ABSTRACT
As maternally administered 5-bromodeoxyuridine (BudR) is very quickly degraded by the liver, a combination of whole embryo culture and organ culture techniques was adopted to expose postimplantation mouse embryos to the analog and to study the effects of long-term treatment on the subsequent differentiation of limb-buds. Early and mid-11th-day mouse embryos were exposed to increasing concentrations of BudR for 12 or 24 h. Forelimbs of the treated embryos were then organ-cultured in drug-free medium and the extent of cartilage development in the expiants examined. Exposure of embryos to 50–150 μg/ml of BudR for 24 h resulted in significant inhibition of chondrogenesis in the subsequent limb cultures and the effect was related to dose. After treatment with 150 μg/ml of the drug, the forelimbs of the early 11-day embryos (somite stage 26–29) showed an almost complete lack of cartilage, while the limbs of mid-11 th-day embryos (somite stage 32–34) were not nearly as sensitive and exhibited about 50 % reduction in the amount of cartilage development. We conclude that if embryos in which the limb development is at a very early stage of development are exposed to BudR, the future course of limb differentiation is permanently and irreversibly damaged, resulting in a partial or even complete suppression of chondrogenesis in the organ. As both the dose and perhaps also the duration of treatment were critical, we suggest that the rather low frequency of reported limb malformations after in vivo injection of teratological doses of BudR may be due to only a small amount of the chemical reaching the embryos.
INTRODUCTION
5-Bromodeoxyuridine (BudR)-an analog of nucleoside thymidine -has recently been described as an ‘ideal teratogen’ by Rutter, Pictet, Githens & Gordon (1975). Applied to a wide variety of cells and tissues in culture, it has been shown to inhibit differentiation both in regard to cellular morphology and to the synthesis of specialized cell products characteristic of the differentiated cells and yet, at least in low concentrations, it is non-toxic and appears to have little effect on cell viability, growth or proliferation (see reviews by Wilt & Anderson, 1972; Rutter, Pictet & Morris, 1973; Levitt & Dorfman, 1974). While BudR is a potent inhibitor of chondrogenesis (Abbott & Holtzer, 1968) and myogenesis (Stockdale, Okazaki, Nameroff & Holtzer, 1964) in cell culture, the only reported limb anomaly after maternal administration of a teratogenic dose was polydactyly in a small proportion of the fetuses (DiPaolo, 1964; Ruffolo & Ferm, 1965; Skalko, Packard, Schwendimann & Raggio, 1971). BudR is very quickly and extensively degraded by the maternal liver (Kriss & Révész, 1962) so that the analog is available to the embryos for only a short period of time (Packard, Menzies & Skalko, 1973). It is possible to increase the incorporation of BudR in the proliferating cells of adult mice by employing partially hepatectomized animals (Hill, Augenlicht & Baserga, 1973) but such a procedure would no doubt complicate the interpretations of in vivo teratological investigations. We have instead employed whole-embryo culture-a ‘mother-free’ system -to study the full potential of BudR teratogenecity.
Postimplantation rat (New, 1967; Cockroft, 1973) and mouse (Kochhar, 1975a) embryos can be grown in culture for a limited period of time. Under suitable conditions, growth and differentiation of embryos in vitro can be maintained almost as well as in vivo (Cockroft, 1976). Whole-embryo culture has not only proved useful in the understanding of normal differentiation processes (Moore & Metcalf, 1970; Deuchar, 1971) and in metabolic studies (Shepard, Tanimura & Robkin, 1970; Tanimura & Shepard, 1970) but also in teratological investigations (Turbow, 1966; Turbow & Chamberlain, 1968; New & Brent, 1972; Morriss & Steele, 1974; Robkin, Shepard & Baum, 1974; Kochhar, 1975b). Teratological studies in whole-embryo cultures have certain advantages in that the effect of a drug can be investigated without the chemical being screened out, degraded or modified in some way by the maternal system. However, the system has the limitation that the embryos can be maintained for only a fraction of their in utero life, so that no long-term effects of a teratogen can be studied. We have tried to alleviate this limitation by combining whole-embryo culture with limb-bud organ culture. In this 2-step culture procedure, isolated mouse embryos were exposed to BudR in vitro for 24 h, after which their limbs were excised and cultured for another 9 days. It was hoped that such an approach might prove to be useful in evaluating the full teratogenic capabilities of BudR, and might provide some information on how BudR treatment of embryos would affect the subsequent development and differentiation of the limbs. The choice of limb as the target organ was deliberate; the mesodermal regions which would differentiate into the various cartilages are already present in their normal seriation in the earliest visible limb-buds (Stark & Searls, 1973) and technique of ]imb-bud organ culture is well established (Aydelotte & Kochhar, 1972; Kochhar & Aydelotte, 1974; Neubert, Merker & Tapken, 1974).
MATERIALS AND METHODS
Animals
Random-bred ICR/DUB mice (obtained from Flow Laboratories, Dublin, Virginia) were maintained on Purina Mouse Chow and tap water ad libitum and kept in the light from 6 a.m. to 6 p.m. The female mice were mated either overnight or, when more precise time of mating was required, for 4 h between 8 a.m. and noon. The presence of a vaginal plug immediately afterwards was regarded as evidence of successful mating; this day was designated as the 1st day of pregnancy.
Culture procedure
To study the effect of the incorporation of BudR by mouse embryos on the subsequent development of their limbs, a ‘two-step culture procedure’ was adopted. Isolated embryos were maintained in vitro for 12–24 h in the presence of the drug. Afterwards, their limb-buds were excised and grown in organ culture for another 9 days.
Whole-embryo culture
On the early or mid-11th or 12th day of pregnancy, female mice were killed by cervical dislocation, dissected and the intact uteri transferred to a dish containing Tyrode saline solution. Using standard sterile techniques, the horns of the uterus were cut open and the conceptuses released into the saline. An attempt was made to select embryos that appeared similar in size and nearly identical in their developmental stage (within 2–3 somites). Reichert’s membrane and the parietal layer of the yolk sac of each individual embryo was dissected away. This operation lasted approximately 30–45 min. Leaving the conceptuses in the saline at room temperature for this period of time did not seem to affect the eventual growth of the embryos in culture, a fact also noted by New (1971). The embryos with their visceral yolk sacs intact were then cultured for 12–24 h in Waymouth’s medium diluted with an equal amount of fetal calf serum, as described in detail elsewhere (Kochhar, 1975a). Embryos were put singly in 10 ml screw-cap vials, each with 2 ml of the medium. The remaining volume was filled with a gas mixture of 95% O2 + 5% CO2. The vials were then clamped on to a motordriven rotator and rotated constantly at 36–40 rev./min. At the time of medium renewal, all of the old medium was removed and replaced with the same amount (2 ml) of fresh medium and the cultures regassed. After enough had been set up in culture, the few remaining embryos were used for staging and their precise somite number recorded. It was assumed that this somite count was representative of all the embryos and is referred to as the ‘somite number at the time of embryo explant’ in the text. The culture medium used was Waymouth’s medium MB 752/1 supplemented with 50% fetal calf serum (both obtained from Flow Laboratories, Rockville, Md.). The well-being of the embryos was monitored at various times during the in vitro growth by checking their heart beat and circulation in the blood vessels of the yolk sac.
Treatment with drugs
BudR (obtained from Sigma, St Louis, Missouri) at various concentrations in Waymouth’s medium was added to the culture media at the initiation of the embryo cultures. Usually 8–12 embryos were used in each experiment; half the embryos were grown in control medium and the other half in medium containing the drug. Enough experiments were conducted to collect samples of 12–24 treated and untreated limbs for comparative studies.
To study the incorporation of labeled BudR, llth-day mouse embryos were cultured in the presence of 2 μCi/ml of [3H]BudR ([6-3H]5-bromodeoxyuridine, specific activity 15–30 Ci/mmole, obtained from New England Nuclear, Boston, Mass.). At suitable intervals, as indicated in Fig. 2, embryos were removed from the culture medium and transfered to chilled saline containing excess ‘cold’ thymidine (500 μg/ml) to stop further incorporation of the isotope. The placentas were trimmed away and yolk sacs separated from the embryos. Both the yolk sac and embryos were rinsed briefly in fresh saline, homogenized separately, and suspended in 10% trichloracetic acid overnight at 4 °C. DNA was extracted by TCA-PCA precipitation method of Ruddick & Runner (1974). The supernate after the hot PCA treatment was assumed to be the DNA fraction. An aliquot of this fraction was removed for isotope counting on a liquid scintillation counter (ISOCAP/300, Nuclear Chicago, III.) and another aliquot was used to quantify DNA employing the diphenylamine reaction (Burton, 1956).
Organ culture of limbs
After exposure of embryos to BudR in vitro, their forelimbs were excised and cultured. Only embryos with good heart beat and blood circulation were used. The cultured whole embryos were briefly rinsed in saline, their remaining membranes dissected away and the forelimbs explanted employing techniques described elsewhere (Aydelotte & Kochhar, 1972; Kochhar & Aydelotte, 1974). The limbs were maintained in vitro for 9 days. The medium (BGJ medium (Difco, Detroit, Michigan) supplemented with 25% fetal calf serum, 12·5 μg/ml streptomycin, 7·5 μg/ml penicillin G and 150 μg/ml ascorbic acid) was changed every third day. Finally the limbs were fixed in Bouin’s fluid, stained in 01 % toluidine blue in 70% ethanol, dehydrated, cleared and stored in cedar wood oil.
‘ Quantitative measurement of the cartilage
As one aspect of the present investigations was to study the dose effect of BudR, the following method was adopted to quantify the amount of differentiated cartilage. Toluidine-blue-stained and cleared limbs were photographed under a low magnification on a Kodachrome color film. After processing, the photographic slides were projected in a Tri-Simplex Micro Projector (Bausch and Lomb, Rochester, N.Y.) onto a graph paper of uniform squares. The outline of the cartilage was drawn on the graph paper and the various cartilage zones (scapula, humerus, radio-ulna and digits) delimited. The number of graph paper squares occupied by each cartilage was counted and this number is refered to as area units for that particular cartilage. Such an approach of quantifying the amount of cartilage has certain built-in errors. In organ culture, the various cartilages of the limb grow in a three-dimensional aspect, rather than two as a photograph would record it. Also sometimes two cartilages would overlap or some parts (especially scapula and humerus) would bend downwards in the growing cultures. The latter sources of error were easier to rectify by comparing the enlarged outline against the original limb and applying proper corrections wherever cartilages overlapped. The amount of cartilage area units in the limb expiants of similarly staged embryos was found to be quite similar and variation in most cases was rather small. In cases where variation was large, the experiment was repeated one or more times to collect a larger sample so as to obtain a statistically more valid mean value. The average cartilage area units in the limbs of similarly staged and treated embryos is the value used in all the tables. Although the measurements were not absolute, such a semi-quantitative approach gave us a basis to compare the effect of various concentrations of BudR and made it easy to visualize if a certain treatment was affecting one particular cartilage segment more than any other.
All measurements were made on toluidine-blue stained preparations. When no metachromasia could be detected, complete suppression of chondrogenesis was assumed to have occurred (Aydelotte & Kochhar, 1975).
RESULTS
A. Factors influencing the differentiation of cartilage in control cultures
In the limbs grown by the 2-step culture procedure, various cartilage segments were recognizable by their relative position in the explant and by their shape (Fig. 1). The scapula developed as a triangular or rhomboidal cartilage at the proximal end of the explant. The humerus and radio-ulna presented themselves as bars of cartilages with the humerus being the longest of the three. The radioulna, when developed, appeared as two rather short and parallel bars, distal to the humerus. Development of digital cartilages was generally poor.
Cultured limbs from control and BudR-treated embryos. Embryos were maintained in whole embryo culture for 12 or 24 h in control or drug-supplemented medium. Forelirnbs of these embryos were then excised and organ-cultured for 9 days in drug-free medium, fixed and stained in toluidine blue. (A) . Early 11-day embryo, control medium 24 h. (B) . Early 11-day embryo, 150 μg/ml of BudR for 24 h. (C) . Mid-ll-day embryo, control medium 12 h. (D) . Mid-ll-day embryo, 100 μg/ml of BudR for 12 h. (E) . Mid-ll-day embryo, control medium 24 h. (F) . Mid-ll-day embryo, 150 μg/ml of BudR for 24 h. In all photographs, the distal tip of the limb is towards the bottom. All photographs are at the same magnification and the bar in the lower right-hand corner represents 1 mm.
Cultured limbs from control and BudR-treated embryos. Embryos were maintained in whole embryo culture for 12 or 24 h in control or drug-supplemented medium. Forelirnbs of these embryos were then excised and organ-cultured for 9 days in drug-free medium, fixed and stained in toluidine blue. (A) . Early 11-day embryo, control medium 24 h. (B) . Early 11-day embryo, 150 μg/ml of BudR for 24 h. (C) . Mid-ll-day embryo, control medium 12 h. (D) . Mid-ll-day embryo, 100 μg/ml of BudR for 12 h. (E) . Mid-ll-day embryo, control medium 24 h. (F) . Mid-ll-day embryo, 150 μg/ml of BudR for 24 h. In all photographs, the distal tip of the limb is towards the bottom. All photographs are at the same magnification and the bar in the lower right-hand corner represents 1 mm.
The effect of embryo age (in terms of somite stage at explantation) and culture conditions (which determined the extent of embryonic growth in vitro)on the development of toluidine blue-stainable cartilage in the subsequent limb cultures is shown in Table 1. The early 11 th day (somite stage 26–29) embryos could be maintained in the same culture medium for at least 24 h during which time they maintained an excellent heart beat and blood circulation. They gained an average of 4–5 somites. When their forelimbs were subsequently cultured for 9 days, the average amount of cartilage development was 171 area units (Table 1), If the medium was changed after 12 h, embryonic growth was markedly improved and they added an average of 9–10 somite pairs and also showed a much better cartilage development in the subsequent limb cultures (Table 1). The mid-11th-day embryos (somite stage 32–34) could be maintained in the same culture medium for only 12 h after which time a rapid deterioration in their heart beat and blood circulation was noted. If these embryos were transferred to fresh medium at this time, many of them survived for another 12 h and altogether gained an average of 10–11 somites pairs (Table 1). In utero, the 11th day mouse embryos add about 12 somite pairs in 24 h (Kochhar & Aydelotte, 1974) so that under suitable culture conditions, the growth and differentiation of 11-day mouse embryos in vitro was very nearly equal to that in utero, at least in terms of gain in the somite pairs.
B. Incorporation of BudR by embryos in culture
Before attempting detailed studies on the effect of BudR on cultured embryos, we investigated if the embryos, in vitro, were able to incorporate the analog into their DNA. For this purpose, 12 embryos from one litter were cultured and after 1 h -to allow the embryos to ‘adapt’ to the culture environment -[3H]BudR was added to the medium. At various time intervals after addition of the isotope, the incorporation of the label was analyzed from a group of embryos (three for each time interval). The results are shown in Fig. 2. As is evident, continuous uptake of the label into embryonic and yolk sac DNA was found throughout the 20 h culture period. At increasing intervals of time during exposure to the isotope, an increase in the incorporation of the label was observed. Towards the end of in vitro growth, there was a slight decrease in the rate of [3H]BudR incorporation in the embryonic tissue. This may be due to slight diminution in the metabolic activities of the embryos as indicated by a somewhat slower heart beat and blood circulation at this time.
Incorporation of [3H]-BudR by the DNA of embryonic and yolk sac tissues after exposure of the 1.1-day mouse embryos, in vitro, to the isotope for varying length of time.
C. Effect of BudR treatment on chondrogenesis
Embryos were exposed to increasing concentrations of BudR in vitro and the effect of this treatment on the differentiation of cartilage in the limb-buds was studied after the latter had been cultured for 9 days in drug-free medium. The BudR treatment of the embryos interfered with the differentiation of cartilage in the limb expiants. The degree of chondrogenic inhibition was found to depend on the drug dose as well as on the developmental stage of the donor embryos. For this reason, great care was taken to ensure that comparisons were made on limbs that had been excised from embryos of similar somite stage and also grown under identical conditions. With this in mind, the embryos were divided into two groups depending on the number of somites at the time of embryo expiants : early 11th day = somite stage 26–29; mid-11th day = somite stage 32–34.
(i) Early 11-day embryos
These embryos were grown in the same medium for 24 h and exposed to various concentrations (0–150 μg/ml) of BudR. The results are presented in Table 2. The data was subjected to an analysis of variance and this coupled with Dunnett Test (Dunnett, 1955) revealed that the treatment of embryos with the drug had a highly significant inhibitory effect (P <0·01) on the differentiation of cartilage in limb cultures. There was no significant effect on the total cartilage area units at the drug level of 25 μg/ml. As the dosage was increased, a ‘thinning out’ of the humerus and a reduction in the size of scapula was observed with the effect being more severe on the long bone. Eventually, at the highest concentration tested ( 150 μg/ml), cartilage development was severely inhibited and a small nodule of metachromasia was all that could be detected in the toluidine-blue-stained preparations (Table 2, Fig. 1 B).
Exposure of early 11-day (somite stage 26–29) mouse embryos to BudR. and its effect on the subsequent chondrogenesis in forelimbs

It was possible to ‘improve’ the growth of embryos in culture by changing the medium after 12 h. As noted earlier, such a procedure resulted in an average gain of 9–10 somites per embryo and the subsequent cartilage development in limb organ cultures was enhanced considerably (Table 1). Exposure of these embryos, in vitro, to BudR (0–150 μg/ml) affected the eventual development of cartilage in limb cultures in a manner similar to that described earlier. The degree of cartilage inhibition was related to dose (Table 3); at the highest dose tested (150 μg/ml), there was almost a 90% reduction in the cartilage development.
(ii) Mid-11th-day embryos (somite stage 32–34)
If grown in the same medium, these embryos could be maintained well for only about 12 h, after which time there was a very rapid deterioration in their heart beat and blood circulation. BudR treatment in these experiments was therefore restricted to 12 h (rather than 24 h as was the case with the early 11 th-day embryos). The treatment of these embryos with BudR (0–100 μg/ml) for 12 h had no significant effect on the subsequent differentiation of forelimb-buds as judged by the development of cartilage in these cultures (Table 4). All limb cartilage components appeared well developed and similar to those observed in control cultures (Fig. 1 C, D). In a few preliminary experiments, doses higher than 150 μg/ml of BudR proved toxic and the embryos failed to survive for longer than 5–6 h in culture.
Exposure of mid-11th-day (somite stage 32-34) mouse embryos to BudR (for 12 h) and its effect on the subsequent chondrogenesis in the forelimbs

We were interested to see if the mid-11 th-day embryos had indeed become refractory to the chemical or whether the treatment was not long enough. To keep the embryos alive for a longer period of time, we transferred the embryos to fresh, pre-warmed medium containing the same amount of BudR that was added to the original medium. Under these culture conditions, many, but not all, of the older embryos could be kept alive for at least another 12 h and all surviving embryos showed a much better growth and gained an average of 10–11 somites (Table 1), almost the same as in vivo (Kochhar & Aydelotte, 1974).
Table 5 summarizes the results of experiments where the mid-11th-day embryos were exposed to a variety of BudR concentrations (0–150 μg/ml) using the modified culture technique. A significant and dose-related inhibition of chondrogenesis in limb cultures was observed. In all cases, the radio-ulna and digits exhibited the maximum sensitivity (Fig. 1 F). Embryos grown in the presence of 150 μg/ml of thymidine in the medium showed an improvement in cartilage development in the subsequent limb cultures (Table 5). High levels of thymidine are toxic to cells in culture (Morris & Fischer, 1960, 1963).
Exposure of mid-11 th-day (somite stage 32-34) mouse embryos to BudR (for 24 h) and its effect on the subsequent chondrogenesis in the forelimbs

Unfortunately, these studies could not be extended to the 12th- or the 13th-day embryos, which proved very difficult to maintain in vitro for longer than 4–6 h. In a few preliminary studies, such a short treatment with BudR did not affect the subsequent differentiation of cartilage in the limb expiants.
DISCUSSION
The 11-day mouse embryos, in vitro, maintained an excellent heart beat and blood circulation at least for the first 24 h. Under suitable culture conditions, they added an average of 9–11 somite pairs after 24 h growth in vitro (Table 1) and this compared well to the gain of 12 somites in uteroduring the same time (Kochhar & Aydelotte, 1974). Cockroft (1976) also found that the growth and differentiation of rat embryos was nearly normal for the initial 24 h in culture.
Because of the limited life span of embryos in vitro, whole-embryo culture has only a limited application in teratological investigations as no long-term effects of a drug can be investigated. The 2-step culture technique used in the present investigations provided a means to extend the BudR treatment of embryos to 24 h and study the long-term effect of the drug on developing limbs. This was a distinct advantage because in vivo the drug is very quickly degraded by the liver (Kriss & Révész, 1962). Although these studies were carried out in vitro, the experimental protocol was similar to the classical teratological approach. Embryos-in a ‘mother-free’ system-were exposed to the drug for only a fraction of their in utero life span after which the organ under study (limb-buds) was allowed to grow and differentiate in the absence of the drug.
Although no systematic investigation was undertaken to study the effect of the length of BudR treatment, there was an indication that this may be important. When mid-11th-day embryos were exposed to up to 100 μg/mi of the analog for 12 h, no significant effect on chondrogenesis in the subsequent limb cultures was observed (Table 4, Fig. 1 C, D). When the treatment was extended to 24 h (which necessitated a slight modification in culture techniques), significant inhibition of chondrogenesis at 100 and 150 μg/ml levels of BudR became apparent (Table 5, Fig. 1F).
In all cases, a dose-related effect of the analog was observed. As the embryos were exposed to increasing concentrations of BudR, there was a corresponding increased inhibition of the cartilage development in the subsequent limb expiants (Tables 2, 3, 5). As our studies show that both the dose and possibly also the length of treatment were critical for the manifestation of chondrogenic inhibition in the limbs, it would appear that the rather mild teratogenic effects of BudR in vivo on the limbs could be due to only a small amount of the drug reaching the embryos in utero. This conclusion is supported by the observation that after maternal administration of a teratogenic dose of BudR in mice, the haff life of the injected drug was short (1 h) and only 2% of the thymine was replaced by bromouracil in the embryonic DNA (Packard et al. 1973). This contrasts with the reported 14–80% replacement in vitro (Djordjevic & Szybalski, 1960; Stellwagen & Tomkins, 1971; Fabian & Wilt, 1973) which could explain why BudR, in culture, is such a potent inhibitor of differentiating processes. Packard, Skalko & Menzies (1974) have argued that ‘…the suppression of cellular differentiation in vitro is (not) analogous to the production of malformations in mammalian embryos.. .(and).. .may indicate a basic dissimilarity between the production of fetal malformations by BrdU in vivo and its ability to inhibit cellular differentiation in vitro …’ In our studies where by employing the wholeembryo culture technique we could treat the embryos to relatively high concentrations of the drug for an extended (24 h) period of time, we were able to reproduce the almost total inhibition of chondrogenesis (Fig. 1B) reported in cultured chondrocytes and limb-bud cells (Abbott & Holtzer, 1968; Levitt & Dorfman, 1972).
Excess thymidine and BudR (10-5M or higher) have been shown to be highly cytotoxic to cells in culture as the conversion of cytidine nucleotide to its deoxynucleotide form was affected (Morris & Fischer, I960, 1963; Reichard, Canellakis & CaneHakis, 1961; Morris, Reichard & Fischer, 1963; Meuth & Green, 1974). BudR at levels of 10-5M or higher was also found to be toxic to preimplantation mouse embryos where cleavage was severely affected and the embryos died soon after (Garner, 1974; Golbus & Epstein, 1974; Pollard, Baran & Bachvarova, 1976). Garner (1974) raised the possibility that the drug may be exerting its influence by competing with and thus restricting the entry of heterologous nucleosides into the nucleotide precursor pool in a manner suggested by Steck, Nakata & Bader (1969). In contrast to the above studies, we did not find BudR or thymidine to be nearly as toxic to the postimplantation mouse embryos grown in vitro . Even in the presence of 100–150 μg/ml of the analog -almost 50-fold higher concentration than that used by the above authors -most of the 11th-day mouse embryos stayed healthy (in as far as no deleterious effect on their heart beat or blood circulation could be detected in the first 24 h of culture), grew and differentiated as indicated by an increase in their somite number. The average gain in somite pairs was nearly the same whether the embryos were grown in control or drug-supplemented medium. In a few preliminary experiments where the embryos were exposed to 200 μg/ml of BudR, the drug proved toxic and only a very few embryos survived in culture for longer than 2–4 h.
In sea urchin eggs, Gontcharoff & Mazia (1967) found that the introduction of BudR during the first 5 h of development prevented the formation of echinochrome pigment which does not appear until 24 h of development. They found it intriguing that ‘. .a known modification of DNA, such as substitution of BU (bromouracil) for thymine, at one stage of development can affect expressions of development that become manifest only at a much later stage.’ In the present study, we have demonstrated a similar ‘delayed effect’ of BudR on the chondrogenesis in the mouse limb-buds. In the 11th-day mouse embryos the limbs are at a very early stage of development (‘protodifferentiated’, Rutter et al. 1968): all the mesenchymal cells appear structurally alike even under the electron microscope and it is not possible to distinguish between the future muscle, cartiiagepr connective tissue cells (Kochhar, 1976). First signs of chondrogenesis do not appear till late 12th day in uteroor the 3rd day of in vitro growth (Neubert et al., 1974; Kochhar, 1976). When these early embryos were treated with 150 μg/ml of BudR for 24 h, an almost complete inhibition of chondrogenesis in the subsequent limb cultures was observed (Tables 2, 3). In some way treatment with the drug had altered or damaged the course of events that lead to the development of cartilage in the limbs. Once affected, chondrogenesis in the organ remained severely suppressed even when the limb was grown in drug-free medium for up to 9 days. It appears that much before the onset of chondrogenesis, the determined (according to Stark & Searls, 1973) but not fully differentiated chondrogenic cells pass through a stage of cytodifierentiation in which they are extremely vulnerable to the teratogen. Levitt & Dorfman (1972) have reported a similar irreversible suppression of chondrogenesis in the BudR-treated cultures of mesenchymal cells taken from very early chick wing-bud.
Whereas the limbs of early 11th-day mouse embryos were very sensitive to the BudR treatment and at appropriate concentrations of the drug exhibited an almost complete inhibition of chondrogenesis, the slightly older mid-11th-day embryos were not nearly as sensitive to the analog. At the highest dose tested (150 mg/μl), only a 51 % reduction in the total cartilage area units was observed (Table 5) in contrast to 95% inhibition that was recorded in the limbs of the younger embryos exposed to the same dose (Tables 2, 3). A stage-dependent effect of BudR has been shown in cleaving echinoderm eggs. Up to 8-cell stage, the sea urchin eggs were able to cleave in the presence of the drug although blastulation was abnormal and the embryos failed to gastrulate. Applied to older embryos (100-cell stage or older), BudR even at 1000 μg/ml level had a minimal effect on embryogenesis (Gontcharoff & Mazia, 1967 ; Tencer & Brachet, 1973). Whether developmentally advanced embryos actually become refractory to the BudR treatment could not be ascertained in these studies. Older embryos (12th and 13th day) proved difficult to maintain in vitro for longer than a few hours. However, it is possible to grow the limbs from these embryos for an extended period of time in organ culture. In the accompanying communication, we studied the effect of BudR on these limbs and found that they were indeed more resistant to the drug.
ACKNOWLEDGEMENTS
We thank Dr P. M. Rodier for her help with the statistical analyses; Mr John Penner for his assistance with photography and Miss Pamela Baker for typing the manuscript. This research was supported by NIH grant no. HD-06550. Some of the results described here were presented orally at the 15th annual meeting of the Teratology Society (May, 1975), Pocono Manor, Pennsylvania.