Paper - Experiments on the development of the amphibian mesonephros (1939)

From Embryology

O'Connor RJ. Experiments on the development of the amphibian mesonephros. (1939) J Anat. 74: 34-44. PMID 17104798

Experiments on the development of the amphibian mesonephros

By R. J. O'Connor

Department of Anatomy, University College, London


In Amblystoma the normal development of the nephric system has been described by Hall (1904) and Field (1891). The pronephric rudiment appears soon after the closure of the neural folds and consists of a collection of cells derived from the mesoderm of the third to seventh body segments, the rudiment lying at the ventro-lateral border of the somites. By vital staining methods (O’Connor, 1938) it has been shown that the portion of the pronephric rudiment derived from the third and fourth segments forms the pronephros. The more caudal portion of the rudiment (derived from segments five to seven) undergoes an independent growth in a caudal direction and unites with the cloaca, thus forming the pronephric duct. At first both the original rudiment and its caudal extension lie at the ventro-lateral border of the somites immediately beneath the ectoderm and so give rise to a swelling visible from the exterior.


Hall’s investigations on the development of the mesonephros show that it appears some little time after the pronephric duct has united with the cloaca, and its differentiation begins with the demarcation of clumps of cells from the surrounding mesoderm in the ninth to eighteenth body segments. These clumps of cells lie on the medial side of the pronephric duct and are arranged segmentally in relation to the posterior quarter of each myotome. In subsequent development the pronephric duct and these clumps of mesonephric cells undergo an inward migration and come to lie close to the mid-line immediately ventral to the notochord. During this migration, however, the close contact between the mesonephric cells and the pronephric duct is maintained. The mesonephric units differentiate into simple tubules which communicate with the pronephric duct which then becomes the draining duct of both the pronephros and mesonephros. The mesonephric tubules become more and more coiled, eventually giving rise to the complicated tubular structure of the mesonephros. As differentiation proceeds the original segmental arrangement of the mesonephric units is lost. Although the mesonephric units appear simultaneously, differentiation proceeds in a cephalo-caudal direction; thus in Amblystoma, after about 30 days’ development, it is possible to illustrate the main stages in the development of the mesonephros. In a section through the caudal end of the mesonephros differentiation of the mesonephric units into tubules has not yet begun and the mesonephros is represented by clumps of cells in contact with the medial aspect of the pronephric duct (PI. I, fig. 1). In the middle of the mesonephric region tubules have begun to form, and this is about the stage at which the mesonephric tubules communicate with the pronephric duct (PI. I, fig. 2). At the anterior end of the mesonephros, tubule formation is well advanced (PI. I, fig. 3).


Thus it is seen that throughout development there is a close functional and topographical relationship between the mesonephros and pronephric duct; consequently the question arises as to whether there is any influence exerted by one structure on the other during development. In order to investigate this question, experiments were devised in which the mesonephros was allowed to undergo its development in the absence of the pronephric duct on the same side. In this way it was possible to test the developmental potencies of the mesonephros in the absence of the pronephric duct and thus gain information in regard to the influence of the pronephric duct in the development of the mesonephros. This type of experiment consisted in preventing the formation of the pronephric duct by obstructing the caudal extension of the pronephric rudiment that gives rise to it.

Material and Methods

The majority of experiments were performed on a species of Amblystoma which is kept in the Zoological Department of University College, London. In the past this species has been referred to as A. tigrinum, but recently doubts have arisen as to the correctness of this name; in this communication, therefore, it will be referred to as Amblystoma without defining the species. The caudal growth of the pronephric rudiment was prevented by a suitably placed transplant at the stage when the pronephric rudiment first became visible as an external swelling. The transplant was then placed at the caudal limit of the swelling and caudal extension thus prevented. Since the transplanted tissue merely acted as a mechanical obstruction, CSO the type of tissue transplanted was not of importance, and neural plate, limb bud, eye vesicle, pronephros and cloaca were used for this purpose. In Amblystoma the appro- O priate stage for the experiment is stage 22 Text-fig. 1. Amblystoma, stage 22. Position (Harrison),! in which the pronephric rudi- of transplant to obstruct caudal ment gives rise to a swelling immediately growth of pronephric rudiment. ventral to the third to seventh somites. The transplant was placed immediately ventral to the seventh and eighth somites, and thus did not disturb the mesonephros which develops in the ninth to eighteenth segments (Text-fig. 1).

The technique of transplantation is well known and the method used was essentially that of Spemann (1920). However, certain modifications have proved useful. A preliminary sterilization of instruments and vessels limited operative mortality. No attempt was made to sterilize the water used, which was taken direct from the tap, but instruments and vessels were kept in 90% alcohol and washed in running tap water immediately before use. This represents a modification of sterilization by formalin vapour as used by Woerderman (1980). In some cases the glass needles of Spemann’s method were replaced by fine knives as described by Brandt (1937).

  • 1 Harrison’s numbered stages refer to the development of A. punctatum, but the development of the species of Amblystoma used is sufficiently similar to warrant their application here.


Results of Obstructing the Caudal Growth of the Pronephric Rudiment

In Text-fig. 2 are shown diagrammatically some of the results of blocking the caudal growth of the pronephric rudiment. The experiment consisted in transplanting the cloaca and surrounding tissues from a donor at stage 22 to a position immediately ventral to somites 7-8 on the left side of an embryo at thesame stage of development. The embryo was killed eighteen days later and examined by serial sections. The accompanying diagram was made by drawing the sections by means of a projection microscope, measuring the width of the structures as well as their distance from the mid-line and plotting these measurements to scale on squared paper. On the operated side the transplant is shown shaded, and it is seen that the pronephric duct ceases at the transplant and is not found immediately caudal to it. However, at the caudal end of the embryo there is a small tag of duct-like structure attached to the cloaca. In a section through this tag (PI. I, fig. 4) its structureis demonstrated and bothin position and histological appearance it resembles the pronephric duct. Although this was not a constant finding, it does suggest the possibility of a contribution from the cloaca to the hindermost portion of the pronephric duct.

The pronephros in this experiment, and in all other similar experiments, shows no modification in development although the natural outlet for its secretion is blocked. A section taken through the pronephros (Pl. I, fig. 5) shows neither atrophy nor dilatation of tubules. The obstructed pronephric duct, on the other hand, undergoes a variety of modifications when the caudal growth of the pronephric rudiment is prevented, and these modifications are illustrated by sections taken from several different experiments. Immediately cranial to the obstruction the pronephric duct may undergo considerable hypertrophy and dilatation (PI. II, fig. 1), while in (PI. II, fig. 2) is shown a communication with an artificially produced diverticulum of the gut. In addition there have been observed communications with the exterior (Pl. II, fig. 8) and with a cloaca transplanted into the flank of the embryo (Pl. II, fig. 4).



Text-fig. 2. Nephric system of Amblystoma. 18 days previously caudal growth of pronephric rudiment obstructed by a transplant. Transplant shown shaded. Levels of P1. I, fig. 4, and Pl. I, fig. 5, indicated.


In all such experiments the development of the host cloaca is unaffected by the absence of the pronephric duct on one side and PI. II, fig. 5, illustrates the symmetry of the cloaca in these circumstances. Particular attention is drawn to the diverticula present on either side. The pronephric duct normally joins these, but even in the absence of the duct the diverticulum forms normally. Its formation is apparently independent of the presence or absence of the pronephric duct.

The Developmental Potencies of the Mesonephros in Amblystoma in The Absence of the Pronephric Duct

The main purpose of the experiments. was to observe the development of the mesonephros in the absence of the pronephric duct. After obstruction of the caudal growth of the pronephric rudiment, embryos were examined at various stages of mesonephric development and a comparison of normal development in the presence of the duct and abnormal development in its absence made at different levels in the mesonephros. Owing to the cephalocaudal differentiation of the mesonephros it was possible to make this comparison at different stages of mesonephric development in a single embryo. To illustrate the findings, sections are taken from an embryo where the caudal growth of the pronephric rudiment on the left side was obstructed by a transplant at stage 22, 25 days before the sections were made. PI. ITI, fig. 1 represents a section through the caudal end of the mesonephros and shows the absence of the pronephric duct on the operated side. The development of the mesonephros on the two sides shows no prominent differences—both are in the form of clumps of cells where tubule formation has not yet begun. Nevertheless, examination with an oil-immersion lens shows certain changes in the mesonephric cells of the operated side suggestive of degeneration. Pl. III, fig. 2 is taken through the middle of the mesonephric region of the same embryo, and the normal side shows the beginning of tubule formation. On the operated side, however, there has been no similar advance and the mesonephric cells are still in the form of clumps of cells. A section through the anterior end of the mesonephros (PI. ITI, fig. 3) shows an accentuation of this difference. There is an advanced formation of tubules on the normal, unoperated side, but on the operated side there is still no advance beyond clumps of mesonephric cells in which degeneration can be seen. 38 R. J. O'Connor It is concluded from these observations that, in the absence of the pronephric duct in the species of Amblystoma used, the mesonephric cells can differentiate themselves from the surrounding mesoderm and form histologically distinguishable clumps of cells. However, in these circumstances they proceed no further in their development and, in the absence of the pronephric duct, do not form tubules.

RESULTS OF RESECTION OF THE PRONEPHRIC DUCT IN AMBLYSTOMA

In the experiments just described the pronephric rudiment is obstructed in its caudal growth so that the pronephric duct does not form caudal to the eighth somite and therefore does not reach the mesonephric region. That is to say, at no stage is there contact between the pronephric duct and the cells of the mesonephros. Another series of experiments were performed which had the object of testing the effect of a temporary contact between the pronephric duct and those cells which would later differentiate into the recognizable cells of the mesonephric units. These experiments were performed on Amblystoma at stage 34, which is the stage at which the caudal growth of the pronephric rudiment, forming the pronephric duct, has reached and united with the cloaca. In growing caudally, the pronephric duct grows past the cells of the mesonephros and herelies in contact with them. At the temperatures prevailing when the experiments were performed, forty-eight hours elapsed between the appearance of the pronephric rudiment and its union with the cloaca. Consequently at stage 34 the pronephric duct has been in contact with the cells of the mesonephros for a period not exceeding forty-eight hours.

At stage 34 the pronephric duct was resected in the region of somites nine to fourteen. The method of resection requires some comment, for Howland (1926) records that resection of the duct at this stage is usually followed by complete regeneration. This regeneration was prevented by leaving the wound open for some hours so as to delay healing. As a consequence of this operation the mesonephros in segments 9-14 underwent its development in the absence of the pronephric duct, but, in contrast to previous experiments, after having had a transient contact with it.

Embryos operated upon in this manner were reared for various periods before examination by serial sections. In the operated area it was found that the development of the mesonephros was retarded as compared with the corresponding level on the normal side, but in spite of this retardation the formation of tubules was observed. In an embryo sectioned 20 days after operation a section through the 11th somite (Pl. ITI, fig. 4) illustrates this point. On the operated side there is absence of the pronephric duct, but there is nevertheless definite evidence of tubule formation although it is not as advanced as on the non-operated side. This represents a striking contrast to the results seen in those experiments where the caudal growth of the pronephric rudiment was prevented. The formation of tubules after duct resection can proceed to the stage where they become functional, and distended by secretion which cannot escape owing to the absence of the pronephric duct (PI. IT], fig. 5).

These results show that contact between the pronephric duct and the mesonephric cells for less than forty-eight hours results in a definite developmental stimulus being imparted to the mesonephric cells which are thus enabled to form tubules. In the development of the mesonephros in the species of Amblystoma used two developmental factors can thus be recognized:

(a) A power of differentiation on the part of the mesonephric cells which is independent of the pronephric duct. This power is responsible for the demarcation of mesonephric cells from the surrounding mesoderm.

(b) A developmental stimulus due to the pronephric duct which is responsible for the formation of tubules from the mesonephric cells.

COMPARISON BETWEEN THE DEVELOPMENT OF THE MESONEPHROS AND THE OPTIC LENS

In the development of the amphibian optic lens many workers (see Spemann, 1988) have shown a co-operation of developmental forces. As a general conclusion it is stated that the self-differentiating power of the lens ectoderm co-operates with the lens-inducing power of the optic cup. This combination of developmental forces is referred to by German authors as “‘doppelte Sicherung”’ of which the accepted English translation is “double assurance”. In the development of the mesonephros in the species investigated, the above experiments show that there is a similar combination of developmental forces. First there is a power of differentiation of the mesonephric cells, and secondly there is a stimulating effect due to the contact of these cells with the pronephric duct. Thus it is justifiable to speak of “double assurance” in the case of the development of the mesonephros with the same meaning as in the development of the optic lens. Incidentally it may be pointed out that the term “double assurance” as usually employed is used in a different sense to that intended by the originator. It was originally introduced by Braus (1906) concerning the eruption of the fore-limb in Bombinator. This eruption takes place through the gill operculum, and in the eruption two forces were demonstrated by Braus. First, a force due to the expansion and growth of the underlying fore-limb, and secondly some property in the gill operculum, for the foramen through which the limb erupts will form even in the absence of the fore-limb. Here the term ‘“‘double assurance” implied the co-operation of two factors either of which would produce the same end-result. Spemann uses the term implying the combination of two forces both of which are necessary for complete development. The application of the term “double assurance” to the development of the mesonephros is in the sense of Spemann and not of Braus.

The term “double assurance” has been applied to the development of the mesonephros in the chick and in the anuran Rana fusca by.Waddington (1937). In both species he found that in the absence of the pronephric duct the mesonephros did not develop beyond clumps of cells. The findings in the Urodele, Amblystoma, are therefore similar to those in Rana fusca and in the chick. This similarity is noteworthy because in the chick the pronephros is a vestigial and non-functional structure, whereas in Amblystoma Howland (1921) has shown it to be functional and essential to the life of the embryo.

A comparison between the development of the optic lens and that of the mesonephros has further implications. When the development of the optic lens in different amphibian species is compared, it is found that there is a considerable variation in the relative importance of the inducing power of the optic cup and the self-differentiating power of the lens ectoderm. The expression of this variation is found in experiments in which the developmental potencies of the lens ectoderm is investigated after removal of the optic cup. This serves as a means of investigating the importance of the self-differentiating power of the lens ectoderm in the formation of the completed lens. All gradations are found between those species where, in the absence of the optic cup, there is no development of the lens at all, and those species where the development of the lens is almost complete in these circumstances. That is to say, on the one hand, there are species where lens development is largely dependent on the self-differentiating power of the lens ectoderm, and relatively independent of the inducing power of the optic cup, while on the other hand there are species where the position is reversed. Between these extremes are all intermediate stages. The similarity of the forces concerned in the development of the mesonephros suggests that a similar variation may exist in the relative importance of the self-differentiating power of the mesonephric cells and the stimulating effect due to the pronephric duct, and that this variation may be expressed in the degree of development undergone by the mesonephros of various species of Amphibia in the absence of the pronephric duct. Such information is available from the work of several authors, but it is obtained by a variety of methods and in making a comparison some caution must therefore be exercised. It has been shown above that even a transient contact between the mesonephric cells and the pronephric duct can impart a considerable developmental stimulus to the mesonephric cells. Consequently, in considering experimental results from this point of view only those results can be considered in which care has been taken to see that there is never any contact between the pronephric duct and the cells of the mesonephros. The methods employed can be classified as follows:

(a) Prevention of the caudal growth of the pronephric rudiment. To achieve this an obstructing transplant can be used, as in the present series of experiments, or the pronephric rudiment can be removed as soon as it appears and before it has extended caudally into the mesonephric region.

(b) Resection of the pronephrie duct. This method has been used by Muira (1930) and Shimasaki (1980) but their results are not relevant to the present discussion as the resection was carried out when the pronephric duct had grown into the mesonephric region and therefore had been in contact with the cells of the mesonephros.

(c) Transplantation of the presumptive mesonephros into heterotopic situations. Here the only results that can be considered are those in which the mesonephros was transplanted before the pronephric duct had reached the mesonephric region, so that the duct had not been in contact with the cells of the mesonephros. Also, it is necessary that the transplant should have been placed in such a situation that the host pronephric duct was not in contact with it.

The results from the literature that seem pertinent are placed in Table I, which indicates the method employed and includes the results found in the chick. The results obtained for Pleurodeles waltlii are from experiments performed by the present writer. Embryos of this species were taken at the stage of the first appearance of the pronephric rudiment, and on the left side the caudal growth of the rudiment was blocked as in Amblystoma. A section taken through the middle of the mesonephros 20 days later shows the result obtained. On the operated side, where the duct is absent there are no mesonephric cells at all (Pl. II, fig. 6), in contrast to Amblystoma where clumps of mesonephric cells were seen. By examination of all the sections of several embryos under similar circumstances it was possible to confirm that in P. waltlii, in the absence of the pronephric duct, the mesonephric cells do not appear at all.


Table I. The development of the mesonephros in the absence of the pronephric duct

Species Author Method used Result Chick Waddington (1937) Obstruction of duct Clumps of mesonephric cells Chick Griinwald (1937) Obstruction of duct Clumps of mesonephric cells Rana fusca Waddington (1937) Obstruction of duct Clumps of mesonephric cells Amblystoma maculatum Humphrey (1928) Transplantation Almost complete development of mesonephros Amblystoma Humphrey (1928) Transplantation Almost complete dejeffersonianum velopment of mesonephros Amblystoma (mainly Burns (1934) Removal of pronephric Occasionalmesonephric tigrinum) rudiment tubule Amblystoma punctatum Burns (1938) Removal of pronephric Some mesonephric turudiment bules Amblystoma ? species = Present paper Obstruction of duct Clumps of mesonephric cells Pleurodeles waltlii Present paper Obstruction of duct No appearance of mesonephros

Examination of this table shows that, in a series of Amphibia, in the absence of the pronephric duct the mesonephric cells undergo a variable development, ranging from no development at all in Pleurodeles waltlii to almost complete development in Amblystoma jeffersonianum and A. maculatum. In Pleurodeles waltlii, therefore, the development of the mesonephros is largely, if not entirely, dependent on the pronephric duct, and the independent power of differentiation of the mesonephric cells is negligible. On the other hand, in Amblystoma jeffersonianum and in A. maculatum the opposite is the case, and the self-differentiating power of the mesonephric cells is such that it can cause complete development in the absence of the pronephric duct. In species such as Rana fusca and the species of Amblystoma here investigated, an intermediate state of affairs is present.

It is finally concluded that, in the development of the amphibian mesonephros, the power of independent differentiation of the mesonephric cells co-operates with the stimulating influence of the pronephric duct, but in any one species the relative importance of these two factors cannot be predicted ; it can only be discovered by experiments on that particular species.

Summary

  1. The developmental potencies of the mesonephros in the absence of the pronephric duct have been tested in Amblystoma by obstructing the caudal growth of the duct. It is found that in the absence of the pronephric duct the mesonephros does not develop beyond the formation of clumps of cells, and tubule formation does not take place.
  2. By resection of the pronephric duct at appropriate stages of development the developmental potencies of the mesonephros were investigated after a contact of less than forty-eight hours between the cells of the future mesonephros and the pronephric duct. In these cases, in spite of the absence of the pronephric duct, in subsequent development the mesonephros forms tubules and undergoes a more or less complete, but retarded, development.
  3. It is therefore concluded that in the development of the amphibian mesonephros two factors operate:
    1. A power of differentiation of the mesonephric cells in virtue of which they become demarcated from the surrounding mesoderm.
    2. A stimulating influence on the part of the pronephric duct which is necessary for tubule formation.
  4. These two factors are compared with the factors operating in the development of the optic lens, and a “double assurance” is suggested for the development of the amphibian mesonephros.
  5. Similar experiments performed on Pleurodeles waltlii showed that, in the absence of the pronephric duct, mesonephric cells did not become demarcated from the surrounding mesoderm. Comparing these results with those in the literature, it is concluded that in the development of the amphibian mesonephros there is a variation in different species of the relative developmental importance of the differentiating power of the mesonephric cells and the stimulating influence of the pronephric duct.
  6. Certain other results of obstructing the caudal growth of the pronephric rudiment are recorded. The development of the pronephros is unaffected, and also that of the cloaca. There is some evidence to suggest that the cloaca may contribute to the formation of the extreme caudal end of the pronephric duct. The blocked pronephric duct may become dilated or hypertrophied, and in certain circumstances it may make abnormal communications with surrounding structures.


I am indebted to Miss R. M. Renton of the Zoology Department, University College for supplying me with the spawn of Amblystoma. Part of the expenses of this research was borne by a grant from the Research Fund of the University of London.

References

Branpt, W. (1937). Enstehlung hen der Gliedma issbildungen, p. 13. Bravs, H. (1906). Morph. Jb. 35, 139.

Burns, R. K. (1934). Anat. Rec. 58, Suppl. 7.

(1938) Proc. Soc. exp. Biol., N.Y., 39, 111.

Fievp, H. H. (1891). Bul. Mus. Comp. Zool. 21, 201.

Grtnwa_p, P. (1937). Roux Arch. Entw. Mech. Organ. 136, 786.

Hatt, R. W. (1904). Bul. Mus. Comp. Zool. 45, 31.

How tanp, R. B. (1921). J. exp. Zool. 82, 355.

—— (1926). J. exp. Zool. 44, 327.

Houmpurey, R. R. (1928). Anat. Rec. 40, 67.

Murra, K (1930). Jap. J. med. Sci. (Anatomy), 1, 105.

O’Connor, R. J. (1938). J. Anat. Lond., 73, 145.

Summasakl, Y. (1930). Jap. J. med. Sci. (Anatomy), 1, 291.

Spemann, H. (1920). Hanb. biol. ArbMeth. 2. Aufl. 1.

—— (1938). Embryonic Development and Induction. Yale Univ. Press.

WapprineTon, C. H. (1937). J. exp. Biol. 15, 371.

WoEpERMAN, M. W. (1930). Roux Arch. Entw. Mech. Organ. 121, 524.


Explanation of Plates I-III

Plate I

Fig. 1. Amblystoma, 30 days’ development. Normal development of the mesonephros. Section through the hinder end of the mesonephros. m, mesonephros; d, pronephric duct. x 150.

Fig. 2. Amblystoma, 30 days’ development. Normal development of the mesonephros. Section through the middle of the mesonephros. Lettering as in fig. 1. x 150.

Fig. 3. Amblystoma, 30 days’ development. Normal development of the mesonephros. Section through the anterior end of the mesonephros. Lettering as in fig.1. x50.

Fig. 4. Amblystoma, experiment of Text-fig. 2. Section in the vicinity of the cloaca. Shows normal right pronephric duct (r.d.), and tag of duct-like tissue on the left side (I.d.). x 27.

Fig. 5. Amblystoma, experiment of Text-fig. 2. Section through the pronephros. The pronephros on the obstructed side (p.) shows no abnormality. x40.

Plate II

Fig. 1. Amblystoma, obstruction to growth of pronephric rudiment. Section immediately cranial to the transplant and shows hypertrophy and dilatation of pronephric duct (d.), on the operated side. x 55.

Fig. 2. Amblystoma, obstruction of caudal growth of the pronephric rudiment. False communication of pronephric duct (d.) with a diverticulum of the gut. x 45.

Fig. 3. Amblystoma, obstruction to caudal growth of left pronephric rudiment. Communication of left pronephric duct (d.) with the exterior. x 65. 44 R. J. O'Connor

Fig. 4. Amblystoma, obstruction to caudal growth of left pronephric rudiment. Communication of left pronephric duct (d.), with transplanted cloaca (c.). x40. Fig. 5. Amblystoma, obstruction to caudal growth of left pronephric rudiment. Normal and

symmetrical development of cloaca in spite of absence of left pronephric duct. r.d. right pronephric duct. x 90.

Plate III

Fig. 1. Amblystoma, obstruction to caudal growth of left pronephric rudiment 25 days previously. Section through the caudal end of the mesonephros. Shows absence of the left pronephric duct and mesonephros approximately of equal development on both sides. r.d. right pronephric duct; r.m. right mesonephros; I.m. left mesonephros. x 110.

Fig. 2. Amblystoma, same experiment as fig. 1. Section through the middle of the mesonephros. Shows retarded development of left mesonephros. Lettering as in fig.1. x 110.

Fig. 3. Amblystoma, ‘same experiment as fig. 1. Section through the anterior end of the mesonephros. Shows retarded development and degeneration of left mesonephros. Lettering as in fig. 1. x 110.

Fig. 4. Amblystoma. Resection of left pronephric duct in segments 9-13 at stage 34, 20 days previously. Section through the 11th segment. On the left side in spite of the absence of the pronephric duct there is tubule formation in the mesonephros. Lettering as in fig. 1. x 130.

Fig. 5. Amblystoma. Resection of the left pronephric duct in segments 9-14 at stage 34, 35 days previously. Section through the 12th segment. On the left side absence of the pronephric duct and distended mesonephric tubules. Lettering as in fig. 1. x 105.

Fig. 6. Pleurodeles waltlii, obstruction to caudal growth of left pronephric rudiment 20 days previously. On the left side no appearance of the mesonephros. Lettering asin fig. 1. x 145.


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