Talk:Paper - Experiments on the development of the pronephric duct (1938)

From Embryology

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EXPERIMENTS ON THE DEVELOPMENT OF THE PRONEPHRIC DUCT

By R. J. O°;CONNOR Department of Anatomy, University College, London

INTRODUCTION

In Amblystoma tigrinum’ and Triton taeniatus the rudiment of the nephric system first appears as a thickening of the somatopleure immediately beneath the ectoderm at the ventrolateral border of certain somites at the anterior end of the embryo. The limits of this thickening are at first difficult to define but in both species it is seen soon after the closure of the neural folds in the region from the third to the seventh somites. At a little later stage of development the thickening becomes more precisely defined and being immediately beneath the ectoderm gives rise to a ridge visible from the exterior. In succeeding stages of development the nephric rudiment extends in a caudal direction, maintains throughout its position at the ventrolateral border of the somites, and eventually reaches the cloaca as a solid column of cells, there being a corresponding extension of the ridge caused by it. The pronephros is formed from the rudiment in the vicinity of the third and fourth somites while the more caudal portion becomes canalized to form the pronephric duct.

The mode of this caudal extension has been a matter of controversy. There are two possibilities; first the rudiment may extend caudally by its own independent growth, or secondly it may be extended by an addition to it from each successive segment. Mollier (1890) could not decide between the two possibilities but was inclined to support the first. Field (1891) however concluded that the extension of the rudiment was due to a proliferation of the somatopleure in situ, his evidence in A. punctatum being the similarity of yolk granules in the nephric rudiment and adjacent somatopleure. Field’s conclusion has been accepted by more modern writers and has been repeated by Goodrich (1930) and Felix (1906), while Burlend (1931) supports the conclusion on comparative grounds. Brauer (1902) described an independent caudal growth of the nephric rudiment in the rare Gymnophonia but in accepting this Burlend states it to be atypical. Therefore it appears that opinion and evidence favour the view that the portion of the nephric rudiment giving rise to the pronephric duct is formed in situ by a contribution from each individual segment.

As a means of studying the mode of formation of the pronephric duct experiments have been performed on 7. taeniatus and A. tigrinum in which

1 T am indebted to Miss R. M. Renton of the Zoology Department, University College, for her kindness in supplying me with the spawn of Amblystoma tigrinum.

Anatomy LXxIII 10 146 R. J. O'Connor

localized portions of the developing nephric system have been stained with Nile blue sulphate in the living embryo.

TECHNIQUE

The technique was adapted from Stone’s (1931) modification of Vogt’s vital staining methods and Nile blue sulphate was applied to the embryos by means of small pieces of agar soaked in the dye. A 2% solution of agar was poured on to a glass plate and evaporation produced thin sheets of a rubbery consistency. After drying these for 2 or 3 days they were soaked in a strong solution of Nile blue sulphate and became impregnated with the dye. The nephric rudiment was exposed by dissecting back a flap of overlying ectoderm using a hair loop and glass needle according to the technique of Spemann and the exposed rudiment was stained directly by the application of a piece of agar corresponding in size to the exposed area. The agar was held in position by means of a fragment of a cover-slip and removed 2 hr. later, the ectodermal flap being allowed to fall back into position. In addition to an intense staining of the exposed tissues stain was also taken up by the ectodermal flap and by the ectoderm at the rim of the wound and this staining of the ectoderm served as a useful means of localizing the stained region in serial sections. The operation wound healed in 3 hr. and subsequent development was always normal.

Experiments were performed at all stages from the first appearance of the nephric rudiment up to its junction with the cloaca. The object was to stain the caudal limit of the rudiment and this could be fairly accurately determined by following the caudal lengthening of the external ridge due to it and developmental tables are available to check such observations. It was found that the caudal limit of the rudiment was always stained when an exposing wound, the width of two somites, was made over the caudal end of the ridge. In PI. I, fig. 1, is shown an embryo of T. taeniatus at stage 25 (Glaesner, 1925) stained in this way. The photograph was taken 5 hr. after operation when healing was complete.

RESULTS OF STAINING EXPERIMENTS

In a high percentage of cases when the caudal limit of the nephric rudiment was stained in the manner described the dye spread in a definite and constant fashion. First there was a general increase in the size of the stained area with a simultaneous decrease in the intensity of staining; no significance is attached to this as it was obviously due to the growth of the embryo. In addition however there occurred a spread of the dye as a narrow streak leading to the cloaca and situated at the ventrolateral border of the somites. The width of this streak of dye was consistent with that of the nephric rudiment, the dye was beneath the ectoderm, and in its extension from the stained area to the cloaca it kept pace with the extension of the ridge of the nephric rudiment of the opposite side. The presumption was therefore that the streak was due to the presence of dye in the developing pronephric duct. This could be confirmed The Development of the Pronephric Duct ~ 147

by dissection and a blue pronephric duct demonstrated leading from the stained area to the cloaca. The characteristics of the extension of the dye are shown in Pl. I, figs. 2 and 8. Fig. 2 is of an embryo of T. taeniatus which had been operated on 2 days previously at stage 22. The caudal limit of the developing nephric rudiment was stained at the level of somites 8 and 9 on the left side. The photograph was taken at stage 26-27 and the streak-like extension of the dye is seen. Actually the dye almost reached the cloaca but in the posterior third of the animal it was too faint to photograph. In Fig. 3 a similar result is seen in A. tigrinum which was stained, in the region of somites 8-11 on the left side, two days before when the embryo was at stage 25 (Harrison).1 The photograph was taken at stage 35 and the streak of dye is seen to reach the cloaca. The actual site of staining is somewhat obscured by the pigmentation of the ectoderm.

External examination and simple dissection left no doubt that these blue streaks were due to dye in the pronephric duct but did not exclude the possibility that it might be in a partially formed lumen. Therefore embryos were examined by serial section. The preservation of Nile blue sulphate during sectioning is a matter of some difficulty as the dye is soluble in alcohol. Stone’s method of adding 0-1% phosphomolybdiec acid to all alcoholic solutions solved the difficulty and it was found an advantage to make up the alcoholphosphomolybdic acid solutions immediately before use. Rapid dehydration did not interfere with the production of good sections and specimens were left 10-15 min. in the alcoholic solutions, and sections were mostly cut at a thickness of 10. In the sections the dye appeared as blue granules and its distribution could be determined. The result of a typical experiment is shown in Text-fig. 1. This diagram was obtained by drawing each section with the aid of the projection microscope and then measuring the width of the structures represented and their distance from the midline; these measurements were then plotted to scale on squared paper and areas where dye granules were present represented by stippling. The experiment represented is one on A. tigrinum. Two days previously the nephric rudiment was stained ventral to somites 8 and 9 at stage 25. At the time of sectioning the embryo was a stage 35 and a streak of dye was present leading from the stained area to the cloaca. In the sections the stained area was recognized by the presence of dye granules in the ectoderm and in this region blue granules were seen in the adjacent somite, in the somatopleure and in the cells of the pronephric duct (PI. I, fig. 4). Caudal to the stained area the dye was confined to the cells of the pronephric duct, there being none in the adjacent structures. This is seen in PI. I, fig. 5 which is two or three sections from the cranial border of the cloaca. In this section the few granules seen in the ectoderm, myotome and somatopleure are due to the pigment of the embryo and microscopically are easily distinguishable from the Nile blue sulphate granules in the pronephric duct. At all levels in the

1 Harrison’s numbered stages refer to the development of A. punctatum but the development of A. tigrinum is sufficiently similar to justify the use of Harrison’s stages. 10-2 148 R. J. O'Connor



Left (operated side)

Right Left (operated side)

-- Ectoderm

—-— Pronephros

—— Pronephric duct



— Pronephric duct

Stained area. Dye granules in duct, somites, somatopleure and ectoderm

— Level of PI. I, fig. 4

Level of Text-fig. 4

Transplant Region where dye has (stippled) extended in pronephric duct. Dye granules in

duct cells only O 125 250%



Level of Text-fig. 5

— —Cloaca Text-fig. 1. Text-fig. 3.

Text-fig. 1. Distribution of Nile blue sulphate (shown as stippling) at stage 35. Left nephric rudiment stained ventral to somites 7-9 two days previously when embryo was at stage 25. Levels of figs. 4 and 5, Plate I, indicated.

Text-fig. 3. Nephric system in A. tigrinum at stage 39-40. Nine days previously transplantation performed as shown in text-fig. 2. Levels of text-figs. 4 and 5 indicated. The Development of the Pronephric Duct 149

pronephric duct the dye was found in the cells alone there being none in any lumen present. .

It is clear that the dye in the cells of the caudal portion of the pronephric duct could not be derived from the unstained adjacent cells and the only possible source could have been the dye placed in the caudal limit of the nephric rudiment which must therefore be the origin of the more caudal portions subsequently developed.

TRANSPLANTATION EXPERIMENTS

The vital staining experiments described above furnish evidence that the nephric rudiment extends caudally by its own independent growth and this evidence is supplemented by experiments where an obstruction is grafted in the path of its caudal extension. Such experiments were performed on both A. tigrinum and T. taeniatus and the hosts chosen from stages where the nephric rudiment is confined to the more anterior segments. A transplant, taken from another embryo, was placed at the ventrolateral border of somites beyond the caudal limit of the rudiment, and Text-fig. 2 illustrates such an experiment.

O


T. taeniatus. Stage 11. A. tigrinum. Stage 23. Text-fig. 2.

A portion of the presumptive neural area from the gastrula of T. taeniatus was transplanted at the ventrolateral border of somites 9-10 in A. tigrinum at stage 28 when the nephric rudiment had not reached beyond the seventh or eighth somite. The host embryo was sectioned at the ninth day at stage 39-40 and the arrangement of nephric system and transplant is represented in Text-fig. 8 which is a diagram made in the same way as Text-fig. 1. The nonoperated side has undergone a normal development. On the operated side the transplant has differentiated into nervous tissue, and on this side the pronephric duct becomes dilated as it comes into the region of the transplant (Text-fig. 4). It then abruptly ceases and caudal to the transplant there is no sign of its formation (Text-fig. 5).

It was immaterial what sort of tissue was used for the transplant and the above result was obtained in both A. tigrinum and T. taeniatus using nervous tissue, phronephros and cloaca as the transplant. Development was followed in these cases up to 30 days and in every case there was absence of the duct caudal to the graft. It is apparent that there is no contribution to the forma150 R. J. O'Connor

tion of the pronephric duct from the more posterior segments and so in normal development it must be entirely formed by a growth caudally of the nephric rudiment.

CONSIDERATION OF A SERIES OF VITAL STAINING EXPERIMENTS

If the conclusion that the pronephric duct is formed by an independent caudal growth of the nephric rudiment be correct then the following results would be expected from vital staining experiments. Firstly when the caudal limit of the rudiment is stained at any stage prior to its union with the cloaca


Text-fig. 4. Transverse section of A. tigrinum at the level shown in text-fig. 3. Cells of transplant (c.t.) and dilated left pronephric duct (J.d.) are seen. x 120.

the dye should extend towards this structure in the cells of the pronephric duct; that is a positive result. Secondly, when the stain is placed some distance cranial or caudal to this point in the line of the development of the rudiment there should be no extension of the dye; that is a negative result. One hundred and twenty-one experiments on 7. taeniatus and A. tigrinum can be divided into two such groups:

(1) Stain in the caudal limit of the rudiment: expected positive. (2) Stain cranial or caudal to the limit: expected negative. The Development of the Pronephric Duct - 51

Table I. Showing the number of cases where expected result was obtained in vital staining experiments

Expected result obtained —_—_—_—— Cases Positive Negative (1) Cases (2) Per cent. A. Tigrinum: (1) Expected positive 24 24 0 24 100 .(2) Expected negative 16 3 13 13 81 Total for A. tigrinum 46 27 13 37 92 T. taeniatus: (1) Expected positive 41 37 4 37 90 (2) Expected negative 40 5 35 35 87 Total for 7. taeniatus — 81 42 39 72 89 Total for whole series 121 69 52 109 90 ,


Text-fig. 5. Transverse section of A. tigrinwm at level shown in text-fig. 3. Absence of left pronephric duct. (Right pronephric duct, r.d.) x 120.

In Table I the results of this series of experiments are shown and it will be seen that in each group for both species the expected result is obtained in over 80 % of cases and in the whole series in 90 %. ® 152 R. J. O Connor

THE QUESTION OF A SEGMENTAL ORIGIN OF THE NEPHRIC SYSTEM

Although Mollier was not able to determine the mode of extension of the nephric rudiment he came to the conclusion that the portion of the rudiment ventral to somites 3-6 in T. alpestris was formed in situ as a proliferation of the somatopleure and so was derived segmentally, and in the original rudiment Field, in A. punctatum, and Glaesner, in T. taeniatus, record some signs of segmentation. Further evidence of a segmental origin of the original nephric rudiment was obtained from vital staining experiments on A. tigrinum and T. taeniatus. In both species the site of the future nephric rudiment was stained in the neurula stage before its appearance. There are no visible somites at this stage of development and so the stain was placed by guesswork and its exact position determined when later the somites and nephric rudiment . appeared.

Two groups of experiments on T. taeniatus can be considered, similar results being obtained in A. tigrinum.

(1) Dye was placed in the vicinity of somites 3 and 4 and confined to these segments. As the nephric rudiments appeared that portion of it in segments 3 and 4 was seen to be stained, but no other portion, the dye remaining where it was placed and not extending into the rudiment of adjacent fifth and sixth segments. That portion of the rudiment ventral to somites 5 and 6 must therefore have come from a different source to that portion ventral to somites 8 and 4. Examination at later stages showed the dye to be confined to the pronephros.

(2) Dye was placed in segments 5 and 6-in the vicinity of the somitic region. The dye in this case could be traced into the portion of the nephric rudiment in association with these segments but not into the more cranial portion. This latter portion, therefore, could not have received a contribution from segments 5 and 6. Examination by serial section or by dissection at later stages showed the pronephros to be free from dye granules, but in some cases there was an extension of dye into the more caudal portions of the rudiment.

In experiments such as these, performed without the guidance of nephric rudiment or somites, accurate placing of the dye is a matter of some difficulty and the fate of dye placed in a single segment cannot be given. However, it is clear that the nephric rudiment of segments 3 and 4 have a different origin to that of somites 5 and 6, and when this evidence is added to the morphological findings it is possible to support the conclusion that the original nephric rudiment is derived in a segmental manner, each of the segments 3-6 and possibly 7 contributing to its formation.

In the second group of experiments where stain was traced into the rudiment of segments 5 and 6 the dye in some cases extended toward the cloaca in the nephric rudiment. The significance of this finding was investigated by a further series of experiments. These were performed on A. tigrinum at stage 22 The Development of the Pronephric Duct 153

and on T.. taeniatus at stage 19. At these stages the nephric rudiment becomes clearly demarcated at the ventrolateral border of somites 4~-7 with the production of an external ridge. This and the appearance of somites enables the nephric rudiment to be stained with a fair degree of accuracy. In each species the experiments were of two types:

(1) Where the rudiment of segments 3 and 4 was stained. (2) Where the rudiment of segments 5—7 was stained.

Table II. Results of staining nephric rudiment when it is first clearly demarcated (A. tigrinum St. 22; T. taeniatus St. 19)

Cases Extension Percentage

Stain in segments 3-4:

(1) A. tigrinum (St. 22) 8 1 12

(2) T. taeniatus (St. 19) 15 2 14 Total 23 3 13 Stain in segments 5-7:

(1) A. tigrinum (St. 22) 9 9 100

(2) T. taeniatus (St. 19) 20 20 100 Total 29 29 100

The results of these experiments are set out in Table II. In both species staining the rudiment in segments 3 and 4 produces less than 15 % of cases where the dye extended into the caudal portions of the rudiment and in the three cases where it did occur the extension was only for a distance of two or three somites and is readily ascribable to an inaccurate placing of the stain. In the negative cases dissection or serial section showed the dye to be confined to the pronephros. On the other hand, in the cases of staining the rudiment of segments 5-7, extension of the dye occurred in 100% of cases. In these experiments no dye could be traced into the pronephros but it could be traced into the pronephric duct. In all cases it extended more than half the length of the duct and in the majority could be traced as far caudally as the cloaca.

Therefore the final conclusion is that the first rudiment of the nephric system is derived in a segmental manner in the region of the third to seventh somites. The cranial portion under somites 3 and 4 forms the pronephros while the more caudal portion under somites 5—7 gives rise to the pronephric duct by its own independent caudal growth.

SUMMARY

1. A technique is described for vitally staining, with Nile blue sulphate, localized portions of the developing nephric system in A. tigrinum and T. taeniatus.

2. It is shown that the pronephric duct in these species is formed by an independent caudal growth of the first formed nephric rudiment. The evidence for this conclusion is:

(a) When Nile blue sulphate is placed in the caudal limit of the nephric 154 R. J. O'Connor

rudiment at any stage prior to its union to the cloaca the dye can be found in the pronephric duct cells from the stained area to the cloaca. (b) When the caudal growth of the nephric rudiment is prevented by a

suitably placed transplant there is no formation of the pronephric duct caudal to the transplant.

8. Evidence derived from vital staining experiments shows that the first formed nephric rudiment is derived in a segmental manner from the third to seventh segments and that while the cranial portion forms the pronephros the

caudal portion grows independently toward the cloaca to give rise to the pronephric duct.

ACKNOWLEDGEMENT

The writer is indebted to Prof. H. H. Woollard for facilities to carry out this work, and for much helpful advice and criticism during the preparation of this paper. Part of the expense of this research was borne by a grant from the Research Fund of the University of London.

REFERENCES

Braver, A. (1902). ‘“ Beitrage zur Kenntnis der Entwicklung und Anatomie der Gymnophonien: III. Entwicklung der Excretionsorgane.” Zool. Jb. Bd. xv1, S. 1.

Burtenp, T. H. (1931). “The origin of the archinephric duct in vertebrates.” Amer. J. Anat. vol. xLvm, p. 261.

Feix, W. (1906). ‘Entwicklung des Harnapparates.”” Hertwigs Handbuch der Vergleichenden und Experimentellen Entwicklungslehre der Wirbeltiere, Jena 1906, Bd. m1, Heft 1, S. 174. Frexp, H. H. (1891). ‘The development of the pronephros and segmental duct in Amphibians.”

Bull. Mus. comp. Zool. vol. xxi, p. 201.

GLaEsNER, L. (1925). Keibel’s Normentafeln zur Entwicklungsgeschichte der Wirbeltiere, Heft 14, “Normentafeln zur Entwicklungsgeschichte des Gemeinen Wassermolchs (Molge vulgaris).”” Jena.

Goopricu, E. S. (1930). Studies on the Structure and Development of Vertebrates, p. 665. London: McMillan and Co.

Mo ttigr, S. (1890). ‘Ueber die Enstehung des Vornierensystems bei Amphibien.”’ Arch. Anat. Physiol. Jahrg. 1890, Anat. Abt. S. 209.

Stong, L. S. (1931). “Selective staining of the neural crest and its preservation for microscopic study.” Anat. Rec. vol. L1, p. 267.

DESCRIPTION OF PLATE I

Fig. 1. Embryo of 7. taeniatus at stage 22. Left nephric rudiment stained five hours previously with Nile blue sulphate. x 25.

Fig. 2. Embryo of 7. taeniatus at stage 26-27. Two days previously at stage 22 nephric rudiment stained with Nile blue sulphate ventral to 8-9 somites. Extension of dye as a streak at ventrolateral border of somites. x 25.

Fig. 3. Embryo of A. tigrinum at stage 35. Two days previously at stage 23 nephric rudiment stained with Nile blue sulphate ventral to somites 8-9. Extension of dye as a streak at the ventrolateral border of somites. x 25.

Fig. 4. Transverse section through left pronephric duct of A. tigrinum at level shown in Fig. 4. Granules of Nile blue sulphate in cells of duct, myotome, somatopleure and ectoderm. (d, duct; m, myotome; s, somatopleure; e, ectoderm.) x 620.

Fig. 5. Transverse section through left pronephric duct of A. tigrinum at level shown in Fig. 4. Granules of Nile blue sulphate in cells of duct only. (Lettering as in Fig. 6.) x 620. Journal of Anatomy, Vol. LX XIII, Part 1 Plate I



O’CONNOR—TuHE DEVELOPMENT OF THE PRONEPHRIC Duct