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TERATOLOGY 56:233–240 (1997)
Effect of Retinoic Acid on Otic Capsule
Chondrogenesis in High-Density Culture
Suggests Disruption of
Epithelial-Mesenchymal Interactions
DOROTHY A. FRENZ1,2* AND WEI LIU1
of Otolaryngology, Albert Einstein College of Medicine, Bronx, New York 10461
2Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461
1Department
ABSTRACT
Previous studies have shown that
in utero exposure of the mouse embryo to nonphysiological levels of all-trans retinoic acid (RA) produces malformations of the epithelial-derived auditory and vestibular
receptors of the inner ear and its surrounding cartilaginous capsule. In this study, we demonstrate the effects
of all-trans RA in high-density cultures of the periotic
mesenchyme fated to form the otic capsule. Our results
demonstrate an inhibition of chondrogenesis in cultured
periotic mesenchyme 1 otic epithelium of embryonic
age E10.5 days (E10.5) in response to all-trans RA
exposure. However, at later stages of development
(i.e., E12, E14), when epithelial-mesenchymal interactions are no longer required for initiation of chondrogenesis, exposure to this teratogen has no effect on the
chondrogenic process. Two analogues of all-trans RA,
i.e., cis-RA and trans-retinol, were investigated for their
biological activity in chondrogenic cultures of inner ear
mesenchyme 1 epithelium. Moreover, we tested the
inductive capability and responsiveness of in utero
RA-exposed inner ear tissues when cultured with inner
ear tissues that were not exposed to this teratogen.
Our results support the hypothesis that all-trans RA
disrupts otic capsule formation by interfering with the
tissue interactions required for its normal differentiation
and development. Teratology 56:233–240, 1997.
r 1997 Wiley-Liss, Inc.
Retinoic acid (RA) exerts a variety of biological effects
on early development. Besides acting as a regulator of
cell differentiation and proliferation during normal
development (Hofmann and Eichele, ’94), RA is teratogenic during embryogenesis, producing multiple malformations of the inner ear, limb, central nervous system,
heart, and craniofacial primordia (Frenz et al., ’96;
Durston et al., ’89; Morriss, ’72). In high-density cultures of craniofacial and limb bud mesenchyme, nonphysiological levels of RA inhibit chondrogenesis (Wedden et al., ’87; Lewis et al., ’78), suggesting that similar
effects on cartilage differentiation in vivo may be responsible for abnormalities in the developing face and limb
(Morriss, ’72; Morriss and Thorogood, ’78; Sulik, ’86;
Satre and Kochar, ’89).
r 1997 WILEY-LISS, INC.
Since interactions with epithelia are required for the
chondrogenic differentiation of craniofacial and limb
bud mesenchyme, the deleterious effects of retinoid
treatment on face and limb development may be directed at epithelial-mesenchymal interactions (Wedden
et al., ’87). Chondrification of periotic mesenchyme to
form the otic capsule is under direct epithelial control
(Frenz and Van De Water, ’91). When interactions
between otocyst epithelium and periotic mesenchyme
are modified in vitro by enzymatic treatment or reduction in mesenchyme volume (Saver and Van De Water,
’84; Van De Water and Galinovic-Schwartz, ’86), dysmorphogenesis of the auditory and vestibular receptors of
the mouse inner ear and the surrounding cartilaginous
capsule occurs (Ruben and Van De Water, ’83). The
malformations resulting from in utero exposure of the
embryonic mouse inner ear to teratogenic levels of
all-trans RA (Frenz et al., ’96) parallel the anomalies
produced by in vitro disruption of epithelial-mesenchymal interactions, raising the question as to whether
exposure to nonphysiological levels of all-trans RA
interferes with the epithelial-mesenchymal interactions required for normal development of the inner ear
(Frenz et al., ’96).
Morphogenesis of the mammalian inner ear results
from a sequence of inductive interactions not only
between otocyst epithelium and periotic mesenchyme,
but also between otic placodal ectoderm and rhombencephalon (Noden and Van De Water, ’86). While it is
speculated that the teratogenic action of all-trans RA
on otic morphogenesis (Frenz et al., ’96) may result
from perturbation of otic epithelial-periotic mesenchymal interactions, disruption of rhombencephalon-otic
anlagen induction may also produce similar dysmorphogenetic effects. Moreover, since interactions between
rhombencephalon-otic ectoderm and periotic mesen-
Contract grant sponsor: NIDCD; Contract grant number: D40D02823.
*Correspondence to: Dr. Dorothy A. Frenz, Albert Einstein College of
Medicine, Kennedy Center, 1410 Pelham Parkway South, Bronx, NY
10461.
Received 14 May 1997; Accepted 22 July 1997
234
D.A. FRENZ AND W. LIU
chyme-otic epithelium represent overlapping inductive
events (Yntema, ’50; Ruben and Van De Water, ’83), the
effects of all-trans RA on inner ear development need
not be mutually exclusive.
To test whether exposure to nonphysiological levels of
all-trans RA can disrupt epithelial-mesenchymal interactions, we have investigated the effects of this teratogen on otic capsule chondrogenesis utilizing highdensity cultures of mouse periotic mesenchyme 1 otic
epithelium to model epithelial-mesenchymal interactions in the developing mouse inner ear (Frenz and Van
De Water, ’91; Frenz et al., ’92, ’94). We show that
exposure of embryonic age E10.5 day (E10.5) periotic
mesenchyme to excess all-trans RA is inhibitory to
chondrogenesis, while at later stages of development,
when interactions of otic epithelium with periotic mesenchyme are no longer required for initiation of chondrogenesis (i.e., E12, E14), exogenous RA (all-trans) has no
effect on this process. We demonstrate the effects of
analogues of all-trans RA on capsule chondrogenesis
and provide evidence that the inhibition incurred by
all-trans RA in E10.5 periotic mesenchyme is specific
for chondrogenic differentiation. Moreover, we demonstrate the inductive capability and responsiveness of in
utero all-trans RA-exposed inner ear tissues when
interacted with normal mouse inner ear tissues in
culture. Our findings support the hypothesis that alltrans RA interferes with the epithelial-mesenchymal
interactions required for normal chondrogenic control
in vitro. We suggest that similar effects of all-trans RA
exposure at a critical period of inner ear development in
utero may account for dysmorphogenesis of the capsule
of the inner ear.
MATERIALS AND METHODS
Experimental animals
C57/BL6 female mice were crossmated by exposure to
CBA males (NCI). Gestational age of embryos was
estimated by the vaginal plug method, with the day of
plug occurrence designated as day 1 (E1). After death of
the gravid females by cervical dislocation, embryos
were excised and immediately placed into Dulbecco’s
phosphate-buffered saline. Embryonic age was determined by a combination of somite count and external
features (Theiler, ’72).
High-density culture
Otocysts were excised with their associated periotic
mesenchyme from embryos of gestation age E10.5, E12,
or E14, and dissociated cells were cultured according to
standard procedures (Frenz and Van De Water, ’91).
Briefly, mesenchymal and epithelial tissues were dissociated with 0.05% trypsin-EDTA (Gibco, Grand Island,
NY), and mesenchymal cells were resuspended in Ham’s
F-12 culture medium (Gibco) supplemented with 10%
fetal bovine serum (FBS) at a density of 2.5 3 107
cells/ml. Equivalent amounts of otic epithelial tissue
per culture were mixed into the cell suspension, and 10
µl droplets of the cell suspension were plated in the
centers of wells of a 4-well tissue culture plate (Nunc,
Naperville, IL). After a 1 hr incubation period at 37°C, 1
ml Ham’s F-12 medium 1 10% FBS with or without
added all trans-RA (1 3 1029 M, 1 3 10210 M, 1 3 10211
M, 1 3 10212 M), trans-retinol (1 3 1029 M), or cis-RA
(1 3 1027 M, 1 3 1028 M, 1 3 1029 M) was added to
each well (days 1–7). In some cultures (n 5 3 per
treatment group), all-trans RA (1 3 1029 M) was added
to the cultures on days 2–7, days 3–7, or days 4–7. All
cultures were maintained in a humidified 5% CO2
atmosphere at 37°C for a 7 day period. Nutrient solution was exchanged every 48 hr.
Quantitation of mesenchymal cell condensations
Cultures were monitored daily by phase contrast and
Hoffman modulation contrast microscopy for identification of condensed mesenchymal cells. On culture day 3,
cell condensations were counted, as described (Frenz et
al., ’89). The aggregation of mesenchyme into condensations was utilized as an early index of the chondrogenic
process (Hall and Miyake, ’92).
Quantitative Alcian blue staining of cultures
Cultures were fixed after 7 days in vitro with a
solution of 10% formalin containing 0.5% cetylpyridinium chloride, and then stained with Alcian blue 8GX
at pH 1.0, as described (Frenz et al., ’92, ’94). After the
cell spots were washed with a 3% solution of acetic acid
(adjusted to pH 1.0 with HCl), bound stain, an index of
accumulated sulfated glycosaminoglycans (S-GAG) (Lev
and Spicer, ’64) in the matrix of chondrifying cells, was
extracted with an 8 M guanidine hydrochloride solution
and measured by spectrophotometric quantitation (Hassell and Horigan, ’82) using a Biotek microplate reader
equipped with a 600 nm wavelength filter.
Detection of type II collagen in culture
After a period of 7 days in vitro, collagen was
extracted from cultures by constant stirring in 0.5 ml of
0.5 M acetic acid at 4°C overnight. Detection of type II
collagen in the extract was determined by indirect
antibody enzyme-linked immunosorbent assay (ELISA)
using an ELISAmate kit (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD), as described (Frenz et al.,
’94). In positive control wells, known reference samples
of type II collagen (bovine; Southern Biotechnology
Associates, Birmingham, AL) in 0.5 M acetic acid were
utilized. In negative control wells, sample or primary
antibody was omitted. Plates were read on a Biotek
microplate reader equipped with a 405 nm wavelength
filter.
In utero RA treatment
A solution of all-trans RA (Sigma, St. Louis, MO) was
made as 5 mg crystalline RA in 0.8 ml absolute alcohol
in 9.2 ml sesame oil and stored in the dark at 4°C.
Pregnant female mice of mean weight 25 g received a
double dose of 25 mg/kg RA on E9, as previously
RA INHIBITS OTIC CAPSULE CHONDROGENESIS
TABLE 1. Effects of RA and RA analogues on otic
capsule chondrogenesis in cultured E10.5 periotic
mesenchyme 1 otic epithelium*
Treatment
Control
All-trans RA
Cis-RA
Trans-retinol
Condensation
number
Alcian
blue stain
26 6 7
0
23 6 1
11 6 2
0.554 6 0.03
0.042 6 0.03
0.548 6 0.06
0.178 6 0.07
*Cultures of E10.5 periotic mesenchyme 1 otic epithelium
were grown in the presence or absence of all-trans RA
(1 3 1029 M), cis-RA (1 3 1029 M), or trans-retinol (1 3 1029
M) for the duration of the culture period. Cell condensations
were counted on culture day 3; optical density of bound Alcian
blue stain was measured spectrophotometrically on culture
day 7. All values represent the mean for 5–7 cultures per
experimental group. Comparable amounts of otic epithelium
were present in each culture.
described (Frenz et al., ’96). Mice were sacrificed on
E10.5 of pregnancy and their embryos examined on a
Wild 3MZ stereomicroscope. Subsequent to gross stereomicroscopic examination of embryos, the otocysts were
microdissected for a more detailed assessment of anomalies (Frenz et al., ’96). Otic epithelium and/or periotic
mesenchyme from otocysts displaying the anomalies
characteristic of RA teratogenicity, as described (Frenz
et al., ’96), were cultured as 1) in utero RA-exposed
periotic mesenchyme 1 in utero RA-exposed otic epithelium; 2) control (i.e., unexposed to RA in utero) periotic
mesenchyme 1 in utero RA-exposed otic epithelium; or
3) in utero RA-exposed periotic mesenchyme 1 control
otic epithelium.
RESULTS
Effects of all-trans RA on chondrogenesis in
cultured periotic mesenchyme 1 otic epithelium
High-density cultures of E10.5 mouse periotic mesenchyme 1 otic epithelium were grown in the presence
(days 1–7) or absence of 1 3 1029 M all-trans RA. On
culture day 3, sites of cellular condensation were counted
(Table 1). In control cultures (i.e., cultures grown in the
absence of exogenous RA), numerous sites of cellular
condensation formed (Table 1), contrasted by cultures
maintained in the presence of all-trans RA (1 3 1029 M),
in which no cellular condensations developed even after
a period of 7 days in vitro (Fig. 1A). Correspondingly,
while extensive chondrogenesis, as measured by binding of Alcian blue stain, pH 1.0 (Table 1), occurred in
control cultures of E10.5 periotic mesenchyme 1 otic
epithelium (Fig. 1B), chondrogenic differentiation did
not occur when cultures were treated with 1 3 1029 M
all-trans RA (days 1–7) (Table 1). Treatment of cultures
with 1 3 1029 M all-trans RA on culture days 2–7 also
resulted in a total inhibition of chondrogenesis (i.e.,
condensation number 5 0), while exposure to all-trans
RA on culture days 3–7 or days 4–7 resulted in only a
partial inhibition of the chondrogenic process (Fig. 2).
In cultures of E12 or E14 periotic mesenchyme 1 otic
235
epithelium, treatment with exogenous all-trans RA
(days 1–7; 1 3 1029 M) had no effect on capsule chondrogenesis (Table 2).
The effects of all-trans RA on chondrogenesis in
cultured E10.5 periotic mesenchyme 1 otic epithelium
were dose dependent. While in the presence of 1 3 1029
M all-trans RA, the chondrogenic response of cultured
E10.5 periotic mesenchyme was totally inhibited, at
1 3 10210 M or 1 3 10211 M, the chondrogenic differentiation of mesenchyme was suppressed by 45% and
34%, respectively (Fig. 3). Following treatment of E10.5
periotic mesenchyme 1 otic epithelium with all-trans
RA at 1 3 10212 M, the extent of chondrogenesis was
comparable to control cultures (Fig. 3).
Effects of all-trans RA on collagen type II
accumulation in cultured periotic mesenchyme
To determine if the inhibitory effects of all-trans RA
on E10.5 periotic mesenchyme were specific for chondrogenic differentiation, the accumulation of collagen type
II, a cartilage-specific macromolecule was examined.
Acetic acid extracts from cultures of E10.5 periotic
mesenchyme 1 otic epithelium, grown in the presence
or absence of all-trans RA (1 3 1029, days 1–7), were
assayed by ELISA as previously described (Frenz et al.,
’94). In the presence of all-trans RA, levels of collagen
type II were only 29% those of untreated cultures of
E10.5 periotic mesenchyme 1 otic epithelium (Table 3).
Treatment of cultured periotic mesenchyme 1
otic epithelium by analogues of all-trans RA
Since analogues of RA might have biologic activity in
cultured E10.5 periotic mesenchyme 1 otic epithelium
which could affect the chondrogenic differentiation of
the mesenchymal cells, we tested the effects of cis-RA
and all-trans retinol in high-density culture. In contrast to the inhibition of chondrogenesis by all-trans RA
at 1 3 1029 M (Table 1), cis-RA (1 3 1029 M) had no
effect on the chondrogenic differentiation of cultured
E10.5 periotic mesenchyme, as indicated by values for
condensation number and bound Alcian blue stain that
were comparable to control values (Table 1). However,
at 1 3 1028 M and 1 3 1027 M, cis-RA suppressed the
chondrogenic process, as evidenced by a decrease, but
not absence, in cellular condensation number (i.e.,
7 6 2 and 4 6 1, respectively) in comparison to controls
(26 6 7). Addition of trans-retinol (1 3 1029 M) to cultured E10.5 periotic mesenchyme 1 otic epithelium
resulted in a 58% and 68% suppression of condensation
number and S-GAG accumulation, respectively, in comparison to control cultures (Table 1). Neither cis-RA
(1 3 1029 M) or all-trans retinol (1 3 1029 M) had any
effect on the differentiation of mesenchyme in cultured
E12 and E14 periotic mesenchyme 1 otic epithelium
(Table 2). Even when tested at 1 3 1028 M, cis-RA did
not suppress the chondrogenic differentiation of cultured E12 periotic mesenchyme, as evidenced by values
for S-GAG accumulation (0.804 6 0.04, n 5 3) that
were comparable to control values (0.802 6 0.07, n 5 3).
236
D.A. FRENZ AND W. LIU
Fig. 1. Cultures of E10.5 periotic mesenchyme 1 otic epithelium (E) were grown in the presence (A) or
the absence (B) of all-trans RA (1 3 1029 M). After 7 days in vitro, a comparison of A to B reveals the
absence of chondrogenic differentiation in the all-trans RA-treated culture. Scale bar 5 35 µm.
TABLE 2. Treatment of cultured E12 and E14 periotic
mesenchyme 1 otic epithelium with all-trans RA
and RA analogues*
Embryonic age
E12
E14
Treatment
Alcian blue stain
Control
All-trans RA
0.441 6 0.15
0.440 6 0.14
Control
Cis-RA
Trans-retinol
0.572 6 0.06
0.530 6 0.01
0.574 6 0.01
Control
All-trans RA
0.492 6 0.05
0.476 6 0.03
Control
Cis-RA
Trans-retinol
0.652 6 0.08
0.627 6 0.06
0.638 6 0.07
*Cultures of E12 and E14 periotic mesenchyme 1 otic epithelium were exposed to exogenous all-trans RA (1 3 1029 M),
cis-RA (1 3 1029 M), or trans-retinol (1 3 1029 M) for the
duration of the culture period. On culture day 7, cultures were
fixed and bound Alcian blue stain was extracted and measured by spectrophotometric quantitation. Values represent
the mean optical density for 3–6 cultures per experimental
group. Comparable amounts of otic epithelium were present
in each group of cultures.
Fig. 2. A comparison of chondrogenesis, as measured by binding of
Alcian blue stain, in control cultures of E10.5 periotic mesenchyme 1
otic epithelium to the inhibitory effects of all-trans RA exposure
(1 3 1029 M) on days 4–7 and days 1–7. In contrast to the total
inhibition of chondrogenesis (i.e., optical density value , 0.05) by
all-trans RA exposure throughout the 7 day culture period (days 1–7),
exposure on days 4–7 resulted in only a partial inhibition of the
chondrogenic process.
Effects of in utero RA exposure on
chondrogenesis in vitro
We sought to investigate whether chondrogenesis is
affected in E10.5 periotic mesenchyme 1 otic epithelium that has been exposed to nonphysiological levels of
RA in utero. Cultures of E10.5 periotic mesenchyme 1
otic epithelium were prepared from the inner ears of
embryos exposed to a double dose of 25 kg/mg of RA.
Although mesenchymal condensations developed in
these cultures, their numbers were decreased by 73% in
comparison to cultures of control periotic mesenchyme 1
otic epithelium (Table 4A).
Suppression of in vitro chondrogenesis by in utero RA
exposure raised the question as to which tissue(s), i.e.,
the periotic mesenchyme and/or otic epithelium, is
affected by exposure to this teratogen. To address this,
we tested the ability of in utero RA-exposed otic epithelium to initiate a full chondrogenic response in un-
RA INHIBITS OTIC CAPSULE CHONDROGENESIS
237
TABLE 4. Effects of in utero RA exposure on
chondrogenesis in cultured E10.5 periotic
mesenchyme 1 otic epithelium*
Condensation
number
A. In utero RA-exposed periotic mesenchyme 1 in utero
RA-exposed otic epithelium
Mesenchyme 1 epithelium
Control
All-trans RA-exposed
37 6 2
10 6 6
B. Untreated (control) periotic mesenchyme 1 in utero
RA-exposed otic epithelium
Epithelium
Control
All-trans RA-exposed
Control
All-trans RA-exposed
Control
All-trans RA-exposed
Control
All-trans RA-exposed
Control
All-trans RA-exposed
Fig. 3. Dose response of cultured E10.5 periotic mesenchyme 1 otic
epithelium to all-trans RA. While in cultures treated with 1 3 10212 M
RA the extent of chondrogenesis was comparable to control cultures,
treatment with 1 3 10211 M RA or 1 3 10210 M RA suppressed the
chondrogenic process. In the presence of 1 3 1029 M RA, the
chondrogenic differentiation of the cultured periotic mesenchyme was
inhibited.
TABLE 3. Effects of all-trans RA on collagen type II
accumulation in cultured E10.5 periotic
mesenchyme 1 otic epithelium*
Treatment
Control
All-trans RA
Quantity
32
0
27
0
26
0
35
2
40
18
C. In utero RA-exposed periotic mesenchyme 1 untreated
(control) otic epithelium
Mesenchyme
Control
All-trans RA-exposed
35 6 3
39 6 3
*Cultures were prepared containing A) in utero RA-exposed
periotic mesenchyme 1 in utero RA-exposed otic epithelium;
B) control periotic mesenchyme 1 in utero RA-exposed otic
epithelium; or C) in utero RA-exposed periotic mesenchyme 1
control otic epithelium. Cell condensation number was utilized as an early index of chondrogenesis in 3–5 cultures per
experimental group. Comparable amounts of otic epithelium
were present in each culture.
42
12
*Acetic acid extracts of cultures that were grown in the
presence or absence of RA (1 3 1029 M) were combined and
separately assayed by ELISA for collagen type II. Quantity of
type II collagen is expressed in ng/ml.
treated E10.5 periotic mesenchyme. In control cultures
(i.e., cultures in which the epithelial and mesenchymal
tissues were unexposed to RA), otic epithelium initiated
chondrogenesis (Fig. 4A), as evidenced by the development of numerous mesenchymal condensations (Table
4B). In contrast, in cultures of untreated periotic mesenchyme (i.e., periotic mesenchyme unexposed to alltrans RA in utero), in utero all-trans RA-exposed otic
epithelium was not as effective an initiator of chondrogenesis. In 3 of 5 cultures, chondrogenesis was not
initiated in response to all-trans RA-exposed otic epithelium, while in the other 2 cultures, chondogenesis was
evoked, but to a limited extent (Table 4B; Fig. 4B).
Since the decreased chondrogenic response in cultures of in utero RA-exposed otic epithelium 1 periotic
mesenchyme (Table 4A) may also reflect an effect of RA
on the competence of the E10.5 periotic mesenchyme,
we tested the ability of in utero RA-exposed periotic
mesenchyme to differentiate into cartilage when cultured with control E10.5 otic epithelium (Fig. 4C).
Comparable numbers of cell condensations formed in
cultures of in utero RA-exposed E10.5 periotic mesenchyme 1 control otic epithelium and cultures of control
E10.5 periotic mesenchyme 1 control otic epithelium
(Table 4C).
DISCUSSION
We have shown previously that in utero exposure of
the mouse embryo to nonphysiological levels of alltrans RA at a critical period of development produces
malformations of the inner ear (Frenz et al., ’96). In this
study, we investigated the effects of this teratogen in
cultures of inner ear mesenchyme and epithelium. Our
results demonstrate an inhibition of chondrogenesis, a
process mediated by epithelial-mesenchymal control, in
response to exposure of cultured E10.5 periotic mesenchyme 1 otic epithelium to teratogenic levels of alltrans RA.
Cartilaginous tissue develops through a sequence of
differentiation events involving the aggregation of mesenchymal cells into condensations. Treatment of cultures of E10.5 mouse periotic mesenchyme 1 otic
238
D.A. FRENZ AND W. LIU
epithelium with 1 3 1029 M all-trans RA on either days
1–7 or days 2–7 inhibited the process of mesenchymal
cell condensation, and correspondingly, the process of
chondrogenesis (Table 1, Fig. 1A). Addition of all-trans
RA (1 3 1029 M) to cultured mesenchyme 1 epithelium
on day 3 or 4, a time by which mesenchymal cell
condensations have begun to form in vitro (Frenz and
Van De Water, ’91), resulted in only a partial inhibition
of chondrogenesis, suggesting that the effects of RA on
otic capsule formation may be targeted at cell condensation events. In cultured chick facial primordia cells,
addition of all-trans RA at 48 hr suppresses chondrogenesis, however, the cultures are less sensitive to RA
treatment than are those treated immediately after cell
seeding (Wedden et al., ’87).
Absence of an otic capsule in mouse inner ears
severely malformed by in utero all-trans RA exposure
(Frenz et al., ’96) is consistent with the inhibitory
effects of all-trans RA exposure on the condensation
process in vitro. Moreover, since cell condensations
represent a template of future cartilaginous and skeletal elements, the abnormal pattern of the developing
otic capsule in moderately malformed, all-trans RAexposed embryos (Frenz et al., ’96) is in accord with
these findings. In cultured periotic mesenchyme 1 otic
epithelium of age E12 and E14, i.e., embryonic stages
corresponding to periods of mesenchymal competence
(E12) and capsular remodeling (E14) (McPhee and Van
De Water, ’86), the lack of an inhibitory effect of
all-trans RA exposure further supports the hypothesis
that RA-induced disruption of epithelial-mesenchymal
interactions occurs during a period of development
critical for initiation of chondrogenesis. Consistent with
these findings are studies which demonstrate that
when mouse embryos are exposed in utero to all-trans
RA during a developmental stage after which induction
of chondrogenesis has occurred, normal development of
the capsule of the inner ear ensues (Frenz et al., ’96).
Decreased accumulation of collagen type II in RAexposed (all-trans) cultures of E10.5 periotic mesenchyme 1 otic epithelium in comparison to control
mesenchyme 1 epithelium cultures demonstrated that
the inhibitory effects of RA were specific for chondrogenesis.
The effects of all-trans RA on chondrogenesis in
cultured E10.5 periotic mesenchyme 1 otic epithelium
were dose dependent (Fig. 3). However, while all-trans
RA at 1 3 1029 M inhibited chondrogenesis in periotic
Fig. 4. Effects of in utero all-trans RA exposure on chondrogenesis in
culture (day 7 in vitro). A: E10.5 control culture of periotic mesenchyme 1 otic epithelium. B: E10.5 culture of control periotic mesenchyme 1 in utero RA-exposed otic epithelium. C: E10.5 culture of in
utero RA-exposed periotic mesenchyme 1 control otic epithelium.
While in the control culture (A) and the culture containing in utero
RA-exposed periotic mesenchyme (C) the extent of chondrogenesis was
comparable, the culture containing in utero RA-exposed otic epithelium (B) demonstrates a significant suppression of chondrogenesis,
with only one region of the culture in which an isolated chondrogenic
focus developed (arrowhead). Scale bar 5 35 µm.
RA INHIBITS OTIC CAPSULE CHONDROGENESIS
mesenchyme 1 otic epithelium cultures (Table 1), cis-RA
(1 3 1029 M) had no affect on chondrogenic differentiation (Table 1). The cis-trans configuration of RA has
been shown to affect the teratogenic activity of this
retinoid (Armstrong et al., ’94), and may account for the
suppression of chondrogenesis by cis-RA at 1 3 1028 M
and 1 3 1027 M. This is in accord with previous findings
in high-density cultures of limb bud mesenchyme
(Kistler, ’87) and whole rat embryo culture (Klug et al.,
’89), where the intrinsic teratogenic activity for 13cis-RA was 5–10-fold lower than that for all-trans RA
(Armstrong et al., ’94). Similarly, inner ear teratogenicity in utero occurs in response to levels of all-trans RA
that are 4-fold lower (Frenz et al., ’96) than that which
produces similar dysmorphogenetic effects in response
to 13-cis-RA (Jarvis et al., ’89). The partial inhibition of
chondrogenesis by all-trans retinol (1 3 1029 M) is
consistent with an in vitro conversion of trans-retinol to
all-trans RA (Armstrong et al., ’94). In mice administered teratogenic levels of retinol, appreciable metabolism of retinol to all-trans RA and all-trans-4-oxoretinoic acid occurs (Armstrong et al., ’94; Kochar et al., ’88;
Eckhoff et al., ’89).
In parallel with our studies of in vitro all-trans RA
exposure in which treatment with exogenous RA
(1 3 1029 M) inhibited chondrogenesis, chondrogenesis
was also suppressed in cultures prepared from periotic
mesenchyme 1 otic epithelium that were exposed in
utero to teratogenic levels of all-trans RA (Table 4A),
suggesting an affect on otic epithelial-periotic mesenchymal interactions. Since exposure to all-trans RA may
alter these tissue interactions by changing the mesenchymal response to an epithelial signal, or by changing
the epithelium itself, we distinguished between the two
by testing the inductive capability and responsiveness
of in utero RA-exposed inner ear tissues when interacted with normal (i.e., untreated) inner ear tissues in
high-density culture (Table 4B,C, Fig. 4B,C). While the
competence of periotic mesenchyme to respond to otic
epithelium was unaffected by in utero RA exposure
(Table 4C), the inductive capability of otic epithelium
was abolished or markedly diminished by maternal
all-trans RA exposure (Table 4B), suggesting that the
teratogenic effects of all-trans RA during inner ear
morphogenesis (Frenz et al., ’96) may be directed at otic
epithelium. Growth factors of the transforming growth
factor beta (TGFb), fibroblast growth factor (FGF), and
bone morphogenetic protein (BMP) families are endogenous to otic epithelium, and mediate otic epithelial
control of periotic mesenchyme chondrogenesis (Frenz
et al., ’92, ’94, ’96) (Frenz and Liu, submitted). Since a
loss of staining for endogenous growth factors, e.g.
TGFb1, has been shown to occur following in utero RA
treatment of neuroepithelial cells, neural crest cells,
and embryonic palatal shelves (Abbott and Birnbaum,
’90; Mahmood et al., ’92), a loss or decrease in inductive
stimuli from otic epithelium may account for the inability or diminished ability of in utero RA-exposed otic
239
epithelium to initiate chondrogenesis in control periotic
mesenchyme.
In summary, our results demonstrate an inhibition of
chondrogenesis by exposure to teratogenic levels of
all-trans RA in cultured E10.5 periotic mesenchyme 1
otic epithelium. Since treatment with all-trans RA has
no effect on chondrogenesis at later stages of capsular
development, our findings support the hypothesis that
exposure to all-trans RA interferes with the event of
initiation of chondrogenesis, a process mediated by
epithelial-mesenchymal control. Given the diminished
capability of in utero all-trans RA-exposed otic epithelium to initiate chondrogenesis in cultured periotic
mesenchyme, and the abnormalities in capsular development that ensue following in utero exposure of the
embryonic mouse inner ear to this teratogen (Frenz et
al., ’96), we suggest that similar effects of all-trans RA
on chondrogenesis in utero may account for dysmorphogenesis of the capsule of the inner ear.
ACKNOWLEDGMENTS
This work was supported by NIDCD research grant
DC/OD02823 to D.A.F.
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