/ . Embryol. exp. Morph. Vol. 36, 3, pp. 639-652, 1976
Printed in Great Britain
639
Direct exposure of mouse embryonic
limb-buds to 5-bromodeoxyuridine in vitro and its
effect on chondrogenesis: increasing resistance to
the analog at successive stages of development
By NARSINGH D. AGNISH 1
AND DEVENDRA M. KOCHHAR 1
From the Department of Anatomy, University of Virginia Medical School, U.S.A.
SUMMARY
The inhibitory effect of 5-bromodeoxyuridine (BudR) - an analog of thymidine - on
embryonic mouse limb-buds was studied in vitro employing an organ-culture system. The
effect was found to be dose-related and also depended on the developmental stage of the donor
embryos. Limbs at an early stage of development (early llth-day embryos, somite stage
26-29) were extremely sensitive to the analog. Treatment with low levels (2-4 fig ml) and for
a relatively short period of time in culture (2-3 days) completely and irreversibly suppressed
chondrogenesis in these explants. Limbs from older embryos (somite stage 40 and up) were
found to be much less sensitive to the inhibitory effect of the drug; a prolonged exposure to
a much higher dose (100-150 fig ml) resulted in an incomplete suppression of chondrogenesis.
Only a 20% inhibition was observed in the cultures of limbs from mid-13th-day mouse
embryos. After continuous growth in vitro, the limbs became progressively resistant to the
analog and towards the end of the culture period had become refractory to the drug. The
time of complete insensitivity appeared earlier in the cultures of the limbs taken from older
embryos than in the explants of younger limbs. These studies show that as limbs continue to
differentiate in vivo or in vitro, they become increasingly resistant to the inhibitory effect of
BudR in at least as far as the effect on the process of chondrogenesis is concerned. It is suggested that the relative sensitivity or insensitivity to the inhibitory effect of BudR may prove
to be a useful parameter in evaluating the developmental stage of an organ.
INTRODUCTION
When mature chondrocytes from chick sternal or vertebral cartilage were
cultured in the presence of low levels of 5-bromodeoxyuridine (BudR), an
analog of thymidine, chondrogenesis was almost completely inhibited (Abbott
& Holtzer, 1968; Lasher & Cahn, 1969; Coleman, Coleman, Kankel & Werner,
1970). The effect, however, was reversible; upon further growth in drug-free
medium, the cells were able to resume synthesis of chondroitin sulfate and
deposit metachromatic matrix. Dorfman and co-workers have, however,
1
Authors' address: Department of Anatomy, Jefferson Medical College, 1020 Locust
Street, Philadelphia, Pennsylvania, 19107, U.S.A.
640
N. D. AGNISH AND D. M. KOCHHAR
reported that chondrogenesis in early chick wing-bud mesenchymal cells in
vitro was irreversibly suppressed after 2-3 day treatment with BudR; once
affected, these cells failed to synthesize chondroitin sulfate even when cultured
in drug-free medium for several days (Levitt & Dorfman, 1972; Levitt, Ho &
Dorfman, 1975). These authors have suggested that the reversible effect of the
drug is characteristic of differentiated cells and that the effect is permanent on
cells that are not yet fully differentiated. In certain cases, differentiated cells
have been shown to become refractory to the analog. Pancreatic cells after
3 days in culture (Wessells, 1964) and erythroblasts that had just begun to
synthesize hemoglobin (Weintraub, Campbell & Holtzer, 1972, 1973) continued to synthesize macromolecules characteristic of their differentiated state
in the presence of BudR.
The present investigations were undertaken to study the effect of BudR on
the differentiation of cartilage in the mouse limb as it passes through various
stages of development and to see if we could find some relationship between the
developmental stage of the organ and its sensitivity to the analog. The choice
of limb as the target organ was made because technique for its organ culture are
well established (Aydelotte & Kochhar, 1972; Kochhar & Aydelotte, 1974) and
extensive literature exists on limb development. Furthermore, developmental
history of the limb encompasses most of the major known morphogenetic processes so that conclusions drawn from response of this organ to BudR may be
broadly applied to other developing systems as well.
For these studies, we have employed an in vitro system which offers several
advantages over the in vivo approach. The developmental stage of the embryos
can be determined precisely by counting the number of somite pairs so that
stage-dependent effects of BudR can be measured with accuracy. The considerable variation that one finds in the size (Barr, 1971) and developmental stage
(Kochhar & Aydelotte, 1974) of the embryos within and between litters from
mothers at the same day of gestation can be eliminated or reduced to an acceptable level. In addition, the in vitro system avoids any modification or degradation (Kriss & Revesz, 1962) of the drug and the treatment can be extended to
a part or the whole of the culture period. For the same reasons, dose-related
studies can be easily carried out. Most of the studies on BudR-mediated interference with developmental processes have indeed been carried out using cellculture techniques. Because a number of developmental processes like cellular
induction, epithelial-mesenchymal interaction and cell movement are known to
play critical roles during limb morphogenesis, we have employed organ-culture
techniques in preference to cell-culture methodology.
Effect of BudR on mouse limb-buds
641
MATERIALS AND TECHNIQUES
The animals and their breeding protocol has been described elsewhere
(Agnish & Kochhar, 1976). The culture procedure was similar to that of
Kochhar & Aydelotte (1974) with the following modifications:
llth-day embryos: the forelimbs were cultured for 9 days and the medium
changed on the 3rd and the 6th day of culture period.
12th-day embryos: the limbs were grown for 6 days with one change of
medium on the 3rd day of culture.
13th-day embryos: the limbs were again cultured for 6 days but as the organ
was very large, the medium was changed every day.
In all cases, the excised limbs were maintained in BGJ medium (Difco,
Detroit, Michigan) supplemented with 25 % fetal calf serum (Flow Laboratories,
Rockville, Md.), antibiotics (12-5/*g/ml streptomycin, 7-5/tg/ml penicillin G)
and ascorbic acid (150/tg/ml). The nucleosides, BudR and thymidine (all
chemicals obtained from Sigma, St Louis, Missouri), were dissolved in BGJ
medium and kept frozen at -20 °C. On the day of culture, these stock solutions
were thawed out and added to the culture medium in appropriate concentrations.
From each embryo, one forelimb was grown in control medium and the other
in BudR-supplemented medium. The average measurements listed in various
tables, unless otherwise specified, were based on 16-24 identically treated limbs
that had been excised from similarly staged (in terms of somite paris) embryos.
In experiments where the effect of short-term exposure to BudR was under
investigation, the drug-supplemented medium was replaced with fresh medium
containing excess thymidine (usually 10-25 /*g/ml) to dilute out whatever BudR
may have been left. After 1-3 days of growth in thymidine-enriched medium, the
cultures were returned to fresh, control medium. The presence of up to 100 [igj
ml of thymidine in the medium did not affect the growth or cartilage development
in these cultures.
The techniques for fixation and staining of the limbs and for quantification
of cartilage has been described earlier (Agnish & Kochhar, 1976). Analysis of
variance coupled with Dunnett test (Dunnett, 1955) were the statistical methods
employed to judge the inhibitory effect of BudR.
RESULTS
A. The extent of cartilage development in control (untreated) limb cultures
Within 24-48 h, the explanted limb-buds attached themselves firmly to the
filter, mostly through an active cell growth at the proximal (cut) end. After
6-9 days in culture, all the cartilage components were easily identifiable by
their shape and position in the explants. The proximo-distal orientation of
cartilages- characteristic of adult limbs-was maintained in vitro even though the
total growth and size of individual cartilages was much smaller (Fig. 1). Scapula
41
EMB 36
642
N. D. AGNISH AND D. M. KOCHHAR
Effect of BudR on mouse limb-buds
643
developed near the proximal end of the explants and appeared triangular or
trapezoid in shape, typically with a prominent acromion process. The successively distal humerus and radio-ulna differentiated as bars of cartilage, often
exhibiting characteristic curvatures. Adequate development of digits occurred
only in the cultures of limbs excised from older embryos.
B. The effect of BudR on chondrogenesis in the cultures of mouse limb-buds
In the presence of up to 100-150/Ag/ml of BudR in the medium, limbs were
able to grow in culture but chondrogenesis was affected to various degrees. The
extent of the suppression was found to be both stage and dose dependent and
was further influenced by the length of drug treatment.
(i) Early and mid-Wth-day embryos. The limbs of early llth-day embryos
(somite stage 26-29) were extremely sensitive to BudR. In the presence of
relatively low levels of the analog (2-4/^g/ml) in the medium for the entire
culture period (9 days) or even for the first 2 days of culture, complete suppression
of chondrogenesis was observed (Fig. IB). Similar results were obtained when
limbs of slightly older, mid-1 lth-day embryos (stage 32-34) were cultured for
9 days in the presence of 10-25 /tg/ml of the drug. As prolonged treatment with
BudR was severely affecting chondrogenesis in these limbs, we then investigated
if the explants were equally sensitive to the analog throughout the culture period.
With this in mind, the limbs from the mid-1 lth-day embryos were exposed to
the drug for a 3-day segment of in vitro growth. Exposure to BudR (25 /*g/ml)
on day 1-3 of culture period followed by growth in drug-free medium for the
next 6 days also resulted in complete inhibition of cartilage differentiation
(Table 1). Treatment on culture day 4-6 had a similar effect although in some
explants small nodules of metachromasia were evident. Delaying the BudR
treatment to the last 3 days of in vitro growth resulted in a minimal inhibition
of chondrogenesis; both scapula and humerus appeared normal but the radioulna was still severely affected (Table 1).
FIGURE 1
Control and BudR-treated limbs of mouse embryos. Forelimbs were organcultured for 6 or 9 days in control or drug-supplemented medium, fixed and stained
in toluidine blue.
(A) Early llth-day limb, cultured in control medium for 9 days.
(B) Early 11 th-day limb, treated with 2 /*g ml of BudR for the first 2 days of culture
period and then grown for another 7 days in drug-free medium.
(C) 12th-day limb, cultured for 6 days in control medium.
(D) 12th-day limb, cultured for 6 days in the presence of 50/Jg/ml of BudR.
(E) Mid-13th-day limb, cultured for 6 days in control medium.
(F) Mid-13th-day limb, cultured for 6 days in the presence of 150/ig/ml of BudR.
In all photographs the distal tip of the limb is towards the bottom. The bar in
each figure represents 1 mm. Outline of scapula in F has been redrawn.
41-2
644
N. D. AGNISH AND D. M. KOCHHAR
Table 1. Treatment of mid-1 lth-day (somite stage 32-34) mouse
embryonic forelimbs with BudR and its effect on chondrogenesis
Extent of cartilage development (in area units)
Duration of
treatment
Scapula
Humerus
Radio-ulna
Digits
Total (±S.D.)
Control
Days 1-3
Days 4-6
Days 7-9
Days 1-9
127
0(-100)*
27 (-79)
127 (0)
0(-100)
63
0(-100)
22 (-65)
74 (+18)
0(-100)
37
0 (-100)
Of-100)
7(-81)
0(-100)
0
0
0
0
0
227 ± 10
0
(-100)
49±10(-78)
208 ±25 (-8)
0
(-100)
Forelimbs were organ-cultured and exposed to 25 /tg ml of BudR for the entire culture
period of 9 days or for a 3-day segment.
* Figure in parentheses is the percentage change in the mean cartilage area units as compared to those in control explants. Each mean value represents 20-24 limbs.
Table 2. One day treatment of mid-1 lth day (somite stage 32-34)
mouse embryonic limbs with BudR and its effect on chondrogenesis
Control
Day 3
Day 4
Day 5
Extent of cartilage development (in area units)
Scapula
Humerus
Radio-ulna
142
21 (-85)*
92 (-35)
139 ( - 2 )
72
33 (-54)
21 (-69)
18 (-75)
28
0(-100)
0(-100)
0(-100)
Digits
o o o o
Day of
treatment
(in vitro)
Total
(±S.D.)
242 ±44
54±13 (-78)
113±20(-53)
157±31 (—35)
Forelimbs were organ-cultured for 9 days and exposed to BudR (25 /tg ml) on either the
3rd, 4th or 5th day of culture period. Before and after the treatment, limbs were grown in
control or thymidine-enriched medium.
* Figure in parentheses is the percentage reduction in the mean cartilage area units as
compared to those in control cultures. Each mean value represents 20-24 limbs.
To pinpoint the period of sensitivity to BudR, the effect of one day's treatment
with the drug on limb explants was investigated. As the explants take 1-2 days
to 'adapt' to the culture environment, during which time there is no net gain
in DNA (Neubert, Merker & Tapken, 1974), the limbs of mid-1 lth-day embryos
were exposed to BudR (25 /*g/ml) for 24 h on the 3rd, 4th, or 5th day of culture.
In each case, cultures were maintained in drug-free medium before and after
the treatment. Treatment on the 3rd day of in vitro growth had the severest
effect and resulted in 78 % reduction in the total cartilage area units (Table 2).
Treatment on the 4th or 5th day resulted in a progressively lesser inhibition of
cartilage differentiation; the scapula gradually became resistant to the drug
while the radio-ulna and humerus still exhibited complete or partial suppression
(Table 2).
(ii) Late 1 lth-day (somite stage 35-38) embryos. The forelimbs of the late
Effect of BudR on mouse limb-buds
645
Table 3. Treatment of late llth-day (somite stage 35-38) mouse
embryonic limbs with BudR and its effect on chondrogenesis
BudR level in
the medium
(fig ml)
0
2
4
10
25
,
Scapula
245
41 (-83)*
2 (-99)
0(-100)
0(-100)
Extent of cartilage development (in area units)
*
Humerus
Radio-ulna
Digits
Total
146
195 (+34)
125 (-14)
6 (-96)
0(-100)
50
35 (-30)
15 (-70)
5 (-90)
0(-100)
0
0
0
0
0
*
(±S.D.)
441 ±42
271 ±24 (-39)
142±34f-68)
11 ±4 (-98)
0
(-100)
Forelimbs were organ-cultured for 9 days in the presence of BudR.
* Figure in parentheses is the percentage change in the mean cartilage area units as compared to those in control explants. Each mean value represents 16-20 limbs.
Table 4. Treatment of 12th-day (somite stage 40-46) mouse embryonic
limbs with BudR and its effect on chondrogenesis
Extent of cartilage development (in area units)
BudR level
in thp mpHiiim
A
111 tilt- 111VU114111
(fig ml)
0
2
5
10
25
50
100
Scapula
Humerus
Radio-ulna
Digits
326
143
111
33
309 (-5)*
154 (-53)
35 (-89)
8 (-98)
K-100)
0(-100)
148 ( + 4)
140 (-2)
111 (-22)
56 (-61)
33 (-77)
23 (-84)
104 (-6)
125 (+13)
55 (-51)
61 (-45)
37 (-67)
29 (-74)
38 (+15)
42 ( + 27)
26 (-21)
28 (-15)
22 (-33)
2 (-94)
Total (± S.D.)
613 ±63
599 ±60 (-2)
461 ±55 (-25)
227 ±48 (-63)
153 ±24 (-75)
93 ±9 (-85)
54±3 (-91)
Forelimbs were organ-cultured for 6 days in the presences of BudR.
* Figure in parentheses is the percentage change in the mean cartilage area units as compared to those in control explants. Each mean value represents 16-20 limbs.
llth-day embryos were able to tolerate small amounts of BudR (2-4/^g/ml) in
the medium and chondrogenesis was incompletely suppressed even when the
limbs were grown for the entire period of 9 days in the presence of the analog
(Table 3). The degree of inhibition of chondrogenesis was found to be doserelated and only at 10-25 /*g/ml level of the drug was the cartilaginous development completely inhibited.
(iii) 12th-day (somite stage 40-46) embryos. In the presence of up to 2 /*g/ml
of BudR in the medium for the entire culture period of 6 days, no significant
effect on chondrogenesis could be detected in the limbs of 12th-day embryos:
all the cartilages appeared normal and comparable in size to those observed in
control cultures (Table 4). At higher doses (5-100/^g/ml), chondrogenesis was
affected and the degree of its inhibition was found to be related to dose. With
increasing concentrations, a corresponding decrease in the total cartilage area
units was seen (Table 4). All the cartilages were not being affected equally. The
646
N. D. AGNISH AND D. M. KOCHHAR
Table 5. Treatment of 12th-day {somite stage 40-46) mouse embryonic limbs with
BudRfor a portion of the culture period and its effect on chondrogenesis
Extent of cartilage development (in area units)
Days of
treatment
Scapula
Humerus Radio-ulna
Digits
Total
(±S.D.)
Control (6 days) 326
143
111
33
613 ±83
Day 1-3
25 (-92)* 77 (-46) 89 (-20) 25 (-24) 216±46 (-65)
Day 4-6
249 (-24) 148 ( + 4)
80 (-28) 20 (-39) 497±69 (-19)
Day 1-6
8 (-98)
56 (-61)
61 (-45) 28 (-15)
153 ±24 (-75)
Control (9 days) 430
266
186
177
1059 ±117
Day 7-9
442 ( + 3) 281 (+6) 234 ( + 26) 181 ( + 2) U38±148 (+8)
Forelimbs were organ-cultured for a segment (as indicated) of the culture period in BudR
supplemented (25 fig ml) medium. Before and after treatment, the explants were grown in
control or thymidine-enriched medium.
* Figure in parentheses is the percentage change in the mean cartilage area units as compared to those in control explants. Each mean value represents 12-16 limbs.
scapula was extremely sensitive and showed a near complete suppression even
at relatively low dose of 10-25 /*g/ml. Surprisingly, the distal cartilages - the
digits - were the least sensitive to the drug except at very high levels of the
analog. Even at the highest concentration tested (100 /*g/ml), chondrogenesis
was not completely inhibited: remnants of humerus, radio-ulna and digits were
still evident (Fig. 1D).
To investigate the reversibility of the BudR effect, these limbs were cultured
for 3 days in the presence of the analog (25 /*g/ml) and then returned to drugfree medium. As can be seen from Table 5, such a treatment still resulted in a
65 % reduction in the total cartilage area units. After 3 days in culture, the
explants were not nearly as sensitive to the drug. The effect on the scapula was
now only moderate. After 6 days in culture, the explants became completely
refractory to the analog and treatment for 3 days at this time (culture day 7-9)
did not produce any observable cartilage inhibition (Table 5). There was a slight,
though statistically insignificant, increase in the amount of cartilage area
units.
(iv) Early ISth-day {somite stage 52-56) embryos. When the early 13th-day
embryonic limbs were cultured in the presence of up to 10 /*g/ml of BudR,
cartilage development was not affected (Table 6). As the concentration of the
drug was increased further (25-150/tg/ml), dose-related inhibition of chondrogenesis became evident. However, even at 150 /ig - the highest dose tested - the
suppression of cartilage was incomplete (Table 6). In all cases, scapula and to a
lesser extent the humerus, exhibited the maximum sensitivity to the analog;
the effect on the radio-ulna and digits was relatively moderate.
The explants were most sensitive to the analog during the first half of their
in vitro growth. Treatment with 25 /*g/ml on day 1-3 of culture period (followed
Effect of BudR on mouse limb-buds
647
Table 6. Treatment of early ISth-day {somite stage 52-56) mouse
embryonic limbs with BudR and its effect on chondrogenesis
BiudR
level
ml) and
days of treatment
Extent of cartilage development (in area units)
(lLg
0
10
25
25
25
50
100
150
Day
Day
Day
Day
Day
Day
Day
A
Scapula
523
534 (+2)*
324 (-38)
500 (-4)
226 (-57)
189 (-64)
151 (-71)
107 (-80)
1-6
1-3
4-6
1-6
1-6
1-6
1-6
Humerus
Radio-ulna
Digits
128
127
121
122
91
78
85
59
130
119 (-9)
125 (-4)
139 ( + 7)
119 (-9)
109 (-16)
103 (-21)
91 (-30)
148
147 (-1)
153 ( + 3)
134 (-10)
137 (-7)
150( + l)
142 (-4)
119 (-20)
(-1)
(-6)
(-5)
(-29)
(-39)
(-34)
(-54)
Total (±S.D.)
929 ±52
927 ±68
723 ± 35
895 ± 54
573 ±61
526 ±27
481 ±27
376 ±23
(0)
(-22)
(-4)
(-38)
(-43)
(-48)
(-60)
Forelimbs were organ-cultured for 6 days and exposed to various concentrations of BudR
for the entire culture period or a segment thereof.
* Figure in parentheses is the percentage change in the mean cartilage area units as compared to those in control explants. Each mean value represents 12-16 limbs.
Table 7. Treatment of mid-13th day (somite stage 58-62) mouse embryonic
limbs with BudR and its effect on chondrogenesis
Extent of cartilage development (in area units)
BudR level in
(/*gml)
0
10
25
50
100
150
Scapula
679
649
659
460
450
457
(-4)
(-3)
(-32)
(-34)
(-33)
Humerus
Radio-ulna
Digits
187
183
166
167 ( — 11)
237 (+30)
195 (+7)
199 ( + 9)
187 ( + 2)
190 ( + 4)
172 ( + 4)
159 (-4)
156 (-6)
160 (-4)
200 (+21)
143 (-24)
190 (+2)
168 (-10)
159 (-15)
Total (± S.D.)
1215 ±126
1225 ± 141 (+1)
1156±153(-5)
1005 ± 52 (-17)
965+ 69 (-21)
1006±70 (-17)
Forelimbs were organ cultured for 6 days in the presence of BudR.
* Figure in parentheses is the percentage change in the mean cartilage area units as compared to those in control explants. Each mean value represents 12-16 limbs.
by growth in control medium) resulted in a 22 % reduction in the total cartilage
area units. When the treatment was delayed to day 4-6 of culture, the explants
had become resistant to the analog (Table 6) and all the limb components
appeared normal and comparable to those observed in control cultures.
(v) Mid-l3th-day (somite stage 58-62) embryos. In the presence of up to
25 /Ag/ml of BudR in the medium, no significant inhibition of chondrogenesis
was observed in the limb cultures of these embryos. At 50 fig, a moderate (17 %)
inhibition was observed. As the concentration of drug was increased further, to
a maximum of 150 /*g/ml (Table 7), no further suppression of cartilage development could be detected. Statistically, the extent of cartilage differentiation in
648
N. D. AGNISH AND D. M. KOCHHAR
the presence of 50,100 or 150 /tg/ml of the analog was similar; the characteristic
dose-related effect noted earlier did not exist. The proximal cartilages were the
only ones being affected; the radio-ulna and digits were refractory (Fig. IF).
DISCUSSION
The presence of BudR in the medium interfered with the differentiation of
cartilage in the explants of mouse embryonic limbs grown in organ culture.
The degree of chondrogenic inhibition was found to depend on the dose and
duration of treatment, the developmental stage of the donor embryo and how
long the organ was allowed to grow in culture before being exposed to the drug.
The limbs of very early llth-day mouse embryos (somite stage 26-29) were
found to be the most sensitive to the analog. Even in the presence of trace
amounts of the chemical (2-4 /tg/ml;~10~5M), chondrogenic differentiation
was completely inhibited. The effect was irreversible; treatment for 3 days in
culture followed by 6 days growth in drug-free medium resulted in no visible
cartilage development (Fig. 1B). These limbs are very small and histologically
all the mesoblastic cells appear alike ('morphologically equivalent', Searls &
Janners, 1969) even under electron microscopy (Kochhar, 1976). The labeling
index (percentages of cells labeled with [3H]thymidine after a 4 h exposure to
the isotope) is virtually 100% (unpublished observations), indicating that all
the cells are actively incorporating DNA precursors and presumably capable
of proliferation. Indeed, no visible signs of differentiation are apparent even
though the position of all the future limb segments is already mapped at this
time (Stark & Searls, 1973). This may be regarded as the 'least differentiated'
(protodifferentiated, Rutter et al. 1968) stage of limb development and as these
studies have shown, a time when the organ is most susceptible to the inhibitory
effects of BudR treatment.
The first recordable signs of differentiation occur in the late llth-day (somite
stage 35-38) mouse forelimbs. While the labeling index was still very high in
the subapical (growth zone) and the proximal-peripheral (myogenic) regions,
in the central proximal zone (the future chondrogenic area), the index was
reduced to about 42% (Kochhar, 1976). The sensitivity of the limbs to BudR
was also affected: 2/^g/ml, which completely inhibited any visible cartilage
development in the limbs of early llth-day embryos, now produced only a
39 % reduction (Table 3). A dose of 10-25 /tg/ml was required for the complete
suppression of chondrogenesis.
Further signs of differentiation become visible in the limbs of 12th- and
13th-day mouse embryos. Mesenchymal condensations in the central proximal
region of these limbs can be clearly seen even at low magnification and the
labeling index in the prospective chondrogenic area is reduced to 7 % (Kochhar,
1976). In addition, the hand plate can be easily recognized. These limbs were
found to be more resistant to BudR, at least as far as the effect on chrondro-
Effect of BudR on mouse limb-buds
649
genesis was concerned. Not only a higher dose but also a longer treatment with
the drug was required to affect the development of cartilage severely. At the
highest dose tested (100 /*g/ml), the inhibition of cartilage development was
still not complete (Table 4) and remnants of humerus, radio-ulna and digits
could still be seen in the limb explants of 12-day embryos. A yet higher level of
BudR (150/Ag/ml) affected the cartilage development in the forelimbs of 13thday embryos only to a moderate degree (Tables 6, 7); all the cartilages were
clearly identifiable and appeared normal except for the scapula which was still
showing a moderate effect (Fig. 1F).
In almost all the cases where limb cultures were able to tolerate at least small
amounts of BudR, the degree of chondrogenic suppression was dose-dependent.
With increasing concentrations of BudR in the medium, a corresponding
decrease in the total cartilage are units was consistently observed in the cultures
of limbs excised from mid-1 lth, 12th and early 13th-day mouse embryos. The one
exception was the limbs of mid-13th-day embryos which did not exhibit the
typical dose effect. Here, inhibition of chondrogenesis was statistically similar
at dose levels of 50, 100 and 150/tg/ml (Table 7).
The limbs of developmentally more advanced embryos required correspondingly higher levels of the drug to show substantial inhibition of chondrogenesis.
Essentially similar results have recently been described by Flickinger (1975) in
the organ cultures of differently staged chick wing-bud mesenchyme.
The increasing resistance to BudR was not exclusive to limbs of the older
embryos. The forelimbs of the llth-day mouse embryos during the course of
their development in culture also became increasingly refractory to the drug.
Whereas treatment on the 3rd day of culture resulted in a 78 % reduction in the
total cartilage area units, similar treatment carried out on the 5th day of in vitro
growth showed only a modest depression (35%) of cartilage development
(Table 2). The maximum effect on chondrogenesis was observed if the limb
explants were exposed to BudR during the early part of their culture history.
Once the limbs were allowed to grow and differentiate in control medium for
3-6 days, the analog, at least in concentrations up to 25 /*g/ml, failed altogether
to inhibit chondrogenesis. The time of total resistance came earlier in older
limbs, after 3 and 6 days in culture of the limbs of 13th- (Table 6) and 12thday (Table 5) embryos respectively. The limbs of llth-day embryos were still
sensitive to BudR after 6 days in culture although the effect was confined to
the distal elements (radio-ulna) only (Table 1).
Total resistance to BudR has been demonstrated in a number of cases. The
synthesis of zymogen granules was inhibited in the cultures of mouse pancreatic
tissue in the presence of BudR during the initial culture period. After 3 days
of growth in control medium, a large number of cells became BudR-insensitive
(Wessells, 1964). Similarly, BudR inhibited hemoglobin synthesis in primitive
erythroblasts was no longer evident once the blast cells had differentiated and
begun to synthesize hemoglobin, even though they continued to divide (Miura
650
N. D. AGNISH AND D. M. KOCHHAR
& Wilt, 1971; Weintraub et ah 1972, 1973; Hagopian, Lippke & Ingram,
1972).
Chondrogenesis was completely suppressed when mature chondrocytes were
cultured in the presence of BudR (Abbott & Holtzer, 1968; Lasher & Cahn,
1969; Coleman et ah 1970). Our studies have shown that such a suppression is
only partial as long as the chondrocytes were in their proper cellular environment. Because of the extensive cartilage development in limb cultures, it is safe
to assume that at some time during this growth, chondrocytes appear in the
explants. Yet the limbs of 12th and especially 13th-day mouse embryos organ
cultured in the presence of BudR (10-150/tg/ml), even for the entire in vitro
period, exhibited varying amounts of cartilage; the inhibition of chondrogenesis
was dose-dependent rather than 'all-or-none'.
Our results differ from those reported by Lasher & Cahn (1969) and Coleman
et al. (1970) in one other important aspect. These authors found that the effect
of BudR on chondrocytes in culture was reversible once the drug was removed
from the medium. Such reversibility was either non-existent or only partial in
our limb organ cultures. Chondrogenesis was irreversibly inhibited when limbs
from llth-day mouse embryos were cultured for 3 days in the presence of the
analog and then returned to control medium. In the case of older limbs (taken
from 12th- and 13th-day embryos), whether the treatment (with 25/tg/ml of
BudR) lasted the entire culture period or was restricted to the first 3 days, the
percentage reduction in the cartilage area units was very similar - 75 %
and 65% in 12th-day limbs (Table 5) and 38% and 22% in 13th-day limbs
(Table 6).
It is generally assumed that the overt differentiation of skeletal and muscle
tissue in developing limbs proceeds in a proximo-distal direction (Saunders,
1948; Stocum, 1975; Wolpert, 1976). As our studies seem to indicate that less
differentiated limbs were more sensitive to BudR, we examined our data to
see if the drug could be used to follow the proximo-distal progression of
differentiation. In llth-day embryonic limbs, all the cartilage components were
sensitive to BudR when exposed to the drug early in culture. However, while
the scapula (presumably the first cartilage to complete its differentiation)
became increasingly resistant to the analog with time in culture, the more distal
radio-ulna (presumably the last one to complete its differentiation) retained
sensitivity throughout culture (Tables 1, 2). The sensitivity pattern of the
humerus was rather inconsistent and did not appear to follow any particular
scheme. In older embryos, the distal cartilaginous elements (humerus and radioulna) were relatively insensitive to BudR whether exposed early or late in
culture and there was no increase in resistance with time in culture (Tables 5, 6).
The scapula was sensitive to the drug when exposed early in culture and less
sensitive to treatment late in culture (Tables 5, 6) as in llth-day limbs.
In general, as differentiation proceeds in a developing organ, more and more
cells withdraw themselves from the proliferation cycle (Cameron & Jeter, 1971).
Effect of BudR on mouse limb-buds
651
The classical example is provided by muscle tissue. Replicating myoblasts do
not synthesize myosin. After 4-5 days in culture, these cells begin to fuse and
form long, multinucleated myotubules and start synthesizing myosin. Around
this time the cells stop multiplying and do not incorporate radioactive DNA
precursors (Stockdale, Okazaki, Nameroff & Holtzer, 1964). In mouse embryos,
as the limb differentiates, there is a slowing down of mitotic activity and a
rapid decrease in the labeling index in the presumptive chondrogenic zone
(Kochhar, 1976). We do not think that the progressive resistance to BudR is
due to cessation of DNA synthesis in the chondrogenic cells of the limb, as we
have on many occasions observed mitotic figures in cells embedded in the
toluidine-blue staining matrix of clearly identifiable cartilage tissue in limbs
cultured for 6-9 days. Also, when 12th-day limbs were cultured for 2-3 days in
the continuous presence of [3H]thymidine, practically all the cells were found
to be labelled (unpublished observations). These observations would indicate
that most, if not all, cells in the limb explants are capable of synthesizing DNA
and undergoing replication.
Searls & Janners (1969) concluded that ' . . . t h e cells from the cartilage
forming area develop an increasingly strong bias as the cartilage-forming area
becomes more distinct.' During this differentiation process, a number of
changes are occurring in the 'protodifferentiated' organ: appearance of
mesenchymal condensations, a drop in the [3H]-thymidine labeling index and
the proliferation rate and, as the present studies have shown, a gradually
increasing resistance to the inhibitory effects of BudR. The effect of the drug on
the limbs of mid-13th-day mouse embryos was minimal (Table 7) and it is
possible that the limbs of 14th-day mouse embryos (by which time the differentiation of chondrogenic cells is complete and irreversible) might turn out to be
completely refractory to the analog. We have not investigated these limbs as
they are very large and in organ culture develop necrotic areas making them
unsuitable for any critical studies.
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.
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{Received 14 June 1976, revised 6 August 1976)