Retinoic Acid Coordinately Proximalizes Regenerate Pattern and Blastema Differential Affinity in Axolotl Limbs

Development 102. 687-698 (1988) 687 Printed in Great Britain © The Company of Biologists Limited 1988

Retinoic acid coordinately proximalizes regenerate pattern and blastema differential affinity in axolotl limbs

KAREN CRAWFORD* and DAVID L. STOCUM

University of Illinois, Department of Cell and Structural Biology, 506 Morrill Hall, 505 S. Goodwin Ave., Urbana, IL 61801, USA

•Present address: Department of Biology, Swarthmore College, Swarthmore, PA 19081, USA

Summary

An assay that detects position-related differences in proximalized the pattern of donor forelimb regener- affinity of axolotl regeneration blastema cells in vivo ates to the level of the girdle and abolished their was used to test whether retinoic acid, which proxi- displacement behaviour on untreated host hindlimbs. malizes regenerate pattern, simultaneously proxi- Conversely, untreated forelimb donor blastemas dis- malizes blastema cell affinity. The assay involved placed distally to their corresponding levels on host autografting or homografting late bud forelimb blas- ankle regenerates, that had been proximalized to the temas derived from the wrist, elbow or midupper arm level of the girdle by retinoic acid. These results levels to the dorsal surface of the blastema-stump indicate that positional memory in regenerating limbs junction of an ipsilateral, medium-bud-stage hindlimb is directly related to blastema cell affinity, and that regenerating from the midthigh level. The grafted very similar or identical sets of level-specific affinity blastemas consistently displaced to their correspond- properties are shared by forelimb and hindlimb cells. ing levels on the proximodistal axis of the host regenerate, indicating the existence of level-specific differ- Key words: differential affinity, positional memory, ences in blastema cell affinity. Retinoic acid retinoids, axolotl, limb, regeneration, pattern formation.

Introduction restoration, via intercalary regeneration, of the pattern normally occupying the space between the two Mesenchymal cells of the amphibian limb regener- levels (Iten & Bryant, 1975; Pescitelli & Stocum, ation blastema inherit from their parent limb cells a 1980; Muneoka & Bryant, 1984). memory of their level of origin. This positional Despite the importance of positional memory in memory (Carlson, 1975) or value (Wolpert, 1971) reestablishing a normal pattern of cell neighbours specifies the proximal boundary of the regenerate by during regeneration, little is known of its cellular or preventing blastema cells forming structures proximal molecular basis. One approach to this problem is to to their level of origin (Stocum, 1984). Since the limb identify cellular properties that exhibit position- regenerates exactly what is amputated from any level, related differences along a limb axis and which therefore might be directly related to positional its proximodistal (PD) axis can be viewed as a series memory. Demonstration of a direct relationship re- of such potential regenerate boundaries. Similar sets quires showing that experimental modification of of level-specific memories appear to exist along the regenerate pattern is accompanied by modification of anteroposterior (AP) and dorsoventral (DV) axes of the cellular property. the limb (Carlson, 1975). The boundary function of Cell affinity is one property that might be related to positional memory in regeneration is seen in exper- positional memory, as shown by an assay of blastema iments that juxtapose cells from different axial levels cell adhesiveness in vitro (Nardi & Stocum, 1983). In (i.e. cells with different positional memories). The this assay, binary combinations of wrist, elbow or result is the dedifferentiation and/or mitosis of cells upper arm blastema mesenchyme were cultured in on either side of the interface, followed by the vitro, with one member of each pair being marked by 688 K. Crawford and D. L. Stocum

[3H]thymidine. The proximal blastema of each pair Materials and methods always engulfed the distal one in hierarchial fashion. This result is consistent with Steinberg's (1963, 1978) Animals and maintenance differential adhesion hypothesis, and suggests the Larval axolotls (Ambystoma mexicanum) were provided by existence of position-related differences in cell ad- the Indiana University Axolotl Colony or were obtained hesiveness along the PD axis of the limb. from spawnings of our laboratory stock. They were reared individually in 50% Holtfreter solution in waxed paper Vitamin A and its derivatives (retinoids) are agents cups, at room temperature (21 °C), and fed freshly hatched that modify regenerate pattern. The effect of reti- brine shrimp or frozen brine shrimp every other day. At the noids is to proximalize regenerate pattern in the PD time of amputation, the animals weighed 2-5 g and were axis, resulting in serial repetition in the regenerate of 6-9 cm in length. structures proximal to the amputation plane (Niazi & Surgical operations Saxena, 1978; Maden, 1982; Thorns & Stocum, 1984). (1) General procedures In the AP axis, retinoids posteriorize the pattern, Animals were anaesthetized in Benzocaine (Sigma) dis- resulting in mirror-image duplications (in regenerat- solved in 100% Holtfreter solution (0-007% w/v). Oper- ing anuran limbs and embryonic chick limbs; Niazi & ations were performed with sharp watchmaker's forceps Saxena, 1978; Maden, 1983; Tickle etal. 1982), or the and iridectomy scissors. Amputations were done in air after completion of the missing posterior pattern following placing anaesthetized animals on Benzocaine-soaked filter amputation of an anterior half limb (in urodeles; Kim paper. Grafting was done in deep Petri dishes lined with filter paper, with the animal submerged in anaesthetic & Stocum, 1986a). All these effects have been inter- solution. After grafting, the animals were maintained in the preted as a reprogramming of the positional memory anaesthetic solution for 5-6 h at room temperature to of blastema cells to a new level. Measurements of facilitate healing, then placed in 100 % Holtfreter solution. endogenous retinoic acid in embryonic chick limb They were left undisturbed in the dark for 2 days and buds have yielded results suggesting that this retinoid special precautions in feeding and changing water were observed for an additional 5 days postgrafting to avoid might be a morphogen that specifies anteroposterior dislodging grafts. All grafts healed in 24 h and blood axial pattern in the limb bud (Thaller & Eichele, circulation in the grafts was observed by 1-2 days post- 1987). operation. Retinoids modify the phenotypic differentiation of a variety of cultured normal and tumour cells. These (2) Differential affinity assay in vivo modifications are accompanied by quantitative Fig. 1 diagrams the assay used to detect level-specific changes in cell-cell and cell-substrate adhesiveness differences in blastema cell affinity in vivo. Forelimbs were that are correlated with qualitative and/or quantitat- amputated bilaterally through the wrist, elbow or distal midupper arm levels, and hindlimbs of the same animal ive changes in cell surface glycoconjugate compo- were amputated bilaterally through the midthigh. The sition (Lotan, 1980; Shapiro & Mott, 1981; Roberts & forelimbs were allowed to regenerate to the late bud stage, Sporn, 1984, for reviews). Taken in conjunction with at which time the hindlimb regenerates were at medium bud the effects of retinoids on regenerate axial pattern, [staging according to Stocum (1979)]. The forelimb blas- these findings suggest that positional memory and temas were autografted to the blastema-stump junction of the ipsilateral hindlimb after removing a piece of blastema blastema cell differential affinity might be directly epidermis and stump skin from the dorsal surface of the related. junction. The AP axis of the grafted blastema was aligned A direct test of this hypothesis requires an assay with that of the host limb. Late bud blastemas rather than that can reveal changes in both blastema cell affinity earlier stages were chosen as donors because they were easier to manipulate and they exhibit a high frequency of and in regenerate pattern in response to retinoid. The autonomous development when grafted to the dorsal fin major disadvantage of an assay for blastema cell (Stocum, 1968). The prediction was that level-specific adhesiveness in vitro (Nardi & Stocum, 1983) is that differences in blastema cell affinity would be detected as the blastema mesenchyme does not undergo morpho- displacement of the grafts to their corresponding levels on genesis in vitro, so correlations between changes in the PD axis of the host regenerate. adhesiveness and pattern in response to retinoid cannot be made. Therefore, we developed an assay (3) Test for coordinate proximalization of which detects level-specific differences in blastema regenerate pattern and blastema cell affinity cell affinity in vivo, and in which blastema morpho- For each experiment, a fresh stock solution of a\]-trans retinoic acid (RA, Sigma) was made by dissolving 50 mg of genesis takes place. Here we describe this assay and RA in 1 ml of dimethyl sulphoxide (DMSO) under subdued use it to show that retinoic acid coordinately proxi- light (to minimize photo-isomerization). Two homografting malizes blastema cell affinity and regenerate pattern. experiments were done, in which either donor or host Cellular basis of positional memory 689

Donor forelimbs regenerating from wrist, their displacement behaviour was correlated with their final elbow or midupper arm morphogenesis. As a control for proximalization of pattern, the donor hindlimbs were allowed to regenerate in situ. Regeneration is inhibited during RA-induced proximalization by approximately 10 days in animals of the size used here, and the late bud stage of an RA-treated donor forelimb was not attained until 20-22 days postamputation instead of the normal 10-12 days postamputation. Host hindlimbs were, therefore, amputated 10 days later than the donor forelimbs, so they would reach the medium bud stage at approximately the same time that the donor forelimbs reached the late bud stage. Host hindlimbs regenerating from midthigh (b) Untreated forelimb blastemas homografted to RA- treated hindlimbs (Fig. 3). In this experiment, host hindlimbs were amputated bilaterally at the ankle level. The animals were injected with RA 4 days postamputation to proximalize regenerate positional memory and allowed to regenerate to the medium bud stage. Donor forelimbs were amputated bilaterally through the wrist, elbow or midupper arm levels and allowed to regenerate to late bud. The forelimb blastemas were then homografted to the ipsilateral blastema-stump junction of the RA-treated host hindlimb and their displacement behaviour correlated with their final morphogenesis. Due to the inhibitory effect of RA, host hindlimbs did Displacement of grafts to their corresponding host levels not reach the medium bud stage until 20-22 days postamputation instead of the usual 10-12 days postamputation. Fig. 1. Scheme and result of the differential affinity assay RA-treated forelimb donors in vivo. Wrist (W), elbow (E), or midupper arm (UA) regenerating from wrist, blastemas were autografted to the blastema-stump elbow or midupper arm junction of hindlimbs regenerating from the midthigh. The dashed oval indicates the area where a patch of blastema epidermis and stump skin was removed from the dorsal surface of the host limb, and the line bisecting this oval indicates the blastema-stump junction. The grafted blastemas displaced to their corresponding host levels. A, ankle; K, knee; T, midthigh of the host hindlimb. Hindlimb hosts regenerating groups of animals were treated with RA prior to their use in from midthigh the differential affinity assay. The animals were injected intraperitoneally, via microlitre syringe, with 100ng of RA per g body weight, at 4 days postamputation (during the accumulation blastema stage). This dose and time of injection have been shown previously to maximize proximalization of pattern in regenerating forelimbs and hindlimbs (Thorns &Stocum, 1984; Niazi el al. 1985; Kim & W, E, UA Stocum, 1986ft). (a) RA-ireatedforelimb blastemas homografted to untreated hindlimbs (Fig. 2). The forelimbs of the donor animals were amputated bilaterally through the wrist, elbow or No displacement midupper arm levels and their hindlimbs were amputated bilaterally at the ankle, knee or midthigh levels. Host hindlimbs were amputated bilaterally through the mid- Fig. 2. Scheme and result of homografting RA-treated thigh. 4 days later, the donor animals were injected with wrist elbow or midupper arm blastemas to the RA to proximalize positional memory. When the donor blastema-stump junction of a host hindlimb regenerating forelimb regenerates reached the late bud stage, they were from the midthigh. Symbols as in Fig. 1. Displacement homografted to the blastema-stump junction of ipsilateral, behaviour was abolished in the grafted wrist and elbow medium-bud-stage host hindlimbs, as described in (2), and blastemas. 690 K. Crawford and D. L. Stocum

Untreated donor forelimbs regenerating Staining of limb skeletons from wrist, elbow, or midupper arm The gTaft-host combinations were allowed to regenerate for 5-7 weeks, harvested and fixed in Gregg's fixative. The limb skeletons were stained in toto for cartilage according to the method of Fox (1982), substituting methylene blue for Victoria Blue B. Donor forelimb regenerates were dis- tinguished from host hindlimb regenerates by their different skeletal patterns (Pescitelli & Stocum, 1980).

Results

Assay for blastema cell differential affinity RA-treated host hindlimbs All of the surviving forelimb blastema grafts devel- regenerating from ankle oped as recognizable forelimb regenerates. 83 % of the wrist and 74 % of the elbow blastemas displaced distally from the host midthigh level to their corresponding levels on the PD axis of the host regenerate, thus eliminating the positional discontinuity between graft and host (Table 1). Hence the fully developed wrist regenerates articulated with the host ankle, and the elbow regenerates articulated with the host knee (Figs 1, 4A,B). 75 % of the midupper arm blastemas did not displace and their regenerates articulated with the host midthigh, which is their corresponding level Displacement of grafts (Figs 1, 4C). Supernumerary autopodial structures to their corresponding host levels were induced between the ventral surface of the graft and dorsal surface of the host regenerate in two cases Fig. 3. Scheme and result of homografting untreated of wrist blastema grafts. wrist, elbow or midupper arm blastemas to the In a few cases, the donor regenerates displaced to a blastema-stump junction of a RA-treated host hindlimb level that was either distal to (elbow or midupper arm regenerating from the ankle level (A). ST, SK, SA refer to the stump thigh, knee and ankle, respectively. DT, blastema grafts) or proximal to (wrist or elbow DK, DA refer to the thigh, knee and ankle, respectively, blastema grafts) their corresponding host level of the serially duplicated regenerate. All other symbols as (Table 1). Displacement of elbow and midupper arm in Fig. 1. grafts distal to their corresponding host level most likely reflects resorption of graft cells destined to form proximal parts, allowing displacement to the Donor forelimbs were therefore amputated at the wrist, level established by the resorption. The reasons why elbow or midupper arm 10 days later than the host some grafted wrist and elbow blastemas failed to hindlimbs, so they would attain the late bud stage when the displace or only partially displaced are unknown. This host regenerates were at medium bud. failure may be related to stage variations in which either the graft or the host, or both, were develop- (c) Controls. Two sets of control experiments were done. mentally too advanced to allow displacement. In First, animals regenerating from the wrist, elbow or midup- several of these cases, however, intercalary regener- per arm of the forelimbs, and from the ankle, knee or ation occurred, restoring the pattern between graft midthigh of the hindlimbs, were injected with 2 y\ of DMSO and host levels. per g body weight at 4 days postamputation and allowed to Visual inspection of the limbs 48 h after grafting regenerate in situ. This control determined whether DMSO often revealed signs that were correlated with dis- alone had any effect on normal regeneration. Second, placement. Wrist and elbow blastemas appeared to animals regenerating from the wrist, elbow or midupper arm levels were injected with 2 (A of DMSO per g body be further distal on the host regenerate than upper weight at 4 days postamputation. When the limbs of these arm blastemas, which remained at the blastema- animals had regenerated to the late bud stage, the blas- stump junction. In addition, the initial 90° angle temas were homografted to the blastema-stump junction of between the ventral surface of the graft and dorsal ipsilateral, medium-bud-stage hindlimb hosts regenerating surface of the host was frequently reduced in wrist from the midthigh, and their displacement behaviour corre- and elbow grafts, but remained constant in upper arm lated with regenerate morphology. This control determined grafts. These observations indicated that displace- whether DMSO by itself would affect blastema cell affinity. ment began by 48 h postgrafting, and possibly earlier. Cellular basis of positional memory 691

Table 1. Displacement behaviour of forelimb wrist, elbow and upper arm blastemas autografted to hindlimbs

Level of displacement

Donor Total 0 F K T

Wrist 32 1 (3-3) 2 (6-7)* 2 (6-7)t 25 (83-3)1: Elbow 23 1 (4-3) 1 (4-3)§ 17 (74) 4 (17-4) Upper arm 12 9(75) 0 3(25) 0

Numbers in parentheses are percentages of the total number of surviving gTafts. 0 = no displacement. F, K, T = displacement to the level of the distal femur, the knee and the tarsus, respectively. * Both cases intercalated zeugopodial and distal stylopodial structures. t Both cases intercalated zeugopodial structures. t Two cases formed supernumerary hands. § Intercalated a distal stylopodial element.

Table 2. Displacement behaviour of RA-treated 20 % of the grafted RA-treated wrist blastemas and elbow, wrist and upper arm blastemas homografted their in situ hindlimb controls serially repeated only a to untreated hindlimbs zeugopodium. These grafts displaced to the knee Level of displacement level of the host regenerate, thus behaving like native elbow blastemas and showing that the degree of Donor Total 0 F K T displacement matched the level of proximalization (Fig. 5C). Wrist 15 12 (80)' 0 3(20) 0 Elbow 11 11 (100) 0 0 0 Upper arm 16 16 (100)t 0 0 0 (2) Untreated forelimb blastemas homografted to RA-treated hindlimbs Numbers in parentheses indicate percentage of total surviving Host ankle regenerates were proximalized by RA to grafts. the level of the proximal femur or pelvic girdle, 0 = no displacement. F, K, T = displacement to the level of the distal femur, the resulting in the serial repetition of girdle, stylopo- knee or the tarsus, respectively. dium and zeugopodium in the regenerate. 100 % of *Two grafts developed as unidentifiable hypomorphic the wrist blastemas were displaced to the ankle level structures. of the host regenerate and 59 % of the elbow blas- tFour grafts developed as unidentifiable hypomorphic temas were displaced to the knee level of the host structures. regenerate (Table 3; Figs 3, 6A,B). 69 % of the upper arm blastemas displaced only slightly, to the proximal or midfemur level of the host regenerate (Figs 3, 6C). Effect of RA on blastema morphogenesis and 41 % of the elbow blastemas and 31 % of the upper differential affinity arm blastemas were displaced beyond their corre- (1) RA-treated forelimb blastemas homografted to sponding level on the host regenerate. In these cases, untreated hindlimbs it is likely that resorption of cells established a more 100 % of RA-treated elbow and midupper arm blas- distal basal level in the graft, allowing it to displace to temas, and 80 % of RA-treated wrist blastemas failed a correspondingly more distal level on the host to displace from the graft site (Table 2). 17 % of the regenerate. nondisplaced wrist blastemas and 25 % of the nondisplaced upper arm blastemas either redifferentiated (3) Controls no cartilage or hypomorphic cartilage elements. The All of the blastemas of DMSO-treated animals regen- remainder of the nondisplaced blastemas developed erated normally in situ. DMSO-treated blastemas forelimb regenerates that were proximalized to the tested in the differential affinity assay behaved like level of the shoulder girdle; i.e. serially repeated a untreated blastemas and displaced distally to their girdle, stylopodium and zeugopodium (Figs 2, corresponding level on the host regenerate in the 5A,B). The control hindlimb regenerates of the majority of cases (Table 4; Fig. 7A-C; compare with donor animals were also proximalized to the level of Fig. 4A-C). The frequency of incomplete displace- the pelvic girdle, indicating that the grafted regener- ment, however, was over twice that for untreated ates developed just as they would have in situ. Hence donor blastemas in the wrist and elbow groups failure of graft displacement was strongly correlated (compare Tables 1 and 4). Intercalary regeneration with proximalization of regenerate pattern. eliminated the pattern discontinuity in all cases of 692 K. Crawford and D. L. Stocum

incomplete displacement. A single upper arm graft blastema cell affinity in vivo, and simultaneously displaced to the distal femur and formed a lower arm allows the morphogenesis and ^differentiation of and hand, again suggesting the establishment of a regenerate pattern to take place. This objective was more distal graft basal level by resorption. accomplished by grafting donor forelimb blastemas from three different PD levels to the blastema-stump Discussion junction of a host hindlimb regenerating from a The in vivo assay reveals position-related differential standard midthigh level. The grafted blastemas devel- affinity of blastema cells oped into normal forelimb regenerates, and each The first objective of our experiments was to develop made host neighbour choices that specifically an assay that detects level-specific differences in matched its own level of origin, thus eliminating

B B Cellular basis of positional memory 693

Table 3. Displacement behaviour of untreated wrist, elbow and upper arm blastemas homografted to the ankle level of RA-treated hindlimbs

Level of displacement

Donor Total 0 F K T Wrist 16 0 0 0 16 (100) Elbow 17 0 0 10 (59) 7(41) Upper arm 16 0 11(69) 3(19) 2(12)

Numbers in parentheses refer to the percentage of total surviving grafts. 0= no displacement. F, K, T = displacement to the level of the proximal or midfemur, the knee and the tarsus, respectively, of the serially repeated host regenerate. positional discontinuity between host and graft. Thus shut. In one experiment, the graft was a mature foot; midupper arm blastemas remained at the graft site, in another, a grafted foot was amputated and allowed whereas elbow and wrist blastemas were displaced to to form a conical blastema before reopening the the knee and ankle of the host regenerate, respect- ventral wound surface. In each case, the ventral ively. wound surface gave rise to a complete regenerate. This assay is a modification of an experiment Although the grafts failed to suppress foot formation designed by Shuraleff & Thornton (1967) to test in the hosts, Shuraleff & Thornton (1967) noted that another hypothesis, namely that grafting a given they were displaced to the ankle level of the host distal structure onto an axolotl hindlimb amputated regenerates, a result that we have confirmed and through the midthigh would suppress the develop- extended in our experiments. ment of that structure during regeneration of the The displacement data strongly suggest that blas- hindlimb. They grafted ankle-level. tissue onto the tema cells derived from different limb levels possess dorsal half of the amputation surface of the midthigh level-specific affinity properties that mediate their and sealed the ventral half of the amputation surface preferential association with cells from like levels.

Fig. 4. Displacement and development of regenerates blastema-stump juntion of untreated hindlimbs after autografting blastemas derived from different levels regenerating from the midthigh. All magnifications are of the forelimb to the blastema-stump junction of host x8 except the insert in (C), which is x6. The arrows hindlimbs regenerating from the midthigh. All indicate the original position of the graft on the host. magnifications are x8. The arrows indicate the original (A) Right RA-treated wrist blastema homografted to position of the graft on the host. right hindlimb, anterior view of hindlimb. The proximal (A) Right wrist blastema autografted to right hindlimb, boundary of the blastema was reset to the level of the dorsal view of hindlimb. The blastema displaced to the shoulder girdle (g), so that the regenerate now formed a tarsal (t) level of the host regenerate and formed several complete forelimb. The graft did not displace. The carpals (c) and three digits, indicated by the asterisks. arrowhead indicates the host tarsals, the level to which an The anterior-to-posterior sequence of host toes is untreated wrist blastema would normally displace. indicated by the numbers 1-5. The tibia, fibula, femur (B) Left RA-treated elbow blastema homografted to and pelvic girdle of the host limb skeleton are indicated left hindlimb, anterior view of hindlimb. The graft was by tb, fb, /and g, respectively. proximalized to the girdle level, formed a complete limb (B) Left elbow blastema autografted to left hindlimb, and failed to displace. The arrowhead indicates the knee anterior view of hindlimb. The blastema displaced to the level, to which an untreated elbow blastema would knee level of the host regenerate (arrowhead) and normally displace. formed a radius and ulna (r), several carpals (c) and four (C) Right RA-treated wrist blastema homografted to digits, plus a supernumerary digit (x). The host femur right hindlimb, anterior view of hindlimb. The graft was and tibia/fibula are indicated by/and tf, respectively. proximalized to the elbow level and formed radius and (C) Left midupper arm blastema autografted to left ulna (z), carpals (c) and four digits. Accordingly, it hindlimb, anterodorsal view. The most proximal element displaced to the knee level (arrowhead) of the host formed by the graft was the distal end of the humerus regenerate. The insert below shows the in situ ankle (h), and it displaced slightly to the corresponding level on regenerate of the donor animal. This regenerate was the host regenerate. The graft radius and ulna are proximalized to the level of the knee. In this limb, the indicated by ru. The host femur, tibia and fibulaar e bar indicates the regenerate-stump junction and the indicated by/ t and fb, respectively. stump tibia and fibula are indicated by t and /, Fig. 5. Result of homografting RA-treated blastemas respectively. derived from different levels of the forelimb to the 694 K. Crawford and D. L. Stocum

The results are consistent with Steinberg's (1963, are shared by forelimb and hindlimb cells. Because 1978) differential adhesion hypothesis, which predicts donor blastemas from different levels 'sort out' along that cell populations differing in adhesivity will the PD axis of a host regenerate according to their exchange neighbours until the intensity of like-cell affinity for cells of corresponding host levels, we have cohesions is maximized. Clearly, very similar or termed the differential displacement behaviour identical sets of PD level-specific affinity properties affinophoresis. Position-related differential cell affinity has also been demonstrated in a number of studies on insect development. Nardi & Kafatos (1976a,b) found that squares of Manduca pupal wing epithelium grafted to different axial positions in the wing rounded up and invaginated, indicating increased cohesion among graft cells and their decreased adhesion to surround- ing cells. The degree of rounding up and invagination was proportional to the distance between the original and final positions of the grafts, suggesting the existence of gradients of adhesiveness along the wing. When dorsal and ventral imaginal discs of Dros- ophila were dissociated, reaggregated in binary combinations and cultured within an adult female host, cells derived from different discs at the same DV level (leg-leg or wing-haltere) failed to sort out, whereas disc cells from different DV levels (wing-leg) sorted out (Fehon & Schubiger, 1985). Identical sorting behaviours were obtained in binary dorsal plus ventral reaggregates of notum, wing and leg discs cul- 1 tured in vitro (Fausto-Sterling & Hsieh, 1987). Finally, Gauger et al. (1985) showed that dissociated disc cells cultured with whole embryos bound prefer- entially to their body segments of origin. These data suggest the existence of dorsal-ventral and segment- specific cell affinities in Drosophila. Our experiments do not address the question of the mechanism whereby blastema displacement occurs

Fig. 6. Result of homografting blastemas derived from different forelimb levels to the blastema-stump junction of RA-treated hindlimbs regenerating from the ankle level. All magnifications are x8. In each case, the host regenerate was proximalized to the level of the proximal femur or pelvic girdle and formed a complete limb angled 90° to the stump. The femur, tibia and fibula of the host r regenerates are indicated by/, / and fb, respectively. I: (A) Left wrist blastema homografted to left RA- treated hindlimb, dorsal view of host regenerate. The blastema displaced to the tarsus level (arrowhead) of the host regenerate and formed carpals and digits. The graft digits are marked with asterisks. (B) Left elbow blastema homografted to left RA- treated hindlimb, dorsal view of host regenerate. The blastema displaced to the knee level (arrowhead) of the host regenerate and formed radius (r), ulna (u), carpals and digits. (C) Right midupper arm blastema homografted to right RA-treated hindlimb, dorsal view of host regenerate. The blastema failed to displace from the graft site and formed a distal humerus (h), radius (/•), ulna (u), carpals and digits. Cellular basis of positional memory 695

on the host regenerate. The grafted blastema might be passively pushed along the host blastema by distal sliding movements of the host epidermis until the corresponding host level is reached, where the strength of cohesion between graft and host mesenchyme cells and/or extracellular matrix is strong enough to anchor the graft. Distal epidermal sliding has been reported to occur in Xenopus and chick limb buds (Tschumi, 1956; Amprino, 1965). Alternatively, active movements of mesenchymal cells at the graft-host interface might provide the motive force. For example, Ettensohn & McClay (1986) have shown that sea urchin primary mesenchyme cells injected into the blastocoel at the animal pole migrate to their normal site of ingression, join host primary mesenchyme cells and develop normally into spic- ules.

RA coordinately proximalizes regenerate pattern and blastema cell affinity t The second objective of our experiments was to test, using the affinophoresis assay, whether retinoic acid coordinately proximalizes blastema cell affinity and B regenerate pattern, and hence to determine whether positional memory is directly related to blastema cell affinity. The results showed clearly that RA-treated wrist and elbow blastemas behaved in the assay like native midupper arm blastemas; they failed to displace from the midthigh level of the untreated host regenerate. Likewise, untreated wrist, elbow and upper arm blastemas displaced on RA-treated host ankle regenerates that were proximalized to the girdle in a manner identical to their displacement on native midthigh regenerates. These data demonstrate that RA coordinately proximalizes level-specific blas- temal cell affinity and positional memory, and allow us to conclude that the two are correlative. There is considerable evidence that cell surface and Fig. 7. Result of homografting DMSO-treated blastemas matrix glycoconjugates (glycoproteins, glycolipids, derived from different forelimb levels to the proteoglycans) mediate cell recognition and affinity blastema-stump junction of host hindlimbs regenerating from the midthigh. All magnifications are x8. The arrows during development (Edelman, 1985; Thiery et al. indicate the original position of the graft on the host. 1985; Gallin etal. 1986; McClay & Ettensohn, 1987). (A) Right DMSO-treated wrist blastema homografted Retinoids alter cell-cell or cell-substrate adhesive- to right hindlimb, posterior view of host regenerate. The ness of fibroblasts, chondrocytes, epithelial cells and graft (G) displaced to the level of the host tarsus various types of carcinoma cells concomitantly with (arrowhead) and formed carpals and digits. changes in their differentiation or morphology. These (B) Left DMSO-treated elbow blastema homografted effects are correlated with qualitative and/or quanti- to left hindlimb, dorsal view of host. The grafted forelimb tative changes in cell surface glycoconjugates, includ- regenerate (C) formed radius, ulna, carpals and digits, ing hormone and growth factor receptors (for re- and shifted with respect to the host so that its ventral views, see Lotan, 1980; Shapiro & Mott, 1981; surface is visible. The ulna of the graft and fibula of the Roberts & Sporn, 1984). In addition, transcriptional host regenerate have fused proximally (arrowhead). (C) Right DMSO-treated midupper arm blastema and translational studies have shown that retinoids homografted to right hindlimb, dorsal view of host alter patterns of keratin gene transcription in cultured regenerate. The blastema displaced slightly toward the human keratinocytes and conjunctival cells (Fuchs & distal end of the femur, and formed a distal humerus (h), Green, 1981; Eckert & Green, 1984). Similarly, it has radius (/•), ulna (u), carpals and digits. been shown that the enhanced synthesis of laminin 696 K. Crawford and D. L. Stocum

Table 4. Displacement behaviour of control DMSO-treated wrist, elbow and upper arm blastemas homografted to untreated hindlimbs Level of displacement

Donor Total

Wrist 15 0 2(13) 4 (27)t 9(6) Elbow 10 2(20)* 0 8(80) 0 Upper arm 10 9(90) 0 1 (10) 0

Numbers in parentheses refer to percentages of total surviving grafts. 0 = no displacement. F, K, T= displacement to the level of the distal femur, the knee and the tarsus, respectively. * Intercalated a zeugopodium and distal tip of stylopodium. t Intercalated a zeugopodium. X Intercalated a zeugopodium and the distal half of a stylopodium. subunits in RA-treated F9 embryonal carcinoma cells reading of the manuscript. This research was supported by is under transcriptional regulation (Carlin etal. 1983; NIH Grant HD 12659. Wang era/. 1985). Taken in conjunction with our data, the evidence References from studies on retinoid-induced changes in cell morphology, adhesion and transcription suggest that AMPRINO, R. (1965). Aspects of limb morphogenesis in positional memory in amphibian limb cells might be the chicken. In Organogenesis (ed. R. L. DeHaan & the result of level-specific differences in gene ex- H. Ursprung), pp. 255-281. New York: Holt, Rinehart pression that affect the molecular structure of the cell & Winston. surface, and that RA modifies positional memory by CARLIN, B. E., DURKJN, M. E., BENDER, B., JAFFE, R. & affecting this expression. Many retinoid-sensitive CHUNG, A. E. (1983). Synthesis of laminin and entactin by F9 cells induced with retinoic acid and dibutyryl cells contain two ligand-specific cytoplasmic proteins cyclic AMP. /. biol. Chem. 258, 7729-7737. (cellular retinol binding protein = CRBP, and cellu- CARLSON, B. M. (1975). The effects of rotation and lar retinoic acid binding protein = CRABP) that bind positional change of stump tissues upon morphogenesis retinol and RA (Liau et al. 1981; Takase et al. 1986; of the regenerating axolotl limb. Devi Biol. 47, Crow et al. 1987; Chytil & Ong, 1984, for review). 269-291. These binding proteins shuttle retinol and RA to the CHYTIL, F. & ONG, D. E. (1984). Cellular retinoid- nucleus and transfer them to a nuclear retinoid binding proteins. In The Retinoids, vol. II (ed. M. B. receptor that is distinct from the cytoplasmic binding Sporn, A. B. Roberts & D. S. Goodman), pp. 89-123. proteins themselves (Daly & Redfern, 1987), which Orlando: Academic Press. are recycled to the cytoplasm (Chytil & Ong, 1984). CROW, J. A., ONG, D. E. & CHYTIL, F. (1987). Specificity A cDNA encoding a retinoid nuclear receptor in of cellular retinol-binding protein in the transfer of retinol to nuclei and chromatin. Arch. Biochem. human breast cancer cell lines has been cloned; the Biophys. 254, 372-375. receptor is a RA-inducible trans-acting enhancer DALY, A. K. & REDFERN, C. P. F. (1987). factor that is structurally related to steroid and Characterisation of a retinoic-acid-binding component thyroid hormone receptors (Petkovich et al. 1987). from F9 embryonal-carcinoma-cell nuclei. Eur. J. A cytoplasmic CRABP has recently been detected Biochem. 168, 133-139. in limb regenerates of axolotls (Keeble & Maden, ECKERT, R. L. & GREEN, H. (1984). Cloning of cDNAs 1986; McCormick et al. 1988). Hence a plausible specifying vitamin A-responsive human keratins. Proc. hypothesis regarding the mechanism by which ex- natn. Acad. Sci. U.S.A. 81, 4321-4325. ogenous retinoids modify positional memory of blas- EDELMAN, G. M. (1985). Specific cell adhesion in tema cells is that after being delivered to the nucleus histogenesis and morphogenesis. In The Cell in by CRBP or CRABP, they bind to and activate a Contact: Adhesions and Junctions as Morphogenetic Determinants (ed. G. M. Edelman & J.-P. Thiery), nuclear receptor, allowing it to bind to the enhancer pp. 139-168. New York: John Wiley and Sons. region of genes that are involved in specifying pos- ETTENSOHN, C. A. & MCCLAY, D. R. (1986). The itional memory. Currently, however, there is no regulation of primary mesenchyme cell migration in the direct molecular evidence for this hypothesis. sea urchin embryo: transplantations of cells and latex beads. Devi Biol. 117, 380-391. We would like to thank David Bergstrom, James Nardi FAUSTO-STERLING, A. & HSIEH, L. (1987). In vitro culture and Shong-Won Norby for their suggestions and critical of Drosophila imaginal disc cells: aggregation, sorting Cellular basis of positional memory 697

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