regeneration of surgically created mixed-handed axolotl ...ventral axis. in this study we have...

J. Embryol. exp. Morph. 82, 217-239 (1984) 2 1 7Printed in Great Britain © The Company of Biologists Limited 1984

Regeneration of surgically created mixed-handed

axolotl forelimbs: pattern formation in the

dorsal—ventral axis

By NIGEL HOLDER AND CHARLESTON WEEKESAnatomy Department, King's College, Strand, London WC2R2LS, U.K.

SUMMARY

The regeneration of surgically created mixed-handed limb stumps is examined in theaxolotl. Operations were performed in the lower arm and upper arm regions and grafts wereallowed to heal for approximately one month prior to amputation or were amputatedimmediately. In the lower arm group both anterior and posterior limb halves were inverted,whereas only posterior halves were inverted in the upper arm group. Almost all the limbsregenerated were normal in the anterior-posterior axis, whereas a range of limb types werefound when the dorsal-ventral axis was analysed using the metacarpal muscle pattern andepidermal Leydig cell number as positional markers. The carpal and forearm muscle patternswere also analysed in order to assess whether the pattern determined from analysis at themetacarpal level reflected that seen at more proximal levels. The results are discussed in termsof the possible role of cell contribution from the stump to the blastema and the relevance ofthe study to models of pattern regulation.

INTRODUCTION

Pattern regulation during limb regeneration is standardly discussed in termsof the three cardinal limb axes; the anterior-posterior axis, the dorsal-ventralaxis and the proximal-distal axis. The recreation of positional values within theblastema has been studied by various types of tissue-grafting operations whichaffect cellular position with respect to one or more of these axes (Tank & Holder,1981). In this paper we further analyse pattern regulation in the dorsal-ventralaxis in the light of recent advances. Unlike the anterior-posterior axis, thedorsal-ventral appears not to rely on a principle of continuity in that limbpatterns bearing clear anatomical discontinuities in this axis are readily obtainedafter certain grafting experiments. To our knowledge no case exists in theliterature where comparable discontinuities have been observed in the anterior-posterior axis following many different types of surgical manipulation.

The discontinuities in the dorsal-ventral axis have been identified in super-numerary limbs following 180° ipsilateral blastema rotations (Maden, 1980,1982, 1983; Maden & Mustafa, 1982; Tank, 1981; Papageorgiou & Holder,1983), and in supernumerary outgrowths formed following skin transplantationand nerve deviation (Reynolds, Holder & Fernandes, 1983; Maden & Holder,

218 N. HOLDER AND C. WEEKES

1984). Four basic categories of limb anatomy have been identified in these typesof supernumerary limbs, two of which bear anatomical discontinuities. These arepart symmetrical-part asymmetrical patterns and mixed-handed limbs in whichone part of the pattern is inverted in the dorsoventral axis with respect to theremainder. The remaining limb types found in these supernumeraries are eithernormal or completely symmetrical double dorsal or double ventral limbs.Neither of these limb types bear anatomical discontinuities.

The existence of discontinuities poses severe problems for any model based ona principle of continuity, irrespective of the method for creating continuouspositional values. For this reason we have performed a series of experimentsdesigned to further elucidate the mechanisms of pattern regulation in the dorsal-ventral axis. In this study we have analysed the regenerative ability of surgicallycreated mixed-handed limbs. As the result of surgical construction we know whatthe initial structure of any outgrowth will be, unlike the situation with super-numeraries. The times of graft healing prior to amputation and the limb levelsat which operations were performed were also varied to test the specific predic-tions of the shortest arc intercalation model for the control of pattern regulationand distal outgrowth (Bryant, 1978; Bryant & Baca, 1978; Bryant et al. 1982;Holder et al. 1980). The results demonstrate a number of points relating to themaintenance and position of discontinuities in the dorsal-ventral axis, the com-plicated diversity of limb anatomies which form following pattern regulation inthis axis and the relationship between this axis and the anterior-posterior axis.

MATERIALS AND METHODS

General

All experiments were performed on larval axolotls (Ambystoma mexicanwn)which were spawned in the colony at King's College. Animals were kept inindividual plastic containers in standing tap water throughout the course of theexperiment and were fed twice a week on chopped heart. All animals wereanaesthetized in MS222 during surgery or harvesting of the limbs. The animalswere between 55 mm and 85 mm in length.

Experimental

Operations were performed on either upper or lower arms. The exact surgicalprocedure will be described for both.

1. Lower arm operations

In experimental cases the limb was split between digits 2 and 3 and a cut madeup to the elbow between the radius and ulna. In one group the anterior half ofthe limb distal to the elbow was removed by disarticulating the radius and cuttingthe anterior tissue free. In a second group the posterior half (digits 3 and 4) was

Regeneration of mixed-handed axolotl forelimbs

v D

D v

219

V D

Fig. 1. Diagrammatic representations of the operations performed. A. Lower armoperations. B. Upper arm operations. In both diagrams the dashed region in the endon views of the amputated limbs represent the grafted tissue. A, anterior; P, pos-terior; D, dorsal; V, ventral; r, radius; u, ulna; h, humerus.

removed after disarticulation of the ulna. The corresponding procedure was car-ried out on the contralateral limb and the separated pieces were exchanged. Thegrafts were then realigned on the opposite limb with the anterior-posterior andproximal-distal axes of graft and host in harmony and the dorsal-ventral axesmisaligned (Fig. 1A). The grafts were then sutured in place using fine thread.Sutures were placed at the proximal end of the graft and in the dorsal and ventralmidline. The limbs were either amputated immediately following the surgery orafter about a month of healing (between 25 and 35 days after initial surgery). Inboth instances, amputation was performed at the mid-forearm level, leaving atleast 2 mm of forearm stump with a dorsal-ventral discontinuity (Fig. 1A).

Control operations involved following the same surgical procedure but theanterior or posterior halves of the lower limb were sutured back into their normalposition without exchange to the contralateral side. These limbs were amputatedeither immediately after surgery or after a month of healing.

2. Upper arm operations

Only posterior tissue was exchanged in this group. The operations involvedcutting out the posterior half of the upper arm. Care was taken to leave theforearm flexor nerve and its adjacent blood vessel intact in the mid-ventral line,and the muscles were removed from the posterior edge of the humerus, leavingit intact in the host limb. The dorsal anconaeus muscle was split into two partsand the triceps muscle was included in the graft. Once removed, the graft was ex-changed with the corresponding posterior upper limb half from the contralateral


limb. The grafts were aligned in the anterior-posterior and proximal-distal axesand misaligned in the dorsal-ventral axis (Fig. IB). The grafts were sutured inplace at the proximal end and in the dorsal and ventral mid-lines. Limbs were thenamputated either immediately after surgery or after one month of healing.

Control operations involved the same surgical procedure but the grafts werereplaced and sutured into their normal positions without contralateral exchange.Control operated limbs were either amputated immediately or after one monthof healing.

All operated animals were observed at weekly intervals to assess the survivalof the graft.

3. Analysis of limb anatomy

All experimental and control operated limbs were removed from the animalafter 6 to 8 weeks of regeneration. They were fixed in Bouin's fluid and decal-cified in EDTA before being dehydrated, stained with Victoria blue and cleared.The skeletons of all limbs were then drawn using a camera lucida. The limbs werethen returned to absolute alcohol and processed for wax embedding. Serial 10 [imtransverse sections were cut and stained with haematoxylin and eosin. Themuscle pattern of each limb was then recorded by examining sections at the planeof the metacarpals, carpals and mid-forearm.

In addition to the musle patterns, the dorsal-ventral asymmetry of Ley dig cellnumber in the epidermis was assessed at the metacarpal level. Ley dig cells arelarge secretory cells which are commonly found in the epidermis of urodeles (seeKelly, 1966, and Hay, 1961, for detailed accounts of their structure and possiblefunction). In order to analyse Ley dig cell number, a series of unoperated controllimbs were fixed, wax embedded and sectioned to assess the normal epidermalanatomy in the forelimb. Leydig cell asymmetries were quantitatively assessedby counting the number of Leydig cells in dorsal and ventral epidermis andexpressing this number as a percentage of total epidermal cell number for dorsaland ventral limb regions.

Fig. 2. Aspects of normal limb anatomy. (A) Camera-lucida drawing of a dorsalview of a normal forelimb regenerate showing the skeleton in a Victoria-blue-stainedspecimen. The dashed lines show the approximate levels at which transverse sectionswere analysed in order to examine muscle patterns and epidermal character. Magni-fication xlO. (B) Camera-lucida drawing of a transverse section at the metacarpallevel of a normal limb. The ventral muscles are marked with asterisks. Magnificationx38. (C) Light micrograph of the dorsal epidermis of a normal limb at the metacarpallevel. The relatively thick epidermis contains many Leydig cells (L) with scatteredepidermal cells (e). A clear basement membrane (bm) is seen and a region of an ebdmuscle. Magnification x 108. (D) Light micrograph of the ventral epidermis from thesame section as C shown at the same magnification. The epidermis is clearly thinner,contains fewer Leydig cells and relatively greater numbers of other epidermal cells.h, humerus; r, radius; u, ulna; r', radiale; u', ulnare; i, intermedium; m, metacarpal;A, anterior; P, posterior; D, dorsal; V, ventral.

Regeneration of mixed-handed axolotl forelimbs 221

• „.» *

Q

EMB82


RESULTS

A. Normal limb anatomy

The skeleton, muscles and Leydig cell numbers were used to assess the patternof the regenerated limbs. The skeleton was used to examine the normality of theanterior to posterior axis. The crucial features of the forelimb skeleton have beenoutlined on numerous occasions (see for example, Tank & Holder, 1978) and anormal limb skeleton is shown in Fig. 2A.

The muscle pattern at the metacarpal level has been used as a dorsal-ventralmarker in several recent studies (see description by Maden 1980,1982). In theseprevious experiments the muscle patterns were examined mostly in supernumer-ary limbs (Maden, 1980,1982; Maden & Mustafa, 1982; Tank, 1981; Reynoldset al. 1983; Papageorgiou & Holder, 1983) where more proximal levels areunclear due to fusion of the supernumeraries with the host limb. In the presentseries of experiments, because the experimental limbs were direct outgrowthsfrom an operated stump, we have assessed the muscle patterns at the carpal andmid-forearm levels for the first time as markers for dorsoventrality in amphibianlimbs. For this reason a brief account of the muscle patterns at these moreproximal limb levels is needed. The normal anatomy of the muscles of theforearm and hand of the axolotl forelimb has been described in detail by Grim& Carlson (1974).

The patterns of muscles in the metacarpal region are clearly dissimilar in thedorsal and ventral positions. The important difference is that in the ventralregion there are a complicated series of five separate muscles which are con-tinuous across the anterior-posterior axis whereas dorsally four separate musclesoccur, one associated with each metacarpal (Fig. 2B, and Maden, 1980, 1982).

At the proximal carpal level the muscle pattern on the dorsal side of the carpalsis similarly less complicated than that on the ventral side. There are two musclesdorsally and six muscles ventrally. The names and positions of these muscles are

Fig. 3. Normal muscle patterns at the proximal carpal and mid forearm levels. (A)A transverse section at the proximal carpal level stained with haematoxylin andeosin. Magnification x34. (B) Camera-lucida drawing of the section shown in A,giving the names of the muscles. The abbreviations are: (1) extensor muscles; edc,extensor digitorum communis; ad 1, abductor minimi 1. (2) flexor muscles; ps, pal-maris superficialis; uc, ulno carpalis; pp 1,2,3, palmaris profundus muscles; abdm,abductor brevis digiti mimimi. The carpals are radiale, intermedium and ulnare fromanterior to posterior (see Fig. 2A). (C) Transverse section at the mid-forearm levelstained with haematoxylin and eosin. Magnification x32. (D) Camera-lucida draw-ing of the section shown in C giving names of the muscles. The abbreviations are: (1)extensor muscles; edc, extensor digitorum communis; ecr, extensor carpi radialis; ear,extensor antebrachii radialis; eacu, extensor antebrachii carpi ulnaris. (2) flexormuscles; ps, palmaris superficialis; uc, ulno carpalis; facr, flexor antebrachii et carpiradialis; feu, flexor carpi ulnaris; pq, pronator quadratus. D, dorsal; V, ventral; A,anterior; P, posterior; r, radius; u, ulna.


Table 1. Ley dig cell/epidermal cell ratio

Generalepidermal cells Leydig cells Total Ratio

Dorsal epidermis 409 ±45* 194 ±26 603 ± 65 0-47 ±0-04Ventral epidermis 525 ±79 80 ±26 605 ±91 0-15 ±0-02

*±S.D.

shown in Fig. 3A,B. The forearm muscle pattern is more complex. The maincomplicating feature is the change in the pattern which occurs at differentforearm levels. For this reason the mid-forearm was selected as the position toassay the pattern (Fig. 3C,D). This region was easily located because of thepresence of a ventral muscle concerned with pronating the limb, the pronatorquadratus (pq), which runs between the radius and ulna in the mid-third of theforearm. In transverse sections the fibres of the muscle are cut tangentiallymaking its identification straightforward (see Fig. 6D). The remaining flexor andextensor muscles in this mid-forearm position are also readily identifiable bytheir individual shape and their position relative to the other muscles and to theradius and ulna. Dorsally just four muscles are found, whereas five muscles occurin the ventral flexor group. The names and positions of these muscles are shownin Fig. 3.

The third marker used to assay dorsoventrally is the presence of Leydig cellsin the epidermis. At the metacarpal level more Leydig cells are found in thedorsal epidermis than the ventral epidermis. This difference has been quantifiedby counting Leydig cells in sections at this level in seven normal limbs, andexpressing Leydig cell number as a ratio of the total epidermal cell count indorsal and ventral epidermis. The results are presented in Table 1. At the lightmicroscope level the remaining epidermal cells appear to be histologicallysimilar and no attempt was made to further categorize them. In Table 1 thesecells are referred to as general epidermal cells. It can be seen that closely similarnumbers of cells are found in dorsal and ventral epidermis but approximatelythree times as many of the total epidermal cells are Leydig cells in the dorsalepidermis. This marker is extremely specific and the numbers are statisticallysignificantly different. The clear structure of the Leydig cells allows them to beeasily visualized and because of their large size relative to the remainingepidermal cells the dorsal epidermis is noticeably thicker than the ventralepidermis (Fig. 2C,D). This feature alone often allows identification of dorsalepidermis after a mere glance at an appropriate section. Unfortunately, thesignificant difference in Leydig cell number at the metacarpal level disappearsat more proximal levels where many Leydig cells are found in dorsal and ventralepidermis.


Table 2. The anatomy of the a-p axis

Level ofamputation

Healingtime

(days) TotalExtra

Normal digits

Normaldigits

Abnorm.forearm

Reduced*digit no.

Spikes/tHalf limbs

No.regn.

Lower armLower armUpper armUpper arm

030

030

18131618

101410

* Normal forearm but only three digits.t Abnormal forearm and reduced digits.

B. Anatomy of experimental limbs

The features of the anterior-posterior and dorsal-ventral axes will be presen-ted in turn.

1. The anterior-posterior axis

The anatomy of the a-p axis was assessed from Victoria-blue-stained wholemounts. It was normal in the great majority of cases, in both upper arm and lowerarm operations following immediate amputation and amputation afterapproximately one month of graft healing. Of a total of 65 operated limbs in allgroups 52 (80 %) had an essentially normal a-p pattern in the hand (Table 2).No clear differences were evident in the lower arm amputations between theanterior (12 cases) and posterior (19 cases) operated groups, so this variation hasnot been entered in Table 2.

In the lower arm amputations, principally in the immediate amputation group,the forearm elements were often abnormal even though a normal hand wasformed. The forearm abnormalities involved fusion of the radius and ulnaproximally with an essentially normal elbow (eight of the ten cases) or an abnor-mal elbow (two of the ten cases). In contrast, when a normal hand was formedfollowing amputation in the upper arm the forearm was invariably normal (Table2). In just two cases, both in the lower arm amputated groups, only three digitswere formed in an otherwise normal limb.

Two categories of limbs were found only in the upper arm groups. These werenon-regenerates, where no outgrowth occurred following amputation (twocases, both in the 30-day healing group), and limbs which had either a singleforearm element and one or two digits, or two forearm elements with one or twodigits (Table 2).

2. The dorsal-ventral axis

A range of limb types was identified following the analysis of muscle and epider-mal markers at the metacarpal level of the regenerates. The results of this analysis


Table 3. Muscle patterns of lower arm operated limbs

CaseHealing time

(days)

Anterior side inverted.1

2

3

4

5

6

7

8

0

35

25

25

34

0

35

30

Posterior side inverted.9

10

11

12

13

14

15

16

17

18

19

20

21

22

30

30

30

30

30

0

0

0

0

0

30

0

0

0

1

DVVDVDVD

v *D *DD *D *VVD *

DVDVDVDVDVDVDVDVDVDVDVDVD *VDV

* represents a point of discontinuity.

Digit number

2

DVVD *v *

D*VV

D*VDVDV

v *V

v *V

D *

v *D*VV*DD*V

V *D*V

v *D*V

v *DVDVDVD *VD *VDVD *VVVD *V

3

DV

V*DVDV

V*DVDVDVDVDV

VDVDVDVDVD

D*VV

D*VV

D*VVVVVVD *VVVV

V*DV

v *

4

DVDVDVDVDVDVDVDV

VDVDVDVDVDVVVVVVVVVVVVDVVDVD

Category

1

2b

2b

2b

2c

3b

5a

5b

2a

2a

2b

2b

2b

3a

3a

3a

3a

3a

3b

5a

5b

5b


Table 4. Dorsal/ventral symmetry of upper arm operated limbs

CaseHealing time

(days)

Posterior side inverted.1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

0

0

0

0

30

0

0

30

30

30

0

0

0

30

0

0

0

30

30

0

0

30

30

30

1

DVDVDVDVDVDVDVDVVVDDDVDVDVDVDVDVDVDVD

v *D

v *DDDDVVDV •

* represents a point of discontinuity.

Digit muscle

2

DVDVDVDVDVD *V *DV

D*VVV

v *D *DD

v *DV *D

v *D

v *DVDVDVDVDDDDDDDDVVD *V

patterns

3

DVDVDVDVDVVDD *v *VVVDVDDDDDDDDDD *VD *VD *VDV *DDDDDDDDVVV

v *

4

DVDVDVDVDVVDVDVVVDVDDDDDDDDDVVVVVVDDDDDDDDDDVVVD

Category

1

1

1

1

1

2a

2c

3a

3a

3a

3a

3a

3a

3a

3b

3b

3b

3b

3c

3c

4

4

4

5b


are shown for the lower arm operations in Table 3 and the upper arm operations inTable 4. Following the description of the different types of limbs their frequency offormation in the different experimental groups will be presented. Finally, theresults of the analysis of forearm and carpal muscle patterns will be discussed.

a. Extra skeletal structures. Of the 65 experimental limbs five (8 %) producedextra skeletal structures in the dorsal-ventral plane. All of these involved theformation of extra digits which emerged on the ventral stump side of the out-growth at the metacarpal level. One of these cases was from a lower arm amputa-tion (case 13, Table 3) and four were from upper arm amputations (cases 2, 6,9 and 19, Table 4). All except one were single extra digits and case 6 in Table 4had two clearly defined digits beneath the inverted digits 3 and 4.

b. Categories of limb types identified from sections. Only limbs with fourdigits in the anterior-posterior axis and an otherwise normal or near normalanterior-posterior anatomy were sectioned. Of these seven lower arm operatedlimbs failed to process correctly following Victoria blue staining. As a result, 22out of 31 lower arm amputations and 24 out of 32 upper arm amputations wereanalysed. These sectioned limbs fell into five basic anatomical classes as deter-mined by the muscle and epidermal markers at the mid-metacarpal level. Theselimb types are described below. The anatomies of each individual limb for lowerarm and upper arm groups are given in Tables 3 and 4, the five classes of limbtypes are represented diagrammatically in Fig. 4 and examples of each class ofabnormal anatomy are shown in Fig. 5.

Type 1. Normal limbs

The anatomy of normal limbs has already been described. An example isshown in Fig. 2B.

Type 2. Mixed-handed limbs

Three subclasses of mixed-handed limbs were identified depending on theposition of the anatomical discontinuity, (a) In the first of these the pattern wasexactly that which was created by surgery, with the line of discontinuity clearlylocated between digits 2 and 3, (Fig. 5A). (b) In some cases the line of disconti-nuity ran at an angle from dorsal to ventral so that one digit bore a hybrid musclewhich changed form from ventral to dorsal. This distinction was coupled with achange in epidermal character at the corresponding anterior-posterior position,(c) The line of discontinuity shifted one complete digit width in this group so thatthree digits had one polarity and one digit was inverted.

Type 3. Part normal/part symmetrical limbs

Again, three sub classes of such limbs were seen which were distinguished bythe position of the discontinuity. Thus, the symmetrical region involved one, twoor three digits with the remainder of the pattern being asymmetrical. Thesymmetrical region involved either dorsal or ventral character, (Fig. 5B,C).

Regeneration of mixed-handed axolotl forelimbsType 1 Type 2

229

Type 3 Type 4

Type 5 TypeS

Fig. 4. Schematic representations of the categories of regenerates seen withreference to the anatomy of the dorsal-ventral axis at the metacarpal level. Fiveclasses are identified based on muscle patterns and epidermal character. Represen-tative sections of each class and some of the sub-classes described in the text areshown. The dashed regions represent dorsal and the clear regions ventral and thesmall circles are metacarpals.

Type 4. Symmetrical limbs

These limbs showed a completely symmetrical character involving eitherdouble dorsal or double ventral symmetry (Fig. 5D).


DD

Fig. 5. Camera-lucida drawings of specimens from the experimental series showingthe skeletons and metacarpal level muscle patterns of the same limb. Abbreviationsin all drawings are; A, anterior; P, posterior; D, dorsal; V, ventral. Numbers 1-4refer to digit numbers, see Fig. 2A. Magnifications of skeletal preparations x9, ofsections x30. (A) A mixed-handed pattern with the discontinuity between digits 2and 3, (type 2a). This limb regenerated after immediate amputation of a right upperarm operation in which the posterior side was inverted (case 6, Table 4). (B) A partdouble ventral/part normal pattern with the posterior two digits symmetrical (type3a). This limb regenerated after immediate amputation of a right lower arm opera-tion in which the posterior side was inverted (case 17, Table 3). (C) A part doubledorsal/part normal pattern with the posterior two digits symmetrical (type 3a). (D)A completely symmetrical double dorsal limb (type 4) regenerated following im-mediate amputation of an operated left upper arm (case 21, Table 4). (E) A limb witha symmetrical ventral region flanked by asymmetrical regions of the same polarity(type 5a). This limb regenerated following immediate amputation of a left lower armoperation in which the posterior side was inverted (case 20, Table 3). (F) A limb witha symmetrical ventral region flanked by asymmetrical regions of opposite polarity(type 5b). This limb regenerated following immediate amputation of a left lower armin which the posterior side was inverted (case 22, Table 3).


D D

Type 5. Limbs with a symmetrical region located between two asymmetricalregions

Two sub-classes of this pattern were found and ventral symmetry was iden-tified in all cases. In the first a double ventral region was flanked by twoasymmetrical regions of the same polarity (Fig. 5E), whereas the second typeshowed flanking regions of opposite polarity (Fig. 5F). These types of limbs havenot been described previously.

c. The frequency of formation of limb types. The frequency of formation ofthe five limb types varied only slightly between forearm and upper arm groups(Table 5). In the upper arm groups part normal part symmetrical limbs were mostfrequent (54 %) , with all three subclasses of this type represented. All classeswere represented in the upper arm operations. In contrast, mixed handed limbswere most frequent in the lower arm operated group (41 %) and part normal/part symmetrical patterns were also commonly found (32%). No completelysymmetrical limbs were formed in the lower arm group.


Table 5. Frequency of limb types found in the regenerates with reference to levelof amputation

Limb type*

12345

Totals:

Upper arm

Total

52

1331

24

* With reference to the categories shown

%

218

54134

100

in Fig. 4.

Lower arm

Total

19705

22

%

441320

23100

Table 6. Frequency of limb types found in the regenerates with reference to timeof graft healing

Limb type*

12345

Immediate amputation

Total

52

1313

%

218

544

13

30 day amputation

Total

19723

%

44132

914

Totals: 24 100 22 100

* With reference to the categories shown in figure 4.

Similar frequencies of limb types are seen if immediate and 20-day periods ofhealing are compared as pooled data from lower arm and upper arm groups(Table 6). Thus, after immediate amputation, part normal/part symmetricalpatterns are the most frequent (54 %) whereas, following graft healing for 30days, mixed handed (41 %) and part normal/part symmetrical limbs (32 %) arethe commonest types regenerated.

Fig. 6. Examples of abnormal carpal and forearm muscle patterns. (A) Lightmicrograph of a section cut at the proximal carpal level of a limb identified as doubledorsal at the metacarpal level (case 22, Table 4). Magnification x32. (B) A cameralucida drawing of the same section identifying the muscles. Abbreviations as for Fig.3A,B. (C) Light micrograph of a section cut at the mid forearm level of a limbidentified as double ventral at the metacarpal level (case 23, Table 4). Magnificationx32. (D) Higher power view of the section in C showing the two copies of pq.Magnification X109. Abbreviations as for Fig. 3C,D.


d. The effect of grafting on the anatomy of regenerates. One consistent aspectof the results is the maintenance of anatomy and polarity in the host, unoperated,side of the limb pattern and the variability of the operated side in the regeneratedpatterns. When the eventual patterns are examined the extreme lateral or medialdigit on the unoperated side (digit 1 for posterior inversions and digit 4 foranterior inversions) is normal in terms of anatomy and polarity in 100 % of thelower arm operations and 79 % (19 out of 24) of the upper arm operations. Inclear contrast, the appropriate extreme medial or lateral digit on the operatedside shows an inverted asymmetrical anatomy in 54 % (12 out of 22 cases) inlower arm operations and 20 % (5 out of 24 cases) in upper arm operations. Thedigit in the middle of the pattern in the unoperated side show less stability dueto local variations in position and extent of the anatomical discontinuity.

The invariability of the host side of the limb is not absolute and five cases occurin the upper arm group which show alterations in anatomy of digit 1. Of these,three were totally symmetrical limbs (cases 21—23, Table 4) and two were partnormal/part symmetrical limbs in which digits 1 and 2 were symmetrical and theoperated side regenerated an inverted asymmetric pattern (cases 10 and 11,Table 4).

e. Analysis of carpal and forearm muscle patterns. The object of analysingmuscle patterns at more proximal levels was to establish whether the patternsdetermined by analysis at the metacarpal level reflected the anatomy of thewhole regenerate. In addition, any alterations in the position of discontinuitiesdown the proximal-distal axis could be established. In the following discussionthe limb types are discussed with reference to their anatomy at the metacarpallevel.

In general, the carpal and forearm muscle patterns are not as informative asthe more distal muscle pattern. This is particularly true of the mixed-handedpatterns where the forearm musculature is extremely complex. However,normal and totally symmetrical limbs and part normal/part symmetrical patternsshow clearly interpretable muscle patterns at more proximal levels.

The most straightforward cases are the normal or totally symmetricalregenerates. In the upper arm group, all of the five normal cases showed normalmuscle patterns at carpal and mid-forearm levels. These patterns are indistin-guishable from those shown in Fig. 3. The three totally symmetrical cases alsoshowed symmetrical muscle patterns at the carpal and forearm levels. The sym-metry at these more proximal levels was appropriate for that seen at the digits.Examples of symmetrical carpal and forearm muscle patterns are shown in Fig.6. These observations clearly show that the normal and totally symmetrical caseshave this anatomy at least from the mid-forearm level and are likely, therefore,to show this anatomy to the amputation plane.

Part normal/part symmetrical patterns with two or three symmetrical digitsare more complicated at proximal levels. In the upper arm group nine limbs ofthis type showed distinct symmetry in the carpals and forearm. In the seven

Regeneration of mixed-handed axolotl forelimbs 235examples with dorsal symmetry five showed dorsal symmetry on the appropriateside in the forearm and thepg, uc and feu were missing (for muscle patterns andmuscle names see Fig. 3), and the opposite side was asymmetrical. Thus theproximal levels again reflect the pattern seen in the digits. One case (number 14,Table 4) had a normal forearm but the carpals appeared to be symmetricaldouble dorsal, leading to the part normal/part symmetrical hand. This limbshowed clear movement of lines of discontinuity down the proximal-distal axis.The remaining case with double dorsal symmetry was uninterpretable. The twolimbs with part ventral symmetry both showed duplication oiabdm at the carpalswith an otherwise normal muscle pattern and one showed two copies of pq at theforearm level with an otherwise normal pattern. In the upper arm group threelimbs showed part normal/part asymmetrical patterns with only one doubleventral digit (type 3d). In all cases (16-18, Table 4) the forearm muscle patternswere clearly normal, yet the carpal pattern reflected the discontinuity in twocases with abdm being duplicated on the posterior side with an otherwise normalpattern.

In the lower arm groups the single normal limb had a clearly normal carpal andforearm musculature. The part normal/part symmetrical cases invariably invol-ved double ventral symmetry. Of the five cases with greater than one symmetri-cal digit, three had two copies of the pq muscle (Fig. 6C), and three hadduplicated sets of uc and feu at the forearm level and all had two copies of abdmat the carpal level.

The duplication of the pq muscle also occurred in many of the mixed-handedpatterns in both upper and lower arm regenerates. This observation, even in theabsence of clarity in surrounding muscles, indicates that the discontinuity in suchlimbs has structural consequences in the dorsal-ventral axis at more proximallevels.

C. Controls

A total of 24 control operations were performed. These include 16 in the lowerarm group, with eight cases amputated immediately and eight amputated after30 days of healing. At each healing time four operations involved removing andreplacing the anterior side and four the posterior side. Eight cases were analysedin the upper arm group, four of which were amputated immediately and fouramputated after 30 days of healing. In all cases the a-p axis was essentiallynormal as assessed from Victoria blue stained wholemounts and the d-v axis wasalso normal at metacarpal, carpal and forearm levels.

DISCUSSION

The results presented in this study provide several insights into the mechanismof pattern regulation in the dorsal-ventral axis. The advantage of examiningregeneration from surgically constructed mixed-handed limb stumps is that the


spatial relationships of cells in the stump prior to amputation are clearly iden-tified. This is in contrast to supernumerary limbs produced following 180°ipsilateral blastema rotation where the eventual anatomy of the extra limbs isused to retrospectively determine the events which led to their formation(Maden & Mustafa, 1982; Maden, 1983). It is reassuring, therefore, that theprinciples of pattern regulation derived from these two types of grafting experi-ment are similar. Two basic points can be made from the results of the presentstudy. (1) It is clear that a number of anatomical limb types can regeneratefollowing amputation of mixed-handed limb stumps. Of the five basic typesidentified (Fig. 4) four have been described in supernumeraries produced eitherfrom blastemal rotation (Maden, 1980, 1982, 1983; Maden & Mustafa, 1982;Papageorgiou & Holder, 1983) or nerve deviation and skin grafting (Reynoldset al. 1983; Maden & Holder, 1984). These are normal limbs, mixed-handedlimbs, part normal/part symmetrical limbs and completely symmetrical limbs.In addition, we have identified a new class of anatomy where a symmetricalregion lies centrally in the pattern and is flanked by asymmetrical digits (Table 5and Figs 4, 5E,F). (2) Anatomical discontinuities where dorsal and ventral cellslie adjacent to one another occur in many of the limbs regenerating from mixed-handed stumps. During regeneration it appears that little interaction occursbetween normally disparate cells in the dorsal-ventral axis, although, in a veryfew cases (8 % of operated limbs) extra digits appeared on the ventral side of theoutgrowth.

A striking feature of the results is that the anatomy of the regenerates on thegrafted side of the limb is far more variable than the anatomy of the unoperatedside (Tables 3 and 4). This is evident irrespective of whether the anterior orposterior limb region is inverted. The significance of this observation with res-pect to the patterning mechanism is unclear at the present time, however, thevariability of regeneration of grafted tissue has been noted previously followingthe amputation of surgically created double anterior and double posterior limbs(Tank & Holder, 1978; Holder et al. 1980; Holder, 1981). In these cases halflimbs regenerate with a frequency of approximately 25 % and the regenerateinvariably arises from the unoperated side of the stump. This result suggests thatdedifferentiation and cell contribution to the blastema maybe severely affectedin the grafted tissue and this conclusion is supported by the histological appear-ance of tissue in the grafted limb region where the muscle and general organiza-tion of other tissues is clearly disrupted (Tank & Holder, 1978). It is possible,therefore, that the pattern of cell contribution from different parts of the stumpplays a role in the production of the different limb types produced in the presentstudy. A complicating factor here is the appearance of the regenerates fromcontrol grafts which invariably have a normal d-v anatomy. It is possible that cellcontribution from the stump is a crucial part of the pattern regulation process andefforts to study this aspect of regeneration are under way at the present time.

The results in this paper have a bearing on two formal models for pattern

Regeneration of mixed-handed axolotl forelimbs 237regulation. The first of these is the boundary model (Meinhardt, 1983) whichproposes that two boundaries or discontinuities exist in the normal limb, one ineach of the transverse axes. Meinhardt has suggested that these two boundariesmust intersect before distal outgrowth will occur. The model does not discuss thenature of the distinction between dorsal and ventral cells but the notion of aboundary between cells in these limb regions in a normal limb is supported bythe appearance of limbs bearing clear discontinuities in this and other studies(see also Maden, 1983). Such discontinuities would represent a shift in positionof the boundary in abnormally patterned limbs. However, the regeneration ofsymmetrical double dorsal and double ventral limbs and limbs which are partsymmetrical on the host posterior side pose problems for the boundary modelbecause, presumably, no d-v boundary is present in such symmetrical regions and,as a result, the interaction between the a-p and d-v boundaries necessary for limboutgrowth cannot occur. Nonetheless, limbs with such patterns do regenerate.Thus, although the idea of a discontinuity in the d-v axis in normal limbs isattractive the formulation of the boundary model as it stands is problematical.

The second formalism to be discussed is the polar coordinate model (Bryantet al. 1981). Any model which predicts the smoothing out of discontinuities andthe subsequent appearance of extra structures fails to explain the maintenanceof pattern discontinuities. At face value, therefore, the results presented herecannot be explained by the polar coordinate model. However, this conclusionmay be thought naive because, as has been discussed previously (Bryant et al.1982; Holder et al. 1980; Holder, 1981), local cell-cell interactions may beconstrained by healing modes which may be governed by wounds or field shape.Such arguments have been used to explain the differential regeneration ofsymmetrical double posterior and double anterior limbs following amputationafter various periods of graft healing and the expansion of the a-p axis followingamputation of double posterior limbs at different limb levels (Holder et al. 1980).In brief, at short healing times cell contact is prevented by the wound, but, ashealing continues cell contact ensues. In terms of the present experiments,therefore, a lack of interaction between dorsal and ventral cells at the sites ofdiscontinuity maybe expected when mixed-handed limbs are amputated immedi-ately after surgery but extra structures would be expected when such limbs areamputated after a month of healing. In fact, extra structures are produced at avery low frequency at both time periods. This result is consequently not consis-tent with a healing constraint governing cell-cell contact. Similarly, expansionof the a-p axis was originally explained by a preferential dorsal to ventral healingmode at the forearm level (Holder etal. 1980; Bryant et al. 1982) and the ellipti-cal shape and cell numbers separating the cardinal axes in the lower arm blastemaare consistent with this notion (Holder, 1981; Holder & Reynolds, 1983,1984).However, such a biased mode of cell contact between dorsal and ventral cellswould preclude the regeneration of symmetrical double dorsal and doubleventral limbs distal to the elbow because cells with like positional values would


contact and intercalation would cease. This is exactly the argument used to explainwhy symmetrical double anterior and double posterior limbs fail to regenerate atlong healing times in upper arm amputations (Holder et al. 1980; Bryant et al.1982). It is evident from the results presented here and those from experimentsinvolving 180 ° blastema rotation (Maden & Mustafa, 1982) that such symmetricallimbs can and do regenerate distally complete limbs which have a normal a-p axis.We must conclude that the arguments of the shortest arc intercalation model(Bryant, 1978; Bryant & Baca, 1978) which underlies the proposals of the polarcoordinate model with respect to amphibian limbs are inconsistent with both a lackof a healing time effect in the regeneration of mixed handed limbs and theregeneration of symmetrical double dorsal and double ventral limbs.

Finally, the results presented in this paper demonstrate that, under the con-ditions imposed by this particular grafting procedure, dorsal-ventral discon-tinuities are stable. However, this is apparently not the case when the dorsal-ventral axis is misaligned following contralateral blastema exchanges whichresults in the formation of supernumerary limbs at dorsal and ventral positionsin the graft-host junction (Bryant & Iten, 1976; Tank, 1978). The reason for thisdifference is unclear at the present time, but the issue will be discussed in detailin a subsequent paper which will describe regeneration from limb stumps bearingdiscontinuities in the anterior-posterior axis which are surgically created in amanner comparable to mixed-handed stumps.

It is a pleasure to thank Malcolm Maden, Rosie Burton, Nigel Stephens and Peter Wigmorefor various comments and criticisms during the course of this work, which was supportedfinancially by the SERC.

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GRIM, M. & CARLSON, B. M. (1974). A comparison of morphogenesis of muscles of theforearm and hand during ontogenesis and regeneration in the axolotl (Ambystomamexicanum). 1. Anatomical description of muscles of the forearm and hand. Z. Anat.EntwGesch. 145, 137-148.

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in the axolotl. Wilhelm Roux. Arch, devl Biol. (in press).MADEN, M. & MUSTAFA, K. (1982). The structure of 180 degree supernumerary limbs and a

hypothesis of their formation. Devi Biol. 93, 257-265.MEINHARDT, H. (1983). A boundary model for pattern formation in vertebrate limbs.

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REYNOLDS, S., HOLDER, N. & FERNANDES, M. (1983). The form and structure of supernumer-ary hindlimbs formed following skin grafting and nerve deviation in the newt, Trituruscristatus. /. Embryol. exp. Morph. 77, 221-241.

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(Accepted 19 March 1984)

regeneration of surgically created mixed-handed axolotl ...ventral axis. in this study we have...

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