1. The cartilaginous limb-bone rudiments of the 6-day-old embryonic chick show a differential growth response to treatment with triiodothyronine (T3) during 8 days’ culture in vitro. The growth in length and wet weight of the tibia is reduced by l · 6 × 10–4g. T3/l. of medium, but the growth of the radius is increased.

  2. Tibia and radius explanted at comparable histogenetic stages respond differently to T3, but the responses are slightly modified by the stage of development at which the rudiments are exposed to the hormone: tibiae from 5-to -day-old embryos show a transitory stimulation of growth in length, followed by retardation, while increase in the growth in length of radii from 7- and 8-day-old embryos is less than that of radii from 6-day-old embryos.

  3. Five small limb-bone rudiments of a similar size (third metatarsus, radius, ulna, third metacarpus, and fourth metacarpus) respond differentially to T3. It is therefore unlikely that the differential growth-response of the tibia and radius is a result of the limited nutritional conditions in vitro.

The proportionate development of the embryonic chick skeleton can be influenced experimentally by a variety of factors such as nutritional deficiencies (Byerly, Titus, Ellis, & Landauer, 1935; Landauer, 1936; Romanoff & Bauernfeind, 1942; Couch, Cravens, Elvehjem, & Halpin, 1948), teratogens (Ancel & Lallemand, 1942; Zwilling & de Bell, 1950; Landauer, 1952, 1953a, 1954) and excess hormones (Willier, 1924; Landauer & Bliss, 1946; Duraiswami, 1950). The leg bones are generally more severely affected than the wing bones, but a comparison of the action of several teratogens on the character of the malformations and on the relative growth of the leg bones indicated that the response of individual bones varies with the different agents (Landauer & Rhodes, 1952; Landauer, 1953 a, b, 1954).

Cartilaginous limb-bone rudiments also respond differentially when they are isolated from the embryo and exposed in culture to various compounds, such as insulin (Chen, 1954), vitamin A, and the thyroid hormones (Fell & Mellanby, 1955, 1956). Each agent has a characteristic histological effect, but the different rudiments are affected to a different extent. Maturation of the cartilage is accelerated in all the long-bone rudiments grown in the presence of additional thyroxine (T4) or triiodothyronine (T3), but the growth response varies; thus the growth in length of the tibia is severely retarded by the hormones while that of the radius is increased. Fell & Mellanby concluded that these differences in response were correlated with differences in the rate of normal diaphyseal differentiation in the different rudiments, and also with the stage of development at which the rudiments are exposed to the hormones.

This differential response of limb-bone rudiments to T3 in culture has been analysed further, since it presumably reflects metabolic differences which may affect the development of the normal proportions of the skeleton. This paper reports experiments on the influence of developmental stage and organ size on the growth response to T3. In subsequent papers the effect of variations in growth rate and hormone concentration on the differential response will be considered.

Tissue culture

Cartilaginous limb-bone rudiments from 5-to 8-day-old chick embryos were cultivated in watch-glasses on a medium of fowl plasma and chick embryo extract (Fell & Robison, 1929; Fell & Mellanby, 1952). Embryo extract was prepared from 13- or 14-day-old embryos (Fell & Mellanby, 1952). One part of embryo pulp was extracted with one part of 1 per cent, glucose Tyrode; one part of the extract was then mixed with three parts of plasma.

0·5 mg. of the sodium salt of 3:5:3′ triiodo-L-thyronine (T3) was weighed with sterile precautions and dissolved in 16 ml. of sterile 0·1 per cent, w/v Na2CO3. This stock solution was prepared once every 2 weeks and was stored in the dark at 4° C. Before use, 0·01 ml. of stock solution was diluted with 1·5 ml. of plasma to give a concentration of 1 ·6 × 10−4g T3/l. in the final medium (Fell & Mellanby, 1956). An equivalent amount of sodium carbonate solution was added to the control medium.

The final volume of medium in each watch-glass ranged from 0·6 to 1·0 ml. according to the size and number of the explants. Not more than three rudiments from one side of an embryo were cultivated in each dish containing T3, and the corresponding rudiments from the other side were grown in a similar dish without the hormone. The explants were transferred to fresh medium every other day and the experiments ended after 8 days.

Measurement of the rudiments

Length

The lengths of the growing rudiments were calculated from daily camera lucida drawings of the explants (Fell & Mellanby, 1952).

Wet weight

Excess moisture was removed from the explants at the end of the culture period by rolling them once on hard filter-paper (Whatman No. 50); they were then weighed in sealed capillary tubes.

Total nitrogen content

Groups of four rudiments from 6-day-old embryos were digested by the method of Ma & Zuazaga (1942). Total nitrogen was estimated by the micro-Kjeldahl method (Markham, 1942); the ammonia was collected in a 2 per cent, w/v solution of boric acid and titrated with 0-005 N HC1 in the presence of Sher’s (1955) indicator.

Estimation of response to T3

The effect of T3 on the growth in length and wet weight of the whole rudiments was estimated in the following ways :

  • The increments of growth in length of the control and treated rudiments were measured every 2 days and the significance of the difference tested by Student’s t-test for paired samples.

  • The response at the end of the culture period was expressed as the ratio of the final lengths, or wet weights, of the treated and control rudiments. Statistical comparison of the response of different bones was made with logarithms of the measurements, i.e. response = log10T—log10C, where T = length or wet weight of the treated rudiment and C = length or wet weight of the paired control. The standard error was calculated from the difference in response of any two bones within the same embryo in order to exclude the variation between embryos.

Fell & Mellanby (1955, 1956) found that T4 stimulated the initial growth in length of humeri in explanted limb blastemata, whereas the growth of rudiments from older embryos was retarded by both T4 and T3. The growth of the radii from these older embryos was stimulated by both hormones, but when the embryos were divided into two groups on the basis of stage of development, Fell & Mellanby found that the growth of the radii of the younger group was stimulated by T3 while the growth of the bones in the older group was unaffected. The conclusion that the differential response was influenced by the stage of development at which the rudiments were exposed to the hormone was supported by the response of the other long bones which were tested. These results suggested that the developmental stage may be the most important factor in eliciting a differential response from the different bones of the same embryo, since the rudiments are at different stages of differentiation at any given time. In the experiments described in this section, this possibility was tested by comparing the response of the tibia and radius to T3 at various stages of development; these two rudiments were chosen because of all the long bones from the 6-day-old embryo they show the most widely divergent response.

Experimental

The embryos from 5-to 8-day-old eggs were divided into groups, roughly according to age, and precisely according to the initial lengths of the bone rudiments and stage of development when viewed through the dissecting microscope (Table 1). Division of the rudiments into groups on the basis of morphological appearance agreed with their subsequent grouping according to initial length, and there was no overlap of initial length between the groups. The radii of any group were at least as mature as the tibiae of the preceding group. No results for group A radii are given, since both treated and control explants developed merely as nodules of small-celled cartilage.

Table 1.

Division of embryos into groups on the basis of the initial stage of differentiation of the tibia and radius

Division of embryos into groups on the basis of the initial stage of differentiation of the tibia and radius
Division of embryos into groups on the basis of the initial stage of differentiation of the tibia and radius

Results

There was a slight stimulation of the growth in length of the group A tibiae treated with T3 during the first 2 days in culture (Text-fig. 1). This increase was statistically significant at the 1 per cent, level, but was slight compared with that of the group B radii with which the group A tibiae were histologically comparable (Text-fig. 1). The hypertrophic areas of the T3-treated tibiae were slightly more prominent and extensive in the living cultures after the first 2 days, and the transitory increase of growth in length may be the result of the slightly earlier onset of hypertrophy, as was found in the young humeri treated with T4 (Fell & Mellanby, 1955). During the last 2 days in culture the T3-treated tibiae in group A showed the retardation in growth characteristic of tibiae from older groups.

Text-fig. 1.

Response of the tibia and radius at different developmental stages. Upper row. tibia; lower row. radius. The dotted lines represent the length of the control rudiments, the full lines the length of the equivalent rudiments treated with T3. The growth of the treated tibiae in all groups is retarded except for a transitory stimulation of the bones in group A. In contrast the growth of the radius is stimulated.

Text-fig. 1.

Response of the tibia and radius at different developmental stages. Upper row. tibia; lower row. radius. The dotted lines represent the length of the control rudiments, the full lines the length of the equivalent rudiments treated with T3. The growth of the treated tibiae in all groups is retarded except for a transitory stimulation of the bones in group A. In contrast the growth of the radius is stimulated.

T3 also stimulated the growth in length of radii in groups C and D, but the effect was less than on radii in group B (Text-fig. 1). The only suggestion of retardation was in radii of group D during the last 2 days of culture, but this was not statistically significant.

Thus tibia and radius at comparable histogenetic stages respond differentially to T3. The precocious onset of hypertrophy produced by T3 in the youngest groups of both bones is probably responsible for the transitory increase in growth in length of the tibia, and the much greater stimulation of the radius as compared with the radii of the older groups.

In organ culture the supply of food to the explant is limited by the periodic replacement of the medium and by the rate of diffusion of materials through the medium. The conditions in such a culture may be more favourable for the growth of a small rudiment with a relatively large surface-volume ratio like the radius, than for the comparatively bulky tibia. The utilization of amino-acids and glucose per unit wet weight of bone rudiment is greater for the radius than for the tibia (Lawson & Lucy, 1961; and Lawson, unpublished observation).

It is possible that the retardation in the growth of tibiae treated with T3 is due to the limitations in food supply to the larger bone.

The response of the tibia was therefore compared with the responses of five small bones: third metatarsus, radius, ulna, third metacarpus, and fourth metacarpus.

Experimental

Rudiments from twelve 6-day-old embryos were cultured, and the initial lengths of the bones were used as an indication of their initial size. The total nitrogen of the rudiments of other embryos from the same batch were determined for comparison. The response to T3 was measured at the end of the culture period.

Results

The responses of the five small bones varied from severe retardation of the growth of the third metatarsus to marked stimulation of the fourth metacarpus (Table 2); the distinctness of the responses of the different bones is indicated in Table 3. When the bones were arranged in a series according to their response to T3, the order was the same whether the response was measured on the final length or on the wet weight of the whole rudiments. Table 2 shows that the predominant response in this series was an increase in wet weight and retardation of growth in length; the epiphyses of all the treated rudiments were much enlarged, and in the ulna and third metacarpus this more than compensated for any loss in weight from the slight reduction in length.

Table 2.

Final response of different bones from the same embryos to T3

Final response of different bones from the same embryos to T3
Final response of different bones from the same embryos to T3
Table 3.

The statistical significance of the differential response to T3

The statistical significance of the differential response to T3
The statistical significance of the differential response to T3

The degree of response was not associated with the initial size of the different bones (Table 2). The absence of association was particularly evident in the metatarsus and radius, which were of the same initial size but showed strikingly different growth-responses (Text-fig. 2); the retardation of the growth of the metatarsus was comparable with that of the tibia (Table 3).

Text-fig. 2.

Differential response of the third metatarsus (left) and the radius (right) to T3. Dotted lines represent the length of control rudiments, the full lines show the length of the equivalent rudiments treated with T3. The two bones were of similar size and at the same histogenetic stage when initially exposed to the hormone.

Text-fig. 2.

Differential response of the third metatarsus (left) and the radius (right) to T3. Dotted lines represent the length of control rudiments, the full lines show the length of the equivalent rudiments treated with T3. The two bones were of similar size and at the same histogenetic stage when initially exposed to the hormone.

A series of control bones arranged in order of final wet weight corresponded with the series of bones arranged in order of their response to T3 (Table 2), except that the reduction of the growth in length of the metatarsus was significantly greater than that of the larger ulna; indeed, the final wet weight of the treated ulna was greater than that of the control, whereas the final wet weight of the treated metatarsus was much less than that of its control (Tables 2, 3). The difference between the order of the bones arranged according to final size and when arranged according to initial size presumably reflects differences in growth rate; the connexion of the differential growth rate with the differential response to T3 will be examined in a later paper.

In this experiment different bones were taken from the same 6-day-old embryos and were therefore at varying stages of development. At this age the developmental stages of the rudiments fall in the same order as their initial sizes. Thus the tibia is the largest and the most advanced, the fourth metacarpus is the smallest and least developed, while the metatarsus and the radius are the same size and at similar stages; any correlation between response and initial bone-size would therefore be enhanced. Although the experiment clearly demonstrated that rudiments of the same size respond differently to T3, it cannot be concluded from the results that the response is not modified at any stage by the size of the treated rudiment.

It may therefore be concluded that the differential growth response to T3in vitro is influenced but not determined by the developmental stage of the rudiments, and that differences in the initial size of different rudiments do not determine the differential response in vitro.

La réaction différentielle de croissance dans les rudiments d’os de membres de Vembryon de Poulet à la triiodothyronine in vitro

I. Stades du développement et taille des organes

  1. Les rudiments cartilagineux des os des membres du poulet embryonnaire de 6 jours montrent une réaction différentielle de croissance au traitement à la triiodothyronine (T3) pendant 8 jours de culture in vitro. La croissance en longueur et en poids du tibia est réduite par l·6× 10−4g. T3/l. du milieu, mais la croissance du radius est augmentée.

  2. Le tibia et le radius explantés à des stades comparables de l’histogenèse ont des réactions différentes à T3, mais la réponse est modifiée légèrement par le stade de développement auquel les rudiments sont exposés à l’hormone: des tibias d’embryons de 5 à jours montrent une stimulation transitoire de la croissance en longueur, suivie d’un retard, tandis que l’augmentation de la croissance en longueur des radius d’embryons de 7 et 8 jours est moindre que celle des radius d’embryons de 6 jours.

  3. Cinq petits rudiments d’articles osseux de taille voisine (le 3e métatarsien, le radius, le cubitus, le 3e métacarpien et le 4e métacarpien) ont des réactions différentes à T3. Il est donc peu vraisemblable que la réaction différentielle de croissance du tibia et du radius soit le résultat des conditions restrictives de nutrition réalisées in vitro.

The author wishes to express her gratitude to Dr. H. B. Fell, F.R.S., for her advice and encouragement throughout the work, and to the Sir Halley Stewart Trust for a Research Studentship. The triiodothyronine used in the work was a gift from Dr. R. Pitt-Rivers, F.R.S.

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