Observations of Dependent Histogenesis in Salamander Limb Development

/. Embryol. exp. Morph., Vol. 11, Part 2, pp. 325-338, June 1963 Printed in Great Britain

by CYRIL V. FINNEGAN1 From the Department of Zoology, University of British Columbia

INTRODUCTION

PREVIOUS work in this laboratory (Finnegan, 1962) had suggested that limb development might be specifically enhanced by experimentally associated somite mesoderm tissue. Detwiler (1938), Swett (1945) and Nicholas (1958) called attention to the influence of this mesoderm in the production of duplications when limb buds were transplanted heterotopically to the superficial somite region and, more recently, Amano (1960) stated that somite tissue was required, inductively and materially, for limb development. In an analysis of development it is assumed that a group of cells, whose histogenesis has been determined by their previous experience, will evidence that histogenesis if placed in an environment in which they continue to develop. Thus, after limb bud transplantation to the flank in urodeles, positive results (that is, histogenesis, proximally, accompanied by growth, distally) have been interpreted as self-differentiation of the limb bud cell mass (see review by Nicholas, 1955). However, the possible synergistic role of the somite mesoderm in the histogenesis of the transplant has not been considered. It may be that greater histogenetic competence has been ascribed to the limb cell mass than it actually posesses. In this respect Wilde (1950) was able to show, by in vitro studies, a dependency of limb differentiation on donor developmental age. The more ventral region of the urodele flank has been considered an unfavour- able site for limb development (Nicholas, 1924; Takaya, 1938) since limb bud transplanted to that region does not continue its development. It seemed desir- able then to use this ventral flank as the transplantation site in order to disclose the actual histogenetic ability of the limb cell mass under environmental conditions which do not specifically assist its development. The same ventral environment was then used to obtain evidence of the possible supportive role of the epibrachial somite mesoderm in the histogenesis of the transplanted limb cell mass. Finally, results of an analysis of the relative supportive capacity of the dorsal somite and of the ventral somite tissue are included in this report. Author's address: Department of Zoology, University of British Columbia, Vancouver 8, Canada. 326 CYRIL V. FINNEGAN EXPERIMENTAL PROCEDURE The mesoderm and overlying ectoderm comprising the limb bud cell mass lying superficial to the somatic hypomere ventral to the pronephric kidney area (see Text-fig. 1) was excised from post-neurula stages of Ambystoma punctatum and Taricha torosa, Stages 25 to 38 inclusive. An incision was made in the ectoderm immediately ventral to this cell mass and the latter was then carefully lifted away from the underlying mesoderm by means of a finely sharpened steel needle. The ectodermal incision was continued around the mesodermal cell mass so that only the material in the cell mass at the time of operation, and not the entire limb disc of 3-4 somites (Nicholas, 1955), was included in the transplant. According to

TEXT-FIG. 1. Lateral view of tail-bud embryo. The areas removed in the operation are indicated by cross-hatching. L = approximate limb area removed. S = approximate area of epibrachial somite removed. P = Pro- nephros. The boundary of the conventional limb disc is indicated by the dotted line. (Modified from Swett, 1945.)

Schwind (1932), this cell mass comprises the limb tissue and contains little or no girdle tissue. A small incision was made in the ventral or ventrolateral ectoderm of the host animals, of approximately the same developmental age as the donors, and the excised limb material was placed in the incision so that the limb mesoderm cell mass was in contact with the hypomeric mesoderm of the host. The initial cut was made small since the wound tended to increase in size rendering subsequent contact of the host and donor ectoderm difficult to achieve. A glass bridge was used to hold the transplant in position until healing had occurred. In the series of experiments involving the addition of somite mesoderm, a portion of the donor's epibrachial somites (somites 3-5) ventral to the lateral line was removed (see Text-fig. 1). This material was placed beneath the host's ventral ectoderm through the incision which subsequently received the donor limb cell mass, HISTOGENESIS OF LIMB 327 and the two transplants were finally placed so that the mesoderm of limb and somite were juxtaposed. These operations involving the ventral flank were difficult to conduct so that complete healing would result, and thus, while over sixty animals were prepared, the results are based on observation of the smaller number (thirty-eight) in which the healing was such as to ensure the retention of the graft in the desired relation to the host's ventral hypomere or the grafted somite tissue. When the limb cell mass was transplanted to the host's somite mesoderm in the final series of experiments, the grafted limb tissue was placed so that the mesoderm of the transplant was in contact with the superficial area of the somite, either dorsal to or ventral to the lateral line. Ambystoma gracile embryos were used as donors in some of these cases and Taricha torosa embryos were used as donors in other cases. The latter species was used as the host animal in all of the seventeen prepared cases. Aseptic techniques were used throughout and the host animals reared in urodele growing medium (Rugh, 1962) at 18° C. Animals were prepared for histological examination by being fixed in Michaelis', sectioned at 8-10 /x and stained with haematoxylin and eosin. TABLE 1 Limb histogenesis in ventral sites Procedure Histogenesis Culture time Donor-host (weeks) Pronephros Cartilage Muscle Den I. T. torosa-T. torosa: (1) Limb bud (stages 25-35) 3 + _ _ + (2) Plus ectoderm (stage 35) 3 — + * — + (3) Plus epibrachial somite (stage 35) 2 — + +t + § + II. A. punctatum-T. torosa: (1) Limb bud (stage 35) 3 + + + + + III. A. punctatum-A. punctatum: (1) Limb bud (stages 30-35) 3 — + — + (2) Plus ectoderm (stage 35) 3+ — + — + (3) Plus epibrachial somite (stage 35) 2+ — + + +$ + + II + IV. T. torosa-A. punctatum: (1) Limb bud (a) Stage 25 8 — + + + — + (b) Stage 30 8 — + + + — + (c) Stage 35 8 — + + + ++ + * + Some concentric cell orientation was observed. t + + Definite cell orientation and a perichondrium were present. % + + + Definite cartilage (cells and matrix) observed. §+ Cell orientation and cytoplasmic acidophilia interpreted as possible myoblastic development. 11 + + A more definite muscle tissue was histologically identifiable. 328 CYRIL V. FINNEGAN RESULTS Heterotopic transplantation to a ventral (hypomere) site The results of both homoplastic and heteroplastic transplantation of limb bud are summarized in Table 1 and are considered further in the following. Limb bud alone as the transplant To facilitate description of the results the donors are grouped as representative of three developmental ages; a Stage 25 group, a Stage 30 group and a Stage 35 group.

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TEXT-FIG. 2. A section through the ventral flank of a T. torosa host at 3 weeks after transplantation of Stage 35 torosa limb cell mass. A pronephric tissue (P) and mesentery (M) have differentiated internally and a reduced aggregation of basiphilic cells is present superficially (LBE-11).

Triturus torosa transplant to Triturus torosa hosts (8 cases). During the first post-operative week the Stage 35 cases developed a projection nearly equal in size to that of the host's anterior limb but the other two donor stages exhibited only a small bump in the graft area. The graft appeared to regress relative to the growth of the host during the second week in all cases, so that, in the third week, the transplant was visible as a cell mass within the ventral body wall. The sectioned material fixed in the third week revealed that a pronephric unit had differentiated in the deeper portion of the graft area in all cases (see Text-fig. 2). This kidney tissue continued from the graft through the mesentery to the ventral wall of the host's gut. In some cases a short pronephric duct had developed HISTOGENESIS OF LIMB 329 either anteriorly or posteriorly from the kidney tissue and in contact with the host's somatic peritoneum. An aggregation of basiphilic cells was always found located immediately superficial to one end of the pronephric unit. The smallest such mass was present in the Stage 25 cases and the largest was in the Stage 35 cases, although this mass in all cases was obviously reduced as compared with the normal limb mesoderm cell mass. Ambystoma punctatum transplant to Ambystoma punctatum hosts (5 cases). The projections initially developed from the transplanted limb cell masses regressed, in the second week, to small mounds on the ventral flank which, when examined histologically, indicated early cartilage-like cell orientation. A thick

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TEXT-FIG. 3. A section through the ventral flank of a T. torosa host at 3 weeks after transplantation of Stage 35 punctatum limb cell mass. A pronephric tissue (P) and mesentery (M) have developed internally and a girdle-like unit (G) with associated oriented cells lies more superficial (LBS-13). dermis, with dorso-ventrally oriented fibroblasts and much ground substance, was present superficial to the procartilage cell mass. In the third week, the graft area was visible macroscopically as a cell mass in the ventral body wall and sectioned material showed that the cartilage-like cell group had a definite perichondrium but little to no matrix production was evident. In some cases a mesentery was present from the graft to the host's gut wall, but no pronephric differentiation was observed. 330 CYRIL V. FINNEGAN Ambystoma punctatum transplant to Taricha torosa hosts {2 cases). The rather large size of the Stage 35 limb transplants relative to that of the host resulted in only two properly oriented surviving cases in this series. These limb transplants developed a projection in the first week which regressed in the second and third weeks so that a small projection remained in one case and no external indication of the original projection could be seen in the second. In the sectioned material it was seen that pronephric tissue had developed in the host's somatic peritoneum and the graft-associated mesentery (see Text-fig. 3). A cartilage-like element was present closely applied to the pronephric tissue and a graft cell mass, immediately superficial to that cartilage girdle-like unit, demonstrated cell orientation and acidophilic cytoplasm suggestive of myoblast differentiation. A second cartilage- like element extended from the girdle region toward the surface, reaching into the small projection in the one case and not reaching the surface in the other. Both such elements were accompanied by apparent myoblastic cells. Taricha torosa transplant to Ambystoma punctatum hosts (6 cases). The Stage 35 transplants produced a projection in the first week but the younger donor cases did not do so until the third week. The Stage 25 cases regressed rapidly in the fourth week while the older transplants did so in the fifth week. After eight weeks, a small piece of cartilage in the host's ventral body wall was revealed histologically in the Stage 25 and Stage 30 cases. The Stage 35 cases, on the other hand, had developed girdle cartilage with superficially associated musculature and an arti- culation with an externally directed humerus-like element was also present. No pronephric differentiation was seen in any of these cases. It appears from these results that when the limb bud cell mass of the two donor species is transplanted to the ventral hypomere environment of Taricha torosa, pronephric differentiation occurs in the deeper region of the transplant-hypomere association and there is an aggregation, or retention, of a basiphilic cell mass immediately superficial to one end of the nephric tissue. When the superficial basiphilic cell mass was developed from transplanted T. torosa tissue no limb histogenesis was observed during the following three-week culture period, but the basiphilic cell mass produced by Stage 35 A. punctatum tissue demonstrated early girdle-limb histogenesis in a similar period. In reciprocal experiments, when A. punctatum was the host, no pronephric differentiation was seen. The grafted tissue showed some degree of chondrogenesis in all cases and some tardy myo- genesis was found in the Stage 35 T. torosa transplants. It is interesting to note that the Stage 35 limb cell mass of both donor species demonstrated better histogenetic competence in heteroplastic than in homoplastic transplantation. Vascu- larization of the graft area was evident in all cases.

Limb bud plus epibrachial somite ventral tips as the transplant The amount of somite tissue added to the limb transplant varied from one to three somite tips but the results did not appear to be affected. Only the homoplastic transplantations were attempted. HISTOGENESIS OF LIMB 331 Ambystoma punctatum transplant to Ambystoma punctatum hosts {6 cases). The projections developed from the graft area in the first week elongated during the second week with one case developing two digits at the same time that the host anterior limb did so (see Text-fig. 5). Examination of the material sectioned at the end of the first week showed an early cartilage cell orientation in the deep portion of the graft, that is, the somite region, and a large more superficial un- orientated basiphilic cell mass extending into the projection. At two weeks, a long

TEXT-FIG. 4. A section through the ventral flanko f an A. punctatum host at 2 weeks after transplantation of Stage 35 punctatum limb cell mass plus epibrachial somite tissue. The girdle-like element (G) lies internally against the peritoneum and the articulating humerus-like element (H) plus associated potential myoblasts continued into the projection (LBE-23). cartilage girdle, with perichondrium and matrix, had developed in the transplanted somite region and a cartilage piece (humerus) was present in the more superficial basiphilic cell mass which articulated with the girdle and continued into the projection (see Text-fig. 4). Material sectioned in the third week showed the latter chondrified further into the projection and also indicated the presence of myoblasts in the girdle-humerus region of the transplant. Taricha torosa transplant to Taricha torosa hosts (2 cases). Again, projections developed in the first week and elongated in the second week. In the sectioned material it could be seen that early cartilage differentiation had occurred in the somite tissue and a concentric cell orientation in the limb cell mass superficial to the above indicated early humerus differentiation. An oriented acidophilic cell group immediately superficial to the ventral end of the girdle element appeared to be myoblastic. No pronephric tissue was found and no mesentery was observed. 332 CYRIL V. FINNEGAN Thus, it would appear that with the addition of the epibrachial somite ventral region to the limb bud transplant there follows limb cell mass retention, elongation (distal growth of the cell mass) and proximal histogenesis (of girdle- humerus tissue) in the ventral hypomere environment. The girdle element is developed, at least in part, from the added somite tissue and apparently consists of the central articulating and more dorsal portions of the shoulder girdle.

Limb bud plus dorsal ectoderm as the transplant In an attempt to increase the amount of dorsal tissue in Stage 35 transplants without adding possible contributing or inductive material, ectoderm lying dorsal to the dorsal limb bud was included in them. First to be considered are those cases in which the donor ectoderm lying between the limb bud and the lateral line, and with no underlying tissue clinging to it, was included in the transplant.

TEXT-FIG. 5. A lateral view of an A.punctatum larva at 2 weeks after transplantation of stage 35 punctatum limb cell mass and epibrachial somite to the ventral flank. A two-digit element was developed from the graft.

Two cases were prepared with Taricha torosa tissue as transplant to T. torosa hosts and two cases with A. punctatum tissue as transplant to A. punctatum hosts. In all cases a projection developed during the first week and appeared to regress in the second week. When examined histologically, the sectioned material showed that a larger basiphilic cell mass was present than had been observed in the limb bud transplants and there was indication of an orientation (whorl) on the central cells of this mass. No pronephric differentiation and no mesentery were observed. In other cases of this series, the donor ectoderm between the limb bud and the dorsal somite, to which a small amount of superficial ventral somite was clinging, was included in the transplant. Again, two cases were prepared with T. torosa tissue as transplant to T. torosa hosts and two cases with A. punctatum tissue as transplant to A. punctatum hosts. The projection, developed in all cases, elongated in the second week with the distal portion elongating further and becoming broader in the third week, though no digits were developed. In the sectioned material a girdle element was found deep in the graft region and there were myoblastic cells immediately superficial to it and its associated humerus-like element. HISTOGENESIS OF LIMB 333 This humerus continued into the projection for some distance as a central cartilage rod surrounded by mesenchyme.

Heterotopic transplantation to a dorsal (epimere) site In these experiments the Stage 35 donor anterior limb buds were placed on the dorsal flank of the host animal with the limb mesoderm in contact with the superficial surface of the somite. Transplantations were made to the more dorsal and to the more ventral region of trunk somites 8 to 10 in an attempt to compare the efficacy of these two sites in supporting limb histogenesis. Taricha torosa was the host in all cases. Limb transplants of T. torosa donors placed in the dorsal somite region (3 cases) produced, in the first week, a projection which was sustained into the third week but which did not appear to elongate further. Limb transplants of A. gracile donors to the dorsal somite site (5 cases) developed a paddle-shaped projection in the first week and indicated some elongation by the end of the second

TABLE 2 Limb histogenesis in dorsal sites Procedure Histogenesis Culture time Donor-host (weeks) Pronephros Cartilage Muscle Dermis I. T. torosa-T. torosa: (1) Dorsal somite 3 — + + +* — + (2) Ventral somite 3+ — + + + +* + II. A. gracile-T. torosa: (1) Dorsal somite 2 — + — + (2) Ventral somite 2 — + + + + * Cartilage and muscle symbols as in Table 1. week. In the cases sectioned at the end of the first week little or no cell orientation in the large basiphilic cell mass was visible while the cases observed in the third week showed a cartilage-like cell orientation {A. gracile tissue) or a large cartilage (T. torosa tissue) present in the dorsal somite region and in intimate contact with the superficial face of the somite. There was usually some indication of cell orientation extending from the above cartilage (girdle) toward and even into the projection. Otherwise unoriented basiphilic cells constituted the internal portion of the projection. When the transplant was placed in the ventral somite region (T. torosa limb in 3 cases and A. gracile limb in 6 cases) a projection developed in the first week and a definite elongation occurred late in the first week or in the second week. The outgrowth, with an internal rod-like structure, continued to elongate into the 334 CYRIL V. FINNEGAN third week. Cases sectioned at the end of the first week showed cartilage-like cell orientation in the girdle area and in the projection, thus evidencing a more distal histogenesis than had been observed in the dorsal somite site in the same culture time. Sections of cases taken from the second week showed definite girdle cartilage immediately superficial to the host's somite tissue and a second cartilage element (humerus) oriented perpendicular to the above and continuing externally into the projection. An oriented, acidophilic cell group was present superficial to the more ventral aspect of the girdle. The cases sectioned in the third week demonstrated the girdle-humerus cartilage with a more definite myoblastic tissue superficial to the girdle and the ventral somite tissue. The humerus cartilage accompanied by a surrounding basiphilic cell group continued some distance externally into the projection. No pronephric differentiation was observed in either of the transplantation sites on the T. torosa hosts.

DISCUSSION Wilde (1950) has described the development of Ambystoma punctatum limb buds explanted in a nutrient medium. If the ventral hypomere environment were a similarly neutral environment, one would expect the older (Stage 35) limb transplants of the present experiments to elongate and to differentiate the shoulder girdle-humerus portion of the limb. In fact, that result was never fully realized and much less limb development actually occurred on the ventral flank than would have been predicted from the developmental age of the donor. Wilde (1950) had emphasized the dependency of histogenesis on the length of culture but the limb histogenesis observed in the ventral flank environment was less than that described by him for a shorter in vitro culture period. Thus, the ventral hypomere environment is not a limb-supporting transplantation site since it does not allow transplanted limb cell mass differentiation even to the point of limb development that should be present as a result of its developmental association prior to transplantation. On the other hand, in all the experimental cases where a portion of the epibrachial somite tissue was placed in the immediate vicinity of the limb cell mass transplanted to the ventral hypomere, the limb histogenesis and growth observed were at least that to be expected from the Stage 35 transplants. This ability to support limb histogenesis and growth is demonstrated by the entire superficial portion of the trunk somites, but more histogenesis and growth is obtained when the limb cell mass is associated with the ventral somite. Also, results obtained when the transplanted tissue mass was increased by adding dorsal ectoderm indicated that the limb cell mass does not show the expected limb histogenesis unless the added tissue is somite mesoderm. In all cases limb cell mass transplanted to the ventral flank of Taricha torosa differentiated pronephric kidney tissue in the deep transplant-host hypomere region. Since nephric differentiation did not occur in limb transplants to Amby- HISTOGENESIS OF LIMB 335 stoma punctatumhosts it appears that this differentiation is a response to local influences of the Taricha torosa ventral hypomere. The reduced limb development associated with this pronephric kidney differentiation would seem to indicate that a limb cell mass is retained, or aggregated, near the pronephros but that its continued development is not necessarily promoted by the pronephric tissue, as had been suggested by Muchmore (1957). In a recent publication (Finnegan, 1962) results were reported which indicated that projections developed when undifferentiated hypomere cells were aggregated in the vicinity of relatively undifferentiated somite tissue, such as exists in the superficial regions of the torosa trunk somite (Finnegan, 1961). The ventral hypomeric mesoderm seemed to lose the ability to respond with limb histogenesis much sooner than did the more dorsal hypomere. Taken with the present results this would seem to indicate that, while the ventral hypomere of torosa loses its competence to respond to any possible chondrogenic influence from the limb cell mass, it exerts an influence toward, and is competent to demonstrate, nephric differentiation. On the other hand the lateral somite tissue retains its competence to respond to a chondrogenic influence for some time (Holtzer & Detwiler, 1953). In the present experiments it would appear that this somite ability to chondrogenesis, which results in the development of a girdle element, initiates the proximo-distal differentiation and growth (elongation) of the limb. The associated limb cell mass then differentiates, that is, chondrogenesis occurs in a specific association with the girdle element. These results, and those of the previous report (Finnegan, 1962), lend themselves to the interpretation that the aggregated limb cell mass, whose aggregation may be in response to adjacent unrelated differentiation, may influence juxtaposed undifferentiated and competent cells to chondrogenesis and that this event then stimulates the differentiation and growth of the limb cell mass iself. In this respect, Nassonov (1936), Goss (1956) and Rubin (1960) have called attention to the stimulatory capacity of implanted cartilage in the production of regenerating, or supernumerary, limbs and Thornton (1938) and Faber (1960) have concluded that the development of the more proximal structures in regeneration is the result of local stump influences on the adjacent dependent histogenesis of the blastema tissues. Recently Pietsch (1961) has demonstrated such dependent chondrogenesis in blastema tissue. Harrison (1921) pointed out that the differentiation of the limb may occur under the direct influence of the tissues of its environment and Amano (1960) concluded that the epibrachial somite region will influence any portion of the neurula hypomere of Urodeles to form limb if the two are brought into contact. Detwiler (1918) and Schwind (1932) have given evidence of the nearly mosaic character of the epibrachial somite portion of the normal girdle (the suprascapular) noting that it developed in situ in the complete absence of the remaining girdle-limb material. It is possible that in the ventral transplantation of limb and epibrachial somite in the present experiments this determined portion of the girdle initiated 336 CYRIL V. FINNEGAN chondrogenesis and the associated limb cell mass was competent to respond. Also, it may well be that because of the chondrogenic competence of the superficial trunk somite tissue, the lateral flank, as pointed out by Amano (1960), can- not be considered a neutral environment for limb development.

SUMMARY 1. The prospective limb portion of the limb disc of Ambystoma punctatum and Taricha torosa tail-bud stages was transplanted homoplastically and hetero- plastically to the ventral hypomere region of similar-aged host animals. The transplants were observed for growth (elongation) and for histogenesis of limb elements and in all cases less limb development was seen, both macroscopically and histologically, than was to be expected from the developmental age of the donors. 2. In a parallel series of experiments a small amount of epibrachial somite tissue was transplanted with the limb cell mass to the ventral hypomere region. The limb histogenesis and growth were enhanced to a considerable degree. It was seen that chondrogenesis (of a girdle) occurred in the somite tissue and the limb cell mass seemed to demonstrate histogenesis (proximally) and elongation (distally) in relation to that development. 3. The limb cell mass was transplanted to various superficial sites on the non- limb trunk somites and it was observed that more advanced growth and histogenesis were obtained when the transplant was associated with the ventral somite region. 4. Pronephric tissue was developed in all cases where limb cell mass was transplanted to the ventral hypomere region of Taricha torosa hosts. When the amount of dorsal tissue was increased, by adding dorsal ectoderm and/or epibrachial somite to the limb cell mass, no pronephric tissue developed in the graft area. No nephric differentiation was seen with similar transplants to Ambystoma punctatum hosts. 5. The results are considered as indicative of a somite-dependent histogenesis in the development of the proximal portion of the transplanted limb.

RESUME Observations sur Us conditions dont depend Vhistogenese du membre d'Urodele au cows de son developpement 1. On a pratique la transplantation homoplastique et heteroplastique de la partie presomptive du membre de l'ebauche correspondante dans la region ventrale de l'hypomere d'embryons du meme age, aux stades du bourgeon caudal, chez Ambystoma punctatum et Taricha torosa. On a observe la croissance (elongation) et l'histogenese des elements du membre dans les transplantats, et dans tous les cas on a observe un developpement du membre moindre que celui qu'on pou- vait attendre d'apres le stade de developpement du donneur, a la fois macro- scopiquement et histologiquement. HISTOGENESIS OF LIMB 337 2. Dans une serie parallele d'experiences, on a transplants une petite quantite de tissu somitique epibrachial avec la masse cellulaire du membre dans la region ventrale de Phypomere. L'histogenese et la croissance du membre se sont accrues a un degre considerable. On a vu que la chondrogenese (d'une epaule) est sur- venue dans le tissu somitique, et la masse cellulaire du membre a paru montrer une histogenese (proximalement) et une elongation (distalement) en relation avec ce developpement. 3. La masse cellulaire du membre a ete transplantee dans divers emplacements superficiels sur des somites troncaux sans rapport avec les membres, et on a observe qu'une croissance et une histogenese plus avancees sont obtenues quand le transplantat a ete associe avec la region ventrale des somites. 4. Du tissu pronephretique s'est developpe dans tous les cas ou la masse cellulaire du membre a ete transplantee dans la region de l'hypomere ventral des notes de l'espece Taricha torosa. Quand la quantite de tissu dorsal a ete accrue, par adjonction a la masse cellulaire du membre dorsal et (ou) de somite epibrachial, il ne s'est pas developpe de tissu pronephretique dans la zone de greffe. On n'a pas observe de differentiation nephretique avec des transplantats semblables sur des notes de l'espece Ambystoma punctatum. 5. On considere que les resultats obtenus indiquent que l'histogenese de la partie proximale du membre transplants depend des somites.

ACKNOWLEDGEMENTS This investigation was supported in part by grants from the U.S. National Institute of Health (RG-6178C2) and the National Research Council of Canada.

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{Manuscript received 1st October 1962)