Internal Factors Influencing Normal and Compensatory Growth of the Axolotl Pronephros

The factors that influence compensatory growth of the pronephros in amphibian larvae may be either mechanical (hydrostatic) or, in a general sense, chemical. On the one hand, coelomic fluid, which enters the complex mass of pronephric tubules along the ciliated nephrostomes, must exert an intra-tubular fluid pressure. On the other hand, pronephroi bathed in blood of the post-cardinal sinus, receiving arterial blood from the dorsal aorta and transmitting coelomic fluid, could well be affected by chemical substances in these fluids.

In the present work an investigation was made of these two possible causal factors in Ambystoma larvae in order to discover whether pronephric growth promoting or inhibiting substances influence normal or compensatory growth in vivo, or whether hydrostatic influences are the more important.

It will be shown that there are no grounds for assuming the presence of specific stimulatory or inhibitory pronephric growth substances in young larvae, though the possibility cannot be ruled out in older stages, and that although the pronephric blastema cells will differentiate into underdeveloped tubules when they have no communication with the coelom, ‘normal’ tubular pattern and tubular hypertrophy (and probably hyperplasia) are determined by intratubular tension.

All operations were performed on decapsulated embryos of A. mexicanum at Harrison stages 24–26 approximately (see Hamburger, 1950), when they were immersed in full-strength Holtfreter solution containing 0·2 per cent, sodium sulphadiazine (Fox, 1955). Recipients received a pronephric blastema graft from either the right-or the left-hand side of donors of the same age (one donor thus supplying two recipients), for there is no difference between the pronephroi of either side. Recipient and donor were arranged side by side on a gelatine base in a solid watch-glass, and, with instruments described by Hamburger (1950), a pronephric blastema (or some portion of it), together with its overlying ectoderm, was transferred from donor to host and inserted with the ectoderm lying uppermost into a surface wound prepared just behind and below the left presumptive forelimb region. Removal and grafting of the small whitish ‘pulpy’ pronephric blastema alone was unsatisfactory, for the tissue either disintegrated in the culture medium or stuck too tenaciously to the glass needle or failed to adhere permanently to the host. A small glass cover-slip delicately laid over the graft prevented movement and facilitated healing, which took place within 3–6 hours. Operated specimens were kept in the above-mentioned medium for 3 days and then they were transferred to glass crystallizing dishes (6 specimens to each dish) containing about 80 ml. of one-tenth-strength Holtfreter solution and 0·2 per cent, sodium sulphadiazine. Solutions were changed every 24 hours and the temperature during development varied between approximately 18° and 23° C.

Group I consisted of 7 control specimens each 10 mm. long, which were treated exactly like the operated ones but suffered neither unilateral pronephrectomy nor grafting. Group II is composed of 9 host specimens whose pronephroi were undisturbed, but each animal received graft tissue; the grafts in these specimens comprise Group IIa. Group III comprises 8 specimens which were unilaterally pronephrectomized only (Howland, 1916, 1921; Fox, 1956). Group IV is composed of 10 host specimens which were unilaterally pronephrectomized like those in Group III, but each one received a pronephric graft. The grafts in these specimens comprise Group IVb. A few of the pronephrectomized specimens did not heal completely in the operated region so that the gut possessed a small opening to the exterior. Most of the grafts were situated behind the pronephric region, ventro-laterally to the lateral plate mesoderm, close to the coelom; all were entirely healed into the body tissue. Only those specimens which possessed graft tubules closed off from the coelom were employed in the main analysis. Graft tubules were thus isolated and probably were never in open communication with the coelomic fluid up to the time of examination.

Specimens were chosen at random from the many prepared, killed in Smith’s fixative 10 days after operation when about 10 mm. long, paraffin-wax blocked and sectioned transversely at 10μ. Other specimens were sectioned horizontally in order to measure nuclear length for the purpose of applying a correction to the pronephric nuclear population (Abercrombie, 1946). Four further specimens were transversely sectioned at 10μ; two of them were like those in Group II (grafted specimens with undisturbed pronephroi) and the others like those in Group IV (grafted specimens unilaterally pronephrectomized), but with the difference that each tubular graft of these 4 specimens was open into the coelom by either one or two nephrostomial tubules.

Histological, sampling, and measuring techniques were identical to those used in earlier papers (Fox, 1956, 1957, 1959). The level of statistical significance used throughout was P<0·05.

• (a) Comparison between pronephric components of Group I (control specimens with undisturbed pronephroi) and those of Group II (grafted specimens with undisturbed pronephroi).

  Means of the total length and of the nose-to-cloacal length of the two groups of larvae and of the various components of the pronephric systems do not differ significantly (Tables 1,2; Plate, figs. A, B) except for the means of the anteroposterior pronephric length and of the diameter of the anterior and posterior nephrostome bases, which are significantly lower in Group II by 6 per cent, and 9 per cent, respectively.

  One control (Group I) and 2 specimens of Group II possess pronephroi each with 3 nephrostomes (see Field, Plate VIII, fig. 57, 1891), and one further specimen of Group II had a pronephros whose anterior nephrostome was divided into two. These pronephroi were not otherwise ‘abnormal’ and only the abnormal nephrostomes were omitted from the analysis.

• (b) Comparison between host pronephroi in specimens of Group II (grafted specimens with paired undisturbed pronephroi) and their grafts (Group Ila).

  Only two components from each group were compared; other comparisons between whole undisturbed pronephroi and a mere portion of pronephric tissue are not applicable. Means of the calculated individual cell volumes of host pronephroi and grafts do not differ significantly, although there is a smaller cell volume in the latter. The second measurement employed was the relationship lumen volume/tissue volume, henceforth termed the ‘tubule ratio’. Use of the tubule ratio is a method of quantifying an obvious morphological feature of the graft tissue. Graft tubules have thickish, somewhat vacuolated walls, the lumina are attenuated and considerably reduced in volume and show no typically expanded ‘normal’ tubular pattern (Plate, figs. C, D, I, L). This abnormal appearance is typical of any graft (whichever group is considered) when not in open communication with the coelom. Only one anomalous graft had a large expanded tubule which ended at the surface of the body, and here its condition encouraged the view that at some time it was open to the exterior. The graft mean tubule ratio (Group IIa) is significantly lower than that of the pronephroi (Group II) by 56 per cent. (Table 1).

• (c) Comparison between pronephroi in specimens of Group I (controls with paired undisturbed pronephroi) and those of Group III (specimens unilaterally pronephrectomized without grafts).

  Pronephric components of Group III showed a significant superiority over those in Group I by the stated percentages in the means of the following measurements: total lumen volume (196 %); overall pronephric volume (tissue volume and lumen volume together) (120 %); tubule ratio (81 %); total cell volume (60 %) ; calculated individual cell volume (54 %) ; internal surface area of the tubules (26 %) (see Plate, figs. A, E, F). Means of the total length and of the nose-to-cloacal length of the larvae were significantly lower in Group III by 5 per cent, and 9 per cent, respectively (Table 1). Howland (1921) has shown that unilateral pronephrectomy may inhibit general growth and a similar result occurred in the other unilaterally pronephrectomized specimens (Group IV). Retardation did not occur in similar circumstances in Triturus cristatus carnifex (Fox, 1956).

  No significant differences were found between the means of the following measurements: antero-posterior pronephric length (6-4 per cent, superiority in Group III over Group I); diameter of the anterior and of the posterior nephro-stomial bases, and of the total nephrostome bases; mean pronephric nuclear population (3–7 per cent, superiority in Group III over Group I). These results may be compared with those of previous work (Fox, 1956, 1957). In unilaterally pronephrectomized T. cristatus carnifex (killed 10–15 days after a stage equivalent to Harrison stage 24), and operated specimens of T. helveticus with unilateral duct blockage (killed 20 days after operation at stages 21–23, Glaesner, 1925), there were the following percentage compensatory responses compared with controls (T. helveticus is shown in brackets). Lumen volume 100 (127); overall pronephric volume 52 (94) ; tubule internal surface area 49 (63) ; total cell volume 34 (78); nuclear population 17 (41); calculated individual cell volume 15 (22); antero-posterior pronephric length 14 (20).

  Calculation from the data for T. helveticus shows the tubule ratio of hypertrophied (compensated) pronephroi to have a significant superiority of 31 per cent, over the controls; i.e. 0-612±0-0610 (compensated pronephroi), 0·466± 0·0380 (controls). For T. cristatus carnifex the tubule ratio of compensated pronephroi 10 days after a stage equivalent to Harrison stage 24 showed a significant superiority of 32 per cent, over controls; i.e. 0·393±0·0280 (unilaterally pronephrectomized specimens), 0·298±0·0150 (controls), and 15–17 days after a stage equivalent to Harrison stage 24 the tubule ratio showed a significant superiority of 49·6 per cent, over controls; i.e. 0·758±0·0856 (unilaterally pronephrectomized specimens), 0·506±0·0141 (controls).

  Howland (1921) in one Ambystoma specimen fixed 9 days after unilateral pronephrectomy at Harrison stages 30–32 found the total cell volume to increase by 63 per cent, when compared with a control specimen, and other increases were nuclear hyperplasia (16 %), tubule length (21 %), internal surface area of tubules (over 100 %), and in addition there was actual cell-volume hypertrophy.

  The results obtained in Ambystoma in this present work, with the exception of the nuclear population and pronephric length, are generally similar to those in the Triturus species, and confirm some of the results of Howland.

• (d) Comparison between pronephroi of Group I (controls with paired undisturbed pronephroi) and host pronephroi of Group IV (grafted unilaterally pronephrectomized specimens).

  Means of the pronephric components of Group IV showed a significant superiority over those in Group I in total lumen volume (236 per cent.) ; overall pronephric volume (139 %); tubule ratio (110 %); total cell volume (60 %); internal surface area of tubules (45 %) ; calculated individual cell volume (39 %) ; nuclear population (15·5 %); antero-posterior pronephric length (13 %) (see Plate, figs. A, G, H, K). Mean nose-to-cloacal length was significantly lower in Group IV by 8 percent, and mean total larval length, though not significantly so, was lower by 4 per cent.

  The means of the diameters of the anterior and of the posterior nephro-stomial tubule bases and of all the nephrostomes together though showing percentage superiorities in Group IV over Group I are not significantly different (Tables 1, 2).

• (e) Comparison between pronephroi in Group III (specimens unilaterally pronephrectomized only) and those in Group IV (grafted unilaterally pronephrectomized specimens).

  No significant differences were found between the pronephric component measurements of the two groups (Tables 1, 2; Plate, figs. E, F, G, H, K).

• (f) Comparison between host pronephroi in grafted unilaterally pronephrectomized specimens (Group IV) and their grafts (Group IVa).

  Means of the individual cell volume and of the tubule ratio show a significant superiority in Group IV over Group IVa by 35 per cent, and 87 per cent, respectively (Table 1).

• (g) Comparison between grafts in specimens with paired undisturbed pronephroi (Group IIa) and grafts in specimens unilaterally pronephrectomized (Group IVa).

No significant differences were found between the means of the individual cell volumes and of the tubule ratios. Although not significant in both groups of grafts, there is a similar lower mean individual cell volume of 5-7 per cent. (Group Ila) and 7·6 per cent. (Group IVa) when compared with controls (Table 1 ; Plate, figs. C, D, I).

In the larval Ambystoma population investigated, pronephric graft tissue, which presumably contained some damaged cells when transferred to a recipient, does not influence growth of undisturbed pronephroi, except for changes in pronephric length and in the relatively unimportant nephrostomial base diameter. Nor does the graft influence pronephric compensatory growth, for compensatory hypertrophy takes place when a specimen is unilaterally pronephrectomized irrespective of whether graft tissue is present or not. Failure in this work to obtain significant increases in the pronephric nuclear population and pronephric antero-posterior length in non-grafted unilaterally pronephrectomized specimens may be an example of the variability of these responses, for compensatory hyperplasia and increase in pronephros length can and normally do take place in urodeles including Ambystoma (Howland, 1921) so treated, and both components show a trend in this direction in the present work. The results may be compared with those in T. helveticus (Fox, 1957), where it was found that pronephric compensatory hypertrophy (and hyperplasia) ensued in an unblocked functional pronephros when the duct of its partner is blocked, though the blocked pronephros, except for some stretching of the tubule walls, appeared undamaged. A pronephros thus compensates in the presence of a blocked but undamaged partner, and the pronephroi do not inhibit one another’s growth—a result which does not support the theory of autoregulation of growth by homologous organs (see Weiss, 1952, 1953, 1955). The evidence in this present work supports the view that it is unlikely that products of normal or of damaged homologous cells influence pronephros cell size or tubule lumen size.

The grafts in unilaterally pronephrectomized specimens, like grafts in specimens with paired undisturbed pronephroi, were situated close to the coelom in intimate association with blood sinuses. If a raised concentration of circulating pronephric growth-promoting substances, however produced, elicited pronephric hypertrophy and hyperplasia, then it may be assumed that there would be a difference in concentration at some time between these substances in grafted unilaterally pronephrectomized specimens possessing a single hypertrophied pronephros, and in grafted specimens with undisturbed pronephroi. Yet the graft tubules in the two groups are similar in tubule appearance and in individual cell volume. Grafts in the two groups do, however, differ in cell number and in total volume. Nevertheless, the means of the total nuclear population, cell volume, lumen volume, and overall volume, were not found to differ significantly. Yet it is impossible to know exactly how much pronephric tissue has been transferred to a recipient, and these numerical differences may be due to the difference in the amount actually transferred, or to a real increase in mitosis (hyperplasia) of graft tissue of Group IVa. The presence of circulating pronephric growth substances which would influence individual cell volume and lumen volume is therefore not supported in this work, but the results do not discount the possibility that an increase in the rate of mitosis has taken place in the grafts of Group IVb and that this increase has been influenced by them.

A difference of major importance between grafts and any pronephros left in situ is that graft tubules are not in open communication with the coelomic fluid. A pronephric blastema graft will differentiate into tubules although it is isolated from the coelom, but the lumina are small and the tubules fail to develop into normal functionally expanded structures. Their condition is similar to that of a pronephric duct remaining after unilateral pronephrectomy, or that portion of a duct distal to a lesion, for the duct is in a collapsed state. Among the collection of experimental animals were 4 specimens each with a graft in the usual ventrolateral position behind the pronephros region, and with an open communication to the coelom by either one or two ciliated nephrostomes (Plate, fig. J). These ‘open’ grafts differed from the blind isolated ones in that their tubules were to a greater or lesser extent expanded (like blocked pronephroi with nephrostome inlets but having no exit) (Fox, 1957). Tubule ratios of these grafts were: 3·467, 1·180, 0·740, and 0·446; all are higher than the means of the tubule ratios of isolated graft tubules in the two groups (IIa and IVa) previously considered. It is clear that intra-tubular fluid pressure, exerted by coelomic fluid which enters by the ciliated nephrostomial tubules, maintained the graft tubules in an expanded state.

The presence of coelomic fluid directed into the pronephric tubules by nephrostomial cilia and exerting an intra-tubular fluid tension would seem to be essential for the maintenance of normal or hypertrophied tubule shape, and to some extent it influences cell size.

Pronephric grafts in either a compensating or non-compensating environment are extremely similar in the appearance of the tubules, and though they differ in cell numbers and total volume, these differences are not significant. In addition the mean individual cell volumes are the same, so it is likely that the mitotic rates of these graft tissues are similar. Yet in pronephric compensation there is usually hyperplasia, and as there is no evidence for the view that cell volume and lumen volume hypertrophy are initiated by pronephric growth-promoting substances, then intra-tubular tension may be the main causal factor eliciting compensatory nuclear proliferation also. This opinion supports the more cautious conclusion reached for the cause of pronephric hyperplasia in T. helveticus larvae each one possessing a blocked and an unblocked hyperplastic pronephros (Fox, 1957), and for the striking results in the paired pronephroi of T. helveticus grown in distilled water (Fox, 1959).

Animals unilaterally pronephrectomized were practically the same in size and shape as controls with paired undisturbed pronephroi. The external surface areas of these animals are generally similar and as the properties of the skin presumably do not differ, similar quantities of fluid will enter these larvae when they develop in media of the same kind. Because both types of larvae appear similar, then approximately the same quantity of fluid must have been evacuated from animals with either a single pronephros or a pair. Intra-tubular tension must therefore be higher in a single remaining pronephros than in either of the paired pronephroi, and it is likely that this super-normal intra-tubular fluid tension elicits all the recognized compensatory pronephric growth phenomena.

Action de facteurs internes sur la croissance normale et compensatrice du pronephros de /’Axolotl

1. Les expériences décrites dans cet article ont eu pour but de distinguer entre les influences chimiques et les influences mécaniques (hydrostatiques) qui s’exercent sur la croissance normale et compensatrice du pronéphros de la larve d’Axolotl.

2. On a fait des mesures sur les pronéphros et les greffes de pronéphros des groupes suivants : (a) spécimens témoins avec pronéphros intacts ; (b) spécimens avec pronéphros intacts et greffons de pronéphros ; (c) spécimens pronéphrec-tomisés unilatéralement; (d) spécimens pronéphrectomisés unilatéralement et contenant des greffons.

3. L’étude des mesures montre que la pression hydrostatique à l’intérieur des tubes du pronéphros est le principal facteur responsable du maintien de la taille et de la forme dans la croissance normale aussi bien que compensatrice.

The Ambystoma embryos used in this investigation were supplied by Professor D. R. Newth whose generosity is warmly appreciated. I am happy to record my thanks to Professor P. B. Medawar and Professor M. Abercrombie for their comments and advice, and to Mr. C. Atherton for his photographic work.