VOL. XIV, No. i JANUARY, 1937

STUDIES ON AMPHIBIAN METAMORPHOSIS

XV. DIRECT TYMPANIC MEMBRANE FORMATION FROMDERMAL PLICAE INTEGUMENT TRANSPLANTED

TO THE EAR REGION

BY O. M. HELFF

Department of Biology, University College, New York University

(Received 19 April 1936)

(With Two Plates)

INTRODUCTION

THE dermal plicae folds or ridges of the metamorphosed anuran constitute strikingintegumentary structures. In most anurans two sets of dermal plicae are present,viz. a dorso-lateral set which run posterior from the dorso-posterior border of eacheye to a point just lateral to the urostyle prominence, and a lateral pair originatingnear the external nares of either side and thence running posterior along the upperjaw ventral to the tympanic membrane to a point just dorsal to the base of the forelimb. The folds, as the term indicates, appear externally as definite elevated ridgesof which the dorsal-lateral pair are usually the more prominent. They are moreoften dull white in appearance although frequently of a somewhat greyish tinge, thelatter condition being more typical of the lateral than of the dorso-lateral pair.Histologically, the folds are seen to be due chiefly to much enlarged mucous andpoison glands, usually closely approximating one another. This necessarily requiresa corresponding thickening of the stratum spongiosum of the integument, in whichlayer the glands are located. In addition, the stratum compactum is also consider-ably hypertrophied, while the epidermis usually contains more cell layers as com-pared with that of ordinary back integument.

The various histological developments leading to dermal plicae formation havepreviously been dealt with by Massie (1894) and also by the writer (1931a).Similarly, the question of whether or not the mucous and poison glands haveseparate or common origins has also been adequately discussed by many workers.In this regard the reader is referred to the papers of Szczesny (1867), Engleman(1872), Calmels (1883), Schultz (1889), Seeck (1891), Heidenhain (1893), Nicoglu(1893), Junius (1896), Ancel (1902), Fano (1903), Esterly (1904), Nirenstein (1908),Weiss (1908 and 1915), Wenig (1913), Dawson (1920), and of the writer (1931a).It is sufficient to state here that at the beginning of larval involution in the anuranthere are no histological elements which distinguish potential dermal plicae integu-ment from ordinary back or side integument. The areas of larval integument which

JEB-XIVl I

2 O. M. HELFF

give rise to dermal plicae structures during metamorphosis are, however, deter-mined prior to the onset of involution. This fact was first demonstrated by thewriter (1931a) in the following manner. Reciprocal autoplastic transplants weremade between integument of dermal plicae regions and integument derived fromthe back, belly, or side of non-metamorphosing larvae. During metamorphosis, thepotential dermal plicae integument developed typical dermal plicae structures,while the reciprocal grafts placed in dermal plicae regions failed to show any markedhistological changes. Thus it was demonstrated that the potentialities for dermalplicae development are localized and determined in certain integumentary areas atsome time prior to the onset of larval involution.

More recently, the writer (1933) has investigated the stability of fully differ-entiated dermal plicae following homoplastic and heteroplastic transplantation tonon-metamorphosing larvae. Dermal plicae transplants obtained from youngR. palustris frogs were transplanted to larvae of the same species, while in othersthe transplantations were made to R. catesbeiana tadpoles. In the former case nohistological regression of dermal plicae structures occurred within 3 weeks of thetransplantation period. Conversely, however, the heteroplastic transplants showedcomplete regression of dermal plicae structures within the same time interval.When allowed to remain long enough on the host, the heteroplastic transplantsunderwent complete integumentary degeneration. It is evident, therefore, that theR. palustris host larvae possessed the necessary influences (hormonal or otherwise)for the preservation of dermal plicae structures which the R. catesbeiana larvaeapparendy lacked.

The fact that complete regression of fully differentiated dermal plicae structurescould be obtained following heteroplastic transplantation gave rise to severalinteresting questions. Did such integument, for example, after having undergonecomplete dermal plicae regression back to normal integument, still possess thepotentialities for redeveloping dermal plicae elements following suitable retrans-plantation ? The answer to this question was partially given in a later paper in whichthe writer (1934a) showed that the retransplantation of such grafts to metamor-phosing R. clamitans larvae failed to redifferentiate typical dermal plicae structures.A second and more interesting possibility presented itself, however, in that theoriginal transplant after having undergone dermal plicae regression back to normalintegument might be induced, following suitable transplantation, to differentiate anentirely different set of histological structures. The writer (1928, 1931&) had pre-viously shown that the tympanic membrane of the anuran is developed under theinfluences of the annular tympanic cartilage and columella. Furthermore, any larvalintegument when transplanted over these cartilaginous components of the middleear will transform into tympanic membrane. Hence, the possibility of tympanicmembrane differentiation from dermal plicae integument suitably retransplantedto the ear region, following the total regression of dermal plicae structures, wassuggested. The results of such experiments (Helff, 1934a) showed quite clearlythat it is possible to obtain tympanic membrane development in this manner.Briefly, the transplantations and results were as follows. Dermal plicae integu-

Studies on Amphibian Metamorphosis 3

mentary grafts were removed from young R. palustris frogs and transplanted tonon-metamorphosing R. catesbeiana larvae. Within 5-6 weeks complete macro-scopic and histological regression of dermal plicae structures had occurred. Thegrafts were then retransplanted to the ear regions of metamorphosing R. clamitansand R. catesbeiana larvae, where they developed fully differentiated tympanicmembranes within 5-7 weeks.

The above-mentioned results served to raise several other interesting questions.Of these, however, the question of whether or not dermal plicae transformationinto tympanic membrane can be obtained following direct autoplastic, homoplastic,and heteroplastic transplantation of fully differentiated dermal plicae integumentto the ear region of metamorphosing larvae, was of most interest to the writer. Theresults of experiments designed to answer this question and to throw light onvarious correlated problems furnish the material for the present paper.

MATERIALS AND METHODS

Three species of metamorphosing larvae were available for the work in hand,viz. R. palustris, R. clamitans, and R. catesbeiana. They were collected during Julyand August in the vicinity of Lake Winnisquam, New Hampshire. The larvae wereusually rapidly undergoing involution when collected, and were isolated in in-dividual bowls in the laboratory until they had attained the proper degree ofmetamorphosis for the particular donor or host stage desired. For the homoplasticand heteroplastic series, the donors used for dermal plicae transplantation werenewly metamorphosed frogs in which all larval characteristics had disappeared withthe exception of small black tail stumps which usually persist for a week or more.The tail stumps of the R. palustris donors varied from 5 to 10 mm. in length, whilethose of the R. clamitans donors measured from 2 to 4 mm. In all other char-acteristics, however, the donors were typically froglike, and presented well-developed dermal plicae folds. The host animals of the three species used were inall cases involuting larvae well advanced in metamorphosis, and at a stage whenthe annular tympanic cartilage has nearly completed its posterior migration to thesite of tympanic membrane formation. The autoplastic transplantations necessitatedthe use of a somewhat different stage of metamorphosis which can be roughlycharacterized as half-way between the stages used for the homoplastic and hetero-plastic donors and hosts. Although the dermal plicae of such individuals wereadmittedly not as fully differentiated as those of the homoplastic or heteroplasticdonors, the annular tympanic cartilages to which the dermal plicae grafts wereautoplastically transplanted were still in a stage during which they exert strongtympanic membrane inductive influences. Had autoplastic transplantations beencarried out with newly metamorphosed frogs, the conditions of the experimentwould have been more seriously affected, since the annular tympanic cartilage atthis stage has begun to lose some of its developmental influence as regards tympanicmembrane formation (see Helff, 1934ft).

4 O. M. HELFF

The transplantation technique employed has previously been described by thewriter (1934a) in some detail. Briefly, it consisted in the removal of a squarepiece of integument so as to include a portion of the dorso-lateral dermal plicaefold, the latter running through the centre of the graft. This was then transplantedover the annular tympanic cartilage of the host animal or of the same animal in thecase of the autoplastic series, after first removing a square of integument from thatregion. The host larvae were then placed in individual aquaria and allowed to com-plete their metamorphosis. The young frogs were then killed and placed in Bouin'sfixative at various intervals following the attainment of complete larval involution,for the purpose of later sectioning and study of the dermal plicae transplants.Representative individuals were also killed and preserved in alcohol for photo-graphic purposes. It may be stated here that Bouin fixed animals do not usuallyphotograph well, due to the yellow coloration imparted by the fixative and theconsequent lack of contrast so desirable for photographic purposes.

RESULTS

Autoplastic R. palustris transplantations

Thirty-two autoplastic R. palustris transplantations were made of which twenty-nine lived until the period of fixation. The transplants healed rapidly, and super-ficial union with the adjacent integument of the head region was always establishedwithin 4 days. As is usual, however, full union of the various integumentarylayers with those of the adjacent integument required a much longer period oftime. In general, the coloration of the transplanted skin remained quite normal.This was characteristic not only of the dermal plicae portion but also of those partsof the transplant adjacent to the dermal fold. The patches of melanophores socharacteristic of back skin during the later stages of R. palustris metamorphosisand which give rise to the pigmentation pattern of the adult, were also maintainedand in some cases even intensified in the transplants. In a few instances, however,the pigment spots were less distinct, due to a generalized darkening of the trans-planted integument as a whole.

Externally, the dermal plicae folds of the transplants remained normal in allrespects. Not only was the coloration unchanged, but, of more importance, theirgeneral shape, width, and height remained the same as when originally trans-planted. In a few cases, however, the folds became somewhat greyish in tint, whichfact was no doubt correlated with the generalized darkening of the graft in question.It is also interesting to note that although the transplants invariably fused com-pletely with the surrounding head integument, the dermal plicae folds in no instancegrew beyond the borders of the transplant.

All host animals were killed on the twenty-sixth day following the initial trans-plantation and either fixed in Bouin's fixative or preserved in alcohol. PI. I, fig. G,illustrates a typical example. Subsequent sectioning and study corroborated themacroscopic findings in that no regressive changes in the dermal plicae folds hadtaken place. Moreover, there were no degenerative changes evident in any part of

Studies on Amphibian Metamorphosis 5

the transplants which might be construed as representing even early signs oftympanic membrane formation. Apparently, therefore, transformation of dermalplicae structure into that of tympanic membrane is not possible following autoplastictransplantation in R. palustris; at least, not under the conditions of the presentexperiment and within the time interval which was available for such transformation.

Autoplastic R. clamitans transplantations

The autoplastic R. clamitans transplantations were made on metamorphosinglarvae in the same stage of involution as selected for the R. palustris series. In suchlarvae the normal tympanic membrane is approximately half-formed, while theannular tympanic cartilage is still active as regards its ability to induce membraneformation. In all, thirty-one transplantations were made of which twenty-sevensurvived for at least 20 days following the autoplastic grafting of the dermal plicaeintegument to the ear region. The animals were killed for fixation or preservationin alcohol between the twentieth and thirty-second day following transplantation.As in the case of the previous series, fusion of the transplant with the surroundinghead integument was achieved, externally at least, within a few days.

In fifteen of the twenty-seven cases, no appreciable diminution in height of the'dermal plicae folds was noticeable, while their width and general contour remainedquite normal (PI. I, fig. A). The integumentary portions of the transplants adjacentto the dermal folds, however, in many cases gave slight external evidence of tym-panic membrane formation in that the coloration underwent a change. Histologicalsections, however, gave little evidence of degenerative changes essential andnormally prerequisite to the later developmental stages of membrane formation,while the component parts of the dermal folds were likewise found to be still intact.

Eight cases exhibited definite external signs of dermal plicae regression. Thesevaried from slight reduction in height of the fold to a condition where the latterwas barely discernible to the eye. A characteristic and proportionate widening ofthe fold accompanied its reduction in height (PI. I, fig. B), while the transplants ingeneral usually assumed a darker shade, although in a few cases a light brown tingewas evident. The histological findings revealed the dermal plicae elements in variousstages of degeneration which were correlated quite closely with the degree ofmacroscopic fold reduction.

The remaining four cases gave external evidence of complete dermal plicaeregression. In one of these, early signs of tympanic membrane formation weredetected, while in another a half-formed membrane actually developed occupyingthe greater part of the transplant (PI. I, fig. C). There were no histological signsindicating the persistence of dermal plicae elements in any of these four cases, whilein the latter two cited definite cellular reorganizations were apparent typical ofearly membrane formation.

The results of the autoplastic R. clamitans series, therefore, were not as uniformas those obtained in the R. palustris series. Aside from the fact that species speci-ficity evidently played a part in determining the two groups of results obtained, thedifferences in results exhibited between the twenty-seven cases of R. clamitans

6 O. M. HELFF

transplantation must be attributed to individual variations in the reactions of thedermal plicae folds to regressive influence or to variations in the inductive influencesof the annular tympanic cartilages. Quite probably both of these factors wereoperative. The length of time during which regressive and inductive influencesfunctioned in each case must also be taken into account. These factors and otherswill be taken up more fully in the discussion section of the present paper.

Homoplastic R. palustris transplantations

Twenty-eight of the thirty-four homoplastic R. palustris transplantations livedfor more than 3 weeks following the grafting of dermal plicae integument over theannular tympanic cartilage. They were killed for fixation or alcohol preservationon either the twenty-sixth or thirty-second day following the transplantation pro-cedure. The hosts were by this time fully metamorphosed young frogs in whicheven the tail stumps had been completely atrophied for at least a week.

The transplants appeared to heal, at their borders with the surrounding hostintegument, in about the same time interval as in the case of the autoplastic series.This rapidity of healing might be considered somewhat unusual in that homoplastichosts, theoretically at least, should not offer as favourable an environment as whenthe transplants were grafted autoplastically. It should be borne in mind, however,that the host larvae in the homoplastic series were in an earlier and more rapidlyinvoluting stage as compared with the autoplastic animals. Hence, the morefavourable growth (healing) influences present probably were sufficient to counter-balance any antagonism towards healing which may have existed between the trans-plant and its homoplastic host.

The pigmentation pattern of the grafts in those regions adjacent to the dermalplicae folds remained quite normal, although it was not as pronounced as in thecase of the autoplastic transplants on the same species. This was probably the resultof the generalized darkening of the transplants which occurred, and which wasmore intense than was true of the autoplastic series. Usually, the transplants gavelittle evidence of superficial disintegration, although in two cases slight epidermalerosion was noticed, while in only one case were coloration changes recordedindicative of tympanic membrane development. Histologically, however, mosttransplants presented disintegrative changes similar to the cellular reorganizationswhich precede normal tympanic membrane formation.

The dermal plicae portions of the transplants presented a striking uniformity intheir reactions. In all cases, without exception, a definite reduction in height of thefolds took place, while the ridges became much broader and diffuse in appearance(PI. I, fig. H). The folds also became less distinct to the eye, due no doubt to acertain amount of pigmentation which developed. This change, resulting in theloss of the characteristic whiteness of fully differentiated dermal plicae, was nodoubt due to the same influences responsible for the generalized darkening of thetransplants as a whole. Histologically, it was quite clear that the various com-ponents of the folds were undergoing rapid disintegration and regression. In noinstance, however, could the histological picture be interpreted as presenting

Studies on Amphibian Metamorphosis 7

developmental changes typical of tympanic membrane formation. The results,therefore, would seem to indicate that homoplastic transplantation in R. palustris isconducive to regressive changes in dermal plicae structure, although transformationinto tympanic membrane is not possible under the conditions and time limits of theexperiment.

Homoplastic R. clamitans transplantations

Thirty homoplastic R. clamitans transplantations of dermal plicae integumentwere made to the developing ear region. Of these, twenty-seven lived for at least28 days following the transplantation procedure. All host animals were finallykilled for fixation or preservation within 3 days of this period. The early historyof the transplants followed quite closely that as described for the homoplasticR. palustris transplantations, especially as regards the rapidity of healing and thegeneralized darkening which occurred. In many cases, however, this initial in-creased pigmentation proved to be but a temporary condition in that its disappear-ance usually followed the early stages of tympanic membrane formation.

The fate of the dermal plicae portions of the transplants was strikingly definiteand uniform, for in all except two cases, complete external and histological regressionoccurred. Externally, the folds were seen to first lose their characteristic whitenessand to assume a dull greyish tinge. Accompanying this coloration change, the foldswere seen to gradually reduce their height and increase in width, becoming ingeneral much more diffuse. In this respect they resembled quite closely the parallelcondition as described for the homoplastic R. palustris transplantations. The speedand extent of the regression was, however, of a greater magnitude as comparedwith the R. palustris series, and resulted finally, as stated above, in complete dermalplicae degeneration. This latter observation was further emphasized by the histo-logical findings which showed that, in all except the two cases cited above, allcellular components of the dermal folds had undergone degeneration; followingwhich the degenerated elements had either been removed or transformed intotympanic membrane structure. In the two exceptions mentioned, the dermalplicae had undergone pronounced degenerative changes but were still visible,externally, as flat, diffuse, greyish bands. The histological pictures of these twocases showed the various structures of the folds in well-advanced stages of degenera-tion. Had another week elapsed before the killing of the host animals, there can belittle doubt but that complete histological regression would have taken place inthese two cases also.

The rapidity of dermal plicae regression was no doubt the result of influencesaffecting to some extent, at least, the entire integumentary graft. In this regard itwas observed that the rest of the transplant (those portions adjacent to the dermalplicae folds) usually became quite soft and delicate. In fact, in three cases rupturingof the epidermis occurred, giving the transplants a somewhat spotty appearance.The generalized softening of the transplants, which apparently was localized in theepidermis, disappeared in all except a few cases following the onset of the develop-mental phase of tympanic membrane formation.

8 0. M. HELFF

In all except the two cases in which complete dermal plicae regression was notobtained, some degree of tympanic membrane formation occurred. The develop-ments attained presented varying degrees of differentiation both as regards externaland histological appearance. All stages of development occurred from early stagesof membrane formation (PI. I, fig. D) to completely formed membranes (PI. I,fig. F) which resembled the tympanic membranes of the opposite or left sides of thehost animals very closely. PI. I, fig. E, represents a case in which the peripheralregions of the membrane are developing, although the central region still presentsthe dark, uniformly pigmented and somewhat delicate integument which is usuallyfound immediately following complete regression of the dermal plicae. There canbe no doubt but that complete membrane development would have occurred inthis case had the host animal been allowed to live for 7 or 10 days longer. In general,therefore, it can be concluded that the conditions for dermal plicae transformationinto tympanic membrane were much more favourable in this series as comparedwith either of the autoplastic series or with the homoplastic R. palustris series.

Heteroplastic R. palustris transplantations

(1) R. palustris dermal plicae integument on R. clamitans hosts.

The results of the heteroplastic transplantation of R. palustris dermal plicaeintegument to the ear region of R. clamitans were quite similar to those as obtainedin the homoplastic R. clamitans series. In general, partial dermal plicae regressionoccurred, although its onset was usually delayed for a considerable period followingthe transplantation procedure. Thus, in one case (PI. II, fig. K) no apparent changehad taken place in the dermal plicae portion of the transplant up to the twentiethday following transplantation. Usually, however, typical macroscopic and histo-logical regressive changes were initiated within 10-14 days following transplan-tation. Externally, the regression was evidenced by a progressive broadening anddiffusing of the fold resulting in a reduction in its height. This degree of regression(PI. II, fig. L) was not surpassed by sixteen of the twenty-one transplants of thisseries. In five cases, however, regression continued in the normal manner, resultingin the complete disappearance of dermal plicae structures in three instances. Earlysigns of tympanic membrane formation were visible in two of these three cases,while in one a half-developed membrane was obtained.

It seems quite logical to assume that, had the host animals survived for a weekor two longer, more examples of complete dermal plicae regression and tympanicmembrane formation would have been obtained, since all individuals were killed forfixation within 29 days following transplantation. It may be parenthetically statedhere that the various hosts were killed as soon as it became evident that deathwould have occurred within a day or two. This procedure was necessary in orderthat fixation of the transplants could be secured before post-mortem changes wouldhave become evident. The development of tympanic membrane structures wasquite probably also greatly inhibited by the pronounced degenerative changes whichfrequently affected all regions of the transplant. This rapidity of degeneration was

Studies on Amphibian Metamorphosis 9

evidenced by the pronounced generalized darkening of the transplant coupled withthe frequent disintegration and sloughing of its epidermis. PI. II, fig. M, forexample, represents a case in which the dermal plicae fold of the transplant hasnearly disappeared, while the entire graft itself is subject to rapid degeneration. Infact, it was difficult to escape the impression that, had the degenerative changesbeen less rapid, the conditions for tympanic membrane development would havebeen more favourable. In conclusion, however, it can be said that the conditionsfor dermal plicae regression and tympanic membrane development were somewhatmore favourable in this series as compared with those of the homoplastic R. palustrisseries.

(2) R. palustris dermal plicae integument on R. catesbeiana hosts.

Thirty-three transplantations of R, palustris dermal plicae integument weremade to the developing ear region of R. catesbeiana hosts. Twenty-nine of the hostanimals survived metamorphosis for at least 3 weeks and were killed on either thethirty-third or thirty-eighth day following the initial transplantation. Dermalplicae regression was obtained in all of these cases and proceeded in a normalmanner until all external and histological signs of the folds had disappeared. Theintegument of the transplants adjacent to the dermal plicae folds usually under-went little if any degeneration, although in a few cases some epidermal disintegrationoccurred following tympanic membrane formation later on. This much delayeddegeneration appeared to particularly affect the epidermis of the tympanic mem-brane (PI. II, fig. N).

The development of tympanic membranes was typical of all twenty-nine casesin which complete dermal plicae regression had occurred. In a few instances,complete membrane formation was not obtained by the time the hosts were killed,but in at least twenty-four cases the membranes which developed were completein all essential respects and presented only slight differences as compared with thenormal left-side membranes on the same animals. These differences were chieflyconcerned with coloration, although many examples were obtained in which thecharacteristic brownish tint and texture of fully differentiated normal membraneswas attained. In a few cases, where complete membrane development did not occur,the peripheral regions of the membranes had differentiated, while the more centralportions were rapidly undergoing transformation.

In general, it can be said that the results of this series were the most uniformand satisfactory as compared with those of the other three heteroplastic series. Theresults, moreover, furnished clear-cut evidence that dermal plicae transformationinto tympanic membrane can be obtained following direct heteroplastic transplan-tation of fully differentiated dermal plicae integument to the developing ear region.It is apparently not necessary, therefore, first to obtain dermal plicae regression bytransplantation to non-metamorphosing larval hosts.

io 0. M. HELFF

Heteroplastic R. clamitans transplantations

(1) R. clamitans dermal plicae integument on R. palustris hosts.

Thirty-six transplantations of R. clamitans dermal plicae integument were madeto R. palustris hosts, of which thirty-one lived for 25 days following the trans-plantation procedure, at which time they were killed for fixation or preservation.The fate of the dermal plicae portions of the transplants was similar in all respectsto that as described for the heteroplastic transplantation of R. palustris dermalplicae to R. catesbeiana hosts, in that total regression eventually occurred in allcases. The onset and rate of regression was subject to considerable variation,however, due no doubt to individual differences in the susceptibility of the trans-plants coupled with possible differences between the host animals as regards theintensity of the regressive influences they exerted.

Approximately one-half of the transplants developed well-differentiated tym-panic membranes (PI. II, fig. O), while in the remaining half the membranesdeveloped were at least 50 per cent, formed as determined by external structureand histological appearance. The membranes were rarely typical as regards colora-tion but were usually darker than normal membranes and of a greyish tint. Severalexcellent cases were obtained, however, in which a typical brownish tinge wasevident, making the membranes indistinguishable from those on the opposite orleft side of the same host animals. There were only two cases in which no signs oftympanic membrane formation could be detected following total regression of thedermal plicae folds. In these two instances the grafts had undergone considerabledisintegrative processes, especially as regards the epidermis which had eroded awayleaving white denuded areas in the centre of the transplants. Apparently thesetransplants were destined to undergo total resorption, while the rapidity of thedegeneration had inhibited whatever inductive influences were present favouringtympanic membrane formation.

(2) R. clamitans dermal plicae integument on R. catesbeiana hosts.

Somewhat more favourable results were obtained in the transplantation ofR. clamitans dermal plicae integument to R. catesbeiana hosts. Thirty-three trans-plantations were made in all. Thirty of the host animals lived for at least 4 weeksfollowing the operation. These were killed for preservation or histological study ofthe transplants from 30 to 47 days following the original transplantation procedure.

The fate of the dermal plicae portions of the transplants closely resembled thatas described for the preceding series, in that an orderly process of regressionoccurred resulting finally in the complete disappearance of all dermal plicaestructures both externally and histologically. The onset and rate of this regression,however, was considerably more uniform as compared with the same series. Tym-panic membrane development was also more uniform and resulted in the differ-entiation of well-formed membranes in approximately 75 per cent, of the cases(PI. II, fig. J). The coloration of these membranes was usually quite normal andcompared very favourably with those on the opposite side of the same animals.

Studies on Amphibian Metamorphosis 11

In the remaining cases various stages of development occurred, resulting usuallyin membranes which could be characterized as being at least half-formed (PI. II,fig. I). The results of the two heteroplastic R. clamitans series are interesting in thatthey show that not only R. palustris but also R. clamitans dermal plicae are capableof undergoing complete transformation into tympanic membrane following suitabletransplantation to the developing ear region.

DISCUSSION

The failure of dermal plicae to undergo regressive changes in most instances,when autoplastically transplanted, can be readily understood in that the transplantswere still subjected to the same general body (hormonic) influences which wereresponsible for their development. The development of tympanic membranestructure, however, was quite probably dependent on the intensity of the inductiveinfluences arising from the annular tympanic cartilages in conjunction with thesensitivity of the transplants towards transformation. Thus, in the case of theR. palustris transplants, the inductive influences were apparently too weak to causedegeneration of the dermal plicae as a necessary stage in the transformation of thesestructures into those of tympanic membrane. The failure of integumentary portionsof the transplants adjacent to the dermal plicae to undergo tympanic membraneformation is more difficult to understand. It seems likely, however, that a certaindegree of resistance may have been imparted to these areas of integument due totheir close association with the developing dermal plicae.

Such acquired resistance may not have been true of the integumentary portionsof the R. clamitans transplants, since in many instances slight signs of externaltympanic membrane formation were recorded in cases where no dermal plicaeregression occurred. As will be recalled, however, there were no histological signsof membrane formation in such grafts, and hence the external signs (colorationchanges, etc.) must have represented merely superficial evidences of the action ofinductive influences at work. In fact, definite histological signs of membraneformation were recorded in only two of the four cases in which complete dermalplicae regression had occurred. It seems logical to conclude, therefore, that theinfluence of the annular tympanic cartilage in the R. clamitans series was in mostcases too weak to bring about either dermal plicae regression or tympanic membraneformation in the adjacent integument. Moreover, in most cases where the de-generative influence was sufficient to degenerate the dermal plicae either partiallyor wholly, the requisite degree of inductive influence for membrane formation wasstill lacking. In the two cases where membrane formation was initiated, the in-ductive influences must have been correspondingly more intense. It is, of course,always possible to postulate that the inductive influences were of the same intensityin all cases and to account for the variable reactions on the part of the transplantsby assuming different degrees of sensitiveness for the latter.

The partial regression of dermal plicae in the homoplastic R. palustris trans-plants was quite probably the result of influences emanating from the annular

12 O. M. HELFF

tympanic cartilage and facilitated, possibly, by a certain degree of antagonismbetween host and transplant. That homoplastic transplantation cannot in itselfinduce regression has been shown by the writer (1933), in that dermal plicaeintegument from newly metamorphosed R. palustris frogs when transplanted to thebacks of non-metamorphosing larvae of the same species failed to undergo de-generative changes. However, it is necessary to realize that two separate influenceswhich might be ineffectual when working separately could bring about dermalplicae regression when acting together. Thus, while the annular tympanic cartilagemay not have been quite powerful enough to induce regression, the added effect ofslight homoplastic antagonism may have been sufficient to induce regressive changes.The fact that full regression of dermal plicae was never attained and no signs oftympanic membrane development became evident, would tend to support the theorythat the influence or influences available were not of maximum intensity. It ispossible, also, that the annular tympanic cartilage was responsible entirely for thedegree of dermal plicae regression that occurred, since it will be remembered thatthe cartilages in the homoplastic and heteroplastic series were in a somewhat earlier andhence more active inductive stage as compared with those of the autoplastic series.

The results of the homoplastic R. clamitans series are subject to the same generalexplanations. It seems quite probable here, however, that a definite antagonismdid exist between host and transplant in that the grafts were at first usually sub-jected to a rapid generalized degeneration. This condition was not only conduciveto dermal plicae regression but favourable to tympanic membrane formation. Theregression of dermal plicae was no doubt also facilitated by the presence of theannular tympanic cartilage which was also, of course, responsible for the varyingdegrees of membrane formation which occurred.

The conditions for dermal plicae regression and tympanic membrane develop-ment were apparently much more favourable in the heteroplastic than in the homo-plastic series. Here, for the first time, the possibility of species specificity andantagonism was a factor and no doubt was responsible for much of the dermalplicae regression which occurred. In this regard the reader is referred to a previouswork of the writer (1933) in which it was shown that very definite regression ofwell-developed R. palustris dermal plicae followed the latter's transplantation tonon-metamorphosing R. catesbeiana larvae. It might be argued that in this caseregression was to be expected in that the non-metamorphosing larval hosts wereexhibiting no dermal plicae developmental influences at the time, while, conversely,the hosts as used in the present heteroplastic series had developed and weremaintaining their own normal dermal plicae folds. However, the possibility ofspecific intolerance to foreign dermal plicae is still quite probable, and the genera-lised disintegration of transplants which occurred in many instances lends supportto this idea. Obviously, further work is necessary before this particular point can beadequately dealt with. It would be interesting in this regard to determine the fateof heteroplastic transplantations of dermal plicae to other regions than that of theannular tympanic cartilage, employing host animals in the same metamorphic stageas utilized for the present work.

Studies on Amphibian Metamorphosis 13

The completeness of dermal plicae regression in most cases of heteroplastictransplantation was no doubt responsible in a large measure for the high degree oftympanic membrane development which usually occurred. As has been statedbefore, the regression of dermal plicae not only reduces a highly differentiatedstructure to a state closely resembling normal integument, but also brings about aslight disintegration of that integument which is similar in most respects to theearly disintegrative phases of normal tympanic membrane formation. Thus, theannular tympanic cartilage is not called upon to induce the early degenerative stepsof membrane formation, but can exercise its full force in the induction of the laterdevelopmental phases. A comparison, moreover, of the results of the variousheteroplastic series would lead one to conclude that the annular tympanic cartilageof R. catesbeiana is more active and efficient as compared with that of R. palustrisor R. clavritans in relation to membrane induction.

The failure of R. palustris dermal plicae to undergo complete regression in mostinstances, when transplanted to R. clamitans hosts, is difficult of explanation.Apparently the regressive influences at work here were of a less intensity as com-pared with those prevailing in the other host species used, since the onset of dermalplicae regression was usually much delayed. In spite of this, however, the integu-mentary portions of the transplants were frequently subjected to rapid disintegrativeprocesses, and it is difficult to escape the impression that the abnormal degenerationpresent may have in this case tended to neutralize the inductive effect of the annulartympanic cartilage towards membrane development. However this may be, it isevident that the host rather than the transplant was mainly responsible for theretarded plicae regression and membrane development which occurred, since thereverse transplantation gave more favourable results, while both the R. clamitansand R. palustris series involving R. catesbeiana hosts resulted in complete plicaeregression and membrane formation.

SUMMARY AND CONCLUSIONS

The writer has previously shown that fully developed anuran dermal plicaeundergo complete regression following heteroplastic transplantation to the larva.Subsequent retransplantation to the ear region of metamorphosing larvae results inthe development of normal tympanic membranes. Whether such transformationwill occur following direct transplantation of dermal plicae to the ear region wastested in the present work by the following operations:

Autoplastic R. clamitans and R. palustris transplantations resulted, in general, inlittle or no regression of dermal plicae.

Homoplastic transplantations resulted in complete regression in R. clamitansand partial regression in R. palustris. In the former species, partial, and in the latter,no tympanic membrane development occurred.

Heteroplastic transplantations from R. clamitans to R. palustris and R. cates-beiana resulted in complete regression in both cases, with the development of well-formed tympanic membranes.

14 O. M. HELFF

Heteroplastic transplantations from R. palustris resulted in partial regression onR. claimtans hosts and usually no tympanic membrane development. Transplantson R. catesbeiana hosts underwent complete regression and tympanic membraneformation.

The conclusions are reached that autoplastic transplantations usually do notresult in dermal plicae regression; homoplastic transplantations result in partial tocomplete regression but without tympanic membrane formation; while hetero-plastic transplantations usually involve complete regression and full tympanicmembrane development.

Initial heteroplastic transplantation to larval hosts is thus not essential, in thatdirect heteroplastic transplantation from young frogs to the ear region of meta-morphosing larvae will result in total regression of dermal plicae structure andcomplete membrane formation.

REFERENCES

ANCEL, P. (1902). Arch. Biol., Paris, 18, 257.CALMELS, M. G. (1883). PflOg. Arch. ga. Physiol. 15, 321.DAWSON, A. B. (1920). J. Morph. 34, 487.ENGLEMAN, T. W. (1872). Pflilg. Arch. ga. Physiol. 5, 498.ESTERLY, C. O. (1904). Univ. Calif. Publ. Zool. 1, 227.FANO, L. (1903). Arch. ital. Anat. Embriol. 2, 405.HBIDENHAIN, M. (1893). S.B. phys.-med. Ges. Wibrzburg, p. 52.HELFF, O. M. (1928). Physiol. Zodl. 1, 463.

(1931a). Biol. Bull. Wood's Hole, 6O, n .(1931 b). J. exp. Zool. 59, 179.(i933)- Biol. Bull. Wood's Hole, 65, 304.(1934a). Anat. Rcc. 59, 201.(1934*). Biol. Bull. Woods Hole, 66, 38.

JUNIUS, P. (1896). Arch. mikr. Anat. 47, 136.MAESIE, J. H. (1894). J. comp. Neurol. 4, 7.NICOGLU, P. (1893). Z. tuiss. Zool. 56, 409.NIRENSTETN, E. (1908). Arch. mikr. Anat. 72, 47.SCHULTZ, P. (1889). Arch. mikr. Anat. 34, 11.SEECK, O. (1891). "t)ber die Hautdriisen einiger Amphibien." Inaug.-Diasert., Dorpat.SZCZESNY, O. (1867). " Beitrflge zur Kenntnis der Textur der Froschhaut." Inaug.-Distert., Dorpat.WEISS, O. (1908). Anat. Ann. 33, 124.

(1915). Arch. mikr. Anat. 87, 265.WENIG, J. (1913). Anat. Anz. 43, 113.

Studies on Amphibian Metamorphosis 15

EXPLANATION OF PLATES I AND II(All photographs approximately x zi)

PLATE IFig. A. Autoplastic R. clamitans graft 31 days following transplantation. No regression of dermalplicae fold. The fold is seen traversing the centre of the graft.Fig. B. Autoplastic R. clamitani graft 29 days following transplantation. Dermal plicae fold hasundergone partial regression. Note increased width and general diffusion of fold.Fig. C. Autoplastic R. clamitans graft 31 days following transplantation. Complete regression ofdermal plicae fold and partial development of tympanic membrane.Fig. D. Homoplastic R. clamitans graft 28 days following transplantation. Complete dermal plicaeregression and early tympanic membrane formation.Fig. E. Homoplastic R. clamitans graft 29 days following transplantation. Complete dermal plicaeregression with peripheral regions of tympanic membrane forming.Fig. F. Homoplastic R. clamitans graft 28 days following transplantation. Complete dermal plicaeregression and fully formed tympanic membrane.Fig. G. Autoplastic R. palustris graft 26 days following transplantation. No regression of dermalplicae fold. The fold is seen traversing the graft just posterior and dorsal to the angle of the jaw.Fig. H. Homoplastic R. palustris graft 26 days following transplantation. Dermal plicae fold hasundergone partial regression. Note increased width and general diffusion of fold.

PLATE II

Fig. I. R. clamitans graft on R. catabeiana host 47 days following transplantation. Complete dermalplicae regression and partial tympanic membrane formation.Fig. J. R. clamitans graft on R. catabeiana host 47 days following transplantation. Complete dermalplicae regression and well-differentiated tympanic membrane.Fig. K. R. palustris graft on R. clamitans host 20 days following transplantation. No regressionof dermal plicae fold.Fig. L. R. palustris graft on R. clamitans host 23 days following transplantation. Partial regression ofdermal plicae fold. Note increased width and general diffusion of fold.Fig. M. R. palustris graft on R. clamitans host 22 days following transplantation. Dermal plicaefold in dorsal part of graft has nearly disappeared while graft, in general, is degenerating.Fig. N. R. palustris graft on R. catabeiana host 38 days following transplantation. Complete dermalplicae regression and well-differentiated tympanic membrane. The epidermis of the membrane hasundergone degeneration.Fig. O. R. clamitans graft on R. palustris host 25 days following transplantation. Complete dermalplicae regression and well-differentiated tympanic membrane.