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Pattern regulation in the embryonic chick limb: Supernumerary limb formation with anterior (non-ZPA) limb bud tissue
DEVELOPMENTAL
BIOLOGY
75, 373-385 (1980)
Pattern Regulation in the Embryonic Chick Limb: Supernumerary Formation with Anterior (Non-ZPA) Limb Bud Tissue LAURIE Department
of Biological
Limb
E. ITEN AND DOUGLAS J. MURPHY Sciences, Purdue
University,
West Lafayette,
Indiana
47907
Received May 29, 1979; accepted in revised form September 14, 1979 The formation of supernumerary limbs and limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. A “wedge” (ectoderm and mesoderm) of anterior or mid donor right wing bud (stage 21) was inserted in a slit made in a host right limb bud (stage 21) at the same position as its position of origin or to a more posterior position. The AER of the donor tissue and host wing bud were aligned with each other. Donor tissue was grafted with its dorsalventral polarity the same as the host’s limb bud or reversed to that of the host’s, Depending on the position of origin of the donor limb bud tissue and the position to which it was transplanted in a host, supernumerary wings or wing structures formed. Furthermore, depending on the orientation of the graft in the host, supernumerary limbs with either left or right asymmetry developed. The results of experiments performed here are considered in light of two current models which have been used to describe supernumerary limb formation: one based on local, short-range, cell-cell interactions and the other based on long-range positional signaling via a diffusible morphogen. INTRODUCTION
The ability of an early embryo or developing organ to regulate and form a complete organism or organ after portions have been removed or the ability of adult animals to regenerate lost parts has fascinated many generations of biologists. This interest in regulation and regeneration, in part, led to the concept of positional information (Wolpert, 1969, 1971). During development and regeneration, cells acquire specific fates according to their position in the embryo or organ which leads to species-typical spatial patterns of cellular differentiation. Regulation and regeneration involve either the replacement of cells with the missing positional values through cell proliferation, there being little change in the positional values of the remaining cells (epimorphosis), or regulation and regeneration involve the respecification of the positional values of the remaining cells such that a miniature but complete pattern is reformed (morphallaxis) (Morgan, 1901). The similarities between the sequence of
events observed during embryonic chick limb development and postembryonic amphibian limb regeneration have been documented elsewhere (Faber, 1971; Stocum, 1975). Briefly, limb buds first appear in the chick embryo as small bulges protruding from the lateral body wall. Each limb bud is a condensation of mesenchyme cells enveloped with an ectodermal jacket; an apical ectodermal ridge (AER) rims the apex of each limb bud. As a limb bud grows in length, differentiation of limb tissues begins proximally and progresses distally resulting in a distinct spatial pattern of differentiated structures. After the amputation of a salamander limb, wound healing and dedifferentiation of stump tissues adjacent to the site of amputation occurs. Cells from the dedifferentiated tissues accumulate at the distal end of the limb stump to form a regeneration blastema which is a mass of mesenchyme cells encased in an epidermal cap. The regeneration blastema, like the chick limb bud, grows in length and differentiation proceeds in a proximal to distal
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DEVELOPMENTAL BIOLOGY VOLUME75, 1980
direction. A basic difference exists between developing and regenerating limbs. In the former, the most proximal structures to be laid down are always the same, whereas, in regenerating limbs, the most proximal structures to be laid down will vary depending on the level of amputation. Both the embryonic chick limb bud and postembryonic regenerating amphibian limb can be operationally defined as a developmental field within which pattern regulation (changes in the presumptive fates of cells) can occur in response to surgical perturbations. Regenerating amphibian limbs, cockroach legs, and Drosophila imaginal disks exhibit epimorphic pattern regulation and there is evidence suggesting that the embryonic chick limb bud, at certain stages of development, also regulates epimorphically. Regulation to form a complete limb can occur following the removal or addition of the central portion of the limb bud (Hansborough, 1954; Barasa, 1964; Amprino and Camosso, 1965; Stark and Searls, 1974) and there appears to be a correlation between cell division and the ability of a chick limb bud to regulate following the removal of its center (Stark and Searls, 1974). That analogous results are obtained from similar experiments with different developing and/or regenerating systems supports the contention that there are fundamental similarities with regards to positional information and the behavior of cells during the construction of a three dimensional pattern of differentiated structures (Wolpert, 1971; French, Bryant, and Bryant, 1976). Experiments done with the embryonic chick limb bud give results analogous to those obtained from similar experiments done with regenerating amphibian limbs, cockroach legs, or Drosophila imaginal disks. For example, removal of a longitudinal strip of cuticle and epidermis from any position around the circumference of the femur of a cockroach leg leads to the cut edges healing together, localized growth, and regeneration of the missing
section of the circumference, i.e., a normal size femur with a complete pattern of cuticular markers results irrespective of the longitudinal strip removed (French, 1978). The same result is obtained with the developing chick limb when a longitudinal slice of wing bud mesoderm and ectoderm is removed. A qualitatively and quantitatively complete wing forms irrespective of the longitudinal slice of wing bud removed (Iten et al., in preparation). When the regulative behavior of complementary anterior and posterior portions of a wing bud is examined, one portion forms limb structures with normal anterior-posterior and dorsal-ventral polarity and the other forms bilaterally symmetrical structures, namely, structures with dorsal-ventral polarity, but without anterior-posterior polarity (Iten, 1977); these results are analogous to the regenerationduplication behavior of complementary Drosophila fragments of regenerating imaginal disks (Bryant, 1971). A characteristic of epimorphic pattern regulation is that supernumerary (extra) structures can form after disharmonic axial opposition of portions of a developmental field. Again, there are striking similarities between the supernumerary limbs obtained with axial reversals with the developing chick limb bud and those obtained with embryonic amphibian limbs and regenerating amphibian and insect limbs (see Bryant and Iten, 1976, for a detailed description of these similarities). The formation of supernumerary limbs and limb structures in amphibians and insects has been described with a simple and unified hypothesis for pattern regulation in epimorphic fields proposed by French, Bryant, and Bryant (1976). In their model they propose that the positional values of cells in an epimorphic field are specified in terms of a two-dimensional polar coordinate system. One component of positional information is a value corresponding to position on a circle, and the second component to position on a radius. The basic rule of cellular behavior proposed by this model is
ITEN AND MURPHY
Pattern
Regulation
that when cells with normally nonadjacent positional values in either the radial or circular sequences are next to each other, growth occurs until cells with intermediate positional values have been intercalated and the discontinuity is eliminated. The polar coordinate model proposes that supernumerary limb formation is the result of local (short-range) cell-cell interactions and cell proliferation. Another model has been proposed by Wolpert and co-workers to describe the formation of supernumerary limb structures in embryonic chick limbs and it is based on long-range positional signaling via a diffusible morphogen (Tickle et al., 1975; Summerbell and Tickle, 1977; Summberbell, 1979). In their model they propose that there is a localized organizing center at the posterior edge of a limb bud called the zone of polarizing activity (ZPA). The ZPA is the source of a diffusible morphogen that specifies a cell’s anterior-posterior coordinate. Simply, a limb bud cell’s anterior-posterior coordinate depends on its distance from this localized source. This hypothesis is based on studies where another ZPA is transplanted to certain position along the anterior-posterior edge of a host limb bud and supernumerary limb structures result. The results of experiments presented here show that anterior (non-ZPA) limb bud tissue transplanted to more posterior positions in a host limb also results in the formation of supernumerary limb structures. MATERIALS
AND
METHODS
Embryos used in these experiments were from a White Leghorn strain of chickens obtained from the Indiana Co-Op Poultry and Breeding Farm (West Lafayette, Ind.). Eggs were incubated at 38°C. At the desired Hamburger and Hamilton (1951) stages, embryos were prepared for surgical operations by withdrawing albumin to lower the yolk and embryo, sawing a window in the shell, and removing the shell membranes. Tissue manipulations were performed with sharpened tungsten needles and fine for-
in the Chwk
Limb
Bud
375
ceps. Stage 21 right wing buds (1ength:width ratio between 2.3 and 2.9) (Hamburger and Hamilton, 1951) were used as donor and host limb buds. Right leg buds (stage 21) were also used as donor limb buds in some of the transplantation operations. Anterior or mid limb bud tissue from a right wing bud was transplanted to the same anteriorposterior level in a host right wing bud or to a more posterior level (Fig. 1). Host wing bud tissue was not removed to prepare a graft site; instead, a slit was cut through the entire thickness of the host wing bud and then a wedge of donor wing bud tissue (ectoderm and mesoderm ca. 150 pm wide at its distal edge and ca. 200 pm long) was inserted into the slit. Host limb bud tissue was not removed to prepare a graft site because host cells that may or may not interact with the cells of the graft would be removed. The wedge of donor tissue was J
1
16
17 I 18 3
1
18 I
19
19 3
24
24
16
J
17 3
17 I
18 1
18 3
19 3
19 3
207
207
FIG. 1. Dorsal view outlines of donor and host stage 21 right wing buds. The position of origin of a wedge of anterior donor tissue and the different positions that it was added to a host are both shown with respect to the adjacent somites. The distance from the center of one somite to the center of the next is approximately 250 pm and the anterior-posterior length of a wing bud at its base is about 1 mm. The left diagram illustrates the different anterior-posterior positions in which a 16/17 wedge was placed in a host limb bud and the right diagram illustrates the same thing, but for a 17/18 wedge of donor tissue.
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DEVELOPMENTAL BIOLOGY
positioned such that the cut surfaces of the graft and host were in contact with each other and the AER of the graft and host were aligned with each other. The position of origin of the wedge of donor tissue as well as the position in the host where the tissue had been added were identified with respect to somites 16, 17, 18, and 19 adjacent to a stage 21 wing bud. For example, 16/17 added to an 18/19 level indicates that a wedge of wing bud tissue adjacent to the junction of somites 16 and 17 was inserted into a slit made in a host at the level of the junction of somites 18 and 19. A carbon mark was placed on the dorsal surface of the wedge of donor tissue so that the orientation of the graft in the host could be recorded. A wedge was added to a host either with its dorsal-ventral axis matching that of the host’s (denoted as dd orientation) or with its dorsal-ventral axis reversed with respect to the host’s (denoted as dv orientation). Observations on the development of a wing bud with its graft were made 1 and 2 days after the transplantation op-
VOLUME 75, 1980
eration and all embryos were sacrificed and fixed at 13 days incubation. The integumentary pattern of resulting wings was recorded at this time. Embryos were then stained with Victoria blue (Bryant and Iten, 1974) and cleared in methyl salicylate to reveal the skeletal pattern of resulting wings. In some cases, wings were embedded in paraffin and sectioned to examine their muscle pattern. RESULTS
The skeletal and integumentary pattern of 266 resulting wings was examined. The wings resulting from the transplantation operations where a wedge of 16/17 or 17/18 donor wing bud tissue was transplanted to the same or more posterior position in a host wing bud have been examined with respect to extra skeletal elements formed (Table 1) and the resulting anterior to posterior sequence of digits (Table 2). The skeleton of the wing can be divided into three proximal to distal segments: stylopodium, zeugopodium, and autopodium. The
TABLE
1
RESULTS OF TRANSPLANTING ANTERIOR OR MID WING BUD TISSUE TO THE SAME OR MORE POSTERIOR POSITION IN A HOST WING BUD Position of origin of graft with respect to somites
Position transplanted in host with respect to somites
Orientation of graft in host”
Total No. resulting wings
No. with extra skeletal element(s)
16/17
16/17
dd du dd
0 (0%)
dv dd do dd dv
10 10 11 11 20 21 25 15
3 4 6 18 20 25 15
(30%) (36%) (55%) (90%) (95%) (100%) (100%)
dd dv dd dv dd dv
11 11 12 14 16 14
0 1 4 7 14 14
(0%) (9%) (33%) (50%) (88%) (100%)
17/18 M/19 19/20
17/18
17/18 H/19 19/20
Extra skeletal elements formed’ S
Z
3 3 5 1 3 1
A
1 1 5 2
Z/A
S/Z/A
9 12 4 2
3 3 20 13
2 3 8 10
4 3
1 1 2
2 3 1
n Graft was inserted in a host with ita dorsal-ventral polarity matching that of the host’s, denoted dd orientation, or with its dorsal-ventral polarity opposite that of the host’s, denoted dv orientation. * S, extra stylopodial element (humerus); Z, extra zeugopodial element(s) (radius, ulna, or both); A, extra autopodial element(s) (digit or digits); Z/A, extra zeugopodial and autopodial elements; S/Z/A, extra stylopodial, zeugopodial, and autopodial elements.
ITEN AND MURPHY
Pattern
Regulation
TABLE
in the Chick
377
Limb Bud
2
DIFFERENT ANTERIOR TO POSTERIOR DIGITAL SEQUENCES OF RESULTING WINGS AFTER ANTERIOR OR MIII WING BUD TISSUE HAD BEEN GRAFTED TO THE SAME OR MORE POSTERIOR POSITION IN A HOST WIKG Bun Anterior to posterior sequence of digits OrientaPosition Position of tion of origin of transplanted II II graft in in host with graft with *:: III respect to respect to host” 1;: 1:: 1:: 1:: 1:: 1::7 1:: IV III somites somites IV IV :z :: :r”r :: & rl”t III III II IV IV 16/17
18/17 17/H 18/19 19/20
17/18
17/M 18/19 19/20
dd dv dd dv dd dv dd dv
10 10 10 10 3 4 1
dd dv dd dv dd dv
11 11 8 8 4
1 1 2 5
1 1 1
2 1
1
2 1 7 4
1 2 12 14 ._____~.
10 6
2 4 15 7
2 3
1 2
Note. Data refer to the number of cases obtained. ” Abbreviations as in footnote a of Table 1.
stylopodium is made up of the humerus which articulates proximally with the shoulder girdle and distally with the radius and ulna of the zeugopodium. The anterior radius and posterior ulna articulate distally with the carpals of the autopodium. Distal to the carpals are the metacarpals and phalanges of the three digits of the wing. Digit II is the most anterior and digit IV is the most posterior. Many of the skeletal elements of the wing have a characteristic morphology which facilitates their identification in resulting wings and aids in the determination of the asymmetry or symmetry of structures formed. Limbs with extra skeletal elements representing one, two, or all three of the proximal to distal segments of the limb, usually next to the same segments of the host wing, form as a result of some of the transplantation operations performed. Extra muscles and feather follicles also formed in some cases, but a detailed description of the muscle and integumentary pattern of resulting wings is not presented here.
Control Transplantations Twenty-one wings resulting from operations where a wedge of 16/17 donor wing bud tissue was added to a 16/17 level slit in a host wing bud with dd orientation (10 cases) or where a wedge of 17/18 donor tissue was added to a 17/18 host site with dd orientation (11 cases) were analyzed. As can be seen from Table 1, these control transplantations clearly show that adding a wedge of anterior or mid wing bud tissue to the same anterior-posterior position in a host wing bud with dd orientation does not result in the formation of supernumerary skeletal elements (Fig. 2a), but extra integumentary structures do form. When a wedge of 16/17 or 17/18 was added to the same respective position in a host wing bud, but with dv orientation, a few limbs resulted in which an extra partial or complete humerus developed next to the host humerus (Table 1). Furthermore, these operations frequently disrupted the normal dorsal and ventral distribution of feather follicles; that is, a region of ventral-like apteria
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DEVELOPMENTAL BIOLOGY VOLUME75. 1980
FIG. 2. Dorsal view of the skeletal pattern of wings formed after a wedge of 16/17 wing bud tissue had been added to the same or more posterior position in a host wing bud. (a) A wedge of 16/17 tissue was added to a 16/ 17 level slit of a host wing bud or (b) to a 17/18 level slit, both with dd orientation, and a normal complement of skeletal elements formed. (c) A wedge of 16/17 tissue was added to an 18/19 level slit in a host wing bud with dd orientation. This limb is an example of a resulting wing with an extra ulna and extra digits Anterior to posterior digital sequence of this wing is II III IV IV III IV. (d) A wedge of 16/17 tissue was added to a 19/20 level slit (posterior edge) of a host wing bud with dd orientation. Arrow points out supernumerary wing structures that formed: humerus, zeugopodial element, and digits This wing has an anterior-posterior digital sequence of II III IV IV III. All x 3.6.
developed in the dorsal wing integument and a corresponding dorsal-like pteryla in the ventral integument. Similar results were obtained when different wedges of posterior limb bud tissue were added to analogous positions in host wing buds (Javois and Iten, in press).
Experimental
Transplantations
When a wedge of 16/17 donor wing bud tissue was added to a 17/18 level slit in a host wing bud with dd orientation, no extra skeletal elements formed in the majority of cases (Fig. 2b), but 3 of the resulting 11 wings had an extra partial or complete humerus and 1 had an extra digit (Table 1). A higher incidence of limbs with an extra humerus or extra digit resulted when the same operation was performed, but the
wedge was grafted with dv orientation (55 vs 36% of the cases, Table 1). When an extra digit formed, it was always a digit II and the anterior to posterior sequence of digits of the resulting wing was II II III IV (Table 2). Depending on the orientation of the graft in the host, extra integumentary structures and/or a disruption in the dorsal and ventral integumentsry pattern resulted. Adding a wedge of 16/17 donor wing bud tissue to an M/19 level slit in a host with dd or dv orientation resulted in limbs with extra skeletal elements in almost all cases, and the majority of these had an extra ulna and digits (Table 1) and an anterior or posterior digital sequence of II III IV III IV or II III IV IV III IV (Table 2, Fig. 2~). However, adding a wedge of 17/18 tissue to an 18/19 host level slit with dd or dv ori-
ITEN AND MURPHY
Pattern
Regulation
entation resulted in a lower incidence of limbs with extra skeletal elements (Table 1) and the majority of resulting wings had a normal II III IV anterior to posterior sequence of digits (Table 2). Again, in these groups of transplantation operations, depending on whether the wedge of donor tissue was grafted with dd or dv orientation, extra integumentary structures and/or a disruption in the dorsal and ventral integumentary pattern resulted. When either 16/17 or 17/M wing bud tissue was added to the posterior edge of a host wing bud (19/20 host slit level) with dd or dv orientation, a separate and identifiable supernumerary outgrowth was seen 2 days later (Fig. 4) and resulting wings had supernumerary structures well separated from the host wing structures in almost all cases. The extra skeletal elements formed and the digital sequence of resulting wings was different depending on the position of origin of the graft. Namely, 16/17 added to the posterior edge of a host limb bud resulted in wings with an extra humerus, extra ulna or radius and ulna, and extra digits in 83% of the cases and a digital sequence of II III IV IV III or II III IV III II in 68% of the cases (Tables 1 and 2; Figs. 2d and 3a and b), whereas 17/18 added to the same position in a host wing bud resulted in wings with an extra ulna and digit and a digital sequence of II III IV III in the majority of cases (Tables 1 and 2; Figs. 3c and d). Furthermore, when 16/17 was added to the posterior edge of a host with dd orientation, the supernumerary wing structures which resulted clearly had left hand asymmetry in 21 of 23 resulting limbs (Fig. 3a) and if grafted with dv orientation, then supernumerary wing structures had right hand asymmetry in 13 of the 15 resulting limbs (Fig. 3b). But when a wedge of 17/18 wing bud tissue was added to the posterior edge of a host wing bud, the extra wing structures formed had neither right nor left hand asymmetry. When grafted with dd or dv orientation, bilaterally symmetric supernumerary structures resulted; the dorsal-
in the Chick
Limb
Bud
379
ventral polarity of these supernumerary structures always coincided with the dorsal-ventral polarity of the graft (Figs. 3c and d). If a wedge of limb bud tissue from the anterior edge of a stage 21 right wing bud (tissue adjacent to somite 16 or adjacent to the junction of somites 15 and 16) was added to the posterior edge of a host wing bud, the extra structures formed were not the same as those formed when a wedge of 16/17 donor tissue was added to the same position in a host limb bud. Transplanting anterior edge wing bud tissue to the posterior edge of a host (dd or dv orientation) resulted in wings with an extra partial proximal or complete humerus (11/25 cases), an extra humerus and an unidentifiable distal skeletal element (lo/25 cases), or no extra skeletal elements (4/25 cases). Similar results were obtained when the AER was removed from a wedge of 16/17 donor tissue before transplanting it to the posterior edge of a host limb bud (Iten et al., unpublished results). Since it has been shown that leg bud tissue in a wing bud will differentiate into leg structures (Saunders et al., 1957), the origin of supernumerary limb structures was examined by transplanting anterior leg bud tissue to the same level or to the posterior edge of a host wing bud. When a wedge of stage 21 anterior right leg bud (tissue adjacent to the junction of somites 27 and 28) was added to the 16/17 level of a host wing bud at the same stage with dd (12 cases) or dv (10 cases) orientation, resulting wings did not have “toes” instead of wing “fingers” or “toes” in addition to their normal set of digits, although their proximal anterior skeletal elements and integument were leg-like (Fig. 5a). However, wings with supernumerary “toe(s)” or a “toe” and wing “finger” did form after anterior leg bud tissue was transplanted to the posterior edge of a wing bud with dd (7 cases) or dv (11 cases) orientation (Fig. 5b). Thus it appears that supernumerary structures arise from the wedge of anterior
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DEVELOPMENTAL BIOLOGY VOLUME75, 1980
FIG. 3.
ITEN AND MURPHY
Pattern Regulation in the Chick Limb Bud
tissue and they may in part arise from the host limb bud. Studies on the origin of these extra structures utilizing the cytological marker available with xenoplastic chick/ quail graft combinations also indicate that the extra structures formed arise from the graft and host wing bud (Cotromanes and Iten, in preparation). DISCUSSION
Results presented in this paper show that when anterior or mid wing bud tissue is transplanted to a more posterior position in a host wing bud, wings with extra structures or a separate supernumerary wing can result. Furthermore, the formation of extra limb structures or a supernumerary limb depends on the position of origin of donor tissue, the position transplanted in a host
381
wing bud, as well as the orientation of the graft in the host. The results of transplanting anterior leg bud tissue to the posterior edge of a host wing bud suggest that both the donor and host contribute to the formation of a supernumerary limb. Experiments similar to those presented here have been briefly reported by Fallon and Thorns (1979). They transplanted anterior edge wing bud tissue (with or without its AER) to the posterior edge of a host wing bud. Host wing bud tissue was removed to prepare a graft site. No extra structures formed in the majority of their resulting wings and when supernumerary structures did form, only a small cartilagenous outgrowth or a single digit-like structure formed. We obtained similar results when a wedge of anterior edge tissue (ad-
FIG. 3. Dorsal view of the integumentary pattern (left photograph) and skeletal pattern (right photograph) of wings formed after wedge of anterior or mid-right wing bud tissue had been added to the posterior edge (19/ 20 level) of a host right wing bud. Arrows identify supernumerary wing structures. (al 16/17 wing bud tissue was added with dd orientation. Resulting wing has an extra humerus next to the host’s humerus, extra ulna, extra digits, and an anterior to posterior digital sequence of II III IV IV III. Supernumerary structures have left asymmetry. (b) 16/17 wing bud tissue was added with du orientation. Resulting wing has an extra humerus (not clearly visible in this view), extra radius and ulna, extra digits, and a digital sequence of II III IV III II. The supernumerary structures have right asymmetry (the ventral surface of the supernumerary structures is seen in a dorsal view). (cl 17/18 wing bud tissue was added with dd orientation or with du orientation (d). Both are examples of a resulting wing with an extra zeugopodial element and digit and a II III IV III digital sequence; the supernumerary structures are bilaterally symmetrical and their dorsal-ventral polarity is the same as the original orientation of the graft in the host limb bud. All x 2.7. FIG. 4. Dorsal view of a right wing bud 2 days after a wedge of anterior wing bud tissue had been added to the posterior edge of this limb bud (left) and a normal right wing bud at approximately the same stage of development (right). Supernumerary outgrowth is indicated by arrow. Both x 14.
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DEVELOPMENTAL BIOLOGY VOLUME75, 1980
FIG. 5.
ITEN
AND
MURPHY
Pattern
Regulation
jacent to somite 16 or the junction between somites 15 and 16) was inserted in a slit made at the posterior edge of a host wing bud. However, when a wedge of 16/17 donor tissue is grafted to the same position in a host limb, very different results were obtained, namely, a separate supernumerary wing developed with left or right asymmetry depending on the orientation of the graft in the host. If the AER is removed from the 16/17 donor tissue, then results similar to those with anterior edge donor tissue are obtained. In our opinion, the results presented in this paper can best be described with a model proposing local cell-cell interactions where cells with intermediate positional values are intercalated after normally nonadjacent cells are placed next to each other, like the polar coordinate model proposed by French, Bryant, and Bryant (1976). Whereas, our results are not easily explained by a mechanism involving longrange positional signaling via a diffusible morphogen from the ZPA at the posterior edge of a limb bud (Tickle et al., 1975; Summerbell and Tickle, 1977). In the recent elaboration of the ZPA model by Summerbell (1979), it could be argued that when a wedge of anterior wing bud tissue was transplanted to a more posterior position in a host wing bud, it was exposed to a higher concentration of morphogen, and in its new position, it was respecified to form certain skeletal parts. The previously specified host limb bud cells would form their appropriate skeletal elements, and the newly respecified grafted cells would form extra skeletal elements due to their exposure to a certain concentration of morphogen emanating from the host’s ZPA. Therefore, one would expect that the more posterior the position in a host the graft was inserted, the greater
in the Chick Limb
Bud
383
the percentage of limbs with extra skeletal elements that should occur, and this was the result obtained in the present study. However, using the same argument, one would expect to find that the same extra skeletal elements would form and in the same percentage of cases when either a wedge of anterior (16/17 wedge) or mid (17/ 18 wedge) wing bud tissue was transplanted to the same posterior position in a host wing bud (an 18/19 or 19/20 host slit level), because the donor tissue in its new position would be exposed to the same concentration of morphogen. This result was not obtained (see Tables 1 and 2). Limbs with different extra skeletal elements and the frequency of limbs with extra skeletal elements was different depending on whether a 16/17 or 17/18 wedge of donor tissue was transplanted to the same posterior position. Furthermore, the results of our transplantation of anterior or mid wing bud tissue to different posterior positions cannot be explained by proposing that there is autonomous development of the graft in the host wing bud, because adding a wedge of limb bud tissue to the same anterior-posterior position in a host wing bud does not result in the formation of supernumerary skeletal elements. By applying the formal rules for cellular behavior proposed by the polar coordinate model we would expect to find supernumerary structures forming after anterior limb bud tissue is transplanted to a more posterior position in a host limb and that the extra structures formed would depend on both the position of origin of the graft and the position transplanted to a host limb bud. These are the results we obtained. Furthermore, we found that the asymmetry (handedness) or symmetry of supernumerary structures formed depends on the ori-
FIG. 5. Dorsal view of the integumentary pattern (left photograph) and skeletal pattern (right photograph) of a resulting wing after a wedge of anterior right leg bud tissue had been added to a 16/17 level (a) or to a 191 20 level (b) in a host right wing bud, both with dd orientation. (a) Leg-like integumentary pattern and skeletal elements are seen in the proximal anterior portion of the resulting wing. Supernumerary leg (large arrow) and wing structures (small arrow) are illustrated in (b). All x 5.7.
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DEVELOPMENTAL BIOLOG Y
entation of the graft in the host limb bud. The polar coordinate model is the only one we know of that can describe how the asymmetry/symmetry of extra structures could arise. The polar coordinate model makes no stipulations about where the cells for supernumerary structures should come from, i.e., graft or host, because extra limb structures are seen as arising as a result of intercalation of cells between the normally nonadjacent cells of the graft and host. With regenerating amphibian limbs, if either the regenerate or adjacent stump is prevented from contributing cells to form a supernumerary limb with X-irradiation, the unirradiated partner can contribute more to the formation of supernumerary structures (Holder et al., 1979). And if posterior edge wing bud tissue from a heavily irradiated donor limb is transplanted to the anterior edge of an unirradiated host, then supernumerary wing structures still form (Smith et al., 1978). Results analogous to these obtained with amphibian and chick limbs have been obtained with the regenerating imaginal disk of Drosophila (Adler and Bryant, 1977; Bryant et al., 1978). In conclusion, the results presented here suggest that at certain stages of development, the embryonic chick limb bud exhibits epimorphic pattern regulation similar to that observed in the postembryonic epimorphically regenerating amphibian limb, cockroach leg, and Drosophila imaginal disk. In addition, the results obtained here can best be described with a hypothesis proposing local, short-range, cell-cell interactions, and they are not easily explained by a mechanism involving longrange signaling via a diffusible morphogen emanating from the ZPA. It is a pleasure to acknowledge the valuable comments and criticisms of Drs. U. K. Abbott, S. V. Bryant, and J. W. Saunders, Jr., Ms. E. Cotromanes, and Ms. L. C. Javois. This investigation was supported by Grant PCM 77-08408, awarded by the National Science Foundation, and in part by an Institutional Grant from the American Cancer Society.
VOLUME 75, 1980 REFERENCES
ADLER, P. N., and BRYANT, P. J. (1977). Participation of lethally irradiated imaginal disc tissue in pattern regulation in Drosophila. Develop. Biol. 60, 29%
304. AMPRINO, R., and CAMOSSO, M. (1965). La regulation d’excedents de l’ebauche de membres du poulet. Arch. Anat. Microsc. Morphol. Exp. 54, 781-810. BARASA, A. (1964). On the regulative capacity of the chick embryo limb bud. Experientia 20,443. BRYANT, P. J. (1971). Regeneration and duplication following operations in situ on the imaginal discs of
Drosophila melanogaster. Develop. Biol. 26, 637651. BRYANT, P. J., ADLER, P. N., DURANCEAU, C., FAIN, M. J., GLENN, S., HSEI, B., JAMES, A. A., LITTLEFIELD, C. L., REINHARDT, C. A., STRUB, S., and SCHNEIDERMAN, H. A. (1978). Regulative interactions between cells from different imaginal discs of Drosophila melanogaster. Science 201,928-930. BRYANT, S. V., and ITEN, L. E. (1974). The regulative ability of the limb regeneration blastema of Notophthalmus viridescens: Experiments in situ. Wilhelm Roul Arch. 174,90-101. BRYANT, S. V., and ITEN, L. E. (1976). Supernumerary limbs in amphibians: Experimental production in Notophthalmus viridescens and a new interpretation of their formation. Develop. Biol, 50, 212-234. FABER, J. (1971). Vertebrate limb ontogeny and limb regeneration: Morphogenetic parallels. Advan. Morphog. 9, 127-147. FALLON, J. F., and THOMS, S. D. (1979). A test of the polar coordinate model in the chick wing bud. Anat. Rec. 193,534. FRENCH, V. (1978). Intercalary regeneration around the circumference of the cockroach leg. J. Embryol.
Exp. Morphol. 47,53-84. FRENCH, V., BRYANT, P. J., and BRYANT, S. V. (1976). Pattern regulation in epimorphic fields. Science 193, 969-981. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88,49-92. HANSBOROUGH, L. A. (1954). Regulation in the wing of the chick embryo. Anat. Rec. 120,698-699. HOLDER, N., BRYANT, S. V., and TANK, P. (1979) Interactions between irradiated and unirradiated tissue during supernumerary limb formation in the newt. J. Exp. Zool. 208.303-310. ITEN, L. E. (1977). Pattern regulation in complementary anterior and posterior portions of the chick wing bud. Amer. 2001. 17,963. JAVOIS, L. C., and ITEN, L. E. Evidence against a zone of polarizing activity in the embryonic chick limb bud. Develop. Biol., in press. MORGAN, T. H. (1901). “Regeneration.” Macmillan, New York. SAUNDERS, J. W., JR., CAIRNS, J. M., and GASSELING,
ITEN AND MURPHY
Pattern
Regulation
M. T. (1957). The role of the apical ridge of ectoderm in the differentiation of the morphological structure and inductive specificity of limb parts in the chick. J. Morphol. 101, 57-87. SMITH, J. C., TICKLE, C., and WOLPERT, L. (1978). Attenuation of positional signalling in the chick limb by high doses of y-radiation. Nature (London) 272, 612-613. STARK, R. J., and SEARLS, R. L. (1974). The establishment of the cartilage pattern in the embryonic chick wing, and evidence for a role of the dorsal and ventral ectoderm in normal wing development. Develop. Biol. 38, 51-63. STOCUM, D. L. (1975). Outgrowth and pattern formation during limb ontogeny and regeneration. Differentiation 3, 167-182. SUMMERBELL, D. (1979). The zone of polarizing activ-
in the Chick
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Bud
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ity: Evidence for a role in normal chick limb morphogenesis. J. Embryol. Exp. Morphol. 50,217-233. SUMMERBELL, D., and TICKLE, C. (1977). Pattern formation along the anteroposterior axis of the chick limb bud. In “Vertebrate Limb and Somite Morphogenesis” (D. A. Ede, J. R. Hinchliffe, and M. Balls, eds.), pp. 41-53. Cambridge Univ. Press, Cambridge. TICKLE, C., SUMMERBELL, D., and WOLPERT, L. (1975). Positional signalling and specification of digits in chick limb morphogenesis. Nature (London)
254,199-202. WOLPERT, L. (1969). Positional information and the spatial pattern of cellular differentiation. J. Theor. Biol. 25, 1-47. WOLPERT, L. (1971). Positional information and pattern formation. Curr. Topics Develop. Biol. 6, 183-
224.
BIOLOGY
75, 373-385 (1980)
Pattern Regulation in the Embryonic Chick Limb: Supernumerary Formation with Anterior (Non-ZPA) Limb Bud Tissue LAURIE Department
of Biological
Limb
E. ITEN AND DOUGLAS J. MURPHY Sciences, Purdue
University,
West Lafayette,
Indiana
47907
Received May 29, 1979; accepted in revised form September 14, 1979 The formation of supernumerary limbs and limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. A “wedge” (ectoderm and mesoderm) of anterior or mid donor right wing bud (stage 21) was inserted in a slit made in a host right limb bud (stage 21) at the same position as its position of origin or to a more posterior position. The AER of the donor tissue and host wing bud were aligned with each other. Donor tissue was grafted with its dorsalventral polarity the same as the host’s limb bud or reversed to that of the host’s, Depending on the position of origin of the donor limb bud tissue and the position to which it was transplanted in a host, supernumerary wings or wing structures formed. Furthermore, depending on the orientation of the graft in the host, supernumerary limbs with either left or right asymmetry developed. The results of experiments performed here are considered in light of two current models which have been used to describe supernumerary limb formation: one based on local, short-range, cell-cell interactions and the other based on long-range positional signaling via a diffusible morphogen. INTRODUCTION
The ability of an early embryo or developing organ to regulate and form a complete organism or organ after portions have been removed or the ability of adult animals to regenerate lost parts has fascinated many generations of biologists. This interest in regulation and regeneration, in part, led to the concept of positional information (Wolpert, 1969, 1971). During development and regeneration, cells acquire specific fates according to their position in the embryo or organ which leads to species-typical spatial patterns of cellular differentiation. Regulation and regeneration involve either the replacement of cells with the missing positional values through cell proliferation, there being little change in the positional values of the remaining cells (epimorphosis), or regulation and regeneration involve the respecification of the positional values of the remaining cells such that a miniature but complete pattern is reformed (morphallaxis) (Morgan, 1901). The similarities between the sequence of
events observed during embryonic chick limb development and postembryonic amphibian limb regeneration have been documented elsewhere (Faber, 1971; Stocum, 1975). Briefly, limb buds first appear in the chick embryo as small bulges protruding from the lateral body wall. Each limb bud is a condensation of mesenchyme cells enveloped with an ectodermal jacket; an apical ectodermal ridge (AER) rims the apex of each limb bud. As a limb bud grows in length, differentiation of limb tissues begins proximally and progresses distally resulting in a distinct spatial pattern of differentiated structures. After the amputation of a salamander limb, wound healing and dedifferentiation of stump tissues adjacent to the site of amputation occurs. Cells from the dedifferentiated tissues accumulate at the distal end of the limb stump to form a regeneration blastema which is a mass of mesenchyme cells encased in an epidermal cap. The regeneration blastema, like the chick limb bud, grows in length and differentiation proceeds in a proximal to distal
373 0012-1606/80/040373-13$02.00/O Copyright All rights
0 1980 by Academic Press, of reproduction in any form
Inc. reserved.
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direction. A basic difference exists between developing and regenerating limbs. In the former, the most proximal structures to be laid down are always the same, whereas, in regenerating limbs, the most proximal structures to be laid down will vary depending on the level of amputation. Both the embryonic chick limb bud and postembryonic regenerating amphibian limb can be operationally defined as a developmental field within which pattern regulation (changes in the presumptive fates of cells) can occur in response to surgical perturbations. Regenerating amphibian limbs, cockroach legs, and Drosophila imaginal disks exhibit epimorphic pattern regulation and there is evidence suggesting that the embryonic chick limb bud, at certain stages of development, also regulates epimorphically. Regulation to form a complete limb can occur following the removal or addition of the central portion of the limb bud (Hansborough, 1954; Barasa, 1964; Amprino and Camosso, 1965; Stark and Searls, 1974) and there appears to be a correlation between cell division and the ability of a chick limb bud to regulate following the removal of its center (Stark and Searls, 1974). That analogous results are obtained from similar experiments with different developing and/or regenerating systems supports the contention that there are fundamental similarities with regards to positional information and the behavior of cells during the construction of a three dimensional pattern of differentiated structures (Wolpert, 1971; French, Bryant, and Bryant, 1976). Experiments done with the embryonic chick limb bud give results analogous to those obtained from similar experiments done with regenerating amphibian limbs, cockroach legs, or Drosophila imaginal disks. For example, removal of a longitudinal strip of cuticle and epidermis from any position around the circumference of the femur of a cockroach leg leads to the cut edges healing together, localized growth, and regeneration of the missing
section of the circumference, i.e., a normal size femur with a complete pattern of cuticular markers results irrespective of the longitudinal strip removed (French, 1978). The same result is obtained with the developing chick limb when a longitudinal slice of wing bud mesoderm and ectoderm is removed. A qualitatively and quantitatively complete wing forms irrespective of the longitudinal slice of wing bud removed (Iten et al., in preparation). When the regulative behavior of complementary anterior and posterior portions of a wing bud is examined, one portion forms limb structures with normal anterior-posterior and dorsal-ventral polarity and the other forms bilaterally symmetrical structures, namely, structures with dorsal-ventral polarity, but without anterior-posterior polarity (Iten, 1977); these results are analogous to the regenerationduplication behavior of complementary Drosophila fragments of regenerating imaginal disks (Bryant, 1971). A characteristic of epimorphic pattern regulation is that supernumerary (extra) structures can form after disharmonic axial opposition of portions of a developmental field. Again, there are striking similarities between the supernumerary limbs obtained with axial reversals with the developing chick limb bud and those obtained with embryonic amphibian limbs and regenerating amphibian and insect limbs (see Bryant and Iten, 1976, for a detailed description of these similarities). The formation of supernumerary limbs and limb structures in amphibians and insects has been described with a simple and unified hypothesis for pattern regulation in epimorphic fields proposed by French, Bryant, and Bryant (1976). In their model they propose that the positional values of cells in an epimorphic field are specified in terms of a two-dimensional polar coordinate system. One component of positional information is a value corresponding to position on a circle, and the second component to position on a radius. The basic rule of cellular behavior proposed by this model is
ITEN AND MURPHY
Pattern
Regulation
that when cells with normally nonadjacent positional values in either the radial or circular sequences are next to each other, growth occurs until cells with intermediate positional values have been intercalated and the discontinuity is eliminated. The polar coordinate model proposes that supernumerary limb formation is the result of local (short-range) cell-cell interactions and cell proliferation. Another model has been proposed by Wolpert and co-workers to describe the formation of supernumerary limb structures in embryonic chick limbs and it is based on long-range positional signaling via a diffusible morphogen (Tickle et al., 1975; Summerbell and Tickle, 1977; Summberbell, 1979). In their model they propose that there is a localized organizing center at the posterior edge of a limb bud called the zone of polarizing activity (ZPA). The ZPA is the source of a diffusible morphogen that specifies a cell’s anterior-posterior coordinate. Simply, a limb bud cell’s anterior-posterior coordinate depends on its distance from this localized source. This hypothesis is based on studies where another ZPA is transplanted to certain position along the anterior-posterior edge of a host limb bud and supernumerary limb structures result. The results of experiments presented here show that anterior (non-ZPA) limb bud tissue transplanted to more posterior positions in a host limb also results in the formation of supernumerary limb structures. MATERIALS
AND
METHODS
Embryos used in these experiments were from a White Leghorn strain of chickens obtained from the Indiana Co-Op Poultry and Breeding Farm (West Lafayette, Ind.). Eggs were incubated at 38°C. At the desired Hamburger and Hamilton (1951) stages, embryos were prepared for surgical operations by withdrawing albumin to lower the yolk and embryo, sawing a window in the shell, and removing the shell membranes. Tissue manipulations were performed with sharpened tungsten needles and fine for-
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ceps. Stage 21 right wing buds (1ength:width ratio between 2.3 and 2.9) (Hamburger and Hamilton, 1951) were used as donor and host limb buds. Right leg buds (stage 21) were also used as donor limb buds in some of the transplantation operations. Anterior or mid limb bud tissue from a right wing bud was transplanted to the same anteriorposterior level in a host right wing bud or to a more posterior level (Fig. 1). Host wing bud tissue was not removed to prepare a graft site; instead, a slit was cut through the entire thickness of the host wing bud and then a wedge of donor wing bud tissue (ectoderm and mesoderm ca. 150 pm wide at its distal edge and ca. 200 pm long) was inserted into the slit. Host limb bud tissue was not removed to prepare a graft site because host cells that may or may not interact with the cells of the graft would be removed. The wedge of donor tissue was J
1
16
17 I 18 3
1
18 I
19
19 3
24
24
16
J
17 3
17 I
18 1
18 3
19 3
19 3
207
207
FIG. 1. Dorsal view outlines of donor and host stage 21 right wing buds. The position of origin of a wedge of anterior donor tissue and the different positions that it was added to a host are both shown with respect to the adjacent somites. The distance from the center of one somite to the center of the next is approximately 250 pm and the anterior-posterior length of a wing bud at its base is about 1 mm. The left diagram illustrates the different anterior-posterior positions in which a 16/17 wedge was placed in a host limb bud and the right diagram illustrates the same thing, but for a 17/18 wedge of donor tissue.
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positioned such that the cut surfaces of the graft and host were in contact with each other and the AER of the graft and host were aligned with each other. The position of origin of the wedge of donor tissue as well as the position in the host where the tissue had been added were identified with respect to somites 16, 17, 18, and 19 adjacent to a stage 21 wing bud. For example, 16/17 added to an 18/19 level indicates that a wedge of wing bud tissue adjacent to the junction of somites 16 and 17 was inserted into a slit made in a host at the level of the junction of somites 18 and 19. A carbon mark was placed on the dorsal surface of the wedge of donor tissue so that the orientation of the graft in the host could be recorded. A wedge was added to a host either with its dorsal-ventral axis matching that of the host’s (denoted as dd orientation) or with its dorsal-ventral axis reversed with respect to the host’s (denoted as dv orientation). Observations on the development of a wing bud with its graft were made 1 and 2 days after the transplantation op-
VOLUME 75, 1980
eration and all embryos were sacrificed and fixed at 13 days incubation. The integumentary pattern of resulting wings was recorded at this time. Embryos were then stained with Victoria blue (Bryant and Iten, 1974) and cleared in methyl salicylate to reveal the skeletal pattern of resulting wings. In some cases, wings were embedded in paraffin and sectioned to examine their muscle pattern. RESULTS
The skeletal and integumentary pattern of 266 resulting wings was examined. The wings resulting from the transplantation operations where a wedge of 16/17 or 17/18 donor wing bud tissue was transplanted to the same or more posterior position in a host wing bud have been examined with respect to extra skeletal elements formed (Table 1) and the resulting anterior to posterior sequence of digits (Table 2). The skeleton of the wing can be divided into three proximal to distal segments: stylopodium, zeugopodium, and autopodium. The
TABLE
1
RESULTS OF TRANSPLANTING ANTERIOR OR MID WING BUD TISSUE TO THE SAME OR MORE POSTERIOR POSITION IN A HOST WING BUD Position of origin of graft with respect to somites
Position transplanted in host with respect to somites
Orientation of graft in host”
Total No. resulting wings
No. with extra skeletal element(s)
16/17
16/17
dd du dd
0 (0%)
dv dd do dd dv
10 10 11 11 20 21 25 15
3 4 6 18 20 25 15
(30%) (36%) (55%) (90%) (95%) (100%) (100%)
dd dv dd dv dd dv
11 11 12 14 16 14
0 1 4 7 14 14
(0%) (9%) (33%) (50%) (88%) (100%)
17/18 M/19 19/20
17/18
17/18 H/19 19/20
Extra skeletal elements formed’ S
Z
3 3 5 1 3 1
A
1 1 5 2
Z/A
S/Z/A
9 12 4 2
3 3 20 13
2 3 8 10
4 3
1 1 2
2 3 1
n Graft was inserted in a host with ita dorsal-ventral polarity matching that of the host’s, denoted dd orientation, or with its dorsal-ventral polarity opposite that of the host’s, denoted dv orientation. * S, extra stylopodial element (humerus); Z, extra zeugopodial element(s) (radius, ulna, or both); A, extra autopodial element(s) (digit or digits); Z/A, extra zeugopodial and autopodial elements; S/Z/A, extra stylopodial, zeugopodial, and autopodial elements.
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Regulation
TABLE
in the Chick
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Limb Bud
2
DIFFERENT ANTERIOR TO POSTERIOR DIGITAL SEQUENCES OF RESULTING WINGS AFTER ANTERIOR OR MIII WING BUD TISSUE HAD BEEN GRAFTED TO THE SAME OR MORE POSTERIOR POSITION IN A HOST WIKG Bun Anterior to posterior sequence of digits OrientaPosition Position of tion of origin of transplanted II II graft in in host with graft with *:: III respect to respect to host” 1;: 1:: 1:: 1:: 1:: 1::7 1:: IV III somites somites IV IV :z :: :r”r :: & rl”t III III II IV IV 16/17
18/17 17/H 18/19 19/20
17/18
17/M 18/19 19/20
dd dv dd dv dd dv dd dv
10 10 10 10 3 4 1
dd dv dd dv dd dv
11 11 8 8 4
1 1 2 5
1 1 1
2 1
1
2 1 7 4
1 2 12 14 ._____~.
10 6
2 4 15 7
2 3
1 2
Note. Data refer to the number of cases obtained. ” Abbreviations as in footnote a of Table 1.
stylopodium is made up of the humerus which articulates proximally with the shoulder girdle and distally with the radius and ulna of the zeugopodium. The anterior radius and posterior ulna articulate distally with the carpals of the autopodium. Distal to the carpals are the metacarpals and phalanges of the three digits of the wing. Digit II is the most anterior and digit IV is the most posterior. Many of the skeletal elements of the wing have a characteristic morphology which facilitates their identification in resulting wings and aids in the determination of the asymmetry or symmetry of structures formed. Limbs with extra skeletal elements representing one, two, or all three of the proximal to distal segments of the limb, usually next to the same segments of the host wing, form as a result of some of the transplantation operations performed. Extra muscles and feather follicles also formed in some cases, but a detailed description of the muscle and integumentary pattern of resulting wings is not presented here.
Control Transplantations Twenty-one wings resulting from operations where a wedge of 16/17 donor wing bud tissue was added to a 16/17 level slit in a host wing bud with dd orientation (10 cases) or where a wedge of 17/18 donor tissue was added to a 17/18 host site with dd orientation (11 cases) were analyzed. As can be seen from Table 1, these control transplantations clearly show that adding a wedge of anterior or mid wing bud tissue to the same anterior-posterior position in a host wing bud with dd orientation does not result in the formation of supernumerary skeletal elements (Fig. 2a), but extra integumentary structures do form. When a wedge of 16/17 or 17/18 was added to the same respective position in a host wing bud, but with dv orientation, a few limbs resulted in which an extra partial or complete humerus developed next to the host humerus (Table 1). Furthermore, these operations frequently disrupted the normal dorsal and ventral distribution of feather follicles; that is, a region of ventral-like apteria
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FIG. 2. Dorsal view of the skeletal pattern of wings formed after a wedge of 16/17 wing bud tissue had been added to the same or more posterior position in a host wing bud. (a) A wedge of 16/17 tissue was added to a 16/ 17 level slit of a host wing bud or (b) to a 17/18 level slit, both with dd orientation, and a normal complement of skeletal elements formed. (c) A wedge of 16/17 tissue was added to an 18/19 level slit in a host wing bud with dd orientation. This limb is an example of a resulting wing with an extra ulna and extra digits Anterior to posterior digital sequence of this wing is II III IV IV III IV. (d) A wedge of 16/17 tissue was added to a 19/20 level slit (posterior edge) of a host wing bud with dd orientation. Arrow points out supernumerary wing structures that formed: humerus, zeugopodial element, and digits This wing has an anterior-posterior digital sequence of II III IV IV III. All x 3.6.
developed in the dorsal wing integument and a corresponding dorsal-like pteryla in the ventral integument. Similar results were obtained when different wedges of posterior limb bud tissue were added to analogous positions in host wing buds (Javois and Iten, in press).
Experimental
Transplantations
When a wedge of 16/17 donor wing bud tissue was added to a 17/18 level slit in a host wing bud with dd orientation, no extra skeletal elements formed in the majority of cases (Fig. 2b), but 3 of the resulting 11 wings had an extra partial or complete humerus and 1 had an extra digit (Table 1). A higher incidence of limbs with an extra humerus or extra digit resulted when the same operation was performed, but the
wedge was grafted with dv orientation (55 vs 36% of the cases, Table 1). When an extra digit formed, it was always a digit II and the anterior to posterior sequence of digits of the resulting wing was II II III IV (Table 2). Depending on the orientation of the graft in the host, extra integumentary structures and/or a disruption in the dorsal and ventral integumentsry pattern resulted. Adding a wedge of 16/17 donor wing bud tissue to an M/19 level slit in a host with dd or dv orientation resulted in limbs with extra skeletal elements in almost all cases, and the majority of these had an extra ulna and digits (Table 1) and an anterior or posterior digital sequence of II III IV III IV or II III IV IV III IV (Table 2, Fig. 2~). However, adding a wedge of 17/18 tissue to an 18/19 host level slit with dd or dv ori-
ITEN AND MURPHY
Pattern
Regulation
entation resulted in a lower incidence of limbs with extra skeletal elements (Table 1) and the majority of resulting wings had a normal II III IV anterior to posterior sequence of digits (Table 2). Again, in these groups of transplantation operations, depending on whether the wedge of donor tissue was grafted with dd or dv orientation, extra integumentary structures and/or a disruption in the dorsal and ventral integumentary pattern resulted. When either 16/17 or 17/M wing bud tissue was added to the posterior edge of a host wing bud (19/20 host slit level) with dd or dv orientation, a separate and identifiable supernumerary outgrowth was seen 2 days later (Fig. 4) and resulting wings had supernumerary structures well separated from the host wing structures in almost all cases. The extra skeletal elements formed and the digital sequence of resulting wings was different depending on the position of origin of the graft. Namely, 16/17 added to the posterior edge of a host limb bud resulted in wings with an extra humerus, extra ulna or radius and ulna, and extra digits in 83% of the cases and a digital sequence of II III IV IV III or II III IV III II in 68% of the cases (Tables 1 and 2; Figs. 2d and 3a and b), whereas 17/18 added to the same position in a host wing bud resulted in wings with an extra ulna and digit and a digital sequence of II III IV III in the majority of cases (Tables 1 and 2; Figs. 3c and d). Furthermore, when 16/17 was added to the posterior edge of a host with dd orientation, the supernumerary wing structures which resulted clearly had left hand asymmetry in 21 of 23 resulting limbs (Fig. 3a) and if grafted with dv orientation, then supernumerary wing structures had right hand asymmetry in 13 of the 15 resulting limbs (Fig. 3b). But when a wedge of 17/18 wing bud tissue was added to the posterior edge of a host wing bud, the extra wing structures formed had neither right nor left hand asymmetry. When grafted with dd or dv orientation, bilaterally symmetric supernumerary structures resulted; the dorsal-
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ventral polarity of these supernumerary structures always coincided with the dorsal-ventral polarity of the graft (Figs. 3c and d). If a wedge of limb bud tissue from the anterior edge of a stage 21 right wing bud (tissue adjacent to somite 16 or adjacent to the junction of somites 15 and 16) was added to the posterior edge of a host wing bud, the extra structures formed were not the same as those formed when a wedge of 16/17 donor tissue was added to the same position in a host limb bud. Transplanting anterior edge wing bud tissue to the posterior edge of a host (dd or dv orientation) resulted in wings with an extra partial proximal or complete humerus (11/25 cases), an extra humerus and an unidentifiable distal skeletal element (lo/25 cases), or no extra skeletal elements (4/25 cases). Similar results were obtained when the AER was removed from a wedge of 16/17 donor tissue before transplanting it to the posterior edge of a host limb bud (Iten et al., unpublished results). Since it has been shown that leg bud tissue in a wing bud will differentiate into leg structures (Saunders et al., 1957), the origin of supernumerary limb structures was examined by transplanting anterior leg bud tissue to the same level or to the posterior edge of a host wing bud. When a wedge of stage 21 anterior right leg bud (tissue adjacent to the junction of somites 27 and 28) was added to the 16/17 level of a host wing bud at the same stage with dd (12 cases) or dv (10 cases) orientation, resulting wings did not have “toes” instead of wing “fingers” or “toes” in addition to their normal set of digits, although their proximal anterior skeletal elements and integument were leg-like (Fig. 5a). However, wings with supernumerary “toe(s)” or a “toe” and wing “finger” did form after anterior leg bud tissue was transplanted to the posterior edge of a wing bud with dd (7 cases) or dv (11 cases) orientation (Fig. 5b). Thus it appears that supernumerary structures arise from the wedge of anterior
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FIG. 3.
ITEN AND MURPHY
Pattern Regulation in the Chick Limb Bud
tissue and they may in part arise from the host limb bud. Studies on the origin of these extra structures utilizing the cytological marker available with xenoplastic chick/ quail graft combinations also indicate that the extra structures formed arise from the graft and host wing bud (Cotromanes and Iten, in preparation). DISCUSSION
Results presented in this paper show that when anterior or mid wing bud tissue is transplanted to a more posterior position in a host wing bud, wings with extra structures or a separate supernumerary wing can result. Furthermore, the formation of extra limb structures or a supernumerary limb depends on the position of origin of donor tissue, the position transplanted in a host
381
wing bud, as well as the orientation of the graft in the host. The results of transplanting anterior leg bud tissue to the posterior edge of a host wing bud suggest that both the donor and host contribute to the formation of a supernumerary limb. Experiments similar to those presented here have been briefly reported by Fallon and Thorns (1979). They transplanted anterior edge wing bud tissue (with or without its AER) to the posterior edge of a host wing bud. Host wing bud tissue was removed to prepare a graft site. No extra structures formed in the majority of their resulting wings and when supernumerary structures did form, only a small cartilagenous outgrowth or a single digit-like structure formed. We obtained similar results when a wedge of anterior edge tissue (ad-
FIG. 3. Dorsal view of the integumentary pattern (left photograph) and skeletal pattern (right photograph) of wings formed after wedge of anterior or mid-right wing bud tissue had been added to the posterior edge (19/ 20 level) of a host right wing bud. Arrows identify supernumerary wing structures. (al 16/17 wing bud tissue was added with dd orientation. Resulting wing has an extra humerus next to the host’s humerus, extra ulna, extra digits, and an anterior to posterior digital sequence of II III IV IV III. Supernumerary structures have left asymmetry. (b) 16/17 wing bud tissue was added with du orientation. Resulting wing has an extra humerus (not clearly visible in this view), extra radius and ulna, extra digits, and a digital sequence of II III IV III II. The supernumerary structures have right asymmetry (the ventral surface of the supernumerary structures is seen in a dorsal view). (cl 17/18 wing bud tissue was added with dd orientation or with du orientation (d). Both are examples of a resulting wing with an extra zeugopodial element and digit and a II III IV III digital sequence; the supernumerary structures are bilaterally symmetrical and their dorsal-ventral polarity is the same as the original orientation of the graft in the host limb bud. All x 2.7. FIG. 4. Dorsal view of a right wing bud 2 days after a wedge of anterior wing bud tissue had been added to the posterior edge of this limb bud (left) and a normal right wing bud at approximately the same stage of development (right). Supernumerary outgrowth is indicated by arrow. Both x 14.
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DEVELOPMENTAL BIOLOGY VOLUME75, 1980
FIG. 5.
ITEN
AND
MURPHY
Pattern
Regulation
jacent to somite 16 or the junction between somites 15 and 16) was inserted in a slit made at the posterior edge of a host wing bud. However, when a wedge of 16/17 donor tissue is grafted to the same position in a host limb, very different results were obtained, namely, a separate supernumerary wing developed with left or right asymmetry depending on the orientation of the graft in the host. If the AER is removed from the 16/17 donor tissue, then results similar to those with anterior edge donor tissue are obtained. In our opinion, the results presented in this paper can best be described with a model proposing local cell-cell interactions where cells with intermediate positional values are intercalated after normally nonadjacent cells are placed next to each other, like the polar coordinate model proposed by French, Bryant, and Bryant (1976). Whereas, our results are not easily explained by a mechanism involving longrange positional signaling via a diffusible morphogen from the ZPA at the posterior edge of a limb bud (Tickle et al., 1975; Summerbell and Tickle, 1977). In the recent elaboration of the ZPA model by Summerbell (1979), it could be argued that when a wedge of anterior wing bud tissue was transplanted to a more posterior position in a host wing bud, it was exposed to a higher concentration of morphogen, and in its new position, it was respecified to form certain skeletal parts. The previously specified host limb bud cells would form their appropriate skeletal elements, and the newly respecified grafted cells would form extra skeletal elements due to their exposure to a certain concentration of morphogen emanating from the host’s ZPA. Therefore, one would expect that the more posterior the position in a host the graft was inserted, the greater
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the percentage of limbs with extra skeletal elements that should occur, and this was the result obtained in the present study. However, using the same argument, one would expect to find that the same extra skeletal elements would form and in the same percentage of cases when either a wedge of anterior (16/17 wedge) or mid (17/ 18 wedge) wing bud tissue was transplanted to the same posterior position in a host wing bud (an 18/19 or 19/20 host slit level), because the donor tissue in its new position would be exposed to the same concentration of morphogen. This result was not obtained (see Tables 1 and 2). Limbs with different extra skeletal elements and the frequency of limbs with extra skeletal elements was different depending on whether a 16/17 or 17/18 wedge of donor tissue was transplanted to the same posterior position. Furthermore, the results of our transplantation of anterior or mid wing bud tissue to different posterior positions cannot be explained by proposing that there is autonomous development of the graft in the host wing bud, because adding a wedge of limb bud tissue to the same anterior-posterior position in a host wing bud does not result in the formation of supernumerary skeletal elements. By applying the formal rules for cellular behavior proposed by the polar coordinate model we would expect to find supernumerary structures forming after anterior limb bud tissue is transplanted to a more posterior position in a host limb and that the extra structures formed would depend on both the position of origin of the graft and the position transplanted to a host limb bud. These are the results we obtained. Furthermore, we found that the asymmetry (handedness) or symmetry of supernumerary structures formed depends on the ori-
FIG. 5. Dorsal view of the integumentary pattern (left photograph) and skeletal pattern (right photograph) of a resulting wing after a wedge of anterior right leg bud tissue had been added to a 16/17 level (a) or to a 191 20 level (b) in a host right wing bud, both with dd orientation. (a) Leg-like integumentary pattern and skeletal elements are seen in the proximal anterior portion of the resulting wing. Supernumerary leg (large arrow) and wing structures (small arrow) are illustrated in (b). All x 5.7.
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entation of the graft in the host limb bud. The polar coordinate model is the only one we know of that can describe how the asymmetry/symmetry of extra structures could arise. The polar coordinate model makes no stipulations about where the cells for supernumerary structures should come from, i.e., graft or host, because extra limb structures are seen as arising as a result of intercalation of cells between the normally nonadjacent cells of the graft and host. With regenerating amphibian limbs, if either the regenerate or adjacent stump is prevented from contributing cells to form a supernumerary limb with X-irradiation, the unirradiated partner can contribute more to the formation of supernumerary structures (Holder et al., 1979). And if posterior edge wing bud tissue from a heavily irradiated donor limb is transplanted to the anterior edge of an unirradiated host, then supernumerary wing structures still form (Smith et al., 1978). Results analogous to these obtained with amphibian and chick limbs have been obtained with the regenerating imaginal disk of Drosophila (Adler and Bryant, 1977; Bryant et al., 1978). In conclusion, the results presented here suggest that at certain stages of development, the embryonic chick limb bud exhibits epimorphic pattern regulation similar to that observed in the postembryonic epimorphically regenerating amphibian limb, cockroach leg, and Drosophila imaginal disk. In addition, the results obtained here can best be described with a hypothesis proposing local, short-range, cell-cell interactions, and they are not easily explained by a mechanism involving longrange signaling via a diffusible morphogen emanating from the ZPA. It is a pleasure to acknowledge the valuable comments and criticisms of Drs. U. K. Abbott, S. V. Bryant, and J. W. Saunders, Jr., Ms. E. Cotromanes, and Ms. L. C. Javois. This investigation was supported by Grant PCM 77-08408, awarded by the National Science Foundation, and in part by an Institutional Grant from the American Cancer Society.
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