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Inhibition of limb chondrogenesis by a Veratrum alkaloid: Temporal specificity in vivo and in vitro
DEVELOPMENTAL
BIOLOGY
111,464-4’70 (1985)
inhibition of Limb Chondrogenesis by a Veratrum Alkaloid: Temporal Specificity in Vivo and in Vitro MARLISSA A. CAMPBELL,*,’ KENNETH S. BROWN,* JOHN R. HASSELL,* ELIZABETH A. HORIGAN,* AND RICHARD F. KEELERt *Laboratory
of Developmental Biology and Anomalies, Natim~al Institute of Dental Research, Natimal Institutes Bethesda, Maryland 20205 and tU S. Department of Agm’culture, Agricultural Research Service, Poisonous Plant Research Laboratory, Logan, Utah 84321 Received February
12, 1985; accepted in revised form Ap.l2$
of Health,
1985
It has been demonstrated that jervine, a steroidal alkaloid derived from plants of the genus Veratrum, exerts teratogenic effects in several animal species. Defects were restricted to structures which depend upon normal chondrogenesis for their development. Here we report studies of the temporal specificity of cellular sensitivity using limb bud mesenchyme cells obtained from Day 10 mouse embryos. These cells, when grown as high-density “spot” cultures, undergo chondrogenesis in vitro. Prior to differentiation, exposure of limb cell cultures to jervine suppressed subsequent accumulation of cartilage proteoglycans. Treatment after differentiation had no significant effect. Additionally, there was a genetic component to jervine sensitivity: C57BL/6J mice were sensitive, whereas NIH Swiss-Webster mice were insensitive. This strain-dependent difference was observed both in vivo and in vitro, supporting the validity of limb mesenchyme spot cultures as a model for jervine-induced teratogenicity. Our studies indicate that jervine acts specifically during an
early phase of the differentiation target tissue of this compound.
of mesenchyme into cartilage. It is likely that a specific stem cell population is the 0 1985 Academic
Press, Inc.
INTRODUCTION
Jervine and cyclopamine are two steroidal alkaloids which are derived from plants of the genus Veratrum (Keeler, 1972). Prenatal exposure to these compounds has been shown to cause craniofacial and limb malformations in sheep, goats, cattle, chickens, and two strains of mice (Keeler, 1971; Binns et ab, 1972; Bryden et ah, 1973; Keeler, 1975; Brown et ab, 1980; F. R. P. Sim, M. L. Omnell, R. F. Keeler, L. C. Harne, and K. S. Brown, in preparation). The types of defects observed were similar for all these species, almost exclusively involving structures which depend upon normal chondrogenesis for their development. Furthermore, induction of specific defects was dependent upon the developmental status of the embryo at the time of its exposure. In sheep, goats, and cattle, gestation Days 12 through 30 comprised the critical period for induction of hypoplastic malformations of the craniofacial complex (Binns et al., 1972). Vera&urn exposure on Days 25-36 resulted in limb reduction defects, while the frequency of facial deformities decreased. Similar sequential sensitivity has been described for chicks (Bryden et al., 1973) and mice (Sim et ab, in preparation). Research on jervine teratogenicity in mice (Brown et al, 1980; Sim et ah, in preparation) established that the types of defects produced were related to the time of treatment, as well as to genetic 1To whom correspondence should be addressed. 0012-1606/85 $3.00 Copyright All rights
0 198.5 by Academic Press, Inc. of reproduction in any form reserved.
464
background and the dose of drug. In two strains of mice, C57BL/6J and A/J, limb defects were induced by exposure to jervine on gestation Days 8, 9, or 10, with a peak frequency on Day 10. In all cases, peak effectiveness of Veratrum-teratogens occurred prior to morphological differentiation of the target tissue. Because of its temporal and tissue specificity, we have investigated jervine sensitivity as a potential marker of early prechondrogenic stem cell populations. Mouse limb development, both in vivo and in vitro, was used as the model system. Much is known about the sequence of limb development in normal mouse embryos. Considering the day of fertilization to be gestation Day 0, the forelimb buds begin to appear on Day 8, and the hindlimb buds on about Day 10 (Theiler, 1972). At this time, the mesenchymal tissue of both sets of limb buds appears to be homogenous. Models of individual limb bones begin to condense out of the mesenchyme during the 11th day. Chondrogenesis also commences at this time, proceeding over the next few days in successively more distal forelimb segments, and in the hindlimb. In order to study the relationship between sensitivity to jervine and state of differentiation at the cellular level, we used an in vitro system that encompasses the transition from undifferentiated mesenchyme to mature cartilage. When plated at high densities, prechondrogenie limb bud mesenchyme cells will undergo chondrogenesis in vitro (Umansky, 1966; Caplan, 1970; Goetinck,
CAMPBELL
ET AL.
Limb Chondrogenesis Inhibiticm
1974; Ahrens, 1977). These cells can be obtained from Day 10 mouse embryos (Lewis et ab, 1978; Hassell et al., 1978; Pennypacker et al., :1978). The progression of chondrogenesis can be demonstrated by staining cartilagespecific proteoglycans with alcian blue at low pH (Lev and Spicer, 1964). Using alcian blue, proteoglycan accumulation (and hence chondrogenesis) can be quantified spectrophotometrically (Hassell and Horigan, 1982). The limb cell culture system has the advantage of allowing introduction of teratogenic agents at different times during differentiation. Therefore, we were able to evaluate the effects of jervine on cellular differentiation in vitro, as well as observing its effects on morphological differentiation in vivo. MATERIALS
AND
METHODS
Animals. C57BL/6J and, A/J mice were obtained from Jackson Laboratories (Bar Harbor). NIH all-purpose mice are derived from Swiss-Webster stock. These mouse strains were chosen because of documented differences in sensitivity to Vera&urn-induced teratogenesis; C57BL/6J and A/J mice are sensitive, whereas NIH Swiss-Webster mice are resistant (Keeler, 1975; Brown et ah, 1980; Sim et ab, in preparation). We wished to determine whether corresponding strain-dependent differences could be documented in vitro. Drug. Jervine was isolated from Veratrum viride roots and rhizomes using methods described by Keeler and Binns (1968). Infrared scans, thin-layer chromatography, and HPLC analysis of the purified material demonstrated a purity of over 96% with traces of other, nonteratogenic, Veratrum alk.aloids. In vivo studies. Females were caged with males overnight, and examined the following morning for the presence of a copulatory plug. The day on which a plug was found was designated as gestation Day 0. Pregnant animals were treated on one of gestation Days 8, 9, or 10. Jervine was administered by gavage, as an aqueous suspension (50 mg/ml). The .amount given to each animal was adjusted to a dosage of 150 mg/kg. Since previous work had demonstrated that the gavage procedure was innocuous (Sim et al., in preparation), vehicle controls were omitted. On gestation Day 17, maternal animals were killed by cervical dislocation, and their uteri were removed by caesarian section. Fetuses were examined with the aid of a dissecting microscope. Limb cell cultures. Limb cell cultures were prepared by the methods of Umansky (1966), Caplan (1970), Goetinck et al. (1974), and Ahrens et al. (1977), as modified by Lewis et al. (1978), Hassell et al. (1978), and Pennypacker et al. (1978). Forelimbs of Day 10 embryos were dissected, pooled, and then dissociated in 0.1% (w/v) trypsin which contained 0.1% EDTA (w/v) in saline G
465
was ( Ca2+, Mg2+-free) for 20 min at 37°C. Trypsinization stopped by the addition of soybean trypsin inhibitor. Cells were suspended by reflux pipetting, and filtered through two layers of 20-pm-mesh Nitex monofilament screen (Tetko, Inc., Elmsford, N. Y.). The cells were then pelleted, and resuspended in 4 ml of CMRL-1066 medium (GIBCO) containing 10% fetal bovine serum (GIBCO) and antibiotics (gentamicin sulfate, 0.25 mg/ml). The concentration of cells was determined using a hemacytometer, and adjusted to 2.5 X 107/ml. “Spot,” also termed “micromass” (Ahrens et al., 1977), cultures were produced by dispensing 20-~1 aliquots on to dry 24-well tissue culture plates (5 X lo5 cells per spot, 1 spot per well). After incubation in a humidified CO2 incubator at 37°C for 1.5 hr, the cells had attached. Then each well was flooded with 0.5 ml of CMRL medium. The day on which cells were plated was designated Day 0. Cultures were exposed to jervine either on Days 1-3, or on Days 4-6. Jervine was dissolved in 95% ethanol to a concentration of 4 mg/ml. This stock was added to culture medium to produce a final concentration of 10 hg jervine/ ml. An equivalent volume of ethanol was added to control cultures. Alcian blue staining. The medium was removed on culture Day 6, and the cultures were stained for 2 hr with alcian blue (0.5% alcian blue in 3% glacial acetic acid, adjusted to pH 1.0 with concentrated HCl). The cultures were then subjected to sequential rinses with 3% acetic acid (pH l.O), 3% acetic acid (pH 2.5), and distilled water. Bound dye was extracted overnight with 6 MguanidineHCl at 4°C. Equal aliquots of the extracted samples were transferred to microtiter plates, and absorbance at 600 nm was determined by a Titertek Multiscan spectrophotometer. Proteoglycan distribution. For experiments involving radiolabeling, limb bud cells from a jervine-sensitive strain (C57BL/6J) were used. Cells were plated as 5 spots per 35-mm culture dish. Control cultures and cultures treated either on Days l-3 or 4-6 were labeled over the 6th night of culture with sodium [35S]sulfate (Amersham) at 20 pCi/ml culture medium. Each experimental condition was performed in triplicate. On the morning of culture Day 7, the media were removed and mixed with solid guanidine-HCl to a final concentration of 4 M. The cell layer was extracted in 4 Mguanidine-HCl containing 0.1 M 6-aminohexanoic acid, 0.01 M EDTA, 5 mM benzamidine-HCl, and 0.5 M sodium acetate (Oegema et ah, 1975). Extraction was allowed to proceed overnight at 4°C. The samples were chromatographed over Sephadex G-25 columns (PD-10, Pharmacia) which had been equilibrated with the above extraction mixture. Fractions containing incorporated radioactivity were pooled, and the total incorporation for each sample was determined by liquid scintillation counting.
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DEVELOPMENTALBIOLOGY
Protein labeling and identijkation of matrix components. Multiple spot cultures on 35mm dishes were labeled for 30 min with 250 &i/ml r5S]methionine (Amersham) contained in an otherwise methionine-free medium. Cultures treated with jervine on Days l-3 were radiolabeled on either Day 3 or Day 6. After treatment on Days 4-6, cells were labeled on Day 6. Following the incubation period, culture dishes were rinsed twice with phosphate-buffered saline (pH 7.2). In preparation for collagenase digestion, labeled cultures were solubilized in 0.5 ml of 4 M guanidine-HCl, 0.02 M Tris (pH 7.0), with 1% CHAPS (3-[3-cholamidopropyldimethylammoniol-1-propane-sulfonate). After a brief sonication, samples were incubated at 37°C for 1 hr, followed by centrifugation to pellet crosslinked collagen. Supernates were dialyzed overnight at 4”C, against 1000 vol of 0.02 M Tris. Samples were divided into two equal aliquots, and each half was mixed with an equal volume of 50 mM Tris-buffered saline (pH 7.4), containing 10 mMNEM (N-ethylmaleimide) and 250 mM CaClz. One aliquot of each sample was subjected to enzymatic digestion; collagenase (Advance Biofactures) was dissolved in the buffered NEM/CaC12 solution at a concentration of 1 mg/ml. After 4 hr incubation at 37”C, samples were precipitated in 10% trichloroacetic acid for 30 min at 0°C. Precipitates were pelleted in a Beckman microfuge, and the pellets were rinsed twice with absolute ethanol chilled to -20°C. The pellets were then solubilized in 0.5 ml of 2% SDS contained in 0.01 M sodium phosphate buffer (pH 7.0). Incorporation of radiolabel was determined by liquid scintillation counting. An equal number of incorporated counts from each sample was reduced, and then electrophoresed in 4 to 10% gradient polyacrylamide slab gels. Gels were fixed in 50% TCA with 0.1% Coomassie blue, destained in 7% acetic acid, and dried on filter paper. Sheets of Kodak X-Omat film were exposed to the dried gels for l-4 days at -70°C. Immunoprecipitations were performed using antibody to the core protein of the cartilage-specific proteoglycan. This antibody precipitates a 370,000 mw precursor protein from chondrosarcoma cells pulse-labeled in culture with [35S]methionine (data not shown). Cultures were labeled as described above, and prepared as described by Ledbetter et al. (1985). Briefly, the cell layer was extracted with 1% deoxycholate, 0.1% SDS, 1% aprotinin (Sigma), and 0.02% NaAzide. Cell lysates were sonicated, centrifuged, and then preabsorbed to protein A-Sepharose and preimmune serum. After centrifugation to remove beads, cell lysates were rotated for 2 hr at 4°C in the presence of protein A-Sepharose coupled to either preimmune or to immune serum. The beads were then isolated by centrifugation, and extracted by 3 min of incubation with 0.01 M sodium phosphate buffer (pH
VOLUME111,1985
7.0) containing 2% SDS/DTT at 100°C. Beads were removed by centrifugation, and the supernatants electrophoresed on 7.5% polyacrylamide gels. Gels were processed as described above, excepting that longer times were required for film exposures. RESULTS
We first examined the effects of in utero exposure to jervine on NIH Swiss-Webster, C57BL/6J, and A/J mice. The 164 viable NIH Swiss-Webster fetuses exposed to jervine in vivo showed no increase over control values in the frequency of malformations. Identical treatment, however, produced a variety of defects in fetuses of the C57BL/6J or A/J strains. At 150 mg/kg maternal body wt, jervine treatment caused abnormalities at frequencies ranging from 40 to 100%) depending upon the day of treatment and the strain of mouse used. In contrast, without treatment, only 5% of 127 C57BL/6J fetuses and 8% of 143 A/J fetuses showed any type of malformation. Limb defects observed included brachydactyly, syndactyly, polydactyly, and oligodactyly (Fig. 1). Because of its occurrence at relatively high frequencies and amenability to objective assessment, oligodactyly was the defect selected as a marker for the temporal specificity of jervine in vivo. Figure 2 shows the frequency of oligodactyly in fore- (solid lines) versus hindlimbs (dashed lines), plotted as a function of day of treatment. For both sensitive strains, forelimb oligodactyly reached a peak frequency following exposure to jervine on gestation Day 9. When exposure was restricted to Day 10, the frequency of forelimb oligodactyly declined significantly, concurrent with a sharp rise in hindlimb oligodactyly. Analogous to the results obtained from the in vivo studies, jervine responsiveness of cultured limb bud cells was dependent upon their strain-of-origin and the time of exposure. Figure 3 shows proteoglycan accumulation of treated cultures as measured by the optical density of bound and extracted alcian blue. In all cases, proteoglycan accumulation was reduced more by jervine treatment on culture Days l-3 (solid bars) than by treatment on Days 4-6 (open bars). Additionally, limb bud cells obtained from C57BL/6J or A/J mice were more sensitive than were cells from NIH Swiss-Webster embryos. For the NIH Swiss-Webster cells, proteoglycan accumulation after treatment on culture Days l-3 was not significantly different from that found after treatment on Days 4-6. This is demonstrated by the overlapping of standard errors for NIH cells treated at the two different times; such overlap was not seen for cells of the other two strains. Proteoglycan synthesis by cultured limb bud cells was also studied using [35S]sulfate incorporation (Table 1). Total [35S]sulfate incorporation was reduced to 12% of
CAMPBELL ET AL.
Limb
Chondrogenesis
467
Inhibition
I
/-
I I A’ I Q
p --- d 8
9
/
hindlimbs -
: 10
9
8
10
DAY OF TREATMENT FIG. 2. The frequency of oligodactyly in fore- (solid lines) and hindlimbs (dashed lines) for two strains of mice (C57BL/6J and A/J). Maternal animals were given jervine (150 mg/kg) on one of gestation Days 8,9, or 10. Data are expressed as a percentage of the total number of viable fetuses after exposure on a given day (numbers in parentheses).
15t)-
FIG. 1. Forelimbs of mouse fetuses on gestation Day 17, subsequent to treatment with jervine. (a) control, (b) oligodactyly, (c) brachydactyly, (d) syndactyly.
control values in cultures exposed to jervine on Days l3. Incorporation by cultures treated only on Days 4-6 was much less affected, reaching 62% of the control level. The distribution of counts between the medium and the cell layer was similar for all three experimental conditions. Incorporation of [35S]methionine indicated the rates of overall protein synthesis. Treated and control cultures were labeled either on Day 3 or on Day 6. When evaluated on culture Day 3, spot cultures which had been treated on Days l-3 demonstrated a 64% depression in [35S]methionine incorporation. By Day 6, protein synthesis had largely recovered, and was similar to the rate
I-
I-
0l-
i NII
I I
C57
FIG. 3. Proteoglycan accumulation by limb bud cell “spot” cultures, as assessed by the optical density of bound and extracted alcian blue. Data are expressed as percentages of the values for concurrent controls. Bars represent the mean values (with standard errors) for a minimum of three separate experiments. Solid bars show results of jervine treatment on culture Days l-3; open bars show the results of treatment on Days 4-6.
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VOLUME 111, 1985
TABLE 1 INCORPORATION OF SODIUM [%]SULFATE BY LIMB CELL CULTURES
CONTROL +
Media Cell layer
Control
Jervine (10 L&ml) Days 1-3
Jervine (10 m/ml) Days 4-6
71.2 + 4.8 (47%) 81.3 k 5.6 (53%)
11.2 f 0.7 (59%) 7.6 k 0.7 (41%)
44.6 k 5.1 (47%) 50.2 2 1.9 (53%)
12%
62%
Total counts as % of control
Note. Limb bud mesenchyme cells from C57BL/6J embryos were plated as five high-density spots per 35-mm culture dish. Control cultures and cultures treated with jervine either on Days 1-3 or 4-6 were labeled over the 6th night of culture with sodium [?8]sulfate (20 &i/ml medium). On Day 7, incorporation was determined by liquid scintillation counting. The data are expressed as cpm/culture dish X10m3t standard error. The numbers in parenthesis show percentages of the total counts per culture.
found for cultures treated only on Days 4-6 (lo-20% below control values). Polyacrylamide gel electrophoresis revealed no apparent qualitative effect of jervine on protein profile, regardless of the treatment and labeling schedule (Figs. 4 and 5). Specifically, neither production of collagenase-sensitive material (Fig. 4) nor of the cartilage-specific proteoglycan core protein (Fig. 5) were prevented by treatment. DISCUSSION
We have studied the effects of jervine on limb development in three mouse strains both in vivo and in vitro. C57BL/6J and A/J mice were sensitive, whereas NIH Swiss-Webster embryos were resistant. The finding that limb mesenchyme cells from NIH Swiss-Webster embryos were relatively resistant to jervine in vitro suggests that teratogenic susceptibility to this compound is a function of embryonic, rather than maternal factors. This hypothesis is supported by preliminary in vivo experiments involving cross-breeding between sensitive and resistant strains. Regardless of the maternal strain, embryos carrying NIH Swiss-Webster genes were unaffected by jervine (unpublished observations). Teratogenie resistance did not, however, extend to all manifestations of Veratrum toxicity. The adult LDsO for jervine in NIH Swiss-Webster mice was 260 mg/kg, just slightly higher than the mean lethal dose of 220 mg/kg found for A/J mice (Sim et al., in preparation). Thus, there may be some distinction between the mechanism of jervine toxicity, per se, and the means by which it exerts its teratogenic effects. It is clear from the results of the in vivo studies that developing skeletal anlagen pass through a transitory phase of jervine sensitivity. Maximal teratogenic insult occurred during the earliest phases of cartilage differentiation, prior to condensation of the prechondrogenic
-
-2‘
TREATED +
-
-
200 92.5 68 43 25.7 FIG. 4. Fluorograms showing proteins of control culture and a culture treated with jervine on Days l-3. Cultures were labeled with [%]methionine on Day 6. Arrow denotes the position of collagenasesensitive material; (+) indicates sample was incubated with enzyme, (-) without enzyme. The migration positions of myosin (H-chain, mw = ZOO,OOO), phosphorylase B (mw = 92,500), bovine serum albumin (mw = 68,000), ovalbuminin (mw = 43,000), and a-chymotrypsinogen (mw = 25,000) are shown.
mesenchyme. In the forelimb, the highest frequency of digital damage was found after treatment on gestation Day 9. Although there is a distinctly delineated forelimb bud at this time, there is as yet no morphological indi-
CONTROL
+
-
TREATED
+
-
25.7 L ‘,,”
FIG. 5. Fluorograms showing immunoprecipitation of the cartilagespecific proteoglycan core protein for control and jervine-treated cultures. (+) Indicates the use of immune serum, (p) preimmune serum from the same animal. The arrow denotes the position of the 370,000 mw protein specified by this antibody. Molecular weight standard proteins are the same as those described for Fig. 4. Equivalent signals for the treated and control autoradiograms were obtained by adjusting exposure times.
CAMPBELL ET AL.
Limb
cation of the future fate of particular regions of limb mesenchyme. On Day 10, the forelimb digital primordia began to be insensitive to insult by jervine while vulnerability of the hindlimlb digits rose steeply. This relationship of timing and response corresponds closely to the extent to which development of the hindlimb lags behind that of the forelimb. In vitro experiments with limb bud mesenchyme were performed in order to focus on the specificity of cellular sensitivity to jervine. Exposure of these cells to jervine on culture Days l-3 significantly suppressed synthesis and subsequent accumulation of cartilage proteoglycans. In contrast, exposure to this agent on culture Days 4-6 had little adverse effect. That the transition from vulnerability to resistance was quantitative, rather than strictly qualitative, is not surprising due to the heterogenous nature of these cultures. Since limb development in vivo proceeds in a proximodistal gradient, limb bud mesenchyme cells isolated on gestation Day 10 are expected to represent a continuum of the differentiation process. Nonetheless, it appears that something happens to the majority of these cells by the third day of culture that renders proteoglycan synthesis immune to inhibition by jervine. Previous reports indicate that during culture Days 3-4 several significant changes take place in these cultures. Morphologically, discrete cartilage nodules become easily distinguishable (Lewis et al., 1978; Biddulph et al, 1984). Biochemically, the onset of chondrogenesis is demonstrated by synthesis of the cartilagespecific proteoglycan (Sawyer and Goetinck, 1981). Biddulph et al. (1984) found that DNA content increased linearly over time, while protein synthesis reached its greatest rate between culture Days 3 and 6. The resulting increase in the ratio of protein to DNA corresponded to the commencement of active synthesis of cartilage matrix components. Based upon the electrophoretic profiles, the teratogenie action of jervine did not appear to be due to selective suppression of specific protein products. Its teratogenic effectiveness, however, would seem restricted to a temporally defined prechondrogenic stem cell population. This was evidenced by the diminishing sensitivity of the limb cell cultures as differentiation progressed. Jervine exposure prior to chondrogenesis led to temporary, as well as to more lasting deficits. The rate of protein synthesis by treated Day 3 cultures, as evaluated by [35S]methionine incorporation, was reduced to less than half of control values. By Day 6, however, this effect of early exposure had been largely compensated for, and incorporation per culture approached control levels. At the same time, ;a depression in accumulation of the cartilage-specific proteoglycan remained evident. The studies on [35S]sulfate incorporation demonstrated that the results obtained with alcian blue staining re-
Chcmdrogenesis
Inhibition
469
fleeted decreased proteoglycan synthesis, rather than changes in its distribution. Assessment on Day 6 showed that the rate of proteoglycan synthesis remained far lower for cultures treated on Days l-3 than for cultures treated on Days 4-6 or for control cultures. It is possible that the transitory interference with overall protein synthesis led to more permanent alterations in the proportion of fully differentiated chondrocytes to other cell types. Such a mechanism could be responsible for the hypoplastic malformations observed after jervine exposure in utero. An interesting comparison can be made between the action and effects of jervine and those of another teratogen, retinoic acid. Retinoic acid also causes limb defects in a proximodistal and cephalocaudal gradient which is dependent upon the time of treatment (Kochhar, 1973). However, no limb defects were found in mouse fetuses after retinoic acid treatment on gestation Days 9 or 10.’ These days were the peak of the critical period for induction of limb abnormalities by jervine. Conversely, retinoic acid exerted maximal interference with limb development on gestation Days 11 and 12, at a time when the teratogenic effectiveness of jervine was declining. Like jervine, retinoic acid also inhibited chondrogenesis is limb bud cell spot cultures (Hassell et ah, 1978; Lewis et al, 1978). In these cultures, however, retinoic acid elicited retention of a specific mesenchymal cell surface protein, beyond the time of its normal replacement by a differentiated cell product (Lewis et al., 1978). This is in sharp contrast to jervine, which appears to perturb a particular cell population in a generalized way. Thus, the similar morphologic abnormalities produced by jervine and retinoic acid would seem to arise from different mechanisms. Detailed study and comparison of the action and effects of teratogenic agents raises the potential for using such compounds in “molecular ablation” experiments. Several teratogens, including jervine, retinoic acid, and thalidomide appear able to recognize specific structural primordia at very early developmental stages. A capacity for selective interference with otherwise unidentifiable cell populations would greatly facilitate study of premorphogenic events of differentiation. REFERENCES AHRENS, P. B., SOLURSH, M., and REITER, R. S. (1977). Stage-related capacity for limb chondrogenesis in cell culture. Dev. Biol. 60, 69-82. BIDDULPH, D. M., SAWYER, L. M., and SMALES, W. P. (1984). Chondrogenesis of chick limb mesenchyme in vitro. Exp. Cell. Res. 153, 2’70274.
’ Timing nomenclature has been adjusted to correspond to the system used in the present study.
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BINNS, W., KEELER, R. F., and BALLS, L. D. (1972). Congenital deformities in lambs, calves, and goats resulting from maternal ingestion of Veratrum californicum: Hare lip, cleft palate, ataxia, and hypoplasia of metacarpal and metatarsal bones. Clin. 2’o.r. 5, 245-261. BROWN, K. S., SIM, F. R., KAREN, A., and KEELER, R. F. (1980). Jervine produced craniofacial and limb anomalies in mice. Terutolo~y 21, 30A. BRYDEN, M. M., PERRY, C., and KEELER, R. F. (1973). Effects of alkaloids of Vera&urn californicum on chick embryos. Teratology 8.19-28. CAPLAN, A. I. (1970). Effects of the nicotinamide-sensitive teratogen 3-acetyl-pyrimidine on chick limb bud cells in culture. Exp. Cell. Res. 62,341-355. GOETINCK, P. F., PENNYPACKER, J. P., and ROYAL, P. D. (1974). Proteochondroitin sulfate synthesis and chondrogenic expression. Eq. Cell Res. 87, 241-248. HASSELL, J. R., PENNYPACKER, J. P., and LEWIS, C. A. (1978). Chondrogenesis and cell proliferation in limb bud cell cultures treated with cytosine arabinoside and vitamin A. Exp. Cell. Rex 112,409417. HASSELL, J. R., and HORIGAN, E. A. (1982). Chondrogenesis: A model developmental system for measuring teratogenic potential of compounds. Terat. Curcin. Mutagen. 2,325-331. KEELER, R. F., and BINNS, W. (1968). Teratogenic compounds of Veratrum califwxicum (Durand) V. Comparison of cyclopean effects of steroidal alkaloids from the plant and structurally related compounds from other sources. Teratology 1,5-10. KEELER, R. F. (1971). Teratogenic compounds of Veratrum calfin-nicum (Durand) XI. Gestational chronology and compound specificity in rabbits. Proc. Sot, Exp, Biol. Med. 136, 1174-1179.
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KEELER, R. F. (1972). Known and suspected teratogenic hazards in range plants. Clin. Toz 5,529-565. KEELER, R. F. (1975). Teratogenic effects of cyclopamine and jervine in rats, mice and hamsters. Proc. Sot. Exp. Biol. Med. 149,302-306. KOCHHAR, D. M. (1973). Limb development in mouse embryos. Analysis of teratogenic effects of retinoic acid. Teratology 7,289-298. LEDBETTER, S. R., TYREE, B., HASSELL, J. R., and HORIGAN, E. A. (1985). Identification of the precursor protein to basement membrane heparan sulfate proteoglycan. J. Biol. Chem., 260, 8106-8113. LEV, R., and SPICER, S. S. (1964). Specific staining of sulphate groups with alcian blue at low pH. J Histochem. Cytochem. 12, 309. LEWIS, C. A., PRATT, R. M., PENNYPACKER, J. P., and HASSELL, J. R. (1978). Inhibition of limb chondrogenesis in vitro by vitamin A: Alterations in cell surface characteristics. Dev. Biol. 64, 31-47. OEGEMA, T. R., HASCALL, V. C., and DZIEWIATKOWSKI, D. D. (1975). Isolation and characterization of proteoglycans from the Swarm chondrosarcoma. J. Biol. Chem. 250,6151-6159. PENNYPACKER, J. P., LEWIS, C. A., and HASSELL, J. R. (1978). Altered proteoglycan metabolism in mouse limb mesenchyme cell cultures treated with vitamin A. Arch. Biochem. Biophys. 186,351-358. SAWYER, L. M., and GOETINCK, P. F. (1981). Chondrogenesis in the mutant nanomelia: Changes in the fine structure and proteoglycan synthesis in high density limb bud cell cultures. J. Exp. Zool. 216, 121-131. THEILER, K. (1972). “The House Mouse.” pp. 44-60. Springer-Verlag, New York. UMANSKY, R. (1966). The effect of cell population density on the developmental fate of reaggregating mouse limb bud mesenchyme. Dev. Biol. 13, 31-56.
BIOLOGY
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inhibition of Limb Chondrogenesis by a Veratrum Alkaloid: Temporal Specificity in Vivo and in Vitro MARLISSA A. CAMPBELL,*,’ KENNETH S. BROWN,* JOHN R. HASSELL,* ELIZABETH A. HORIGAN,* AND RICHARD F. KEELERt *Laboratory
of Developmental Biology and Anomalies, Natim~al Institute of Dental Research, Natimal Institutes Bethesda, Maryland 20205 and tU S. Department of Agm’culture, Agricultural Research Service, Poisonous Plant Research Laboratory, Logan, Utah 84321 Received February
12, 1985; accepted in revised form Ap.l2$
of Health,
1985
It has been demonstrated that jervine, a steroidal alkaloid derived from plants of the genus Veratrum, exerts teratogenic effects in several animal species. Defects were restricted to structures which depend upon normal chondrogenesis for their development. Here we report studies of the temporal specificity of cellular sensitivity using limb bud mesenchyme cells obtained from Day 10 mouse embryos. These cells, when grown as high-density “spot” cultures, undergo chondrogenesis in vitro. Prior to differentiation, exposure of limb cell cultures to jervine suppressed subsequent accumulation of cartilage proteoglycans. Treatment after differentiation had no significant effect. Additionally, there was a genetic component to jervine sensitivity: C57BL/6J mice were sensitive, whereas NIH Swiss-Webster mice were insensitive. This strain-dependent difference was observed both in vivo and in vitro, supporting the validity of limb mesenchyme spot cultures as a model for jervine-induced teratogenicity. Our studies indicate that jervine acts specifically during an
early phase of the differentiation target tissue of this compound.
of mesenchyme into cartilage. It is likely that a specific stem cell population is the 0 1985 Academic
Press, Inc.
INTRODUCTION
Jervine and cyclopamine are two steroidal alkaloids which are derived from plants of the genus Veratrum (Keeler, 1972). Prenatal exposure to these compounds has been shown to cause craniofacial and limb malformations in sheep, goats, cattle, chickens, and two strains of mice (Keeler, 1971; Binns et ab, 1972; Bryden et ah, 1973; Keeler, 1975; Brown et ab, 1980; F. R. P. Sim, M. L. Omnell, R. F. Keeler, L. C. Harne, and K. S. Brown, in preparation). The types of defects observed were similar for all these species, almost exclusively involving structures which depend upon normal chondrogenesis for their development. Furthermore, induction of specific defects was dependent upon the developmental status of the embryo at the time of its exposure. In sheep, goats, and cattle, gestation Days 12 through 30 comprised the critical period for induction of hypoplastic malformations of the craniofacial complex (Binns et al., 1972). Vera&urn exposure on Days 25-36 resulted in limb reduction defects, while the frequency of facial deformities decreased. Similar sequential sensitivity has been described for chicks (Bryden et al., 1973) and mice (Sim et ab, in preparation). Research on jervine teratogenicity in mice (Brown et al, 1980; Sim et ah, in preparation) established that the types of defects produced were related to the time of treatment, as well as to genetic 1To whom correspondence should be addressed. 0012-1606/85 $3.00 Copyright All rights
0 198.5 by Academic Press, Inc. of reproduction in any form reserved.
464
background and the dose of drug. In two strains of mice, C57BL/6J and A/J, limb defects were induced by exposure to jervine on gestation Days 8, 9, or 10, with a peak frequency on Day 10. In all cases, peak effectiveness of Veratrum-teratogens occurred prior to morphological differentiation of the target tissue. Because of its temporal and tissue specificity, we have investigated jervine sensitivity as a potential marker of early prechondrogenic stem cell populations. Mouse limb development, both in vivo and in vitro, was used as the model system. Much is known about the sequence of limb development in normal mouse embryos. Considering the day of fertilization to be gestation Day 0, the forelimb buds begin to appear on Day 8, and the hindlimb buds on about Day 10 (Theiler, 1972). At this time, the mesenchymal tissue of both sets of limb buds appears to be homogenous. Models of individual limb bones begin to condense out of the mesenchyme during the 11th day. Chondrogenesis also commences at this time, proceeding over the next few days in successively more distal forelimb segments, and in the hindlimb. In order to study the relationship between sensitivity to jervine and state of differentiation at the cellular level, we used an in vitro system that encompasses the transition from undifferentiated mesenchyme to mature cartilage. When plated at high densities, prechondrogenie limb bud mesenchyme cells will undergo chondrogenesis in vitro (Umansky, 1966; Caplan, 1970; Goetinck,
CAMPBELL
ET AL.
Limb Chondrogenesis Inhibiticm
1974; Ahrens, 1977). These cells can be obtained from Day 10 mouse embryos (Lewis et ab, 1978; Hassell et al., 1978; Pennypacker et al., :1978). The progression of chondrogenesis can be demonstrated by staining cartilagespecific proteoglycans with alcian blue at low pH (Lev and Spicer, 1964). Using alcian blue, proteoglycan accumulation (and hence chondrogenesis) can be quantified spectrophotometrically (Hassell and Horigan, 1982). The limb cell culture system has the advantage of allowing introduction of teratogenic agents at different times during differentiation. Therefore, we were able to evaluate the effects of jervine on cellular differentiation in vitro, as well as observing its effects on morphological differentiation in vivo. MATERIALS
AND
METHODS
Animals. C57BL/6J and, A/J mice were obtained from Jackson Laboratories (Bar Harbor). NIH all-purpose mice are derived from Swiss-Webster stock. These mouse strains were chosen because of documented differences in sensitivity to Vera&urn-induced teratogenesis; C57BL/6J and A/J mice are sensitive, whereas NIH Swiss-Webster mice are resistant (Keeler, 1975; Brown et ah, 1980; Sim et ab, in preparation). We wished to determine whether corresponding strain-dependent differences could be documented in vitro. Drug. Jervine was isolated from Veratrum viride roots and rhizomes using methods described by Keeler and Binns (1968). Infrared scans, thin-layer chromatography, and HPLC analysis of the purified material demonstrated a purity of over 96% with traces of other, nonteratogenic, Veratrum alk.aloids. In vivo studies. Females were caged with males overnight, and examined the following morning for the presence of a copulatory plug. The day on which a plug was found was designated as gestation Day 0. Pregnant animals were treated on one of gestation Days 8, 9, or 10. Jervine was administered by gavage, as an aqueous suspension (50 mg/ml). The .amount given to each animal was adjusted to a dosage of 150 mg/kg. Since previous work had demonstrated that the gavage procedure was innocuous (Sim et al., in preparation), vehicle controls were omitted. On gestation Day 17, maternal animals were killed by cervical dislocation, and their uteri were removed by caesarian section. Fetuses were examined with the aid of a dissecting microscope. Limb cell cultures. Limb cell cultures were prepared by the methods of Umansky (1966), Caplan (1970), Goetinck et al. (1974), and Ahrens et al. (1977), as modified by Lewis et al. (1978), Hassell et al. (1978), and Pennypacker et al. (1978). Forelimbs of Day 10 embryos were dissected, pooled, and then dissociated in 0.1% (w/v) trypsin which contained 0.1% EDTA (w/v) in saline G
465
was ( Ca2+, Mg2+-free) for 20 min at 37°C. Trypsinization stopped by the addition of soybean trypsin inhibitor. Cells were suspended by reflux pipetting, and filtered through two layers of 20-pm-mesh Nitex monofilament screen (Tetko, Inc., Elmsford, N. Y.). The cells were then pelleted, and resuspended in 4 ml of CMRL-1066 medium (GIBCO) containing 10% fetal bovine serum (GIBCO) and antibiotics (gentamicin sulfate, 0.25 mg/ml). The concentration of cells was determined using a hemacytometer, and adjusted to 2.5 X 107/ml. “Spot,” also termed “micromass” (Ahrens et al., 1977), cultures were produced by dispensing 20-~1 aliquots on to dry 24-well tissue culture plates (5 X lo5 cells per spot, 1 spot per well). After incubation in a humidified CO2 incubator at 37°C for 1.5 hr, the cells had attached. Then each well was flooded with 0.5 ml of CMRL medium. The day on which cells were plated was designated Day 0. Cultures were exposed to jervine either on Days 1-3, or on Days 4-6. Jervine was dissolved in 95% ethanol to a concentration of 4 mg/ml. This stock was added to culture medium to produce a final concentration of 10 hg jervine/ ml. An equivalent volume of ethanol was added to control cultures. Alcian blue staining. The medium was removed on culture Day 6, and the cultures were stained for 2 hr with alcian blue (0.5% alcian blue in 3% glacial acetic acid, adjusted to pH 1.0 with concentrated HCl). The cultures were then subjected to sequential rinses with 3% acetic acid (pH l.O), 3% acetic acid (pH 2.5), and distilled water. Bound dye was extracted overnight with 6 MguanidineHCl at 4°C. Equal aliquots of the extracted samples were transferred to microtiter plates, and absorbance at 600 nm was determined by a Titertek Multiscan spectrophotometer. Proteoglycan distribution. For experiments involving radiolabeling, limb bud cells from a jervine-sensitive strain (C57BL/6J) were used. Cells were plated as 5 spots per 35-mm culture dish. Control cultures and cultures treated either on Days l-3 or 4-6 were labeled over the 6th night of culture with sodium [35S]sulfate (Amersham) at 20 pCi/ml culture medium. Each experimental condition was performed in triplicate. On the morning of culture Day 7, the media were removed and mixed with solid guanidine-HCl to a final concentration of 4 M. The cell layer was extracted in 4 Mguanidine-HCl containing 0.1 M 6-aminohexanoic acid, 0.01 M EDTA, 5 mM benzamidine-HCl, and 0.5 M sodium acetate (Oegema et ah, 1975). Extraction was allowed to proceed overnight at 4°C. The samples were chromatographed over Sephadex G-25 columns (PD-10, Pharmacia) which had been equilibrated with the above extraction mixture. Fractions containing incorporated radioactivity were pooled, and the total incorporation for each sample was determined by liquid scintillation counting.
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Protein labeling and identijkation of matrix components. Multiple spot cultures on 35mm dishes were labeled for 30 min with 250 &i/ml r5S]methionine (Amersham) contained in an otherwise methionine-free medium. Cultures treated with jervine on Days l-3 were radiolabeled on either Day 3 or Day 6. After treatment on Days 4-6, cells were labeled on Day 6. Following the incubation period, culture dishes were rinsed twice with phosphate-buffered saline (pH 7.2). In preparation for collagenase digestion, labeled cultures were solubilized in 0.5 ml of 4 M guanidine-HCl, 0.02 M Tris (pH 7.0), with 1% CHAPS (3-[3-cholamidopropyldimethylammoniol-1-propane-sulfonate). After a brief sonication, samples were incubated at 37°C for 1 hr, followed by centrifugation to pellet crosslinked collagen. Supernates were dialyzed overnight at 4”C, against 1000 vol of 0.02 M Tris. Samples were divided into two equal aliquots, and each half was mixed with an equal volume of 50 mM Tris-buffered saline (pH 7.4), containing 10 mMNEM (N-ethylmaleimide) and 250 mM CaClz. One aliquot of each sample was subjected to enzymatic digestion; collagenase (Advance Biofactures) was dissolved in the buffered NEM/CaC12 solution at a concentration of 1 mg/ml. After 4 hr incubation at 37”C, samples were precipitated in 10% trichloroacetic acid for 30 min at 0°C. Precipitates were pelleted in a Beckman microfuge, and the pellets were rinsed twice with absolute ethanol chilled to -20°C. The pellets were then solubilized in 0.5 ml of 2% SDS contained in 0.01 M sodium phosphate buffer (pH 7.0). Incorporation of radiolabel was determined by liquid scintillation counting. An equal number of incorporated counts from each sample was reduced, and then electrophoresed in 4 to 10% gradient polyacrylamide slab gels. Gels were fixed in 50% TCA with 0.1% Coomassie blue, destained in 7% acetic acid, and dried on filter paper. Sheets of Kodak X-Omat film were exposed to the dried gels for l-4 days at -70°C. Immunoprecipitations were performed using antibody to the core protein of the cartilage-specific proteoglycan. This antibody precipitates a 370,000 mw precursor protein from chondrosarcoma cells pulse-labeled in culture with [35S]methionine (data not shown). Cultures were labeled as described above, and prepared as described by Ledbetter et al. (1985). Briefly, the cell layer was extracted with 1% deoxycholate, 0.1% SDS, 1% aprotinin (Sigma), and 0.02% NaAzide. Cell lysates were sonicated, centrifuged, and then preabsorbed to protein A-Sepharose and preimmune serum. After centrifugation to remove beads, cell lysates were rotated for 2 hr at 4°C in the presence of protein A-Sepharose coupled to either preimmune or to immune serum. The beads were then isolated by centrifugation, and extracted by 3 min of incubation with 0.01 M sodium phosphate buffer (pH
VOLUME111,1985
7.0) containing 2% SDS/DTT at 100°C. Beads were removed by centrifugation, and the supernatants electrophoresed on 7.5% polyacrylamide gels. Gels were processed as described above, excepting that longer times were required for film exposures. RESULTS
We first examined the effects of in utero exposure to jervine on NIH Swiss-Webster, C57BL/6J, and A/J mice. The 164 viable NIH Swiss-Webster fetuses exposed to jervine in vivo showed no increase over control values in the frequency of malformations. Identical treatment, however, produced a variety of defects in fetuses of the C57BL/6J or A/J strains. At 150 mg/kg maternal body wt, jervine treatment caused abnormalities at frequencies ranging from 40 to 100%) depending upon the day of treatment and the strain of mouse used. In contrast, without treatment, only 5% of 127 C57BL/6J fetuses and 8% of 143 A/J fetuses showed any type of malformation. Limb defects observed included brachydactyly, syndactyly, polydactyly, and oligodactyly (Fig. 1). Because of its occurrence at relatively high frequencies and amenability to objective assessment, oligodactyly was the defect selected as a marker for the temporal specificity of jervine in vivo. Figure 2 shows the frequency of oligodactyly in fore- (solid lines) versus hindlimbs (dashed lines), plotted as a function of day of treatment. For both sensitive strains, forelimb oligodactyly reached a peak frequency following exposure to jervine on gestation Day 9. When exposure was restricted to Day 10, the frequency of forelimb oligodactyly declined significantly, concurrent with a sharp rise in hindlimb oligodactyly. Analogous to the results obtained from the in vivo studies, jervine responsiveness of cultured limb bud cells was dependent upon their strain-of-origin and the time of exposure. Figure 3 shows proteoglycan accumulation of treated cultures as measured by the optical density of bound and extracted alcian blue. In all cases, proteoglycan accumulation was reduced more by jervine treatment on culture Days l-3 (solid bars) than by treatment on Days 4-6 (open bars). Additionally, limb bud cells obtained from C57BL/6J or A/J mice were more sensitive than were cells from NIH Swiss-Webster embryos. For the NIH Swiss-Webster cells, proteoglycan accumulation after treatment on culture Days l-3 was not significantly different from that found after treatment on Days 4-6. This is demonstrated by the overlapping of standard errors for NIH cells treated at the two different times; such overlap was not seen for cells of the other two strains. Proteoglycan synthesis by cultured limb bud cells was also studied using [35S]sulfate incorporation (Table 1). Total [35S]sulfate incorporation was reduced to 12% of
CAMPBELL ET AL.
Limb
Chondrogenesis
467
Inhibition
I
/-
I I A’ I Q
p --- d 8
9
/
hindlimbs -
: 10
9
8
10
DAY OF TREATMENT FIG. 2. The frequency of oligodactyly in fore- (solid lines) and hindlimbs (dashed lines) for two strains of mice (C57BL/6J and A/J). Maternal animals were given jervine (150 mg/kg) on one of gestation Days 8,9, or 10. Data are expressed as a percentage of the total number of viable fetuses after exposure on a given day (numbers in parentheses).
15t)-
FIG. 1. Forelimbs of mouse fetuses on gestation Day 17, subsequent to treatment with jervine. (a) control, (b) oligodactyly, (c) brachydactyly, (d) syndactyly.
control values in cultures exposed to jervine on Days l3. Incorporation by cultures treated only on Days 4-6 was much less affected, reaching 62% of the control level. The distribution of counts between the medium and the cell layer was similar for all three experimental conditions. Incorporation of [35S]methionine indicated the rates of overall protein synthesis. Treated and control cultures were labeled either on Day 3 or on Day 6. When evaluated on culture Day 3, spot cultures which had been treated on Days l-3 demonstrated a 64% depression in [35S]methionine incorporation. By Day 6, protein synthesis had largely recovered, and was similar to the rate
I-
I-
0l-
i NII
I I
C57
FIG. 3. Proteoglycan accumulation by limb bud cell “spot” cultures, as assessed by the optical density of bound and extracted alcian blue. Data are expressed as percentages of the values for concurrent controls. Bars represent the mean values (with standard errors) for a minimum of three separate experiments. Solid bars show results of jervine treatment on culture Days l-3; open bars show the results of treatment on Days 4-6.
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VOLUME 111, 1985
TABLE 1 INCORPORATION OF SODIUM [%]SULFATE BY LIMB CELL CULTURES
CONTROL +
Media Cell layer
Control
Jervine (10 L&ml) Days 1-3
Jervine (10 m/ml) Days 4-6
71.2 + 4.8 (47%) 81.3 k 5.6 (53%)
11.2 f 0.7 (59%) 7.6 k 0.7 (41%)
44.6 k 5.1 (47%) 50.2 2 1.9 (53%)
12%
62%
Total counts as % of control
Note. Limb bud mesenchyme cells from C57BL/6J embryos were plated as five high-density spots per 35-mm culture dish. Control cultures and cultures treated with jervine either on Days 1-3 or 4-6 were labeled over the 6th night of culture with sodium [?8]sulfate (20 &i/ml medium). On Day 7, incorporation was determined by liquid scintillation counting. The data are expressed as cpm/culture dish X10m3t standard error. The numbers in parenthesis show percentages of the total counts per culture.
found for cultures treated only on Days 4-6 (lo-20% below control values). Polyacrylamide gel electrophoresis revealed no apparent qualitative effect of jervine on protein profile, regardless of the treatment and labeling schedule (Figs. 4 and 5). Specifically, neither production of collagenase-sensitive material (Fig. 4) nor of the cartilage-specific proteoglycan core protein (Fig. 5) were prevented by treatment. DISCUSSION
We have studied the effects of jervine on limb development in three mouse strains both in vivo and in vitro. C57BL/6J and A/J mice were sensitive, whereas NIH Swiss-Webster embryos were resistant. The finding that limb mesenchyme cells from NIH Swiss-Webster embryos were relatively resistant to jervine in vitro suggests that teratogenic susceptibility to this compound is a function of embryonic, rather than maternal factors. This hypothesis is supported by preliminary in vivo experiments involving cross-breeding between sensitive and resistant strains. Regardless of the maternal strain, embryos carrying NIH Swiss-Webster genes were unaffected by jervine (unpublished observations). Teratogenie resistance did not, however, extend to all manifestations of Veratrum toxicity. The adult LDsO for jervine in NIH Swiss-Webster mice was 260 mg/kg, just slightly higher than the mean lethal dose of 220 mg/kg found for A/J mice (Sim et al., in preparation). Thus, there may be some distinction between the mechanism of jervine toxicity, per se, and the means by which it exerts its teratogenic effects. It is clear from the results of the in vivo studies that developing skeletal anlagen pass through a transitory phase of jervine sensitivity. Maximal teratogenic insult occurred during the earliest phases of cartilage differentiation, prior to condensation of the prechondrogenic
-
-2‘
TREATED +
-
-
200 92.5 68 43 25.7 FIG. 4. Fluorograms showing proteins of control culture and a culture treated with jervine on Days l-3. Cultures were labeled with [%]methionine on Day 6. Arrow denotes the position of collagenasesensitive material; (+) indicates sample was incubated with enzyme, (-) without enzyme. The migration positions of myosin (H-chain, mw = ZOO,OOO), phosphorylase B (mw = 92,500), bovine serum albumin (mw = 68,000), ovalbuminin (mw = 43,000), and a-chymotrypsinogen (mw = 25,000) are shown.
mesenchyme. In the forelimb, the highest frequency of digital damage was found after treatment on gestation Day 9. Although there is a distinctly delineated forelimb bud at this time, there is as yet no morphological indi-
CONTROL
+
-
TREATED
+
-
25.7 L ‘,,”
FIG. 5. Fluorograms showing immunoprecipitation of the cartilagespecific proteoglycan core protein for control and jervine-treated cultures. (+) Indicates the use of immune serum, (p) preimmune serum from the same animal. The arrow denotes the position of the 370,000 mw protein specified by this antibody. Molecular weight standard proteins are the same as those described for Fig. 4. Equivalent signals for the treated and control autoradiograms were obtained by adjusting exposure times.
CAMPBELL ET AL.
Limb
cation of the future fate of particular regions of limb mesenchyme. On Day 10, the forelimb digital primordia began to be insensitive to insult by jervine while vulnerability of the hindlimlb digits rose steeply. This relationship of timing and response corresponds closely to the extent to which development of the hindlimb lags behind that of the forelimb. In vitro experiments with limb bud mesenchyme were performed in order to focus on the specificity of cellular sensitivity to jervine. Exposure of these cells to jervine on culture Days l-3 significantly suppressed synthesis and subsequent accumulation of cartilage proteoglycans. In contrast, exposure to this agent on culture Days 4-6 had little adverse effect. That the transition from vulnerability to resistance was quantitative, rather than strictly qualitative, is not surprising due to the heterogenous nature of these cultures. Since limb development in vivo proceeds in a proximodistal gradient, limb bud mesenchyme cells isolated on gestation Day 10 are expected to represent a continuum of the differentiation process. Nonetheless, it appears that something happens to the majority of these cells by the third day of culture that renders proteoglycan synthesis immune to inhibition by jervine. Previous reports indicate that during culture Days 3-4 several significant changes take place in these cultures. Morphologically, discrete cartilage nodules become easily distinguishable (Lewis et al., 1978; Biddulph et al, 1984). Biochemically, the onset of chondrogenesis is demonstrated by synthesis of the cartilagespecific proteoglycan (Sawyer and Goetinck, 1981). Biddulph et al. (1984) found that DNA content increased linearly over time, while protein synthesis reached its greatest rate between culture Days 3 and 6. The resulting increase in the ratio of protein to DNA corresponded to the commencement of active synthesis of cartilage matrix components. Based upon the electrophoretic profiles, the teratogenie action of jervine did not appear to be due to selective suppression of specific protein products. Its teratogenic effectiveness, however, would seem restricted to a temporally defined prechondrogenic stem cell population. This was evidenced by the diminishing sensitivity of the limb cell cultures as differentiation progressed. Jervine exposure prior to chondrogenesis led to temporary, as well as to more lasting deficits. The rate of protein synthesis by treated Day 3 cultures, as evaluated by [35S]methionine incorporation, was reduced to less than half of control values. By Day 6, however, this effect of early exposure had been largely compensated for, and incorporation per culture approached control levels. At the same time, ;a depression in accumulation of the cartilage-specific proteoglycan remained evident. The studies on [35S]sulfate incorporation demonstrated that the results obtained with alcian blue staining re-
Chcmdrogenesis
Inhibition
469
fleeted decreased proteoglycan synthesis, rather than changes in its distribution. Assessment on Day 6 showed that the rate of proteoglycan synthesis remained far lower for cultures treated on Days l-3 than for cultures treated on Days 4-6 or for control cultures. It is possible that the transitory interference with overall protein synthesis led to more permanent alterations in the proportion of fully differentiated chondrocytes to other cell types. Such a mechanism could be responsible for the hypoplastic malformations observed after jervine exposure in utero. An interesting comparison can be made between the action and effects of jervine and those of another teratogen, retinoic acid. Retinoic acid also causes limb defects in a proximodistal and cephalocaudal gradient which is dependent upon the time of treatment (Kochhar, 1973). However, no limb defects were found in mouse fetuses after retinoic acid treatment on gestation Days 9 or 10.’ These days were the peak of the critical period for induction of limb abnormalities by jervine. Conversely, retinoic acid exerted maximal interference with limb development on gestation Days 11 and 12, at a time when the teratogenic effectiveness of jervine was declining. Like jervine, retinoic acid also inhibited chondrogenesis is limb bud cell spot cultures (Hassell et ah, 1978; Lewis et al, 1978). In these cultures, however, retinoic acid elicited retention of a specific mesenchymal cell surface protein, beyond the time of its normal replacement by a differentiated cell product (Lewis et al., 1978). This is in sharp contrast to jervine, which appears to perturb a particular cell population in a generalized way. Thus, the similar morphologic abnormalities produced by jervine and retinoic acid would seem to arise from different mechanisms. Detailed study and comparison of the action and effects of teratogenic agents raises the potential for using such compounds in “molecular ablation” experiments. Several teratogens, including jervine, retinoic acid, and thalidomide appear able to recognize specific structural primordia at very early developmental stages. A capacity for selective interference with otherwise unidentifiable cell populations would greatly facilitate study of premorphogenic events of differentiation. REFERENCES AHRENS, P. B., SOLURSH, M., and REITER, R. S. (1977). Stage-related capacity for limb chondrogenesis in cell culture. Dev. Biol. 60, 69-82. BIDDULPH, D. M., SAWYER, L. M., and SMALES, W. P. (1984). Chondrogenesis of chick limb mesenchyme in vitro. Exp. Cell. Res. 153, 2’70274.
’ Timing nomenclature has been adjusted to correspond to the system used in the present study.
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BINNS, W., KEELER, R. F., and BALLS, L. D. (1972). Congenital deformities in lambs, calves, and goats resulting from maternal ingestion of Veratrum californicum: Hare lip, cleft palate, ataxia, and hypoplasia of metacarpal and metatarsal bones. Clin. 2’o.r. 5, 245-261. BROWN, K. S., SIM, F. R., KAREN, A., and KEELER, R. F. (1980). Jervine produced craniofacial and limb anomalies in mice. Terutolo~y 21, 30A. BRYDEN, M. M., PERRY, C., and KEELER, R. F. (1973). Effects of alkaloids of Vera&urn californicum on chick embryos. Teratology 8.19-28. CAPLAN, A. I. (1970). Effects of the nicotinamide-sensitive teratogen 3-acetyl-pyrimidine on chick limb bud cells in culture. Exp. Cell. Res. 62,341-355. GOETINCK, P. F., PENNYPACKER, J. P., and ROYAL, P. D. (1974). Proteochondroitin sulfate synthesis and chondrogenic expression. Eq. Cell Res. 87, 241-248. HASSELL, J. R., PENNYPACKER, J. P., and LEWIS, C. A. (1978). Chondrogenesis and cell proliferation in limb bud cell cultures treated with cytosine arabinoside and vitamin A. Exp. Cell. Rex 112,409417. HASSELL, J. R., and HORIGAN, E. A. (1982). Chondrogenesis: A model developmental system for measuring teratogenic potential of compounds. Terat. Curcin. Mutagen. 2,325-331. KEELER, R. F., and BINNS, W. (1968). Teratogenic compounds of Veratrum califwxicum (Durand) V. Comparison of cyclopean effects of steroidal alkaloids from the plant and structurally related compounds from other sources. Teratology 1,5-10. KEELER, R. F. (1971). Teratogenic compounds of Veratrum calfin-nicum (Durand) XI. Gestational chronology and compound specificity in rabbits. Proc. Sot, Exp, Biol. Med. 136, 1174-1179.
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KEELER, R. F. (1972). Known and suspected teratogenic hazards in range plants. Clin. Toz 5,529-565. KEELER, R. F. (1975). Teratogenic effects of cyclopamine and jervine in rats, mice and hamsters. Proc. Sot. Exp. Biol. Med. 149,302-306. KOCHHAR, D. M. (1973). Limb development in mouse embryos. Analysis of teratogenic effects of retinoic acid. Teratology 7,289-298. LEDBETTER, S. R., TYREE, B., HASSELL, J. R., and HORIGAN, E. A. (1985). Identification of the precursor protein to basement membrane heparan sulfate proteoglycan. J. Biol. Chem., 260, 8106-8113. LEV, R., and SPICER, S. S. (1964). Specific staining of sulphate groups with alcian blue at low pH. J Histochem. Cytochem. 12, 309. LEWIS, C. A., PRATT, R. M., PENNYPACKER, J. P., and HASSELL, J. R. (1978). Inhibition of limb chondrogenesis in vitro by vitamin A: Alterations in cell surface characteristics. Dev. Biol. 64, 31-47. OEGEMA, T. R., HASCALL, V. C., and DZIEWIATKOWSKI, D. D. (1975). Isolation and characterization of proteoglycans from the Swarm chondrosarcoma. J. Biol. Chem. 250,6151-6159. PENNYPACKER, J. P., LEWIS, C. A., and HASSELL, J. R. (1978). Altered proteoglycan metabolism in mouse limb mesenchyme cell cultures treated with vitamin A. Arch. Biochem. Biophys. 186,351-358. SAWYER, L. M., and GOETINCK, P. F. (1981). Chondrogenesis in the mutant nanomelia: Changes in the fine structure and proteoglycan synthesis in high density limb bud cell cultures. J. Exp. Zool. 216, 121-131. THEILER, K. (1972). “The House Mouse.” pp. 44-60. Springer-Verlag, New York. UMANSKY, R. (1966). The effect of cell population density on the developmental fate of reaggregating mouse limb bud mesenchyme. Dev. Biol. 13, 31-56.