effect of augmentation of nerve supply upon limb regeneration in the chick embryo

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 221:49-59 (1982)

Effect of Augmentation of Nerve Supply Upon Limb Regeneration in the Chick Embryo

IRA FOWLER AND BETTY F. SISKEN Department of Anatomy, University of Kentucky, MN224 Medical Center, Lexington, KY 40536 (I.F.) and WennerGren Research Laboratories, Depart- ment of Anatomy, University of Kentucky, Veteran’s Administration Medical Center, Lexington, Kentucky 40506 (B.F.S.)

ABSTRACT Limb regeneration was induced in 4-day chick embryos by implanting stage 15 neural tube into the amputated stump of the limb. In control animals, amputation alone or amputation with implant of either notochord or somites resulted in development of proximal wing segments only. In experimental animals, more than one-fourth of the animals containing viable neural tube implants developed proximal, middle, and distal limb segments. These regenerated limb segments contained muscle groups, cartilage models, and nervous structures that approximated the normal situations. Since some new parts regenerated in more than one-fourth of the cases, it may be concluded that augmentation of nerve supply is an effective method of inducing regeneration in limbs of chick embryos. Volume measurements of the neural tube implant indicate that a “critical mass” of nerve tissue may be necessary for regeneration to occur.

Induction of limb regeneration in higher vertebrates that do not normally regenerate re- mains a challenge to biologists working in this area. The chick embryo offers advantages over other warm-blooded animals for the study of regeneration since the embryo is readily acces- sible, cheap, and plentiful.

The use of embryos in the study of the events following limb amputation provides a system in which the main factors of growth, pattern formation, and differentiation that are respon- sible for normal limb development occur rapid- ly without the delay experienced in the use of adult vertebrates. The limb bud can respond quickly to environmental changes, facilitating the study of extrinsic factors upon development of the amputated limb stump.

The classical experiments of Saunders (’48) demonstrate that the chick embryonic limb dif- ferentiates in a well-defined proximo-distal sequence under the influence of the thickened apical ectodermal ridge (AER). Summerbell (’74) has emphasized that the AER is of critical importance in normal limb development. He removed the AER from the early limb bud and found that the structures that develop following excision of the AER are correlated with the stage at which the operation i s performed. Only proximal structures, such as girdle and humerus, were formed following excision of the AER at early stages; more distal structures de-

veloped when the operation was done at later stages.

Simple removal at stage 24 (Hamburger and Hamilton, ’51) of the apical zone (defined as that portion of the wingbud distal to a sagittal plane passing through the cranial and caudal limits of the ectodermal ridge) results in development of a limb with normal skeletal elements only to the elbow; the radius and ulna, if present at all, are very small, while hand parts are missing. Fate maps derived from such experiments (Saunders, ’48; Amprino et al., ’58; Saunders et al., ’62) reveal that, even at stage 21, the regions that will form the major parts of the proximal and middle segments are already determined in the wingbud; if these regions are removed by amputation of the limb bud, the corresponding limb parts do not form. In his analysis of chick limb development following excision of the AER only, Summerbell (’74) found that at stage 24, which was used in our experiments, the wingbud will develop skeletal elements that include the wrist. In our experiments, mesoderm of the apical zone was

Address reprint requests to Ira Fowler, Ph.D.. Department of Anat. omy. Universityof Kentucky. MN224 Medical Center. Lexington. KY 40536.

This report is based upon data that was presented. in part. at the 1981 meeting of the American Association of Anatomists (Anat. Rec. i99:238).

0022-104X/82/2211-0049$03.50 0 1982 ALAN R. LISS, INC.

50 I. FOWLER AND B.F. SISKEN

t Longitudinal Slit B

Implant

al Zone

Fig. 1. Diagram of stage 24 limb buds showing the procedure for amputation of the apical zone (A). In iBI the procedure used for the control embryos is shown. In (C) the orientation of the neural tube implant within the longitudinal slit is represented.

extirpated along with the AER resulting in limbs that never contained wrist elements in the absence of stimulation by extrinsic factors. We report experiments demonstrating that some development of distal limb parts can occur with the addition of neural tissue to the stump of the amputated wingbud.

MATERIALS AND METHODS Amputation of the Wingbud

In the present experiments, amputations were performed on chick embryos incubated for 4 to 4.5 days when some of the embryos were at stage 25, but most were at stage 24. The wingbuds at stage 24 extended laterally and ventrally as rods of uniform diameter, of greater length than width, but with no indica- tion of an elbow joint. The embryo usually lay on its left side with the right wingbud directly beneath the shell where it could be adequately exposed by removing a piece of shell and shell membrane approximately 1 cm square. Local application of Nile blue defined the apical ectodermal ridge clearly facilitating the demarca- tion (Fig. 1A) of the apical zone (defined as that portion of the wingbud distal to a sagittal plane passing through the cranial and caudal limits of the ectordermal ridge). With the use of

fine tungsten needles (Dossel, '58) the apical zone was extirpated and a longitudinal slit was made in the center portion of the limb stump parallel to its long axis (Fig. 1B). Amputated limb tips of all embryos were examined gross- ly, and many histologically, to determine that extirpation of the apical zone was complete. Representative examples of embryos that were photographed with their extirpated limb tips are shown in Figure 2. As further investigation of the uniformity of the surgical procedure, surface area measurements were made of nine limb tips extirpated at stage 24 and 10 at stage 25. These measurements showed a variation of less than 10% in surface area of the tips. The surface areas of the amputated tips were closely correlated with the length of the wingbuds; i.e.. the wingbuds of the larger embryos of stage 24 had longer wingbuds with larger apical zones than did the morphologically smaller embryos of the same stage.

Neural tube implants To augment the amount of nervous tissue in

limbs in the experimental group, a segment of neural tube six somites in length (six spinal cord segments) was dissected from either a cer- vical, brachial, or thoracic region of a donor embryo at stage 15 (2 days of incubation) and inserted into the longitudinal slit of the amputated wingbud of each host embryo, leaving the tip of the neural tube implant protruding at the amputation surface (Fig. 1C). No attempt was made to be consistent in the cranial-caudal orientation of the implant in relation to the limb. In 10 of 58 specimens receiving neural tissue, the implant consisted of a lateral half of the neural tube segment with adjacent neural crest and scattered mesodermal cells adhering to the neural tissue. The remaining 48 specimens received an implant that included the en- tire neural tube segment, the underlying notochord, adjacent neural crest, and scattered mesodermal cells.

In an attempt to determine whether the observed effects upon development of the amputated limbs were due to the neural tube or to adherent tissues, strips of notochord or of SO- mites without neural tube were implanted in amputated limbs. Since there were no observable effects of these implants and no detectable differences in response of the limbs to neural tube implants with and without notochord, the latter two groups will be described together.

The treatment of the control groups was the same except that no implant was inserted into

LIMB REGENERATION IN THE CHICK EMBRYO 51

Stage 25 Stage 24

\ I Limb stumps

Fig. 2. Stage 25 and stage 24 embryos photographed after amputation of the apical zone. The respective ampu-

the longitudinal slit of the amputated wingbud (Fig. 1B). After the surgical procedures were completed, the opening in the eggshell was closed by replacing the piece of shell and seal- ing it with melted paraffin. The eggs were incubated for an additional period of 2 ,6 , or 9 days prior to fixation.

Histological preparation

The embryos were usually fixed in 10% neu- tral formalin, and most were photographed be- fore the limbs were removed for hisotological examination. Microscopic examination of the implant (neural tube and other tissues) was considered essential in this investigation; therefore, the specimens were not stained in toto for the demonstration of the skeletal elements present. The specimens were prepared for histological study by excising a segment of the body that included the portion of upper thorax to which the wings attached (Fig. 3). Each segment, including both limbs, was em- beded in paraffin, sectioned serially along the longitudinal axis of the limbs at 6 or 8 p, and stained with hematoxylin and eosin. Some of the embryos were fixed in formalin and im- pregnated with silver (Cajal) to study the distribution of nerve fibers within the regenerating limb.

tated limb tips are located adjacent to each embryo. Magnification: x 5.

Volume determinations The volume of the humerus of the amputated

and of the unamputated limb of the same embryo was determined from serial histological sections. Both limbs, contained on the same slide, had been processed together, thus min- imizing differential treatment, including tissue shrinkage. Using a MOP 111 Unit (Carl Zeiss, Inc.) attached to a Zeiss microscope, the outlines of both humeri and neural implant of every tenth section were traced at the same magnification. After all the tracings were completed, areas were determined with the same MOP 111 Unit; the total was obtained and cor- rected to absolute mm’ by adjusting according to the magnification used. In order to obtain the total volume of the humeral and the neural implants, all areas were multiplied by 0.077 mm to adjust for the thickness of the sections (0.007 mm) and for the intervening sections that were not traced.

RESULTS

For the purpose of analyzing the results, the normal wing is described as consisting of three segments (Fig. 4), and the bones are named using the terminology of Nickel et al. (’77). The proximal segment containing the humerus is the arm; the middle segment with the radius

52 I . FOWLER AND B.F. SISKEN

Fig. 3. Cross section of trunk with attached wings of a Type 111 embryo. Note that the regenerated right limb (R)

and the unoperated left limb (L) consist of three segments: P (proximal), M (middle), D (distal). Magnification: x 6.

s carpometacarpale I Ulna

MIDDLE SEGMENT

arpometacarpale

0 s carpametacarpale III

OISTAL SEGMENT Fig. 5. Photograph of a 10-day chick embryo ( x 35) that received a neural tube implant. Note the welldifferentiated neural tube Int) containing ependyma (ep), gray and white matter, and nerve roots (nr) emerging from the neural tube.

Fig. 4. Diagram of normal, adult right wing (Nickel et al.. '77).

and ulna as skeletal elements is the forearm; and the distal segment, the anatomical hand, consists of carpals, fused carpometacarpals, and the phalanges of digits I, 11, and 111.

Fifty-eight embryos received an implant of neural tube. In the control group, five received a strip of somites, 5 received a segment of notochord, and 38 had no implant. In the experimental group, the neural tube implant was found in each specimen, although the amount of surviving tissue and state of differentiation

varied. Nerve fibers grew from the implant into the host tissues apparently at random (Fig. 5). The skeletal components of the limbs that received neural tube implants were more variable than those of controls.

Classification of regenerating limbs On the basis of reconstructions of serial sec-

tions, the limbs were classified into three types (Table 1). In 16 of 58 specimens, there was no evidence of stimulation of growth or of differ-


TABLE 1. Responses o f amputated limbs to implants

Treatment of amputated limb Morphological type I' 112 1 1 1 ~ Total

Control 38 0 (no implant) (100%) (0%)

(100%) (0%)

(100%) (0%)

Notochord implanted 5 0

Somites implanted 5 0

Neural tube 16 25 imdanted 127.6%) 143.1%)

0 38

0 5

0 5

17 58

(0%)

(0%)

(0%)

129.3%)

'Type I specimens showed no evidence of stimulation of growth or of regeneration. The proximal segment never exceeded the length of the proximal segment of the unoperated limb of the same embryo. Cartilages of the middle segment, when present, were small fragments. 'Type I 1 specimens had proximal segments containing cartilage in excess of that present in the proximal segments of the unoperated limb of the same embryo. The excess cartilage was present as either: (a) a humerus longer than normal, (b) a bifid humerus, or (c) an apparently duplicated humerus. 3Type I11 specimens had three segments in the amputated limbs. The proximal and middle segments were approximately of normal length. The distal segment contained from one to four cartilages with an average of two cartilages per segment. Although Type 111 specimens varied greatly in their morphology, they are classed as a single type because the limbs include skeletal components that clearly demonstrate three segments (proximal. middle and distal).

' Implant

Fig. 6. Diagram of Type I response following amputation. The proximal segment varied but never exceeded the normal length. The middle segment was absent or contained small remnants of radius and ulna.

entiation of structures not found in controls (Fig. 6) . The proximal segment never exceeded the length of the proximal segment of the unoperated limb of the same embryo. Cartilages of the middle segment, when present, were small fragments. The embryos appeared to be similar to those described by Summerbell ('74) in which he excised the AER from limbs of stage 24 embryos. None of the control embryos without implant or with implants of either somites or notochord showed any evidence of stimulation.

Twenty-five of 58 embryos were classified as Type I1 in which the proximal segment contained cartilage in excess of that present in the proximal segment of the intact limb of the same embryo. The excess cartilage was present as either: (a) a humerus that was longer than normal, usually in the shape of a curved

rod that encircled the neural tube implant (Fig. 7A); (b) an apparently duplicated humerus that remained separate so that the proximal limb segment had two bones (Fig. 7B); or (c) a bifid or partially duplicated humerus that fused at one or more points with the centrally posi- tioned humerus (Fig. 7C). The abnormal shapes of the skeletal elements in these embryos may be due to a mechanical influence of the implant, or to failure of the limb as a whole to keep pace with growth of the cartilage rods, resulting in mechanical forces that cause the rods to curve or fuse with one another. The abnormal growth in length could be due to a failure in the general property of intercalation to achieve continuity. See the polar coordinate model proposed by Bryant et al. ('81). If the new cells produced failed to acquire appropriate positional values, then growth could continue without transformation into appropriate distal structures.

Seventeen of the 58 specimens, in which a segment of neural tube was implanted, showed evidence of regenerative development of distal segment skeletal components (Fig. 8). The proximal and middle segments were approximately of normal length. The distal segment in these embryos contained from one to four cartilages with an average of two cartilages per segment. Figure 8 is a diagram of a reconstruction of the specimen that shows the most nearly complete regeneration that was obtained in this study. The neural tube implant extended from the shoulder region to near the elbow, passing obliquely between the humerus of the proximal limb segment and the portion


Implant

A. PROXIMAL SEGMENT

-., Humerus

{Duplicate of Humerus

B PROXIMAL SEGMENT

\ Bifid Humerus

c. PROXIMAL SEGMENT

Fig. 7. Diagram of Type I1 response following amputation. In (A), only the proximal segment is present. The humerus exceeds the length of the humerus in the unoperated left wing. In (B) the humerus is duplicated. In (C) the appar- ent duplicate fused with the humerus.

PROXIMAL \ SEGMENT

MIDDLE SEGMENT

of distal segment

Fig. 8. Diagram of Type 111 response. The proximal segment is of normal length. The middle segment contains radius and ulna of normal length although the radius is of- ten fused with the humerus. The distal segment contains from two to four skeletal elements.

of the humerus that extended into the middle limb segment. The extension of the humerus into the middle segment may represent fusion of the humerus with the radius and would have

prevented movement at elbow joint. The four cartilages distal to the ulna are in the distal segment and, although they are abnormal morphologically, they must be considered to be hand parts.

Morphology of the regenerating limbs The gross appearance of the specimen which

showed the maximum degree of development is illustrated in Figure 9A. The partially regenerated wing in this embryo is slender but consists of three segments and approaches normal length in comparison with the unoperated left wing of the same embryo (See Fig. 3). The specimen (Fig. 9A) may be compared with the amputated control limb shown in Figure 9B whose only segment (proximal) is the same length as the proximal segment of the unoperated left wing.

Figure 9C is a photomicrograph of the operated right wing of the specimen depicted in Figure 9A. The implant, labeled I in the illus- tration, consists of neural tube, notochord, and vertebral cartilages located near the distal end of the humerus of the proximal wing segment. The cartilage in the middle segment is the same length as the ulna of the left unoperated wing of the same embryo. An additional cartilage rod that may represent the radius which was not included in the section extended into the proximal segment where it fused with the humerus. See Figure 8 for a diagram of a reconstruction of the wing of this embryo. The distal segment of the section, labeled H in Figure 9C, contains three small cartilage profiles that are in the distal segment and presumably represent hand elements. Reconstruction of the distal segment (Fig. 8), revealed one relatively long and three short cartilages. The presence of skeletal elements in middle and distal segments contrasts sharply with the absence of such elements in the controls. An example of a typical control specimen in which the humerus is present in the proximal segment as the only skeletal element of the amputated wing is shown in Figure 9D. In some of the control embryos, two small cartilages that presumably represent proximal portions of the radius and ulna were present.

Morphology of the neural tube graft The position of the neural tube implant with-

in the regenerating limb varied from the super- ficial location where it protruded through the skin to a deeper position closely related to the humerus. In no case did the implant extend distal to the elbow, although nerve fibers origi-

A

LIMB REGENERATION IN THE CHICK EMBRYO

B

55

D Fig. 9. Photographs of IOday embryos illustrating re-

generating limbs. (A) Amputated limb received neural tube implant. x 3. (B) Amputated limb control, x 3. (C) Photomi- crograph of histological section of limb shown in (A), x 10. Note the longitudinal extent of the regenerated ulna in the middle segment and the new joint between the middle seg ment and the distal segment. This picture demonstrates the

presence of new muscle and feathers as well as bone and cartilage. (D) Photomicrograph of histological section of control limb shown in (B), x 10. (1,2,3) indicate proximal middle, distal limb segments. (I) refers to implant, (nj) to new joint, (H) for hand parts, (M) for skeletal muscle. The arrows indicate the approximate level of amputation.

C

nating in the implant extended into the middle segment. Nerve fibers grew from the neural tube apparently at random (Fig. 5). Silver preparations (Fig. 10) demonstrated nerve fibers in limb muscles. However, it could not be determined whether all of these fibers originated from donor neural tube.

Volume of the humerus and neural tissue

In Table 2 the volume of the humerus in unamputated and amputated limbs as well as the volume of the neural tissue implant are presented for four control and ten experimental embryos. In the control group, the volume of


Fig. 10. Photographs of neural tissue implants. Impreg- nated with silver, Cajal method. (A) Note the two donor sen- sory neurons (DSN), x 250. (B) Note the donor neural tube (DNT) from which nerve fibers (NF) extend into the host limb tissues, x 200.

the humerus of the amputated limb was less than that of the same embryo’s unamputated limb, ranging in volume from 0 (complete absence) to 76.8% of the humerus of the unamputated limb. This may be compared with the results observed when the amputated limbs received neural tissue implants. In these specimens, the volume of the humerus ranges from 17.5% in a Type I specimen to 192% in a Type I11 specimen when compared with the volume of the humerus of the unamputated limb of the same embryo. In the limited data available, there is a good correlation of the amount of neural tissue present with the morphological type of the amputated limb. For instance, in an embryo containing a neural implant of 0.188mm3, the limb was Type I and its proximal segment was much smaller than that of its unoperated counterpart. Further, the data presented in Table 2 suggests that a neural implant of 0.2mm3 or greater stimulated growth of the amputated limbs yielding Type I1 or I11 embryos. Larger amounts of neural tissue in- creased the chance of regenerating middle and distal limb segments (Type 111). The mean volume of neural tissue in Type I11 specimens (0.912) exceeded by a factor of four the mean volume of Type I1 embryos (0.215).

DISCUSSION

In the present study, implantation of a segment of neural tube in the stump of amputated

wingbuds stimulated varying degrees of mesodermal growth and differentiation in 72% of the specimens (Types I1 and 111). In the remaining 28% (Type I), there was no significant stimulation. Volume measurements of the neural tissue implants suggested that the failure to stimulate growth in Type I embryos was due to failure of an adequate mass of the neural tube implant to survive. The available data indicate that a “critical mass” of neural tissue is necessary in order to have an observable effect upon the growth of limb tissues.

In the embryos in which there was a quantitative effect upon limb skeletal components, do the effects represent stimulation of growth of structures already determined in the limb buds of stage 24 embryos or have components not previously determined been stimulated to differentiate? In the embryos with an elon- gated humerus, as shown in Figure 7A, there appears to have been excessive growth of the humerus that was already present; this should not be considered regeneration. Similarly, the anlaga of the humerus in the embryos that have a bifid or duplicated humerus, as illustrated in Figure 7B,C, could have been split during the process of implanting the neural tube. Stimulation of growth of the divided primordia could then have produced duplicated humeri that were almost normal in size.

In our experiments, there was no evidence of the formation of the AER following the operation. Skin epithelium closed the wound within two days after excision of the apical zone, in a process similar to that described by Scadding (’81) for some salamanders, but did not appear morphologically to organize as an AER. Janners and Searles (’71) have shown that following excision of the AER, extensive cell death spreading proximally from the distal tip shortly after excision contributes to the reduc- tion in length of the limb. In our experiments, presence of the neural tube implant appears not only to have prevented the waves of cell death following excision of the AER, but actu- ally led to excessive growth of skeletal tissue within the wing. With the removal of the apical zone which includes the progress zone of Sum- merbell et al. (’73), the development of the sequential proximodistal axis of the limb pattern ceases at the stage of the operation. As described by Tank and Holder (’81), cells that are proximal to the progress zone have their positional values fixed and differentiate into proximal structures. In embryos of Type I1 in our experiments, i t appears that the influence of the neural tube implant was inadequate to alter the positional values of the cells of the


TABLE 2. Influence of the volume of neural implant upon the volume of the humerus in the amputated wing

Volume of Volume of % of Volume of Experimental unamputated amputated unamputated neural tissue

condition Age Type humerus (mm’)’ humerus (mm’) humerus bm’)

Control (no implant) 10 days I 0.92 0 0 10 days I 1.47 0.69 46.8% 10 days I 3.45 2.65 76.8% 13 days I 5.20 2.12 40.8%

Experimental (Neural Implant) 10 days I

10 days I1 10 days I1 10 days 11 10 days I1 13 days I1 10 days 111 10 days 111 13 days 111 13 days 111

3.18 1.58 2.06 2.17 0.803 3.35 0.766 1.65 3.15 2.83

0.55 1.72 2.06 1.25 0.982 3.18 0.482 1.132 6.05 3.18

17.5% 0.188 108.8% 0.067 100.0% 0.127 57.6% 0.186

115.5% 0.451 94.9% 0.246 62.9% 0.672 68.6% 1.25

192.0% 0.828 112.5% 0.90

‘Volumes were calculated from tracings of histological sections using a MOP 111 unit (Zeiss). The percentage of unamputated humerus was determined by dividing the volume of the amputated humerus by the volume of the unamputated humerus and multiplying by 100.

wing stump. Thus, only a single or duplicated skeletal component resembling a humerus was produced. Continuity of cells with appropriate positional values (Bryant et al., ‘81) was not achieved; excessive growth of the humerus was the result.

In the limbs classified as Type 111, it is clear that skeletal elements of the distal limb segment developed that were never observed in control embryos. Both the gross and the microscopic morphology of these specimens indicate that these skeletal components are new parts and are not mere extensions of previously determined limb parts. Although distal limb seg ment components have not been determined in stage 24 wingbuds (Summerbell, ’74), excision of the apical zone removes cells that would normally differentiate into the distal limb seg ment. We believe the development of these components represents true regeneration and that the stimulus for initiation of the regeneration is the neural tube implant. Notochordal and somitic tissues implanted with the neural tube do not appear to be effective in initiating regeneration.

The distal limb segments of embryos of Type I11 contained all the tissues found in the distal segments of limbs of normal chick embryos but were somewhat abnormal morphologically. This suggests that the neural tube implant was capable of influencing growth and differentiation of cells in amputated chick limbs but had little effect upon pattern formation. As a result, cells divided and differentiated without the sequential proximodistal and cir-

cumferential organization normally dictated by the AER.

Role of nerve supply in limb regeneration The capacity to regenerate complex struc-

tures such as limbs, in which formation of a blastema is a prerequisite for replacement of lost parts, occurs readily in some adult tailed amphibians (urodeles) but can occur only in lar- val stages in most amphibians without tails (anurans). In higher vertebrates such as the chick, limb regeneration never occurs even in the very young, unless stimulated by extrinsic factors. Limb regeneration in all animals and at all ages requires the presence of nerve fibers at the amputation surface (Singer, ’52). Al- though the specific target of the neural influence has not been identified, target cells seem to be stimulated to proliferate until a “critical mass” is obtained; subsequent cellular interactions within the mass and with adjacent tissues then regulate the differentiation process (Singer, ’78). The nature of the role of neural tissue is not known, but it may consist of a release of a substance at ends of nerve fibers similar to that which occurs normally at the chemical synapse (Rzehak and Singer, ’66). The neural agent may be a peptide of low molecular weight similar in action to nerve growth factor, which “alters the rate of ongoing events” but does not seem to change the qualitative nature of events (Singer, ’78). Evidence from experiments on the lizard suggest that the effective agent may be produced by the ependyma (Simpson, ’61). Our experiments do not contrib-


Ute to the resolution of this question since ependyma was present in the implants along with nerve fibers. The stimulation of limb outgrowth by nerve growth factor applied to young rat limbs has been reported (Sisken et al., '79) and suggests that nerve growth factor may act by increasing the neural elements in the amputated limbs.

A critical mass of nervous tissue may be required for a neurotropic effect to be detectable. In forms that do not regenerate (frog, lizard, mouse), the amputated limb contains only about one-sixth the nerve mass per area of amputation wound of that found in the newt which regenerates readily (Singer, '54). In our study, some regeneration of the limb occurred in most embryos with a neural tissue implant of approximately 0.672mm3 (see Table 2) and was usually unsuccessful with 0.45mm3 or less of neural tissue. However, there is not enough data to estimate precisely the critical mass of neural tissue required to induce complete regeneration.

The relative position of the neural tissue within the amputated limb is probably an im- portant factor in the absolute volume required to initiate the regeneration process. Its location in or near special limb bud tissue, such as the zone of polarizing activity (Saunders and Gasseling, '68; Balcuns et al., '70; MacCabe et al., '72) or the progress zone of Summerbell et al. ('73) and Summerbell ('74), may be essential if the neural tissue is to influence growth and form of the limb. Summerbell and Tickle ('77) has presented evidence that the presumptive fate of chick limb bud cells is not fixed and that embryonic regulation can occur. If our Type I11 specimens were the result of the process of embryonic regulation, it would be expected that the proximal and middle segments would be considerably reduced in size in comparison with the normal limb. Since the humerus of the amputated limb was approximately equal to that of the unoperated side, it seems likely that cells were not recruited from the humerus to contribute to the development of the more distal skeletal components of the regenerating limb.

It seems possible that the neural tissue implant affects the course of development of the amputated limb by altering the positional values, as described by Tickle et al. ('75), of limb mesoderm of the amputated stump. Failure of the distal segments to develop normally suggests that any influence upon positional information was less than that required for deter- mination of size and form. The neural tissue

implant may thus be a partial substitute for the apical ectodermal ridge. The implant seems to be able to replace AER function in regu- lating growth and differentiation more com- pletely than that of dictating positional values. Further work will be necessary to determine the role of neural tissue in providing positional information.

Limb regeneration has been induced in young adult anurans by rerouting the sciatic nerve from the hindlimb to augment the nerve supply of the forelimb (Singer, '54). Similar experiments in the rat failed to yield any evidence of induction of regeneration (Bar-Maor and Gitlin, '61). However, experiments in the opossum, a non-regenerating mammal, have clearly demonstrated that limbs of young mammals can respond to the presence of nervous tissue in the regenerative process (Mizell, '68). Implanted homologous or heterologous (frog) brain tissue in hind limbs of newborn opossums prior to amputation of the limbs induced new growth, including elements of the foot and digits. In the study of limb regeneration in young animals, care must be taken to extirpate primordia of the distal limb parts. Otherwise, regeneration that occurs may be due to reorganization of remaining undifferen- tiated material instead of true replacement of lost parts. Since Mizell claimed that cartilage models of bones of the foot and digits were excised in the opossum, the interpretation that reformation of foot parts and digits represents true regeneration seems justified. Recently Fleming and Tassava ('81) have expressed doubt that Mizell removed primordia of foot parts and digits. However, they did not transplant neural tissue to the amputated limbs. Without augmentation of nervous tissue, Mizell failed to obtain limb regeneration in the opossum. The hindlimbs of newborn opossums correspond to human embryonic limbs at 8 weeks of gestation. Our experiments on chick wingbuds were performed on embryos of stage 24, which correspond to limb buds of the human of 4 weeks of development.

In all our cases, muscle and other soft tissues were associated with the cartilages of the middle and distal wing segments. However, these tissues were less voluminous than those in the contralateral unoperated limb. The growth and differentiation of skeletal components of both the middle and distal segments is interpreted as true replacement of lost parts and not just stimulation of growth of remaining cells. Since new parts regenerated in more than one-fourth of the cases, it may be con-


eluded that augmentation of nerve supply is an effective method of inducing partial regeneration in limbs of chick embryos.

ACKNOWLEDGMENTS

Supported in part by Veterans Administra- tion Research Program. We are indebted to Ms. M. J. Gumbert and Mr. J. Starrs for the histological preparations and to Ms. Elsie Barr for the camera lucida tracings and volume measurements.

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Amprino, R., and M. Camosso (1958) Analisi sperimentale dello sviluppo dell'ala nell'embrione di pollo. Roux Arch. Entwicklungsmech., 150:509-541.

Balcuns, A,, M.T. Gasseling, and J.W. Saunders, Jr. (1970) Spatio-temporal distribution of a zone that controls antero posterior polarity in the limb bud of the chick and other bird embryos. Amer. Zool., 10:323.

Bar-Maor, J.A.. and G. Gitlin (1961) Attempted induction of forelimb regeneration by augmentation of nerve supply in young rats. Transplant. Bull.. 27t460-461.

Bryant, S.V., V. French, and P.J. Bryant (1981) Distal Re- generation and Symmetry. Science, 212993-1002.

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effect of augmentation of nerve supply upon limb regeneration in the chick embryo

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