1. Growth and degeneration of various components of the pronephric system of R. temporaria were investigated qualitatively and quantitatively in a series of stages ranging from 29 to 54, i.e. stages equivalent to those for R. dalmatina (Cambar & Marrot, 1954).

  2. All pronephric components except individual cell volume increase up to stage 47 (specimen 33 mm. long), and the first signs of degeneration are recognizable at stage 49, i.e. the stage preceding that at which the animal possesses an externally visible left forelimb.

  3. Progressive degeneration of the pronephros and duct continues until stage 54 (the animal having just metamorphosed), by which stage the organ has almost disappeared. The glomus likewise degenerates pari passu with the pronephros, but between stages 29 (specimens 9 mm. long) and 47 (specimens 33 mm. long) it barely changes in size and shape.

The growth and metamorphosis of the common frog Rana temporaria, though complex, are well known. The dramatic changes involved embrace profound alterations in larval size and shape together with the development of new organ systems and the loss of others. Many structures enlarge during larval development but ultimately undergo regression either wholly or in part, e.g. the tail, external gills, gill-slits, arterial blood-vessels, branchial skeleton, and the first spinal nerve. Among these structures is the pronephros.

The fact that the pronephros of R. temporaria degenerates—as it does in all amphibia—at some time before metamorphosis and is superseded by the mesonephros or adult kidney has long been known (Hoffmann, 1886; Marshall & Bles, 1890), yet details of the overall growth and degeneration patterns of the pronephric components, in particular the quantitative changes pursued by them, and knowledge of the exact stage of development reached by the pronephros in arbitrarily chosen but well-recognizable larval stages, are not clearly understood. This contribution is an attempt to refine our existing knowledge of the larval amphibian excretory system and to provide a basis for any future investigation of the causal factors involved.

The quantitative measurements of the pronephric system which were made during its growth and degeneration included the pronephric length, nuclear population, total volumes of the tubule cells and of the tubule lumen, tubule internal surface area and individual cell volume, and the length, total volume, and external surface area of the glomus.

Pronephric growth and degeneration were examined in a chosen group of R. temporaria specimens ranging from 9 mm. long to the just fully metamorphosed condition. To relate pronephric structure to age or to total length of the animal (Marshall & Bles, 1890; Matsukura, 1935) is unsatisfactory and to some extent misleading. Rate of development of amphibian larvae is variable and depends in part on temperature and on feeding (Dempster, 1933; Twitty & Schwind, 1931). Furthermore, R. temporaria larvae of different parentage and year have been found to differ—probably genetically—in their rate of development. It will be apparent from the data (vide infra) that quite different developmental stages are of approximately the same length, and growth is revealed by profound external changes in body-shape in addition to those changes which take place internally. In order to obviate any ambiguities on this score, pronephric development is here expressed in terms of distinct larval stages equivalent to members of the comprehensive series for R.dalmatina (Cambar & Marrot, 1954).

Pronephric components of the following equivalent stages were investigated. (Hatching takes place when the specimen is about 5–6 mm. long, 7–8 days after spawning. The number in brackets refers to the number of specimens, if more than one, of that particular stage was examined.)

Stage 29, 9 mm. long, no fore- or hind-limb blastemata or open gill-clefts; stage 31, 11 mm. long and similar to the preceding stage but with open gillclefts; stage 41 (2), 16 mm. long, tiny hind-limb buds visible externally, forelimb buds present beneath the operculum; stage 45,28 mm. long; stage 47 (2), 33 mm. long; stage 49, 33 mm. long. The three latter stages show a progressive development of the externally visible hind-limbs although the fore-limbs are likewise growing beneath the operculum. Stage 50, 33 mm. long, left fore-limb has emerged through the opercular aperture. In R. temporaria this stage is a fleeting one and in some cases fixation leads to a premature spasmodic extrusion of the right fore-limb through the opercular wall. Stage 51, 32 mm. long, both forelimbs, like the large hind-limbs, visible; stage 52, 25 mm. long, tail shortened; stage 53, 14 mm. long, tail merely a small stump; stage 54 (2), 12–13 mm. long, no tail rudiment and the animal has completely metamorphosed. The whole process including metamorphosis takes between 3 and 4 months and the time from about stage 48 until completion of metamorphosis is approximately 10 days.

All specimens were killed in Bouin’s fluid, blocked in paraffin wax, sectioned transversely at 10 μ, and stained with Ehrlich’s haematoxylin and counterstained with aqueous eosin. One further 15-mm. specimen (stage 41 approximately) was sectioned horizontally at 10 μ in order to measure nuclear length at right angles to the transverse plane and so to correct the crude count of the pronephric nuclear population. Previous experience of this kind of analysis revealed that pronephric nuclei do not significantly change in length during larval development; pronephric nuclear length was 11 μ (mean length and standard error for 300 pronephric nuclei chosen at random was 11·08±0·102 μ) and this measurement was used in the correction formula (see Abercrombie, 1946). Measuring techniques were generally the same as those used in previous investigations (Fox, 1955, 1956), but serial sections were projected at a linear magnification. ×200, for this was found to be the most convenient value for specimens of the size used.

The earliest recognizable pronephric anlage is found in specimens about 2-5 mm. long (Fürbringer, 1877), but the earliest pronephros which can be measured with any degree of accuracy is present in specimens about 9 mm. long. At stage 29 (9 mm.) R. temporaria possessed well-developed paired and measurable pronephroi each composed of tubules with thickish yolky-celled walls and lightly staining nuclei (Plate, fig. A). Each of three slightly pigmented and internally ciliated nephrostomial tubules opens by a separate nephrostome into the coelom in the dorso-mesial region of the pronephros, and at the other end leads into the main collecting tubule (Fürbringer, 1877,1878; Marshall & Bles, 1890), but a vestigial nephrostome IV was recognized in each of the paired pronephroi of another unmeasured specimen of the same stage. On each side a pronephric duct with a lumen leads backwards from the pronephros ventro-laterally to the somites, to open separately into the cloaca.

Pronephric tubules are similar but larger in stage 31 (11 mm.), and in the specimen shown in the Plate, fig. B, they are somewhat dilated in cross-section —a condition which no doubt is of common occurrence.

In stage 41 (16 mm.) the pronephric tubule walls are more sharply differentiated, thinner and contain little or no yolk, and the lumen volumes are larger than in the previous stages. The right pronephros of one specimen possessed confluent nephrostomes II and III, and nephrostome I was separate. Each of the other three pronephroi included three separate nephrostomial units. Pronephric ducts lead posteriorly each one ventro-mesial to the somites to open separately into the cloaca.

Pronephric tubules in stage 45 (28 mm.) have clearly defined walls with well-stained nuclei and larger lumen than those of stage 41. Nephrostomes I and II are confluent and nephrostomial unit III is separate. The pronephric duct in the region of the mesonephros is now quite clearly also a mesonephric duct, the mesonephros having utilized the original pronephric duct to discharge its products to the exterior.

The pronephros continues to enlarge up to stage 47 and, size apart, is similar to that of the previous stage (Plate, fig. C). One of the two specimens examined possessed paired pronephroi, each of which had a blind vestigial nephrostomial unit IV like the paired pronephroi of the unmeasured specimen of stage 29 (see Fox, 1962).

The first signs of degeneration—exemplified by granular degeneration tissue of tubule origin—are recognizable in pronephroi of stage 49. The pronephric tubules are smaller than those of stage 47, although doubtless they are still functional; the ill-defined walls are more deeply stained than tubules of earlier stages, and those of the posterior end of the one pronephros had little or no lumen (Plate, fig. D) and were similar in appearance to the strands of later stages; this suggests that degeneration begins posteriorly and progresses cranial-wards (Matsukura, 1935). One pronephros of stage 49 included nephrostomial units like those of stage 45, but the other had three separate ones which led into a common confluent nephrostome. The pronephric duct in the region between the pronephros and mesonephros is recognizable along most of its length as a dorso-ventrally flattened and luminated duct. Presumably this ‘intermediate’ duct, like the pronephros, has also commenced to degenerate, but the remainder is large and well developed in the dorso-lateral margin of the mesonephros, where it is distinguishable from the mesonephric tubules by having more distinct walls and more intensely stained and plentiful nuclei.

By stage 50 recognizable pronephric tubule tissue is reduced to solid nucleated strands embedded in a mass of lymphocytes (Plate, fig. E). Presumably some pronephric tissue has been invaded by lymphocytes and leucocytes (Kerr, 1919) during the degeneration process. Nephrostomial units I and II share a common nephrostome and the third is separate. All open at one end into the coelom and at the other into the degenerate pronephric tissue. In the ‘intermediate’ duct anterior to the mesonephros either no lumen was recognized in some serial sections or it was very small in others.

Further pronephric degeneration has occurred by stage 51. Vestigial nucleated pronephric strands are surrounded or invaded by lymphocytes (Plate, fig. F), but small open ciliated nephrostomial tubules like those of stage 50 still persist. A delicate lumen is recognizable along the whole length of the ‘intermediate’ duct in this particular specimen; it is thus likely that the degeneration of the anterior duct is a variable process which, particularly in the case of the lumen, cannot be referred to any one stage but progresses from stages 49 to 53.

The pronephros of stage 52 consists of solid strands invested by lymphoid cells, but pronephric nuclei can still be distinguished from nuclei of the latter, for they are slightly larger in cross-section. The three nephrostomial tubules are minute and almost solid, but in one pronephros they open into the coelom by a common nephrostome (Plate, fig. G), and in the other nephrostomes I and II are confluent and the third is separate. The pronephric duct situated between the degenerate vestigial pronephros and the large functional mesonephros is now probably not functional for it is in part a solid vestigial strand or it retains merely a tiny lumen.

Most of the pronephros has disappeared in the specimen of stage 53, though the remaining relics still possess faintly stained nuclei. A small vestigial nephrostome, probably derived from nephrostomes I and II, is still recognizable at the extreme posterior end and there is a ‘lymphoid tail’ about 200 μ long to each pronephros. The ‘intermediate’ duct is reduced to a vestigial solid strand.

In specimens which have just finished metamorphosis (stage 54) the p aired pronephroi are merely minute masses of tissue about 100 μ long (Plate, fig. H), which ultimately would disappear within a year (Marshall & Bles, 1890). In stages 53 and 54 the pronephric vestige is related to spinal nerves II and III. and this would leave little doubt as to its correct identification.

Paired glomeri originate as outgrowths from the dorsal aorta. Each glomus is situated in the upper margin of the body cavity opposite the nephrostomes (Plate, figs. B, G), and is an oval-shaped capillary mass across whose glomerular surface a glomerular transudate diffuses into the coelomic cavity. Size, shape, and structure do not change substantially from stage 29 onwards, but it becomes smaller after stage 47, until by stage 54 (in the fully metamorphosed animal) merely a vestigial (paired) glomus averaging about 115μ long is recognized. It is present but very small after one year and absent in second-year frogs (Marshall & Bles, 1890).

Mesonephric blastemata were not found at stage 29 of R. temporaria (9 mm. long), but incipient mesonephric cell masses situated between the dorsal aorta and the pronephric duct were recognizable at stage 31 (11 mm.); found in specimens between 10 mm. and 12 mm. (Marshall & Bles, 1890), earlier than in the 16-mm. specimens reported by Fürbringer (1878). These results are in general similar to those found in R. esculenta, in which early mesonephric blastemata are recognizable at 9 mm., with incipient tubules at 10 mm. and open nephrostomes (peritoneal funnels) in a 12-mm. larva (Fox, 1961a). Incipient mesonephric tubules and open ciliated nephrostomes are recognized at stage 41 (16 mm.) of R. temporaria, first reported in young and adult frogs by Spengel (1876) and Meyer (1875) and subsequently confirmed by Marshall & Bles (1890); these nephrostomial tubules open into the renal veins in R. fusca, R. esculenta, Bufo calamita, and Alytes obstetricans (Nussbaum, 1880, 1886), and in R. temporaria (see Gray, 1930). At stage 41 small paired gonadal (genital) ridges are likewise incipient. At stage 45 mesonephroi are considerably larger and more complex, with well-formed tubules and incipient glomeruli, and well-developed glomeruli in both the gonad and the post-gonad mesonephric region are recognizable in stage 47. Mesonephroi and gonads continue to enlarge as development proceeds. When the intermediate pronephric duct has disappeared the expanded remainder is termed the mesonephric (or Wollfian) duct of the mesonephric (or Wolffian) body.

Quantitative analysis of the pattern of pronephric growth and degeneration supports the conclusions reached from histological examination. Measurements of the pronephric nuclear population and length, tissue volume, lumen volume, overall volume (tissue and lumen volumes together), and tubule internal surface area all steadily increase in size up to stage 47. At stage 49, and at succeeding stages, there is a sharp fall in the size of these components until the almost complete disappearance of the pronephros at stage 54 (Table 1; Text-figs. 1-4). At the latter stage, pronephric nuclei were not recognizable. Mean pronephric nuclear length (at right angles to the transverse plane) was found to be about 11 μ with significant difference (P < 0·05) from the mean length of 12 μof R. escalenta, and this could well be due to fixation or other histological treatment. Correction of the crude nuclear count (Abercrombie, 1946) results merely in a difference of about 4·5 per cent, in nuclear population and calculated individual cell volume when either of these two correction measurements are used. Calculated individual cell volume in R. temporaria was found to vary between 4,150 and 7,000 μ3 up to stage 47; later, at a time when tissue was degenerating, it extended within a range of 2,300-5,600 μ3 (Table 1; Text-fig. 5). Tn R. escalenta pronephric individual cell volume of specimens between 12 mm. and 28 mm. long was practically constant around 7,000 μ3 (Fox, 1961a). Cell volume is thus larger and would appear to be more stable in normally growing pronephric cells than in degenerating ones, a result which might well have been expected.

Table 1.

Various measurements of pronephric components in the relevant stages of R. temporaria; the upper measurements of each pair are from the left and the lower from the right pronephros

Various measurements of pronephric components in the relevant stages of R. temporaria; the upper measurements of each pair are from the left and the lower from the right pronephros
Various measurements of pronephric components in the relevant stages of R. temporaria; the upper measurements of each pair are from the left and the lower from the right pronephros
Text-fig. 1.

Mean pronephros length in mm. (ordinate) related to host larvae of R. temporaria, equivalently staged according to the convention of Cambar & Marrot (1954) for R. dalmatina (abscissa). Each of the pronephric measurements of stages 41, 47, and 54 is the mean of four and all the other stages the mean of two.

Text-fig. 1.

Mean pronephros length in mm. (ordinate) related to host larvae of R. temporaria, equivalently staged according to the convention of Cambar & Marrot (1954) for R. dalmatina (abscissa). Each of the pronephric measurements of stages 41, 47, and 54 is the mean of four and all the other stages the mean of two.

Text-fig. 2.

Mean pronephric nuclear population related to host larvae of R. temporaria. Scheme is identical to that for Text-fig. 1.

Text-fig. 2.

Mean pronephric nuclear population related to host larvae of R. temporaria. Scheme is identical to that for Text-fig. 1.

Text-fig. 3.

Mean pronephric tissue volume (A), pronephric lumen volume (B), and pronephric overall volume (tissue and lumen volumes together) (C) in mm.3, related to host larvae of R. temporaria. Scheme is identical to that for Text-fig. 1.

Text-fig. 3.

Mean pronephric tissue volume (A), pronephric lumen volume (B), and pronephric overall volume (tissue and lumen volumes together) (C) in mm.3, related to host larvae of R. temporaria. Scheme is identical to that for Text-fig. 1.

Text-fig. 4.

Mean pronephric tubule internal surface area in mm.2 related to host larvae of R. temporaria. Scheme is identical to that for Text-fig. 1.

Text-fig. 4.

Mean pronephric tubule internal surface area in mm.2 related to host larvae of R. temporaria. Scheme is identical to that for Text-fig. 1.

Glomus length, volume, and external surface area are fairly stable up to stage 47; thenceforth the glomus regresses and it is vestigial in the metamorphosed form (Table 1; Text-figs. 6-8).

Text-fig. 5.

Mean calculated pronephric individual cell volume in μ.3, related to host larvae of R. temporaria. Scheme is identical to that for Text-fig. 1.

Text-fig. 5.

Mean calculated pronephric individual cell volume in μ.3, related to host larvae of R. temporaria. Scheme is identical to that for Text-fig. 1.

Text-fig. 6.

Mean glomus length in mm., related to host larvae of R. temporaria. Scheme is identical to that for Text-fig. 1.

Text-fig. 6.

Mean glomus length in mm., related to host larvae of R. temporaria. Scheme is identical to that for Text-fig. 1.

Text-fig. 7.

Mean glomus volume in mm.3, related to host larvae of R. temporaria. Scheme is identical to that for Text-fig. 1.

Text-fig. 7.

Mean glomus volume in mm.3, related to host larvae of R. temporaria. Scheme is identical to that for Text-fig. 1.

Text-fig. 8.

Mean glomus external surface area in mm.2, related to host larvae of R. temporaria. Scheme is identical to that for Text-fig. 1.

Text-fig. 8.

Mean glomus external surface area in mm.2, related to host larvae of R. temporaria. Scheme is identical to that for Text-fig. 1.

Within the range of development studied mesonephric length increased from about 1-2 mm. (stage 41) to 3·2 mm. (stage 50). Thereafter it shortens slightly up to stage 54. The modest reduction may well be bound up with the shortening of the body as a whole as the final stages of metamorphosis are reached. Afterwards the mesonephros continues to lengthen to reach its adult form.

Metamorphosis in Rana is merely a stage of normal development from a larval aquatic form to an adult terrestrial one. One way of arbitrarily defining its actual onset is that stage when the forelimbs are first visible externally and when the animal actually begins to use all its limbs for walking. Other external changes ultimately result in alteration in the shape of the body and regression of the tail, and the lungs are more active for breathing air (Helff, 1931). Metamorphosis in R. temporaria could thus be considered to commence at a stage of development equivalent to stages 49–51 (approximately) for R. dalmatina (Cambar & Marrot, 1954). A fully developed larva 29 mm. long (head to trunk 13 mm.) is considered to be the metamorphosis stage (see de Beer, 1937), which would support this view, though Cambar and Marrot (1954) consider stage 39—when the limb blastemata are incipient—as the starting-point.

If thebeginning of terrestrial life signals the onset of metamorphosis, then the first signs of pronephric degeneration portend and just precede this significant developmental period. Actual pronephric degeneration in Rana would thus form part of the programme of metamorphosis, and the evidence*of necrosis of the pronephros and various other organs, e.g. the tail and the branchial skeleton, which proceeds in all of them at a similar time, suggests that they may be causally interrelated.

It is known that in amphibian larvae the anterior portion of the pronephric duct degenerates, but that the portion in the region of the mesonephros hypertrophies to become the mesonephric duct. Extirpation of the pronephros or blocking a pronephric duct leads to regression of either the whole or part of the duct behind the lesion (Fox, 1957), though presumably it ultimately can enlarge again to become a functional mesonephric duct.

Resection of the three pronephric nephrostomial canals in Bufo hastens pronephric tubule atrophy (Shimasaki, 1930), and isolated pronephric grafts transplanted postero-laterally from donor to host Axolotls, and which were not in open communication with the coelom, have stunted tubules (Fox, 1960).

Functional continuity is thus essential to maintain typical duct or tubule structure during the normal life-span of the organ.

Nevertheless, pronephric degeneration begins before the ‘intermediate’ pronephric duct has degenerated into a solid strand of cells. It is thus unlikely that duct blockage is the initial cause of pronephric malformation. Neither can nephrostomial blockage be invoked, for the nephrostomial tubules and nephrostomes remain open and presumably functional until the final stages of degeneration (Hoffmann, 1886), a result confirmed in the present work.

Furthermore, no evidence was forthcoming to support the conclusion reached by Marshall & Bles (1890) that tubule dilation precedes tubule degeneration. In this present work excessive tubule dilation was not apparent from or before stage 47. Nor was it described in Bufo (Matsukura, 1935); in fact tubules were somewhat dilated in pronephroi of a specimen of stage 31 in the early period of tubule development.

Tubule dilation certainly need not precede tubule degeneration, for after unilateral pronephrectomy in young larvae of Axolotl (Howland, 1921), Rana and Bufo (Miura, 1930), and Triturus (Fox, 1956) the tubules and duct of the remaining pronephros are dilated. The same is true in ipsilateral and contralateral pronephric tubules of Triturus after unilateral pronephric duct blockage, and in tubules of specimens of Triturus grown in distilled water (Fox, 1957, 1959). In all cases there were no recognizable phenomena of pronephric tubule degeneration.

It is thus unlikely that the immediate cause of normal pronephric degeneration is due to interference of pronephric function by blockage either of the pronephric duct or of the nephrostomes. It is more likely that functional activity diminishes as the tubules and anterior duct progressively degenerate.

Croissance et dégénérescence du système pronéphrétique chez Rana temporaria

  1. On a examiné qualitativement et quantitativement la croissance et la dégénérescence de divers composants du système pronéphrétique de R. temporaria, sur une série de stades allant du st. 29 au st. 54, équivalents à ceux de R. dalmatina (Cambar & Marrot, 1954).

  2. Tous les composants pronéphrétiques, excepté le volume cellulaire individuel, s’accroissent jusqu’au stade 47 (individu de 33 mm. de longueur), et les premiers signes de dégénérescence sont reconnaissables au stade 49 (stade précédant celui auquel l’animal possède un membre antérieur gauche visible).

  3. La dégénérescence progressive du pronéphros et de son canal se poursuit jusqu’au stade 54 (animal venant de se métamorphoser), l’organe ayant alors presque disparu. De même le glomus dégénère conjointement avec le pronéphros, mais change à peine de taille et de forme entre les stades 29 (individus de 9 mm.) et 47 (individus de 33 mm.).

I am grateful for the advice received from Professors M. Abercrombie and P. B. Medawar, and for the photographic assistance of Mr. C. Atherton.

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.

Photomicrographs of transverse sections of R. temporaria larvae, all at 10 p. Specimens are staged according to the scheme for R. dalmatina (see Cambar & Marrot, 1954).

Fig. A. Stage 29; 9 mm. long; nose-to-cloaca 3·62 mm. Section through pronephroi 0·20 mm.(left) and 0·19 mm. (right) from the anterior end of the pronephros.

Fig. B. Stage 31; 11 mm. long; nose-to-cloaca 3·65 mm. Section through pronephroi 0·19 mm. (left) and 0·20 mm. (right) from the anterior end of the pronephros.

Fig. C. Stage 47 (a); 33 mm. long; nose-to-cloaca 10·38 mm. Section through left pronephros in region of nephrostomial unit II (nephrostomes I and II are confluent), 0·26 mm. from the anterior end of the pronephros.

Fig. D. Stage 49; 33 mm. long; nose-to-cloaca 9·36 mm. Section through hind region of right pronephros (0·56 mm. long), 0·50 mm. from its anterior end.

Fig. E. Stage 50; 33 mm. long; nose-to-cloaca 10·10 mm. Higher-power section through hind region of left pronephros (0·40 mm. long), 0·35 mm. from its anterior end.

Fig. F. Stage 51; 32 mm. long; nose-to-cloaca 10·09 mm. Section through left pronephros 0·20 mm. from its anterior end and just behind the 3 nephrostomial tubules which open into the coelom.

Fig. G. Stage 52; 25 mm. long; nose-to-cloaca 9·83 mm. Higher-power section through nephrostomial unit III (nephrostomes I, II and III are confluent), 0·21 mm. from the anterior end of the left pronephros (0·23 mm. long).

Fig. H. Stage 54 (a); 13 mm. long; nose-to-cloaca 8·10 mm. Higher-power section through left vestigial pronephric rudiment 30 μ from its anterior end (pronephric vestige length 90 μ).

Abbreviations, a. lg., anterior region of lungs just preceding their separation; co., coelom; g., glomus; ga. IX, X, ganglia of glossopharyngeus and vagus; ga. s.n. II, ganglion of spinal nerve II; in., intestine; lg., lung; 1.1., lymphoid tissue ;n. II, III, nephrostome II and III; n. c., nerve-cord; ne. Ill, nephrostomial tubule III; not., notochord; o., oesophagus; p., pronephros; p.c.s., post-cardinal sinus; p.g., pectoral girdle; p.s., pronephric strand; p.t., pronephric tubule; r.n. II, rib of neural arch II; s.n. II, III, spinal nerves II and III; som., somite.

Photomicrographs of transverse sections of R. temporaria larvae, all at 10 p. Specimens are staged according to the scheme for R. dalmatina (see Cambar & Marrot, 1954).

Fig. A. Stage 29; 9 mm. long; nose-to-cloaca 3·62 mm. Section through pronephroi 0·20 mm.(left) and 0·19 mm. (right) from the anterior end of the pronephros.

Fig. B. Stage 31; 11 mm. long; nose-to-cloaca 3·65 mm. Section through pronephroi 0·19 mm. (left) and 0·20 mm. (right) from the anterior end of the pronephros.

Fig. C. Stage 47 (a); 33 mm. long; nose-to-cloaca 10·38 mm. Section through left pronephros in region of nephrostomial unit II (nephrostomes I and II are confluent), 0·26 mm. from the anterior end of the pronephros.

Fig. D. Stage 49; 33 mm. long; nose-to-cloaca 9·36 mm. Section through hind region of right pronephros (0·56 mm. long), 0·50 mm. from its anterior end.

Fig. E. Stage 50; 33 mm. long; nose-to-cloaca 10·10 mm. Higher-power section through hind region of left pronephros (0·40 mm. long), 0·35 mm. from its anterior end.

Fig. F. Stage 51; 32 mm. long; nose-to-cloaca 10·09 mm. Section through left pronephros 0·20 mm. from its anterior end and just behind the 3 nephrostomial tubules which open into the coelom.

Fig. G. Stage 52; 25 mm. long; nose-to-cloaca 9·83 mm. Higher-power section through nephrostomial unit III (nephrostomes I, II and III are confluent), 0·21 mm. from the anterior end of the left pronephros (0·23 mm. long).

Fig. H. Stage 54 (a); 13 mm. long; nose-to-cloaca 8·10 mm. Higher-power section through left vestigial pronephric rudiment 30 μ from its anterior end (pronephric vestige length 90 μ).

Abbreviations, a. lg., anterior region of lungs just preceding their separation; co., coelom; g., glomus; ga. IX, X, ganglia of glossopharyngeus and vagus; ga. s.n. II, ganglion of spinal nerve II; in., intestine; lg., lung; 1.1., lymphoid tissue ;n. II, III, nephrostome II and III; n. c., nerve-cord; ne. Ill, nephrostomial tubule III; not., notochord; o., oesophagus; p., pronephros; p.c.s., post-cardinal sinus; p.g., pectoral girdle; p.s., pronephric strand; p.t., pronephric tubule; r.n. II, rib of neural arch II; s.n. II, III, spinal nerves II and III; som., somite.