1. The effects of treatment with phenylthiourea and of thyroidectomy and hypophysectomy on the embryo of Eleutherodactylus are described, mainly with reference to changes within the nervous system.

  2. In Eleutherodactylus the events at organ level during metamorphosis which are controlled by the thyroid are reduced to loss of the tail and of the pronephros. The primary sensory neurones of the cord, the Rohon-Beard cells, are also lost at the same time.

  3. The normal loss of cells from the ventral horns during development is not affected by any treatment to which the embryo has been submitted. Development is retarded by treatment with phenylthiourea, and also by hypophysectomy even when the pituitary is not wholly removed. In embryos submitted to this operation there is a loss of affinity for silver in the neurones of the ventral horn, which is most severe when the pituitary is completely excised.

  4. The addition of thyroxine to thyroidectomized embryos has been shown to increase the number of fibres within a muscular and also a cutaneous nerve.

  5. The number of fibres in a muscular nerve may be reduced by thyroidectomy or by hypophysectomy. It is suggested that in muscular nerves the loss of fibres is unaffected by thyroid hormones but that the accession of new fibres is sensitive to their action.

  6. The movement of supernumerary grafted limbs is much enhanced by general retardation of development with phenylthiourea.

In a recent paper (Hughes, 1965 a) the ontogeny of the peripheral nervous system in Eleutherodactylus martinicensis was studied by counting the numbers of constituent fibres in the nerves of the hind limb at several stages of development. Eleutherodactylus is one of the smallest of living tetrapods and such quantitative studies are aided by the comparatively small numbers of fibres within its peripheral nerves. The next step in the analysis of the various changes which occur simultaneously in the normal embryo is to attempt to disengage them by some treatment which affects the general course or development. Interference with the endocrine system of the embryo is one obvious line of investigation, more particularly as a number of events in the development of the nervous system in other vertebrates have been shown to be influenced by thyroid hormones.

The action of the thyroid in Eleutherodactylus has already been investigated by Lynn & Peadon (1955). In this Anuran development is wholly embryonic and direct; of the metamorphic events at organ level in larval Amphibia, only the loss of the tail and of the pronephros remains. The loss or retention of the tail in Eleutherodactylus provides a valuable index of the extent to which the influence of the thyroid gland has been reduced under experimental treatment, especially as only limited information can be obtained from the histology of the gland itself in this animal. In these pages, the results are described of treatment with an anti-thyroid agent, and of both thyroidectomy and of hypophysectomy on a number of features of development, mainly with reference to events within the nervous system. The results obtained are summarized in Table 1.

Table 1.
graphic
graphic

The source of the embryos of E. martinicensis and the methods of culturing and observing them are the same as those used in previous studies (Hughes, 1962, 1964a, b, 1965a, b, c). Unoperated embryos of Eleutherodactylus shed from their envelopes develop equally well in either water or saline.

Relatively crude methods of thyroidectomy and of hypophysectomy were adequate for Eleutherodactylus embryos, which suffered no special inconvenience from mutilation of the oral region. For the former operation the lower jaw was removed as far back as the border of the third aortic arch, and behind the zone of the developing thyroid. In all of thirteen embryos submitted to this opération and subsequently examined, regenerated follicles were seen 6-7 days later, atypical both in number, size and distribution. The partial absence of the developing gland, however, in a critical period during the first few days after operation was effective in preventing atrophy of the tail.

Hypophysectomy was attempted on embryos at 7-9 days (stages ‘a’ to ‘d’ of Table 2) by excising tissue from the roof of the mouth to a level extending dorsalwards to the floor of the mid-brain. Of 48 operated embryos, the pituitary was completely removed in 21. The remainder served as sham-operated controls.

Table 2.

Stages of development of Eleutherodactylus martinicensis at 25-30 °C

Stages of development of Eleutherodactylus martinicensis at 25-30 °C
Stages of development of Eleutherodactylus martinicensis at 25-30 °C

In Eleutherodactylus embryos towards the end of development the various regions of the pituitary can be recognized, though histological differentiation within each region has not progressed very far. Where however the organ has not been completely removed, and some hypophysial tissue is present in distorted relationships, the region to which it belongs cannot be recognized with certainty, particularly as the constituent cells-are less differentiated than in a normal embryo. In some examples there was evidently a regenerated anterior lobe. However, all embryos in which hypophysectomy was incomplete are so described in succeeding pages with no attempt to identify the nature of the residual tissue.

These glandular extirpations were performed at later stages of development than are used for the corresponding operations on larval amphibia (Rugh, 1948), for at comparable stages the embryo of Eleutherodactylus is inaccessible.

In one series of embryos an arm was severed and then grafted near an undisturbed leg, in the same way as in previous studies on supernumerary limbs (Hughes, 1962, 1964a, b).

On several occasions embryos were treated with thyroxine dissolved in saline at concentrations of 1:107 or 1:108. The sodium salt of L-thyroxine was used, supplied by British Drug Houses Ltd.

Rate of development and external appearance

Close observations were made on the rate of development of the experimental embryos. One set of landmarks were provided by their progress in reflex behaviour (Hughes, 1965,b). This can be correlated with changes in external form as is set out in Table 2. This information supplements, and in some details corrects, that given in an earlier paper (Hughes, 1962, Table 1), in which the appearance of the egg tooth is wrongly placed later than that of the first xanthophores. A further well-marked stage is when the xanthophores on the head form an ‘interorbital line’ separating a dense anterior zone from a sparser area which extended caudally behind the head. Melanophores first appear in the tail at the time when its shrinkage begins.

No differences were observed between the development of intact embryos of Eleutherodactylus shed from their envelopes and immersed in either water or Holtfreter saline. The skin thus appears to be largely impermeable to ions.

In Text-fig. 1 the course or development at 26-30 °C of control embryos freed from their envelopes at various stages and then immersed in saline is shown. Allowance is made in this figure for instances when an embryo shows one feature only of a particular stage (e.g. leg extension without xanthophores).

Text-fig. 1.

Development of control embryos in saline; stages as in Table 2 with isopleths of tail length of 2·0 mm and 3·0 mm respectively.

Text-fig. 1.

Development of control embryos in saline; stages as in Table 2 with isopleths of tail length of 2·0 mm and 3·0 mm respectively.

The rate of development is independent of the stage at which the embryo is removed from its envelopes. However, in those removed relatively early (at stages ‘a’ or ‘b’) the tail remains at its maximum size of about 4·0 mm in length for 3-4 days, while in those animals placed in saline at later stages (‘d e ‘) the tail begins to shrink by the end of the first day of immersion. Thus in Text-fig. 1, where the dotted lines represent isopleths of tail length, those for 3·0 and 2·0 mm tails respectively bulge obliquely towards the top right of the figure.

The effect of 0·01 % phenylthiourea (PTU)

Lynn & Peadon (1955) showed that treatment of Eleutherodactylus embryos with PTU caused a general slowing of development. In larval amphibia, on the other hand, in which development extends over a much longer time-scale, antithyroid agents not only inhibit metamorphosis but also promote growth of the larva beyond normal limits by suppressing the ‘feed-back’ of the thyroid to the pituitary. This effect was shown with thiourea on Rana pipiens by Gordon, Goldsmith & Charipper (1943); a similar action is seen with larvae of Xenopus treated with PTU (Prestige & Hughes, unpublished).

The observations of Lynn & Peadon on embryos of Eleutherodactylus treated with this substance have been confirmed in the present experiments, in which the development of thirty-nine embryos submitted to the action of PTU was observed (Text-fig. 2). The slope of the time-development curve for treated embryos decreased with time, and by the fourth or fifth day there is little or no further progress in development. The stage at which this arrest occurs depends upon that at which the embryo was placed in PTU; embryos at stage ‘a’ are halted somewhere between stages ‘d’ and ‘e’. Xanthophores appear, but the legs show little or no extensor thrust, and the tail shows only the first signs of decrease in size. In embryos immersed in PTU from stage ‘d’ onwards, the extensor reflex appears, and they are subsequently able to maintain continuous swimming, but the shrinkage of the tail is halted at a length of about 1-0 mm, although the rate of the earlier phases of tail reduction are normal.

Text-fig. 2.

Development of embryos in saline containing 0·1 % PTU; stages as in Table 2 with isopleths of tail length as in Text-fig. 1.

Text-fig. 2.

Development of embryos in saline containing 0·1 % PTU; stages as in Table 2 with isopleths of tail length as in Text-fig. 1.

As was described by Lynn & Peadon (1955), melanin is lost from pigment cells under the influence of PTU. Such embryos become paler than the corresponding saline controls on the second day of treatment. The extent of the reaction is similar in embryos treated at any stage between ‘a’ and ‘e’.

The effect of thyroidectomy

The general retardation of development in Eleutherodactylus with PTU shows some differences from the effects of removal of the thyroid gland. When this operation is performed at stage ‘d’ the subsequent development of the embryos continues at a normal pace (Text-fig. 3); operated at stage ‘b’, however, while the development of pigmentation and of leg behaviour proceed normally for the next 48 h, the embryo retains the tail at full length even though some thyroid follicles regenerate. There is thus a critical period, at or near stage ‘c’, when the presence of the thyroid is necessary for development to reach the juvenile stage. In such embryos the addition of 1:108 thyroxine 4 days after the operation results in shrinkage of the tail at the normal rate.

Text-fig. 3.

Development of thyroidectomized embryos in saline; stages as in Table 2 with isopleths of tail length as in Text-fig. 1. Embryos operated at stage ‘d’ reach stage ‘i’ with complete loss of tail; those operated at stage ‘b’ are halted in development. One such is treated with thyroxine on 4th day (arrow) and loses tail within the next 3 days. All embryos regenerate follicles.

Text-fig. 3.

Development of thyroidectomized embryos in saline; stages as in Table 2 with isopleths of tail length as in Text-fig. 1. Embryos operated at stage ‘d’ reach stage ‘i’ with complete loss of tail; those operated at stage ‘b’ are halted in development. One such is treated with thyroxine on 4th day (arrow) and loses tail within the next 3 days. All embryos regenerate follicles.

The effect of hypophysectomy

The effect on an Anuran larva of extirpation of the pituitary was first shown by Smith (1916) in Rana boylei. He demonstrated a hypoplasia of the thyroid gland and a reduction in the general rate of growth. The action of the amphibian pituitary on the thyroid gland is still imperfectly understood, however, for ‘the control of thyrotropic hormone production and its release by the tadpole pituitary is as yet a mystery’ (Kollros, 1961).

In the present experiments on Eleutherodactylus, development was slowed in all embryos in which removal of the pituitary was attempted, even though the whole or part of the gland was present. The initial slowing of development may thus be attributed to the effects of operative interference, in which disturbance of the pituitary stalk may be partly responsible. These embryos, however, showed a special vigour in their limb movements.

There were however differences to be observed between those in which the pituitary was wholly removed and those in which some or all of the gland remained. In the former, the tail remained largely constant in length for periods up to 17 days after operation. In others, however, a slow decline in its length was observed ten or more days after the operation (Text-fig. 4). After fixation and sectioning these animals were found to have a pituitary, even though there was much disturbance at the base of the mid-brain, often with damage to the optic chiasma. In three embryos which were fixed 18-25 days after the operation, a small undifferentiated group of hypophysial cells was found, which suggested some degree of regeneration. The tail remained at constant length in only one of these three embryos.

Text-fig. 4.

Lengths of tail with time in (a) saline control embryos; (b) embryos with incomplete hypophysectomy, treated with thyroxine; (c) untreated incompletely hypophysectomized embryos; (d) completely hypophysectomized embryos. All at stage ‘b’ to ‘c’ at day 0.

Text-fig. 4.

Lengths of tail with time in (a) saline control embryos; (b) embryos with incomplete hypophysectomy, treated with thyroxine; (c) untreated incompletely hypophysectomized embryos; (d) completely hypophysectomized embryos. All at stage ‘b’ to ‘c’ at day 0.

During the course of observation from day to day, thirteen embryos became lighter in colour than normally, and in them melanophores were seen to have contracted to small round dots. In all of these it was afterwards found that the pituitary was wholly absent and the effect on the pigment cells could be ascribed to the absence of the melanophore-stimulating hormone (Barrington, 1964). In two others which showed a partial contraction of the melanophores, some regenerating pituitary cells were present. The earliest stage at which melanophore contraction was observed was in an embryo at stage ‘e’, 2 days after operation.

Eight hypophysectomized embryos were treated with thyroxine at a concentration of 1:107. Five died, but in the three survivors the tail began to dwindle 1-3 days after the operation at rates much greater than were seen in other animals of this group with no treatment with the hormone. In all three of these embryos some hypophysial cells were subsequently found. In them, however, the loss of the tail began much earlier than in any other embryo with a reduced pituitary (Text-fig. 4b, c). Two of these three embryos showed some contraction of melanophores during life.

The length of the foot

An index of the growth of the embryo is given by the length of the foot, measured from the tarso-metatarsal joint of the tip of the second digit. In the normal embryo, from stage ‘f’ to the hatching period, this increases from 1-0 to about T8 mm. The experimental embryos were measured daily and thus it was possible to detect any inaccuracy in individual values, as on occasions when movement of the embryo interfered with measurement. In Text-fig. 5 averaged data are given for a group of 3 or 4 individuals under three sets of circumstances, namely (a) unoperated embryos developing in saline, and embryos after complete (b) and incomplete (c) removal of the pituitary. There is a clear distinction between the two latter categories in this respect. In some partially hypophysectomized embryos which were fixed 14-25 days after operation, well after normal embryos of the same age had hatched, the foot increased in length beyond that of the normal embryonic range. It is possible that here the differences caused by complete removal of the pituitary are due directly to the action of a hypophysial growth hormone. In the three partially hypophysectomized embryos which were treated with thyroxine it did not seem that growth of the foot was thereby increased.

Text-fig. 5.

Growth in length of foot with time in (a) average of four saline control embryos; (b) average of three incompletely hypophysectomized embryos; (c) average of four embryos with complete hypophysectomy.

Text-fig. 5.

Growth in length of foot with time in (a) average of four saline control embryos; (b) average of three incompletely hypophysectomized embryos; (c) average of four embryos with complete hypophysectomy.

After thyroidectomy, the growth of the foot is much slowed down, equally in embryos which retained the tail and in those where it atrophied. Treatment of thyroidectomized embryos with thyroxine did not make any appreciable difference in this respect. In embryos immersed relatively early in PTU-saline, and where development was halted at stage ‘e’, the length of the foot was below that of a normal embryo of the same stage.

The thyroid gland

Lynn & Peadon (1955) have described the effect of treatment with PTU on the histology of the thyroid in Eleutherodactylus embryos. After 4 days the height of the follicular epithelium is increased. Hypertrophy of the gland is seen at 5 days, with the disappearance of much of the colloid from the follicles. Later, the follicular cells become highly vacuolated. If an embryo is transferred from PTU to water the normal condition of the thyroid is regained within a short time. The present observations confirm these results of Lynn & Peadon. All groups of embryos treated with PTU save one showed the effects described by these authors. Again, the normal appearance of the gland reappeared on cessation of the treatment. Colloid reappeared in the glands of embryos treated first with PTU for 5 days, and then for 2-3 days with both PTU and thyroxine. Lynn & Peadon also found that thyroxine added to the water in which embryos were developing caused a flattening of the follicular epithelium, and an increase in the amount of stored colloid. In the present experiments, however, no constant differences of this kind resulted from treatment with exogenous thyroxine, but the whole gland in such embryos seemed somewhat smaller than normal.

In all animals in which hypophysectomy was attempted the follicular epithelium became progressively flattened, though no general differences could be detected between those in which the pituitary was wholly removed and those in which some residual tissue was present. Empty follicles were observed in some operated embryos of both groups.

In animals which were thyroidectomized and fixed from 6-7 days after operation, thyroid follicles regenerated. These were small in number but abnormally large in size and were seen both in those embryos in which the tail atrophied and in those in which it remained. In the latter group, although the regenerated follicles appeared too late to influence the tail, some internal changes were arrested, as described below.

The pronephros

Lynn & Peadon (1955) found in Eleutherodactylus that PTU inhibited the normal degeneration of the pronephros, the loss of which is among the few events at organ level which is controlled by thyroid hormones.

In embryos taken from their envelopes and then fixed, the first signs of degeneration of the pronephros are seen at stage ‘e’, which is characterized by the kick reflex of the legs and the first appearance of xanthophores over the body. The tail begins to shrink a day or so later. A slight vacuolation of the pronephric tubules is seen, with the vascular spaces of the surrounding mesenchyme dilated with blood. In other embryos at stage ‘e’, degeneration may be further advanced, with a flattening of the tubular epithelium, the accumulation of cell debris in the lumen, and the appearance of necrotic cells around the tubules. Often the pronephroi on both sides of the embryo are not in the same phase of degeneration. This second phase is reached in all embryos in which the tail has been reduced to 2 mm in length. Before the tail has entirely disappeared the site of the pronephros is recognizable only as an area of reticular connective tissue. In embryos which have been developing in saline the disappearance of the pronephros is more advanced relative to that of the tail, which may still be at its full size when the pronephros already contains necrotic cells.

When PTU has been added to the saline in which an embryo develops, the extent to which the pronephros degenerates, as with the general development of the embryo, depends on the age at which the treatment began. With those embryos placed in PTU relatively late (from ‘d’ onwards) and which subsequently reach the juvenile stage, the disappearance of the pronephros bears the same relation to that of the tail as in a control embryo. When treatment begins at stages ‘a’ to ‘c’ and the tail remains unresorbed, some pronephric tubules become dilated, but no further changes are seen.

In embryos thyroidectomized at stage ‘b’ and in which development is arrested 3-4 days later with little or no shrinkage of the tail, degeneration of the pronephros nevertheless proceeds, and only small lengths of tubular epithelium remain intact. The regenerated follicles in these embryos must produce enough thyroid hormones to affect the pronephros, but not the tail.

In hypophysectomized embryos, degeneration of the pronephros is again seen without shrinkage of the tail. Here the vacuolation of the epithelial cells is especially marked, equally where the pituitary was entirely removed and in those with some residual hypophysial tissue. Vacuolization is seen as early as the second day after operation. The tubules subsequently break down at varying rates, which show a slight correlation with the presence or absence of the pituitary, and among the former group with decrease in size of the tail. In all embryos of this group only reticular traces of the pronephros remain by the 13th to 14th day after operation.

The Rohon-Beard cells

In Eleutherodactylus the life-history of the Rohon-Beard (RB) cells, the primary sensory neurones of the trunk, is somewhat different from that in larval Amphibia, where these cells develop and function long before the appearance of the dorsal root ganglia, and are superseded by them at a stage well before metamorphosis—in Xenopus at about stage 49, when the limb buds are still very small (Hughes, 1957). In Eleutherodactylus, on the other hand, the spinal ganglia appear before the RB cells (Hughes, 1959), and the latter do not vanish until the tail dwindles.

In Text-fig. 6 the spinal cord with its RB cells is reconstructed in a series of embryos of Eleutherodactylus. Each RB cell is represented in these reconstructions. Fifteen embryos were studied in this way. The reconstructions include the first 100 μ of the tail region of the cord in which the RB cells are the last to disappear. Text-fig. 6a-c represent the normal course of events from stages ‘d’ to ‘g’ in embryos taken from their envelopes and then fixed. By the time that the tail has begun to dwindle the number is already halved and continues to decrease while the tail degenerates further. In normal embryos the loss of the RB cells thus proceeds pari passu with the degeneration of the pronephros. This synchrony is maintained in embryos which have been treated with PTU. If thyroidectomy is performed early enough to prevent loss of the tail, the disappearance of RB cells begins some 7 days later. It proceeds to completion with loss of the tail in similar embryos treated with thyroxine (Text-fig. 6g, h).

Text-fig. 6.

Diagrammatic reconstructions from medulla to tail (upper border of figure) of neural tube in embryos to show Rohon-Beard cells, (a) Normal embryos at stage ‘d’ (Table 2); (b) at stage ‘e’; (c) at stage ‘g’; (d) 10 days after complete hypophysectomy; (e) 17 days after complete hypophysectomy; (f) 10 days after partial hypophysectomy; (g) 7 days after thyroidectomy at stage ‘b’, degenerating cells in black; (h) 7 days after thyroidectomy at stage ‘b’, with thyroxine on last 3 days. The tail was shrinking in embryos (c) and (f) and was lost in (h).

Text-fig. 6.

Diagrammatic reconstructions from medulla to tail (upper border of figure) of neural tube in embryos to show Rohon-Beard cells, (a) Normal embryos at stage ‘d’ (Table 2); (b) at stage ‘e’; (c) at stage ‘g’; (d) 10 days after complete hypophysectomy; (e) 17 days after complete hypophysectomy; (f) 10 days after partial hypophysectomy; (g) 7 days after thyroidectomy at stage ‘b’, degenerating cells in black; (h) 7 days after thyroidectomy at stage ‘b’, with thyroxine on last 3 days. The tail was shrinking in embryos (c) and (f) and was lost in (h).

In all ‘hypophysectomized’ embryos, decrease in the length of the tail and in the number of RB cells is closely related to the presence of residual pituitary tissue. The full number of RB cells may be maintained for 10 days after a compíete hypophysectomy, and more than half may still be present a further 7 days later (Text-fig. 6 d, é). In two such examples the pronephros was already in an advanced stage of degeneration.

In all ‘hypophysectomized’ embryos in which the length of the tail decreased and in which some pituitary cells were found, the RB cells were confined to the tail region of the cord. In the embryo in which the period between operation and fixation was longest, namely 25 days, the tail remained constant in length, even though there was evidence of a regenerated anterior lobe. RB cells were found only in the tail region of the cord.

The degenerative changes which have been described in the tail, the pronephros, and in the RB cells are all related to metamorphosis on the greatly reduced scale of this event in Eleutherodactylus, and in all thyroid hormones are concerned. Below, the effects of their withdrawal on several features of the development of the nervous system which are unrelated to metamorphosis are described.

The ventral horn cells (the lateral motor column)

Between stages ‘c’ and ‘d’ of the present series in the normal ontogeny of E. martinicensis, some ventral horn cells are differentiating rapidly. The linear dimensions of the nucleus enlarge by about 30%, the contents of the nucleus become sparser, with one nucleolus within a largely clear sap. The distinction between developing ventral horn cells and those of the mantle layer becomes very clear. At the same time the cell enlarges as a whole. Hitherto visible cytoplasm has been restricted to polar caps but now the nucleus is surrounded by an evident layer of cytoplasmic material with marked expansions at the poles. The development of the lumbar ventral horn is at first in advance of that of the brachial horn.

From the beginning, the cell axon reacts to silver as to a lesser extent do the polar caps of cytoplasm. As the cell enlarges the argyrophilic material within the cell assumes a fibrous appearance against an unstained background. It is certain that these ‘neurofibrillae’ are greatly altered by fixation (Hughes, 1954) and by accretion of silver metal on whatever was their original form in the living cell. Yet they represent some component of the neuronal cytoplasm in which changes are seen in the course of development and which is susceptible to endocrine influences (Plate 1, fig. A). In the maturing neurone the course of the main bundle of neurofibrillae is usually to one side of the nucleus from where they sweep dorsally into the dendritic processes of the cell, which branch and extend widely.

During the later stages of embryonic life not all ventral horn cells have reached this stage. At the caudal pole of the ventral horn all the cells are relatively young. This disparity among the ventral horn cells of developing Anura suggests that new cells are being recruited to the ventral horn as others degenerate. In comparing embryos developing under different conditions with respect to the degree of differentiation of ventral horn cells, one difficulty is that the depth of impregnation with silver is not constant from slide to slide. One can, however, reject any specimen in which the fibres of the white matter and of the peripheral nerves are not deeply stained, and in the remainder it is possible to compare the most advanced cells with the others.

The early stages of differentiation of ventral horn cells can be accelerated by adding thyroxin to the medium in which the embryos are developing. This effect has already been described for tadpoles of Rana pipiens by Beaudoin (1956) and by Reynolds (1963). In Eleutherodactylus, 1:107 thyroxin administered between stages ‘b’ and ‘c’ results in a general increase in the size of the nuclei of cells in the ventral horn. Treatment with PTU sufficiently early to arrest general development at stage ‘f’ or before retards their differentiation. Some effect was seen as the result of thyroidectomy.

After hypophysectomy there are marked effects within the ventral horn. The differentiation of the nuclei of the ventral horn cells is retarded and the extent to which these neurones stain with silver is greatly reduced by complete extirpation of the pituitary. As early as 2 days after operation a difference can be seen between embryos with full and with partial removal of the gland. In one embryo with full hypophysectomy the loss of argyrophilia was complete 6 days after operation. Of eleven embryos in which the pituitary was wholly removed, all showed a loss of argyrophilia in the ventral horn cells (Plate 1, fig. C). In ten of these embryos melanophores were seen contracted during life.

This failure to stain on silver impregnation is confined to the cell body of the neurone, distal to which the axon is as black as in the normal embryo. There is an intermediate condition in which the poles of the cell take up silver, with no impregnation near the nucleus. In those embryos in which some pituitary cells still remained, the degree of reduction of argyrophilia was variable (Plate 1, fig. B); some were similar in this respect to embryos with complete hypophysectomy. In the three embryos which were partially hypophysectomized and subsequently treated with thyroxine, the degree of argyrophilia in ventral horn cells was similar to that of normal embryos, and was greater than that of comparable partially hypophysectomized embryos untreated with the hormone.

In embryos which were treated with PTU or were thyroidectomized, there were signs of a loss of argyrophilia among ventral horn cells; those neurones which were most differentiated showed a normal impregnation, while many of the remainder showed little affinity for silver.

The primary motor neurones of the cord, which innervate the axial musculature, are similar to the ventral horn cells in reduction of argyrophilia after complete hypophysectomy. In contrast to the effect of the operation on motor neurones, those cells of the dorsal root ganglia which are fully differentiated continue to take up silver even when the adjacent motor cells within the cord have lost all reactivity. Rohon-Beard cells within the cord also retain their affinity for silver (Plate, 1, figs. D, E).

The numbers of ventral horn cells

In normal development, parallel changes are seen in the numbers of cells in both brachial and lumbar ventral horns (Hughes, 1962, 1965a). About a thousand neuroblasts are present in each horn at 7-8 days of development, and this number is reduced to some 400 cells at the hatching stage. The decline in numbers is most rapid during the tenth and twelfth days, at stages ‘e’ to ‘f’ of the present series. Degenerating cells are then common in the ventral horns. The thyroid has apparently little or no influence in this decrease in the number of ventral horn cells for in embryos thyroidectomized at stage ‘b’, early enough to prevent any shrinkage of the tail during the subsequent 6 or 7 days, the numbers of ventral horn cells were similar to those of the normal juvenile. Addition of thyroxine to the saline in which such embryos were developing made no difference in this respect.

Again, addition of thyroxine to unoperated embryos was without effect. In embryos treated with 1:10* 7 thyroxine from stages ‘a’ to ‘e’, ventral horn cell numbers were similar to those of the corresponding control embryos in saline, even though the differentiation of the individual neurones was accelerated. Treatment with thyroxine at the same concentration prolonged as far as the hatching stage was without effect. In embryos treated with PTU from stage ‘a’ for 8 days, and arrested in development at stage ‘d’ from the fourth day, the number of ventral horn cells was again that of a normal juvenile, though there were indications of further differentiation of new cells in other embryos similarly treated for the first 4 days and then transferred either to saline or to saline with 1:108 thyroxine. During this second phase of treatment these embryos resumed development as far as stage ‘f’.

The changes in numbers of ventral horn cells in hypophysectomized embryos are shown in Text-fig. 7. The operation was performed at stage ‘e’ at a time when 700-800 cells are present in the ventral horn. Although the further development of those embryos in which the pituitary had been wholly removed did not progress beyond stage ‘f’, yet the number of ventral horn cells was found to diminish to levels even below those of the normal juvenile. This decrease was also seen in those embryos where a pituitary was present, either through incomplete removal at the time of operation, or by regeneration. There was no correlation between the number of ventral horn cells and the size of the tail, for no difference can be seen between those embryos in which it remained at its original length and those where a sudden decrease took place in the last few days before fixation. In those hypophysectomized embryos which were subsequently treated with thyroxin it appears that treatment with the hormone had no effect on the numbers of ventral horn cells.

Text-fig. 7.

Numbers of cells in lumbar ventral horns of embryos operated as indicated, plotted against time after operation. In five examples the total number of lumbar ventral root fibres is also counted.

Text-fig. 7.

Numbers of cells in lumbar ventral horns of embryos operated as indicated, plotted against time after operation. In five examples the total number of lumbar ventral root fibres is also counted.

In all embryos in which hypophysectomy was attempted development was largely arrested 4 days after the operation and remained at the same stage for periods up to 3 weeks. At this time, control embryos had reached juvenile stages. While in the operated embryos the numbers of ventral horn cells ultimately fall to levels below that of a normal juvenile, the rate of loss is less than in unoperated controls.

In normal development the decrease in the population of ventral horn cells is accompanied by an increase in the number of ventral root fibres to which they give rise. The proportion of developing neurones which send an axon into a lumbar ventral root rises from about one in five to a nearly equal ratio (Hughes, 1965 a). In embryos with either complete or partial hypophysectomy there was no indication of any rise in the number of lumbar ventral root fibres with time.

Peripheral changes in numbers of nerve fibres

In the development of embryos taken from their envelopes and then fixed it has been shown that at a time when the tail first shows signs of shrinkage the number of fibres in many nerves within the leg is at a maximum. That this is a general feature of this phase of development is suggested by the fact that the embryo is then often larger than before this stage or during the subsequent day before emergence from the envelope. In embryos developing in saline this peak in the numbers of nerve fibres is less clear. Here it is not seen in terminal branches in the thigh; in the main sciatic nerve at the entry into the limb it is somewhat obscured (Text-fig. 8), but is still recognizable distally at the bifurcation into tibial and peroneal nerves.

Text-fig. 8.

Numbers of fibres in the sciatic nerve at the point of entry into the limb in embryos operated as indicated, plotted against time after operation, together with corresponding data for four stages of saline control embryos.

Text-fig. 8.

Numbers of fibres in the sciatic nerve at the point of entry into the limb in embryos operated as indicated, plotted against time after operation, together with corresponding data for four stages of saline control embryos.

Why there should be this difference between embryos developing under these two circumstances is obscure. It is possible that the small volume of fluid within the egg envelope may contain some growth-promoting substance, secreted by some organ or tissue, which is diluted below its effective range when the embryo is in saline. It is certainly true that the rapid decline on the further side of the normal peak occurs after the envelope has been ruptured. The addition of thyroxine to unoperated embryos developing in saline makes no difference in this respect.

Observations of the effects of thyroidectomy and of hypophysectomy on the number of fibres in peripheral nerves have been made at three points; high in the sciatic trunk (Plate 2, figs. F, G, H), in the Ramus profundus anterior which supplies the triceps femoris muscle (Plate 2, figs. I, J, K), and in the crural cutaneous. The two latter are the largest nerves of their respective kinds in the leg.

In the sciatic nerve at the point of entry into the limb, no differences are seen in the number of fibres between embryos with full and partial hypophysectomy (Text-fig. 8). In those thyroidectomized at stage ‘b’, early enough to prevent loss of the tail, the corresponding numbers of fibres fall well below this range, and are low enough to suggest that there may be some actual loss of fibres in the days following the operation. Embryos of this group treated with thyroxine 4 days before fixation show a marked increase in this respect to levels within the range of saline controls, thus providing a quantitative demonstration of the influence of thyroxine on the outgrowth of fibres of the peripheral nervous system.

The ratio of number of nerve fibres to muscle volume is plotted for the triceps femoris under various conditions in Text-figs. 9-11. At stages ‘c’ and ‘d’ there are 40-50 fibres in the nerve which supplies the triceps, and the volume of the muscle is from 0·01 to 0·02 mm3. In normal development, the number of nerve fibres and the volume of the muscle increase markedly during stages ‘g’ and ‘h’. The variance for both parameters is then high, although the ratio between them is less variable. By stage ‘i’, when the embryo hatches, both quantities drop sharply, the number of nerve fibres to a value little above that of the embryo at stage ‘c’ or ‘d’, and the volume of the muscle to 0·06-0·09 mm3. The ratio between these quantities then becomes less than at stages ‘g’ or ch’. From this time the muscle grows without increase in the number of innervating fibres, for this remains at the same level in an adult animal. The number of fibres in cutaneous nerves, on the other hand, increases greatly during post-embryonic growth.

Text-fig. 9.

Numbers of fibres in the nerve to the triceps femoris plotted against volume of muscle. The stage of each embryo at fixation is shown.

Text-fig. 9.

Numbers of fibres in the nerve to the triceps femoris plotted against volume of muscle. The stage of each embryo at fixation is shown.

The new fibres which enter the nerve before stage ‘g’ are all small in calibre, as are those which are lost between stages ‘h’ and ‘i’. These changes suggest that in normal development there is a peripheral turnover of nerve fibres at stages after that of the first movements of the limb. It seems likely that during development there is both an ingrowth of new fibres into a peripheral nerve and a concomitant loss. The two processes vary separately in rate during development, and, after the peak in numbers, loss overtakes gain.

The changes in muscle volume seem more obscure. There may be some turnover in the constituent muscle fibres but it seems that the increase in volume of the triceps femoris is partly due to intake of water, for in sections it can be seen that the individual fibres at stages ‘g’ and ‘h’ are then proportionately larger in cross-section, with the myofibrils separated by clear spaces.

In embryos freed from their envelopes and which are developing in saline, the triceps femoris shows no peaks either in the number of nerve fibres which enter it or in the volume of the muscle itself (Text-fig. 9). The effects of exogenous thyroxine in these respects on such embryos is variable. Some are slightly retarded in development and in them the number of nerve fibres which enter the triceps femoris is below normal. Deprivation of thyroid hormones also reduces the number of nerve fibres which enter the muscle. It falls to between 25 and 30 in embryos thyroidectomized late in stage ‘b’, which is sufficiently early to prevent loss of the tail. This decrease is proportionately greater than that seen in the main sciatic nerve. If exogenous thyroxine is added to such operated embryos 4 days later at stages ‘e’ to ‘f’, the tail subsequently disappears and the number of fibres in the R. profundus anterior rises to near that of saline controls (Text-fig. 11). When thyroidectomy is postponed until stage ‘d’ the operation leads to no loss of fibres.

A critical period after which deprivation of thyroxin has no effect can similarly be demonstrated by treatment of the embryo with PTU (Text-fig. 10). It was mentioned above that immersion in PTU saline early in stage ‘b’ usually results in an arrest of development between stages ‘d’ and ‘e’. In such embryo after 8 days of treatment, the number of nerve fibres which enters the triceps femoris is about 35 (Text-fig. 10). No change is seen if after the first 4 days in PTU-saline the embryo is then transferred to saline without PTU, or even into saline containing thyroxine. On the other hand, if embryos are immersed in PTU-saline from stage ‘c’ or ‘d’ they can continue development nearly to the end of the embryonic period and then show little or no difference from saline controls in respect to the volume of the triceps femoris or the number of nerve fibres which enter the muscle. With thyroidectomy the critical period comes later in stage ‘b’ than with treatment with PTU which necessarily acts more slowly in the removal of thyroxine from the tissues of the embryo.

Text-fig. 10.

Numbers of fibres in the nerve to the triceps femoris, plotted against volume of muscle, mainly for embryos treated with 0·01 % PTU during the stages of development as indicated. The data for saline control embryos of Text-fig. 9 are given for comparison.

Text-fig. 10.

Numbers of fibres in the nerve to the triceps femoris, plotted against volume of muscle, mainly for embryos treated with 0·01 % PTU during the stages of development as indicated. The data for saline control embryos of Text-fig. 9 are given for comparison.

Embryos which had been ‘hypophysectomized’ subsequently fell into two groups with respect to changes in the volume of the triceps femoris and the number of nerve fibres which entered it (Text-fig. 11). In one there was growth of the muscle, but the number of nerve fibres steadily decreased with time after operation, though at a rate somewhat slower than that seen after thyroidectomy. These were the embryos in which there was no pituitary and in which the tail remained constant in length. The only exception was the oldest, fixed 25 days after operation, in which a regenerating anterior lobe was found, but where the tail nevertheless remained unchanged. In this embryo the number of nerve fibres which entered the muscle is among the lowest of all. In all embryos of this group the volume of the muscle increased steadily with time after operation.

Text-fig. 11.

Numbers of fibres in the nerve to the triceps plotted against volume of muscle. For hypophysectomized embryos the number of days between operation and fixation is given. The thyroidectomized embryos were those operated at stage ‘b’ and fixed 7 days afterwards, which lost their tails only on treatment with thyroxine. The data for saline control embryos of Text-fig. 9 are given for comparison.

Text-fig. 11.

Numbers of fibres in the nerve to the triceps plotted against volume of muscle. For hypophysectomized embryos the number of days between operation and fixation is given. The thyroidectomized embryos were those operated at stage ‘b’ and fixed 7 days afterwards, which lost their tails only on treatment with thyroxine. The data for saline control embryos of Text-fig. 9 are given for comparison.

In the second group, which consists of embryos in which some pituitary tissue was found, and where the tail decreased in length in those fixed ten or more days after the operation, the number of fibres which entered the triceps femoris was within the range of saline controls (45-55). Those partially hypophy-sectomized embryos which were treated with thyroxine fall also into this group.

The effects of thyroidectomy and of hypophysectomy which have just been described presumably concern both motor and proprioceptor fibres in the nerve to the triceps femoris. As with turnover in normal development, fibres of small diameter are lost, while those of larger calibre remain, although the R. profundis anterior is the only muscular nerve which has been studied systematically in the course of this work. Comparison suggests however that while this nerve is specially sensitive to thyroid hormones, in others finer fibres also tend to disappear after thyroidectomy.

In order to test whether the exteroceptive fibres of cutaneous nerves show any corresponding effect, the number of fibres in the crural cutaneous has been counted under various conditions, and compared with the length of the foot, which can be used as an index of the general growth of the hind limb (Text-fig. 12). For a complete investigation it would be necessary to measure the area of skin surface supplied by each cutaneous nerve, the difficulties of which would be formidable.

Text-fig. 12.

Numbers of fibres in the crural cutaneous nerve under various conditions plotted against length of foot. The thyroidectomized embryos were those operated at stage ‘b’ and fixed 7 days afterwards, which lost their tails only on treatment with thyroxine.

Text-fig. 12.

Numbers of fibres in the crural cutaneous nerve under various conditions plotted against length of foot. The thyroidectomized embryos were those operated at stage ‘b’ and fixed 7 days afterwards, which lost their tails only on treatment with thyroxine.

Only in embryos thyroidectomized relatively early is there no correlation with growth in length of the foot, when the number of nerve fibres apparently remains the same as at the time of operation, though without actual decrease. In such embryos treated with thyroxine 3 days before fixation the number of fibres in the crural cutaneous increases to levels similar to those of saline controls. In both groups of ‘hypophysectomized’ embryos, as in controls, the numbers of fibres in the crural cutaneous increases with length of the foot, but no general differences in this respect are seen between these three groups of embryos within the range of lengths of foot common to all.

The calibre of fibres in muscular nerves

In Hughes (1965 a) mention is made of the enlargement of the diameter of individual nerve fibres which occurs towards the end of development in Eleutherodactylus. Further observations have shown that in the R. profundus anterior this increase in calibre is associated with the onset of function of the triceps femoris. In a limb paralysed by deafferentation of the cord the fibres in this nerve remain relatively small in diameter (Hughes, 1965 c).

In normal embryos up to stage ‘d’ when the leg can retract but not extend, no fibre in the nerve reaches a diameter of more than half a micron, and most are considerably smaller. At stage ‘e’, however, when the leg is first able to kick and to use this muscle, the calibre of the largest fibres exceeds 1·0 μ (Plate 2, fig. I). This increase in diameter begins at the entry of the nerve into the muscle and progresses proximally with time. The group of relatively large fibres may include both motor axons and proprioceptive fibres. Gray (1957) has shown that in the nerve to an adult frog muscle, spindle afferents are among the larger fibres present.

If embryos are treated with 0·1 % PTU at stage ‘a’, then development slows down before they acquire the kick reflex and the leg shows little or no signs of extension. In such embryos fixed after 8 days of treatment no fibres in the nerve to the triceps femoris were found to be as large as a micron in diameter (Plate 2, fig. J). After 4 days of immersion in PTU saline, similar groups of embryos were transferred to saline without PTU, or to saline containing 1:107 thyroxine. In such embryos the kick reflex appeared 2 days after the cessation of treatment with PTU, and after a further 2 days progressed as far as stage ‘g’, with the first signs of decrease in length of the tail. After fixation, fibres up to 1·5 in diameter were found in the nerve to the triceps femoris (Plate 2, fig. K).

In a further experiment, a group of embryos were treated with PTU saline for 5 days from stage ‘a’. In them, xanthophores characteristic of stage ‘e’ appeared, but the action of the leg did not progress beyond the withdrawal reflex. These embryos were then divided into groups, in one of which treatment with PTU was continued, while in another 1:107 thyroxine was added to the PTU. After a further 2 days the extension reflex of the leg appeared in all embryos of the second group, while it was doubtful whether there was any trace of leg extension in the former. Two other groups of embryos were transferred from PTU to water, or to water with thyroxine. In embryos given the hormone during this second phase of treatment mortality was very high, but in one which survived for 2 days in PTU-thyroxine with the appearance of leg extension there was enlargement of some nerve fibres to the triceps femoris.

Deprivation of thyroid hormones either by thyroidectomy or by hypophysectomy in the present experiments had no effect either on the behaviour of the hind limb or on the calibre of its nerve fibres at the periphery, even in embryos in which there was no shrinkage of the tail, though some difference in this respect was seen in the sciatic trunk in thyroidectomized embryos after treatment with thyroxin. The growth in diameter of individual fibres in nerves of supply is apparently a separate process from increase in total number, for the two are affected by different degrees of deprivation of thyroid hormones.

The effect of PTU on the movement of supernumerary limb grafts

In embryos of Eleutherodactylus, if a limb is grafted near to an undisturbed member the transplant may become innervated, depending on the age of the embryo at operation (Hughes, 1962). The nerve fibres to the muscles of the graft are however progressively lost towards the end of the embryonic period (Hughes, 1964a). Since the withdrawal of innervation may occur at much the same time as the atrophy of the tail, it seemed of value to test the effect of treatment with PTU on embryos bearing supernumerary limb grafts. Accordingly, forelimbs were autografted near hind limbs in a group of embryos, much as in a previous series of experiments (Hughes, 1964 a), except that in the present group the operation was performed one day later, at 6 days of development. These grafts when innervated usually show some movement with the nearby leg on light stimulation. Such observations when repeated from day to day serve as a means of gauging the degree of innervation of the grafts in the living animal.

In Text-fig. 13 the history of eleven such grafts is followed over periods of 8-11 days from the time of operation. Movement of the grafts may be seen as early as 3 days after operation but only in one example was this maintained continuously until the end of the experiment. In others, movement was intermittent and for much of the time was recorded as ‘slight’. The stage of development of the embryo as a whole was noted and in Text-fig. 13 dotted lines are drawn through those points on each record at which the embryo reached stages ‘d’, ‘e ‘, ‘f’ and ‘g’ respectively. The operation itself results in a slowing of development to a variable extent but only in one instance did the embryo not reach stage ‘f’ after 11 days of observation. In unoperated embryos developing in saline these stages succeed each other approximately at daily intervals.

Text-fig. 13.

Records of movement in supernumerary grafted forelimbs, with stages of development of host embryo as in Table 2. (a) Eleven untreated embryos; (b) corresponding embryos treated with 0·01 % PTU (arrows).

Text-fig. 13.

Records of movement in supernumerary grafted forelimbs, with stages of development of host embryo as in Table 2. (a) Eleven untreated embryos; (b) corresponding embryos treated with 0·01 % PTU (arrows).

In Text-fig. 13 b the results are shown for seven similar embryos which were immersed at stage ‘c’ or ‘d’in 0-01 % PTU at either 3 or 4 days after grafting an arm near a leg, and fixed 7 or 8 days later. The embryos reached stage ‘e’ much at the same time as in the untreated group, but none advanced any further. Embryos without grafts treated with PTU at stages ‘c’ or ‘d’ reach stage ‘f’ with continuous swimming within the same period. The extent of this arrest of development must therefore be due to the combined effects of the operation and the treatment with PTU, for, as already mentioned, limb grafting by itself retards the development of the host embryo. Furthermore, the thyroid glands in the present series of embryos were found to be much less affected by the PTU than in corresponding ungrafted embryos, for follicles containing colloid were still present.

The movement of the grafted arm in treated embryos was from the first much greater than in those of the corresponding untreated embryos. Once movement had begun in the supernumerary arm it continued until fixation in six out of seven of these embryos. Frequently the movement was described in the experimental record as ‘good’ or ‘+ + ‘.

It seems that there is an inverse relationship between the continued action of a supernumerary arm and progress in the movement of the nearby normal limb. A comparable result was seen in a previous experiment (Hughes, 1964b) where the nerve to the adjacent leg was cut at the time of grafting the supernumerary, with the result that both were innervated by axons from a much reduced number of regenerated ventral horn cells. Here both the innervation and the development of movement in the leg was greatly restricted, yet the action of the supernumerary arm was much greater than if the nearby leg had received a full nerve supply.

Where a transplanted limb shares in the unreduced innervation of another member, into which nerve fibres have already penetrated at the time of operation, the graft becomes innervated by axon collaterals which are withdrawn towards the end of development in untreated embryos. In embryos of the present series which were treated with PTU, and from which successful silvered preparations were obtained, it is possible to trace these collateral fibres inwards to the lumbar plexus as far as the region of the spinal ganglia, where there is always some residual axonal branching in juvenile animals.

It was observed that the fibres to the muscles of a supernumerary arm which was motile at fixation were very few in number, some five or six at the most. The fate of these fibres as judged by the behaviour of the graft during life is thus a sensitive test of any changes in the extent of fibre branching within the limb plexus. The errors involved in counts of the total number of fibres at various levels conceal differences of this order.

The influence of thyroxine on the developing nervous system of Anuran amphibians has been studied in several ways. Exogenously administered thyroxine has been shown to hasten certain normal features of development including some events which occur during metamorphosis, such as the acquisition of the eye retraction reflex (Kollros, 1943). To these we may now add the disappearance of the Rohon-Beard cells in Eleutherodactylus. Events within the nervous system unrelated to metamorphosis have been shown to be accelerated by thyroxine, such as the rate of mitosis in the neural tube of larval Rana temporaria (May & Mugard, 1955) and the maturation of ventral horn cells in R.pipiens (Beaudoin, 1956; Reynolds, 1963). Such immature neurones respond by increase in nuclear size. The action may be a direct one, as when the hormone is applied as pellets close to the spinal cord, or indirectly through the mediation of the periphery when the substance is inserted into the tissues of the limb (Beaudoin).

Another procedure has been to observe the effect of the hormone on hypophysectomized larvae. Kollros & McMurray (1956) found that in the differentiation of the mesencephalic nucleus of the trigeminal in Rana pipiens the hormone could fully compensate for the loss of the pituitary. Ventral horn cells, however, do not develop normally under these conditions. In hypophysectomized larvae the legs develop only as far as the late limb bud and the ventral horn remains at the stage with large numbers of small cells. The effect of thyroxine on such a larva is to promote limb growth as well as the loss of cells from the ventral horn, without however any concomitant differentiation of the remainder (Kollros & Race, 1960; Kollros, 1961; Race, 1961). In their experiments, decrease in cell number exhibited a lower threshold of response to thyroxine than did increase in cell size.

A comparable result emerges from the present observations, for no treatment of Eleutherodactylus embryos which depressed the activity of the thyroid was found to reduce the loss of cells from the ventral horn. The differentiation of the individual cells however was largely inhibited but was resumed after treatment with thyroxine. In the embryo of Eleutherodactylus, unlike the larval Anuran, the development of the limbs is not suppressed by thyroidectomy. The effects of deprivation of thyroxine on developing ventral horn cells in Eleutherodactylus may thus be ascribed to a direct effect on the hormone.

The only experimental observations on the peripheral effects of deprivation of thyroid hormones on the development of nerve fibres which have hitherto been described are those of Eayrs and his colleagues which concern the neuropil of the cerebral cortex of thyroidectomized infant rats (Eayrs, 1955). The failure under these circumstances to develop the correlative neural mechanism of higher centres has far-reaching consequences on the behaviour of these animals, with striking similarity to mental retardation in the human cretin.

In Eleutherodactylus thyroidectomy at a stage well before the end of embryonic life is necessary in order to cause any inhibition of the development of the peripheral nervous system. For a cutaneous nerve such treatment results in failure to increase the number of constituent fibres, while there is an actual decrease in number within a muscular nerve. This contrast can be explained on the hypothesis that fibre turnover in the latter is especially active, or is particularly sensitive to thyroxine. The number of fibres will decrease if loss continues normally after thyroidectomy, without replacement by newly developed axons. There are two separate pieces of evidence that developing nerve fibres to muscles are more unstable than those of cutaneous nerves in Eleutherodactylus, namely the effects of thyroidectomy, and secondly the withdrawal of motor innervation from supernumerary limb grafts which leaves cutaneous nerves largely unaffected (Hughes, 1964a).

La thyroïde et le développement du système nerveux chez Eleutherodactylus martinicensis: une étude expérimentale

  1. On décrit les effets d’un traitement à la phénylthiourée, et de la thyroïdectomie et de l’hypophysectomie, sur l’embryon à’Eleutherodactylus, surtout dans leurs rapports avec des modifications à l’intérieur du système nerveux.

  2. Chez Eleutherodactylus, au niveau des organes, les phénomènes contrôlés par la thyroïde au cours de la métamorphose se réduisent à la perte de la queue et du pronéphros. Les neurones sensoriels primaires de la moelle épinière, les cellules de Rohon-Beard, disparaissent aussi en même temps.

  3. La disparition normale des cellules des cornes ventrales au cours de développement n’est affectée par aucun des traitements auxquels l’embryon a été soumis. Le développement est retardé par un traitement à la phénylthiourée, et aussi par l’hypophysectomie, même quand l’hypophyse n’est pas complètement extirpée. Chez les embryons soumis à cette opération, il se produit une perte d’affinité pour l’argent dans les neurones de la corne ventrale, plus importante quant l’hypophyse est complètement excisée.

  4. L’adjonction de thyroxine à des embryons thyroïdectomisés augmente le nombre de fibres dans un nerf musculaire et aussi un nerf cutané.

  5. Le nombre de fibres dans un nerf musculaire peut être réduit par la thyroïdectomie ou par l’hypophysectomie. On suppose que, des deux composants du renouvellement rotatif des fibres dans les nerfs musculaires, la perte de fibres n’est pas affectée par les hormones thyroïdiennes, mais, l’arrivée de nouvelles fibres est sensible à leur action.

  6. Le mouvement de membres surnuméraires greffés est fortement accru par un retard général du développement, avec la phénylthiourée.

I am again grateful to Professor Ivan Goodbody and his colleagues at the Zoology Department of the University of the West Indies for all the help which they continue to provide for these researches. I am much indebted to my colleague Martin Prestige for frequent discussion of common problems. The expenses of the work were partly met by a grant from the Medical Research Council.

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All figures are from sections through embryos of Eleutherodactylus, stained by Palmgren’s method. The horizontal line in each figure represents 10 μ.

Plate 1

Fig. A. Part of transverse section of spinal cord in lumbar of saline control embryo near hatching stage, to show ventral horn cells, fully argyrophilic.

Fig. B. Part of transverse section of spinal cord of embryo incompletely hypophysectomized for 14 days, to show loss of argyrophilia in ventral horn cells.

Fig. C. Part of transverse section of spinal cord embryo completely hypophysectomized for 16 days, to show entire loss of argyrophilia in ventral horn cells.

Figs. D, E. Rohon-Beard cell in same section as in fig. C, to show retention of argyrophilia after complete hypophysectomy. D is at level of nucleolus; E at level of axon.

Plate 1

Fig. A. Part of transverse section of spinal cord in lumbar of saline control embryo near hatching stage, to show ventral horn cells, fully argyrophilic.

Fig. B. Part of transverse section of spinal cord of embryo incompletely hypophysectomized for 14 days, to show loss of argyrophilia in ventral horn cells.

Fig. C. Part of transverse section of spinal cord embryo completely hypophysectomized for 16 days, to show entire loss of argyrophilia in ventral horn cells.

Figs. D, E. Rohon-Beard cell in same section as in fig. C, to show retention of argyrophilia after complete hypophysectomy. D is at level of nucleolus; E at level of axon.

Plate 2

Fig. F. Section through sciatic nerve near entry into limb of saline control embryo near hatching stage. There are about 1000 fibres within the nerve, the largest about 2·0μ in diameter.

Fig. G. Section through sciatic nerve near entry into limb of embryo thyroidectomized for 7 days, with retention of tail. There are about 500 fibres within the nerve, all less than 1μ in diameter. The larger objects within the nerve are impregnated Schwann cells.

Fig. H. Section through sciatic nerve near entry into limb of embryo thyroidectomized for 7 days, and treated with thyroxine for last three, with loss of tail. There are about 840 fibres in the nerve, the largest of which are just over 1 μ in diameter.

Fig. I. Oblique section through nerve to triceps femoris muscle of normal embryo at kicking stage. The largest fibres are about 1·0μ in diameter.

Fig. J. Transverse section of nerve to triceps femoris muscle of embryo treated with 0·01 % PTU for 8 days, and halted in development before kicking stage. The largest fibres in the nerve are about 0·7 μ in diameter.

Fig. K. Oblique section through nerve to triceps femoris muscle of embryo treated for 4 days with 0·1 % PTU and then transferred for a further 4 days to thyroxine-saline. Development advanced to the kicking stage, and some fibres in the nerve enlarged to a diameter of 1·6μ.

Plate 2

Fig. F. Section through sciatic nerve near entry into limb of saline control embryo near hatching stage. There are about 1000 fibres within the nerve, the largest about 2·0μ in diameter.

Fig. G. Section through sciatic nerve near entry into limb of embryo thyroidectomized for 7 days, with retention of tail. There are about 500 fibres within the nerve, all less than 1μ in diameter. The larger objects within the nerve are impregnated Schwann cells.

Fig. H. Section through sciatic nerve near entry into limb of embryo thyroidectomized for 7 days, and treated with thyroxine for last three, with loss of tail. There are about 840 fibres in the nerve, the largest of which are just over 1 μ in diameter.

Fig. I. Oblique section through nerve to triceps femoris muscle of normal embryo at kicking stage. The largest fibres are about 1·0μ in diameter.

Fig. J. Transverse section of nerve to triceps femoris muscle of embryo treated with 0·01 % PTU for 8 days, and halted in development before kicking stage. The largest fibres in the nerve are about 0·7 μ in diameter.

Fig. K. Oblique section through nerve to triceps femoris muscle of embryo treated for 4 days with 0·1 % PTU and then transferred for a further 4 days to thyroxine-saline. Development advanced to the kicking stage, and some fibres in the nerve enlarged to a diameter of 1·6μ.