proportion of single-digit regenerates. The variation in the completeness of the pattern is possibly due to variations in the position of the medial cut made in constructing these stumps.

Dorsal and ventral lower arm halves showed approximately equal regenerative potential. Many of these limbs were similar to regenerates from upper arm operations in having only dorsal or ventral muscle. It is likely that the dorsoventral axis is specified in a similar way in both the upper and lower parts of the arm. The ability to regenerate the pattern of the normal limb is thought to be due to the holding of different positional values by cells in different parts of the limb. A possible explanation for the difference between upper and lower arms in the regeneration of the anteroposterior axis suggested by Maden (1979) is that positional values are clustered in the posterior dorsal quadrant of the upper arm but are more diffusely distributed in the lower arm. The present results fit this interpretation but further work is required to define the interactions between different regions of the limb in making the normal pattern.

I would like to thank Rosie Burton, Dick Glade and Nigel Holder for many useful comments on this work and Meena Jesani for her expert technical assistance. The project was supported financially by the Science and Engineering Research Council.