Paper - An experimental study of the development of the amphibian cloaca
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An Experimental Study of the Development of the Amphibian Cloaca
AN EXPERIMENTAL STUDY OF THE DEVELOPMENT
OF THE AMPHIBIAN CLOACA /
By R. J. O°>CONNOR Department of Anatomy, University College, London
SPEMANN (1988) and others have shown that in the development of the amphibian optic lens there is a combination of the self-differentiating power of the lens ectoderm with the lens-inducing power of the optic cup. Further, in different amphibian species it has been shown that there is a considerable variation in the relative importance of these two developmental factors. Recent work on the development of the mesonephros (Waddington, 1937; O’Connor, 1939) has shown that in the development of this structure there is a similar co-operation of developmental forces. The development of the mesonephros is dependent partly on the self-differentiating power of the mesonephric cells and partly on a stimulating influence due to the contact of these cells with the pronephric duct. Here, as in the case of the optic lens, there is a variation in different species of the relative importance of the self-differentiating power and the stimulating effect of the adjacent structure.
The two structures concerned in the development of the optic lens on the one hand and the mesonephros on the other are in a close functional and topographical relationship, and the possibility is suggested that when any two structures are in such a relationship they may have a similar interaction in development. Such a relationship exists between the pronephric duct and the cloaca in Amphibia. In Urodeles the pronephric duct is formed by a caudal growth of the pronephric rudiment which appears in the third to seventh segments. The pronephric duct meets the cloaca at the dorsal aspect of its cranial wall and there forms a patent union. At the point of union there is a thinning of the cloacal wall, giving rise to a diverticulum of the cloacal cavity on either side. Subsequently these diverticula become more pronounced, involving both the cloacal wall and the cloacal cavity, and receive the pronephric ducts at their apices. The formation of these diverticula would thus appear to be closely related to the union of the pronephric ducts with the cloaca and experiments were performed to investigate the development of the cloaca in the absence of one or both pronephric ducts and particularly to investigate the development of the cloacal diverticula in such circumstances.
Anatomy LXXIVv 20 302 R. J. O Connor
MATERIAL AND TECHNIQUE
The material used consisted of the embryos of Triton taentatus, Pleurodeles waltlit and a species of Amblystoma the specific name of which is not certain.
Experiments performed were of two main types:
(a) Localized excisions and transplantations of tissues. In this case the technique used has been described in a previous paper (O’Connor, 1939). In these experiments the commonest procedure was to prevent the caudal growth of the pronephric rudiment that gives rise to the pronephric duct. A transplant was placed at the caudal limit of the pronephric rudiment soon after it appeared and when it was confined to the third to seventh segments. The situation of the transplant is indicated in Text-fig. 1. It consisted of any convenient tissue taken from another embryo.
(6b) Vital staining experiments. Nile blue sulphate was applied to localized regions of the embryo by means of small portions of agar soaked in the dye. The method of application is described by Stone (1931).
Text-fig. 1. Amblystoma at the stage of the first appearance of the pronephric rudiment. The shaded area indicates the site of the transplant to prevent the formation of the pronephric duct.
DEVELOPMENT OF THE CLOACA IN THE ABSENCE OF ONE PRONEPHRIC DUCT
The successful placing of a transplant as described above resulted in the non-formation of the pronephric duct on the operated side and consequently the development of the cloaca could be followed in the absence of one pronephric duct. In all such experiments the cloaca developed as a symmetrical structure. Thinning of the cloacal wall took place at the same site and at the same time as on the normal side, and the formation of the diverticula of the cloacal wall was equal on the two sides (Pl. I, fig. 1). A similar result was obtained in all of the three species examined. Therefore, in the absence of the pronephric duct on one side, the cloaca undergoes a normal development, and in all three species examined the formation of the cloacal diverticula is independent of the pronephric duct on the same side.
DEVELOPMENT OF THE CLOACA IN THE ABSENCE OF BOTH PRONEPHRIC DUCTS The possibility exists, however, that in the absence of one pronephric duct the symmetrical development of cloacal diverticula may be due to some influence exerted on both sides of the cloaca by the remaining pronephric Study of the development of the amphibian cloaca 303
duct. Although this would seem somewhat unlikely its exclusion is possible by examining the development of the cloaca in the absence of both pronephric ducts. Experiments for this purpose were most conveniently performed by taking embryos at the stage of the first appearance of the pronephric rudiment and dividing them by a dorso-ventral cut in such a way that the caudal portion contained none of the pronephric rudiment. The two portions were then reunited with the caudal half reversed as is indicated in Text-fig. 2. Healing was usually successful, and the abnormal arrangement of the structures in the embryo prevented the extension of the pronephric ducts into the caudal portion of the embryo so that the cloaca underwent subsequent development in the absence of both pronephric ducts.
In all cases and in all species examined the cloaca underwent a normal development and cloacal diverticula were formed as in normal embryos (Pl. I, fig. 2). It is certain therefore that, in the three species of Amphibia examined, the development of the cloaca is independent of the pronephric ducts and the cloacal diverticula to which the ducts join are formed independently of them. These observations suggest that in all amphibian species
O po Text-fig. 2. Division and reunion of an embryo to prevent the formation of both pronephric ducts in the caudal portion.
these cloacal diverticula are formed independently of the pronephric ducts. However, such a generalization should be conditional on similar findings being recorded for other species, especially as the work of Boyden (1924) shows that differentiation of the middle chamber of the avian cloaca depends on the contact of the pronephric or Wolffian ducts.
ROLE OF THE CLOACAL DIVERTICULA IN THE UNION OF THE PRONEPHRIC DUCT AND THE CLOACA
The modifications of the cloacal wall that lead to the formation of diverticula begin at the time the pronephric duct meets the cloaca, and it would thus appear that the formation of diverticula is essential to the patent union of the pronephric duct and the cloaca. Experiments were therefore performed to investigate in more detail the manner of union of the cloaca and pronephric duct and to investigate the role of the diverticula in the communication of the two structures. These investigations have been planned to answer the following two questions:
(a) Can the pronephric duct form a patent union with other structures than the cloaca?
20-2 304 R. J. O'Connor
(b) Can the pronephric duct form a patent union with any other portion of the cloacal wall than the site of formation of the cloacal diverticula?
(a) Union of the pronephric duct with structures other than the cloaca. Many experiments were performed of the type indicated in Text-fig. 1 in order to prevent the caudal growth of the pronephric duct. These resulted in certain modifications of the pronephric duct immediately cranial to the transplant. In some cases these modifications consisted of a patent union of the pronephric duct either with the exterior or with the gut. These abnormal unions have been described and figured in a previous paper (O’Connor, 1939). However, the occurrence of such communications was unusual and they were only found in 4 % of 150 cases examined; thus they do not indicate more than an extremely limited capacity of the pronephric duct to unite with abnormal structures. These results are supported by an experiment in which an embryo of Triton taeniatus was divided as shown in Text-fig. 2. The caudal portion was removed and the raw surface of the cranial half covered with a flap of ectoderm. As the pronephric ducts grew caudally they came up against the ectoderm and could grow no further caudally. The embryo was fixed 7 days after the operation and sectioned longitudinally. A section passing along the left pronephric duct is shown in PI. I, fig. 3, where it is seen that the pronephric duct comes up against the ectoderm but has made no communication with it. This observation was confirmed by examination of all the sections, when it was seen that the same findings were present in the case of the duct of the opposite side. These results, then, confirm the conclusion that the pronephric duct has not any great power to unite with structures other than the cloaca.
(b) Union of the pronephric duct with the cloacal wall. In this investigation experiments were performed where the cloaca and surrounding tissues from one embryo were transplanted into another in the situation and at the stage indicated in Text-fig. 1. As the pronephric duct of the host embryo grew caudally on the operated side it came into contact with the transplant and its further growth was prevented. In some cases the surrounding tissues transplanted with the cloaca prevented contact between the duct and the cloacal wall, but in twenty-five cases such a contact between the two structures resulted, these consisting of twelve cases in Amblystoma and thirteen in Triton taeniatus. In ten cases there was a patent union between the two structures, while in the remaining fifteen there was merely a contact between the two structures and the pronephric duct did not open into the transplanted cloaca. These cases were examined in detail, and since the findings were similar they can be illustrated by one case from each group. Where a patent union formed it was found that the transplanted cloaca formed diverticula and that the pronephric duct opened into one of these diverticula. This is illustrated by a series of camera lucida drawings of sections showing the entrance of the pronephric duct into a transplanted cloaca (Text-fig. 3). On the other hand where there was contact between the two structures without a patent union, a series of camera lucida drawings shows that the pronephric duct came into contact Study of the development of the amphibian cloaca 305
with a portion of the cloacal wall where a diverticulum had not formed (Text-fig. 4). :
These results show that, besides only having a very limited ability to unite with structures other than the cloaca, the pronephric duct only unites with the cloaca at a specific locality in the cloacal wall where certain modifications take place leading to the formation of cloacal diverticula.
Text-fig. 3. Amblystoma, camera lucida drawings. The cloaca from another embryo was transplanted into the left flank 15 days previously. The drawings represent the relationship of the transplanted cloaca and left pronephric duct as seen in consecutive transverse sections cut at 10yu. Drawing (a) represents the most cranial section. The pronephric duct (stippled) approaches the transplanted cloaca (shaded) and opens into it via a cloacal diverticulum (div.).
THE GROWTH OF THE PRONEPHRIC DUCT SUBSEQUENT TO ITS UNION WITH THE CLOACA
Many experiments in this and other papers have been recorded where the caudal growth of the pronephric rudiment has been prevented. In experiments allowed to develop up to 15 days after the operation the result has been constant—there was no formation of the pronephric duct caudal to the transplant. However, when development was followed for longer periods, it was found that, although the pronephric duct ceased at the transplant and was not seen for a considerable distance caudal to it, there was a structure attached to the cloaca on the operated side, which in its attachment to the cloaca, its position, and histological structure, is identical with the pronephric duct on the non-operated side (see O’Connor, 1939, Text-fig. 2 and Pl. I, fig. 4). The presence of this structure was confined to Amblystoma and was not found in a similar series of experiments on Pleurodeles waltlit and Triton taeniatus. In 306 R. J. O'Connor
Amblystoma this structure was not found before the eighteenth day after operation (representing 22 days’ total development) but it became increasingly more frequent in embryos reared for longer periods and was found in all of fifteen cases reared for more than 25 days after operation. The maximal length found in this structure was about 650 p, and this, as in all cases, represented less than 20 % of the total length of the pronephric duct on the non-operated side. This tag of tissue could not have been derived from the pronephric rudiment, and its position, as well as its time of appearance, makes it extremely unlikely that it is derived from the mesodermal structures at the hinder end of the embryo. It does not appear until the eighteenth day or later, when the mesodermal tissues in the vicinity have already differentiated into muscular and fibrous tissue. The transformation of these into a duct-like structure is searcely possible, and it is therefore concluded that the cloaca is the most likely source of this structure.
Text-fig. 4. Amblystoma, camera lucida drawings of same type of experiment as in Text-fig. 3. The pronephric duct (stippled) approaches the transplanted cloaca (shaded) and comes into contact with a region of the wall where no diverticulum has formed. There is contact between the two structures but not a patent union. (div. cloacal diverticulum.)
Attempts were made to test this opinion by the use of vital staining methods. The object of these experiments was to stain the cloaca with Nile blue sulphate prior to the union of the pronephric ducts and to see if subsequently any of the dye placed in the cloaca extended into the hinder ends of the pronephric ducts. Embryos of Amblystoma were stained in the earliest neurula stage and the stain applied to the ventral half of the blastopore and the adjacent area as indicated in Text-fig. 5. In the tail-bud stage and later, the dye could be seen in the cloaca, in the region of the tail, and in the myotomes. In sections the dye appeared as blue granules and its precise distribution could be determined. Following this staining procedure a series.of embryos was examined soon after the time of union of the pronephric duct with the cloaca. Dye granules could be found throughout the wall of the cloaca as well as in the Study of the development of the amphibian cloaca 307
caudal somites and adjacent mesoderm. None however could be found in the pronephric ducts. Other embryos were allowed to develop for longer periods, but this led to difficulties in examination as the staining decreased considerably in intensity. Its accurate distribution was thus difficult to determine and negative observations could not be relied upon. However, two cases were found where after twelve days’ total development dye was found in the hinder end of the pronephric ducts extending cranially for about 70u. It may be mentioned that Bijtel (1931) performed similar experiments and mentions one where dye was found in the pronephric ducts for a distance of about five somites from the cloaca, this being seen after about 12 days’ total development. Although these vital staining experiments cannot be regarded as conclusive, they are in accordance with the findings in experiments where the caudal growth of the pronephric rudiment is prevented. At the time of meeting of the pronephric duct and cloaca there is no formation of a duct-like tag of
Text-fig. 5. Amblystoma, early neurula stage. Stippled area indicates the region where the Nile blue sulphate was placed in staining experiments.
tissue, nor is there any extension of the dye into the pronephric duct from the stained cloaca. At later stages of development there is a correspondence between the formation of a tag of duct-like tissue and the presence of dye extending from the cloaca into the hinder end of the pronephric ducts. Therefore in Amblystoma it appears justifiable to conclude that, although the pronephric duct is entirely formed by the caudal growth of the pronephric rudiment, there is some evidence to warrant the opinion that the cloaca may subsequently add to its caudal portion. SUMMARY
1. At the site of union between the cloaca and pronephric duct in Amphibia the cloaca forms diverticula. In Pleurodeles waltlii, Triton taeniatus and a species of Amblystoma, these diverticula are formed independently of the pronephric duct and can be demonstrated in its absence.
2. ‘The pronephric duct has been shown only to unite with the cloaca at the site of formation of these diverticula.
3. In the species of Amblystoma investigated there is evidence to show that, although the pronephric duct is primarily formed by the caudal extension of the pronephric rudiment, it may subsequently receive a contribution to its hinder end from the cloaca. 308 . R. J. O’ Connor
The spawn of Amblystoma was kindly supplied by Miss R. M. Renton of the Zoological Department, University College, London. This paper is based on a portion of a thesis accepted for the degree of M.D. by the University of Adelaide, South Australia.
BrgTEL, J. (1931). Roux Arch. Eniw. Mech. Organ. 125, 448.
Boypen, E. A. (1924). J. exp. Zool. 40, 437.
O’Connor, R. J. (1939). J. Anat., Lond., 74, 34.
SPEMANN, H. (1938). Embryonic Induction and Development. Yale Univ. Press. Stongz, L. 8. (1931). Anat. Rec. 51, 267.
WapvpinaTon, C. H. (1937). J. exp. Biol. 15, 371.
EXPLANATION OF PLATE I
Fig. 1. Amblystoma, transverse section through the cloaca 15 days after operation to prevent the formation of the left pronephric duct. Symmetrical development of the cloaca and cloacal diverticula. (r.p. right pronephric duct; d, cloacal diverticulum.) x 120. .
Fig. 2. Triton taentatus, transverse section through the cloaca 12 days after operation as indicated in Text-fig. 2. Normal development of the cloaca in the absence of both pronephric ducts. (d, cloacal diverticulum.) x 140.
Fig. 3. Triton taeniatus, longitudinal section 7 days after operation as described in text. The pronephric duct comes up against the ectoderm but does not unite with it. (p, pronephric duct.) x 120.
Fig. 4. Amblystoma, transverse section through the cloaca 40, cranial to the entrance of the pronephric ducts. 9 days previously embryo stained with Nile blue sulphate as indicated in Text-fig. 5. Nile blue sulphate seen as granules in the cloacal wall (c) and in the right pronephric duct (p). x 300.