ABSTRACT
Autotransplants of pigmented (melanophore-bearing) skin on a white region, and of white skin on a pigmented region both exhibit a strict specificity.
There is no migration of the melanophores in any case of transplantation.
Transplants of pigmented skin lose their colour-changing power immediately on being completely disconnected from neighbouring tissues, and regain it slowly as nerves regenerate.
The melanophores are supplied largely by nerves which reach them directly through the underlying tissues. If these nerves are cut from a given area of skin, without separating the area from the surrounding skin, it at first loses the power of colour change, but the neighbouring integument appears able, after a delay of a few days, to control the denervated area.
Areas of melanophore-bearing skin, isolated by incision from surrounding skin, but with their subcutaneous connections intact, can act normally with the rest of the animal in the colour-changes.
Adrenalin, in producing the green state in pigmented grafts which have regained the colour-changing function, acts as it does upon normal integument. The action is indirectly through the sympathetic nervous system, and not directly upon the melanophores.
Homoiotransplants, either pigmented or white, are absorbed. The absorption occurs after the grafts have well healed in place. The scutes disappear, leaving the area covered by thin scaleless integument.
New scutes regenerate wherever there are areas barren of them. Scute regeneration progresses centripetally, suggesting some formative influence exerted by the surrounding normal integument.
1. Introduction
The large amount of work on skin grafts in vertebrates has concerned itself for the most part with either amphibians or mammals. I have been able to find only one recorded case of skin grafting in reptiles. It is that of Winkler (1910) on Lacerta. However, his account is not very clear concerning this lizard. He simply states that he used skin from the throat region for the graft, and does not give the subsequent history of the transplant, but includes whatever results he obtained along with those reported for amphibians.
W. H. Cole (1922) has recently summarised the evidence concerning the specificity of skin transplants and the migration of melanophores in transplanted skin, including both autotransplants and homoiotransplants. From his own observations on frog tadpoles, supplemented by the earlier report of Schöne (1912), he has come to the conclusion that in frog tadpoles, mice, and human beings autotransplants show a local specificity of integument. He says (p. 406) that in frog tadpoles the integument is “locally specific, and is self-differentiating when transplanted to new soil on the same animal.”
Nevertheless, he reports that in frog tadpoles white belly skin grafted on to the back, whether in autotransplants or in homoiotransplants, eventually acquires melanophores. This agrees with the results obtained by Winkler (1910) on frogs and tadpoles, and with those of Dawson (1920) in autotransplants on Necturus maculosus. With guinea-pigs a similar pigmentation of white grafts on a black region was reported by Loeb (1897) and by Seelig (1913).
Evidence concerning the reactions of pigmented grafts on a white region is not so clear. Dawson (1920) reported that an autotransplant on Necturus maculosus, after several weeks, became lighter at its centre, the area of lightening extending centrifugally. The light appearance was due to the absence of the usual dermal chromatophores, as the epidermal chromatophores were still present. Carnot et Deflandre (1896) noted that in both autotransplants and homoiotransplants on guinea-pigs the graft persists and the pigment invades the surrounding white skin when the graft is made on animals which have some pigmented areas on their coats, but it disappears if skin from a pigmented animal is grafted on to albinos. Loeb (1897) also reported the spreading of pigment from the black graft to the surrounding white skin of guinea-pigs, while Sale (1913), repeating Loeb’s experiments, found that this occurred only in autotransplants ; homoiotransplants became gradually lighter in colour.
To determine the reactions of highly specialised chromatophores to transplantation, I chose the so-called Florida or American chameleon, Anolis carolinensis, Cuv. This lizard offers several points of great value in this connection. As is well known, its skin can go through a fairly wide range of colour phases, the most common and conspicuous of which are an emerald green and a mahogany brown. These changes were first described by Lockwood (1876), and the histology of the skin was worked out by Carlton (1903), but especially well in the masterful paper of von Geldern (1921). Anolis, in its pigmentation, differs greatly from those forms on which skin grafting had been done previously. Loeb and Strong (1904) have pointed out that in the frog skin there are two layers of chromatophores, one dermal, the other epidermal, while in the guinea-pig the cutis has no well-developed chromatophores, and its pigment is distributed irregularly in layers or clumps. In Anolis, on the other hand, there are no epidermal chromatophores, and the dermal melanophores are highly specialised. Instead of branching irregularly in several directions, as is usually the case, they have dendritic processes which are always directed toward the epidermis. Whatever the secondary factors may be in the colour changes, both Carlton (1903) and von Geldern (1921) have conclusively shown that the main change is a migration of the pigment granules toward the epidermis to produce the brown state, while a cellulipetal movement of the pigment granules produces the green state.
While working on the main problem, several collateral questions arose, and I have incorporated the work which I put on them into the present paper.
I take great pleasure in acknowledging here my debt of gratitude to Professor H. W. Rand, at whose suggestion the work was begun, and under whose inspiring guidance it was carried on.
2. Material and Methods
Animals were procured from a dealer in New Orleans. They were kept in a thermostat chamber at a temperature of 23·5° C. At this temperature the animals thrive if the air in the chamber is kept humid. This is best accomplished by keeping a wet sponge in the vivarium. The sponge is used also as a drinking supply, since the lizards take water only in the form of droplets. They were given insects for food.
Anæsthetisation was found to cause so many fatalities that it was discarded. In operating, all antiseptic precautions must be taken, as the lizards are very liable to infection. The areas of skin which were to be cut out were first marked with india ink, so that the graft would fit as closely as possible into the area which was to receive it. Pieces of skin 0.3 to 0.8 centimetre square were used, and reciprocal grafts were usually made, whether autoplastic or homoioplastic. The best results were followed when the edges of the skin to be grafted were applied closely to the edges of the skin at the place of reception. This was sometimes difficult. The skin of Anolis is connected with the underlying muscles only by a very loose layer of connective tissue, so that when cut there is little resistance to its tendency to shrink, and the area for reception of the graft becomes often much larger than desired. After applying the smoothed-out graft, a very little celloidin was placed on the edges, both to hold the graft and to prevent infection. The operative shock caused the lizards to be very quiet for several hours. This lapse of time was enough to allow the celloidin to harden completely, and for a vascular supply to be established for the grafts.
Lizards of various sizes, but apparently all adult, were used for the experiments. I was successful in thirty-one cases of grafting. Of these, twenty-one were autotransplants and ten were homoiotransplants.
3. Autotransplants
a. Pigmented Grafts on a White Region
In Anolis, as in many reptiles, the under side of the body contains very few melanophores, so that the venter and throat are whitish. Some individuals do show a very slight change in shade of the under side when the animal changes colour. This shading appears as an extremely light green when the lizard is green, and a very light brown when it is in the brown state. In most individuals, however, this does not occur, so that the venter and throat are whitish when the animal is in either the brown or the green state. In the following descriptions, I shall use the term “pigmented skin” to mean that portion of integument containing numerous melanophores.
In the first series of experiments pigmented skin from the back or sides of the body was grafted on to the belly or throat. The results are the same in all cases.
When the patch to be grafted is cut from the surrounding skin and is pulled up from the subcutaneous connective tissue it becomes immediately either green or brownish green, if the lizard when operated on is in the brown state. If, however, the lizard is in the green phase during the operation, then the patch remains green. The brownish - green colour of the isolated skin, when it occurs, is only transitory. Ten to fifteen minutes after the patch is attached to its place of reception, it is always bright emerald green. Furthermore it has completely lost the power to change from green to brown. The rest of the integument retains the capacity of colour change, so that in the brown state the graft stands out in strong contrast on the white belly or throat as a green area (fig. 1), while the back and sides of the lizard are brown.
When the graft “takes,” a vascular supply for it is established within a few hours. A graft for which a blood supply is not established becomes wrinkled and soon dies. The fact that such a supply exists in the case of successful transplants can be easily ascertained by pulling up one of their corners. The area under it is richly bathed in blood. Nevertheless the graft remains green for days, and is unaffected by the changes in colour of the animal as a whole. In several cases some of the scutes of the graft, or even a good portion of it, acquired a straw yellow colour after two or three days. This colour, however, did not change when the animal as a whole changed, but seemed to be static, like the remaining green colour of the transplant.
I examined my grafted animals daily to determine the first appearance of the brown state in the grafted skin. This comes on very gradually, and its mode of appearance varies somewhat in different lizards. Usually beginning twenty or twenty-one days after the operation, a few of the scutes—as a rule, two or three at one edge of the graft—are found to be brown when the animal as a whole is brown, and synchronously become green when it becomes green. In the following days more of the scutes acquire this capacity. The area within the graft reacquiring the power of colour change grows very irregularly and with no definite pattern. Twenty-four to thirty-three days after the operation the entire graft has fully acquired the power to change from green to brown, and vice versa, with all the intermediate colours, and it does so synchronously with the animal as a whole.
After the reacquisition of the capacity for colour change in the graft, there is no further change. The pigmented skin transplanted to a region with no melanophores now behaves in exactly the same way that it did when it was in its normal place on the back or side of the lizard.
The specificity of the integument is complete. There is no invasion of the surrounding white skin by the melanophores, and the line of demarcation between pigmented and white areas of the integument remains perfectly definite. Lizards which were observed for seven months showed at the end of that time as sharp a line of distinction between the graft and the surrounding skin as on the day of the operation (figs. 3, 5).
As von Geldern (1921) has noted, different areas of the integument have scutes of different shapes and sizes. Without going into the details of these differences, for which the reader is referred to von Geldern’s paper, it can be stated that there has never been the slightest change in the appearance of back skin when transplanted to an area on the belly or throat. The scutes of transplanted back or side skin can easily be distinguished from the surrounding larger and more closely arranged scutes of the venter, or from those of the throat skin where there are wide spaces between the scutes.
To ascertain whether the change of position of the skin on the animal had anything to do with the behaviour which it subsequently underwent, pieces of skin were cut out and replaced on the same area from which they had been excised. The resulting changes in colour behaviour were exactly of the same nature as when integument was transplanted to another area of the body.
There is, then, no influence of the surrounding skin upon the graft. It shows exactly the same changes whether it is surrounded by pigmented or by white skin.
b. Experiments on the Nervous Control of the Melanophores
Carlton (1903) injected solutions of nicotine into Anolis to poison the sympathetic ganglia. A result was the change of brown animals to green. Isolated pieces of skin in which the brown state had been induced by mechanical stimulation were permeated with a nicotine solution, but did not become green. He showed that the spinal nerves were not concerned with the colour changes, since these took place after pithing the spinal cord. He concluded that the effect of the nicotine was not on the melanophores directly, but through the sympathetic nervous system. Since poisoning the sympathetic ganglia with nicotine produced the green state, he postulated that the brown state is ordinarily maintained through a tonus established by the sympathetic nerves. Fuchs (1914, p. 1648) has questioned this result, claiming that it is not proven that nicotine does not have a direct effect on the chromatophores or on their nerve supply, irrespective of the ganglia.
The present experiments afford added evidence in support of Carlton’s contention. By grafting a piece of skin, we destroy all its nerve connections and thereby abolish the function of the nerve endings. It is true that so far there has been no anatomical proof of the existence of nerve endings in relation to the melanophores of Anolis. Von Geldern failed to get them by the chloride of gold method ; I failed likewise, using the methods of Golgi and of Bielschowsky. But that nerve endings, such as Ballowitz (1893) and Eberth und Bunge (1895) demonstrated for the chromatophores of fishes, exist likewise in the melanophores of Anolis, seems almost certain. The sudden change of excised skin to the green state, and the fact that a vascular supply fails to bring back the colour-changing reaction, are indications of a nervous control. But the best evidence for it is in the gradual regaining of full function by the melanophores, a change which can be correlated only with the slow regeneration of nerves.
Hooker (1912a) found that a three and a half months’ old regenerated tail in Lacerta agilis was innervated by two pairs of nerves which had grown out from the stump of the tail. These nerves, which normally innervated about 2 mm. of the tail, now took care of the whole 30 mm. of regenerated tip. Whether, in the case of skin grafts, the new innervation of the melanophores is a result of an extension of the nerves from the skin surrounding the graft, or whether the new innervation is by nerves growing up through the subcutaneous connective tissue is, of course, not proven. It is likely, however, that as in Lacerta, it is the nerves from the immediate vicinity which extend into the new region. In the case of a pigmented patch on the venter, we have the curious example of nerves which had supplied a region where almost no melanophores were present, growing into skin with numerous melanophores, and assuming then the colour-changing function which up to that time they had exercised either not at all, or at most only in very small degree.
In order to determine whether in Anolis the melanophores are supplied by nerves going vertically up to them through the subcutaneous tissue, or whether the innervation is by a plexus which joins all portions of the integument, two types of experiment were performed. In the first a sharp scalpel was inserted under the dermis in a pigmented area, and a small area of integument was wholly separated from the underlying tissues, thus severing any nerves which came to the skin from that direction. However, excepting the incision made by the insertion of the scalpel, there was no separation from the surrounding skin. The animals were operated on while in the brown state. The results of this experiment showed some variation,, but in general the cases were similar. Usually the separated skin immediately became green, but in some cases it remained brown for several hours. After a day, in both instances, the operated area was composed of irregular blotches of green and yellow, with usually some brown scutes. The green and yellow spaces, as time went on, became more and more restricted, so that after four or five days the operated area was totally brown when the lizard was in the brown state.
The other experiment consisted in leaving the area of skin attached to the subcutaneous tissue, but cutting it off from the surrounding skin. In this case, the severing of relations with the surrounding integument seemed to have little effect on the melanophore control. The cut area was brown when the animal was in that state, and changed colour synchronously with the rest of the skin. The only change that could be noticed was that sometimes there appeared patches on the edges of the area somewhat lighter brown than the remaining integument.
If the two operations were combined, so that three or four days after the separation of an area of skin from the underlying connective tissue, this same area was also isolated from the surrounding skin, it immediately turned green and remained so. The further changes, with the reacquisition of the colour-changing capacity, were the same as those noted for transplants of pigmented skin. The reapparition of the brown colour came on in the same irregular and slow fashion.
We see, then, that three conditions were observed. When a portion of the integument was separated from the subcutaneous tissue alone it became green, then blotched, and regained the capacity of colour change within four or five days. When an area of skin was separated from the surrounding integument, but not from the subcutaneous layer, no important change occurred ; the area showed normal colour behaviour. When finally an area which had been separated from the subcutaneous layer, and had regained the power of colour change, was subsequently cut off from the surrounding skin, it became green and reacquired completely normal behaviour only twenty-four to thirty-three days later.
What the factors are which operate in these conditions I am unable to say with certainty. It seems, however, that the surrounding skin can exert some influence in bringing about the normal state in an area lacking most of its innervation. This is shown by the fact that such an area which had regained the capacity of colour change loses it when separated from the neighbouring integument.
From these experiments, I feel that we are justified in concluding that—
The normal innervation of the melanophores is largely from nerves which reach them directly from beneath and not through a plexus in the skin.
Any melanophore-bearing portion of the skin isolated, by incision, from surrounding skin but with its subcutaneous layer intact, can act normally with the rest of the integument in its colour changes.
An area whose direct (i.e., subcutaneous) innervation has been cut off regains, after a delay of three to five days, control of its melanophores, apparently by acquiring some relation with the surrounding skin.
c. The Effect of Adrenalin on Grafts
Redfield (1918) has stated that injections of adrenalin brought about the green state of the skin in Anolis carolinensis. It seemed to me of interest to determine whether the effect would be the same on grafts which had fully regained their colour-changing power. To test this question I injected 0.1 c.c. of a solution made up of one part of adrenalin chloride in either 1000, or else 100,000, of Ringer’s solution, subcutaneously into lizards which were in the brown state. One and a half to two minutes after the injection the animal as a whole and the graft had both turned green. There is then no difference as regards the reaction to adrenalin between normal skin and grafts.
That the effect of adrenalin in the case of the melanophores of Anolis is not directly upon them, but indirectly through the sympathetic nervous system was well shown. Skin was cut out, placed on filter paper bathed with Ringer’s solution, and mechanically stimulated so as to bring it into the brown state, as Carlton had done. If now a solution made up of adrenalin, one part in either 1000 or 100,000 of Ringer’s solution, is added, so that the brown skin is bathed in it, there is no change in the coloration, even after an immersion of one hour’s duration. The state induced by mechanical stimulation persists so that the skin remains brown, or at most shows only such minor changes in its colour as are also shown by controls bathed merely in Ringer’s solution.
Two lizards upon which pigmented skin had been transplanted several months previously had their spinal cord pithed. They were allowed to recover from the operative shock for two hours; the graft as well as the rest of the pigmented skin became brown. Adrenalin was then injected, and the usual change to green was shown by both the graft and the melanophore-bearing integument.
The effect of adrenalin in causing the proximal migration of the pigment granules in the melanophores, in pigmented grafts as well as upon normal skin, is shown as a result of these experiments to take place indirectly through the sympathetic nervous system. These results agree with those of Carnot (1897) on the frog. He notes that section of the spinal cord did not inhibit the colour changes of the skin in the paraplegic portion of the animal. Isolated portions of the skin failed to show their normal colour changes when treated with drugs. Thus, detached light skin failed to darken when bathed in amyl nitrite, and dark skin failed to lighten when treated with aniline hydrochloride. In the case of skin normally attached to the animal, however, amyl nitrite causes a centrifugal movement of the pigment granules in light skin, while aniline hydrochloride causes a centripetal movement of the granules in dark skin. These drugs, as in the case of adrenalin in Anolis, can produce their effect on the melanophores of the frog only through the action of intact sympathetic nerves.
d. White Grafts on a Pigmented Region
Skin taken from the white venter or throat and grafted on to the pigmented sides or back of an Anolis shows a strict specificity. Lizards observed for six and a half months did not show during that time the slightest evidence of blending of the grafts with the surrounding skin. The line of demarcation between the white graft and the surrounding skin was absolute (figs. 2, 4, 6). There was not the slightest indication of migration of melanophores into the white graft, and the latter was in no way influenced by the surrounding skin. The highly specialised melanophores of Anolis have apparently lost the power of migration which is possessed by the chromatophores of amphibians and at least some mammals. As in the case of pigmented grafts, the white transplants showed a strict specificity as regards the region from which they were taken. Belly skin retained the characteristic larger scutes, while throat skin could easily be recognised by the wide intervals between the scutes, and by its loose connection with the subcutaneous tissues. The other characteristics of the scutes from the different regions of the lizard’s body, such as shape and the presence or absence of a central keel, were also retained unchanged.
It appears, therefore, that both pigmented and white skin, when transplanted autoplastically to regions of the opposite kind, retain their own characters. Lacerta likewise (Winkler, 1910) shows a specific character of different portions of the integument. Furthermore in Anolis the melanophores have no power of movement independently of the nervous system, such as Hooker (1912b) found in the melanophores of the corium of Rana fusca, nor is there, in the lizard, any evidence of migration of any kind.
4. Homoiotransplants
a. Transplants
With one notable exception, all homoiotransplants were absorbed, although they usually “took” at first. The following data, taken from my notes, on a white graft transferred to the back of another Anolis, illustrate the usual run of events.
The interval of ninety days here reported between the grafting operation and the total absorption of the scutes is somewhat longer than usual. The interval in the other cases was from sixty-one to seventy-seven days. Pigmented skin transferred to the venter of another lizard was absorbed in the same way as white skin, and in about the same time.
In only one case of homoioplastic grafting did I get results unlike those described above. One lizard which had white skin from another Anolis grafted on to its back, and skin from the other animal’s back grafted on to its belly was observed for four months. During that time there was not the least indication of absorption. The grafts healed well. The white graft did not differ from an autoplastic graft of the same character. The pigmented graft, however, never reassumed complete colour change. Two and a half months after the transplantation irregular areas of the graft, or even the greatest portion of it, had assumed a light brown colour quite distinct from the deeper brown of the animal as a whole. These light brown areas showed synchronous change to green with the rest of the pigmented integument. They were not, however, constant in position on the graft, but changed from day to day, or even temporarily disappeared from it. The graft would then appear green, mottled with straw yellow, in the brown state of the animal. This graft thus showed striking differences from other pigmented transplants, both autoplastic and homoioplastic.
The Anolis from which the grafts were taken in this case was of exactly the same size as the recipient. Both came from New Orleans in the same lot. There is, then, the possibility that I was, in this instance, dealing with syngenesio-transplants. This would perhaps explain why the behaviour of the graft was different from that of both autotransplants and homoiotransplants.
In general, my homoioplastic cases showed, as before noted, complete destruction of the grafted skin after an interval of from sixty to ninety days. These results differ markedly from those of Winkler (1910) on Lacerta and amphibians. He says that “Es spielt dabei keine Rolle, ob die zur Transplantation verwendeten Hautstückchen von demselben Tiere stammen, oder ob die Haut eines andern Tieres benutzt wird.”
b. The Growth of Scales
As has been noted, absorbed homoiotransplants were replaced by a grey membrane. When autotransplants were accidentally thrown off, a grey membrane, presumably new integument, likewise was seen covering the surface. These membranes showed ecdysis along with the rest of the integument.
The somewhat similar phenomenon of scute regeneration occurring when the lizard breaks its tail and regenerates a new one led me to compare the growth of scutes in both situations. In the case of regenerated tail tips, the first outgrowth is a blackish cone, covered by thin, smooth skin. When this undergoes ecdysis, the scutes are found completely formed underneath, and very regular in shape and size.
The mode of regeneration of scutes on the back or belly differs from that on the tail. Wherever scuteless integument has replaced a graft, whether after absorption of homoiotransplants, or to cover open wounds, after two or two and a half months there can be noticed on the edges of the regenerated integument a formation of new scutes. These, as fig. 7 shows, are very irregular in shape and size, some being larger than normal scutes, while others are much smaller. When surrounded by pigmented skin the regenerated scutes are copper-coloured, and show no change of colour when the animal changes. In white areas they are white. In both cases we find that at successively later stages of scute regeneration they are found nearer the centre. After five months they have completely invaded the area formerly denuded of scales. They are then pressed close together, having lost the interspaces which characterised them at first. The white scales blend in very well with the surrounding skin, but the copper-coloured regenerated scutes never have, in my experience, acquired the colour-changing capacity.
We see, then, that the regenerated scutes differ in pigmentation according to whether they appear near melanophore-bearing or white integument. The fact that these scutes always appear first at the edges of the old skin seems to justify the belief that the neighbouring old skin has a direct influence in their regeneration.
Acknowledgements
The photographs for this paper were kindly taken for me by Mr E. C. Cole, and the coloured plates were made by Mr E. N. Fischer.