Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, BP 10142, 67404 Illkirch Cedex, CU de Strasbourg, France
* Author for correspondence (e-mail: marek{at}igbmc.u-strasbg.fr)
Accepted 5 February 2003
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SUMMARY |
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Key words: Retinoic acid embryopathy, Synthetic retinoids, Nuclear receptors, Embryo cultures, Endoderm, Branchial arches, Pharyngeal pouches, Mouse, Synergy, Agonists, Antagonists, Teratogenicity
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INTRODUCTION |
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RA is indispensable for early morphogenesis and for organogenesis as these
are dramatically disturbed when the physiological level of RA is lowered
(Niederreither et al., 1999)
and when RAR/RXR-mediated signalling pathways are abrogated by genetic (see
above) or pharmacological approaches
(Wendling et al., 2000
;
Wendling et al., 2001
;
Schneider et al., 2001
).
However, RA is also a potent teratogen that, at pharmacological
concentrations, can induce congenital defects in all vertebrate species as
well as in certain invertebrates (Soprano
and Soprano, 1995
; Collins and
Mao, 1999
; Escriva et al.,
2002
). In humans, oral intake of Accutane (13-cis RA) during
gastrulation and early organogenesis (gestational weeks 2-5) results in a
spectrum of malformations referred to as retinoic acid embryopathy (RAE)
(Lammer et al., 1985
).
An external ear malformation is the most frequent defect observed in human
RAE and in mammalian models of RAE (Lammer
et al., 1985; Webster et al.,
1986
; Wei et al.,
1999
). The external ear and the other craniofacial components
commonly deficient in RAE syndromes, the mandible and the middle ear
(Lammer et al., 1985
;
Coberly et al., 1996
;
Mallo, 1997
), originate from
the first two branchial arches (BAs), the first branchial cleft and the first
pharyngeal pouch (Larsen,
1993
). BAs are transient bulges of the embryonic surface that
flank the oral and pharyngeal cavities, and develop in a cranial to caudal
sequence. Each consists of a mesenchymal core covered externally by ectoderm
and lined internally by endoderm. Grooves of the BA ectoderm (branchial
clefts) and evagination of the pharyngeal endoderm (pharyngeal pouches)
separate the BAs from one another. The mesenchyme of the first two BAs is
largely made of neural crest cells (NCC) that have emigrated from the caudal
midbrain and the anterior hindbrain
(Lumsden et al., 1991
;
Serbedzija et al., 1992
).
Maternal exposure of mouse embryos to RA at embryonic day (E) 8.0 results in
fusion and hypoplasia of the first two BAs. It has been assumed that such BA
defects could account for alterations of the external ear and mandible
displayed at birth by RA-exposed embryos, and that neural crest is the primary
target tissue of RA-induced teratogenesis in the BA region
(Goulding and Pratt, 1986
;
Webster et al., 1986
;
Pratt et al., 1987
;
Lee et al., 1995
;
Wei et al., 1999
).
The aim of the present study was to gain insights into the cellular and molecular mechanisms underlying the RA-induced BA defects and more specifically to: (1) evaluate the relative contributions of RARs, RXRs and individual RAR isotypes to the generation of these defects; and (2) characterize the pathogenetic events underlying RA-induced teratogenesis. To this end, we have analyzed the effects of various synthetic agonistic and antagonistic retinoids on BA formation in wild-type and Rarb-null embryos in culture.
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MATERIALS AND METHODS |
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Embryos collected at E8.0 (two- to four-somite stage) were cultured, as
described previously (Copp and Cockroft,
1990), for 12, 24, 30 or 48 hours. Retinoids were diluted in
ethanol and added to the culture medium to give a final concentration of 0.1%
(vol/vol). In control cultures, the ethanol vehicle was added at the same
final dilution. Mouse lines carrying the Rarb2-lacZ as well as the
Rare-hsp68-lacZ RA-reporter transgenes have been described previously
(Mendelsohn et al., 1991
;
Rossant et al., 1991
).
Rarb-null embryos were generated from intercrosses between
Rarb+/ mice and were genotyped at the end of the
culture, as described (Ghyselinck et al.,
1997
).
External morphology, histology, ink injection and in situ
hybridization
Cultured embryos were fixed in Bouin's fluid for 5 hours, rapidly rinsed in
70% ethanol and then in phosphate-buffered saline (PBS), stained for 3 minutes
in Acridine Orange (10 µg/ml in PBS, Sigma) according to Zucker et al.
(Zucker et al., 1995), and
photographed. Embryos were then post-fixed in Bouin's fluid for 16 hours, and
processed for histology according to standard procedures. Staining for
ß-galactosidase activity was carried out as described
(Mendelsohn et al., 1991
).
Embryos were post-fixed in 4% buffered paraformaldehyde (PFA) for 16 hours at
4°C and processed for histology. To visualize the aortic arches, embryos
cultured for 48 hours were injected with Pelikan Ink (number 17) via the yolk
sac vasculature using a 1 mm capillary tube, and then fixed in 4% PFA for 16
hours and cleared according to Waldo et al.
(Waldo et al., 1990
).
For in situ hybridization, cultured embryos were fixed by 4% PFA in PBS (1
hour; 4°C). The digoxigenin-labelled antisense riboprobes were synthesized
by T7 polymerase using Rarb (all isoforms)
(Ruberte et al., 1991),
Hoxa1 (Duboule and Dollé,
1989
), Hoxb1 (Frohman
et al., 1990
) and Pax1 (Deutsch et al., 1998) cDNA as
templates. Whole-mount in situ RNA hybridization was carried out as described
(Décimo et al., 1995
),
except that 100 mM maleic acid, 150 mM NaCl, 0.1% Tween 20 was used for the
washes. In situ hybridization was performed according to Myat et al.
(Myat et al., 1996
) with the
following modifications: 10 µm paraffin wax-embedded sections were
rehydrated in water and 120 µl of the heat inactivated RNA probe (diluted
1/100 in hybridization buffer) was applied on each slide. The anti-DIG
antibody (Roche, Germany) was diluted 1/2500 in blocking solution. Sections
were incubated in NBT/BCIP (the two substrates for alkaline phosphatase;
Boehringer Mannheim, Germany) for 48 hours, with one change of the staining
solution after 24 hours.
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RESULTS |
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Wild-type embryos (n=95) cultured in the presence of
107 M RARß-selective agonist BMS453 reproducibly
displayed an external defect consisting of a 1-2BAFH (compare B1 and B2 in
Fig. 2A with B1-2 in
Fig. 2B) with a small or absent
first branchial cleft (arrowhead in Fig.
2A, compare with Fig.
2B). Variable degrees of fusion between the first two BA arteries
(i.e. the first and second aortic arches; A1 and A2 in
Fig. 2C) were seen following
injection of ink into the embryonic vasculature (A1-2 in
Fig. 2D), and on serial
histological sections (compare A1 and A2 in
Fig. 2E with A1-2 in
Fig. 2F). The first and second
pharyngeal pouches were markedly hypoplastic on flat mounts of isolated
pharyngeal endoderm (P1 and P2, compare
Fig. 3G,I,K with
Fig. 3H,J,L). In situ
hybridization with a Pax1 probe was used to investigate pharyngeal
pouch development. Similar to what is observed during normal embryogenesis,
vehicle-treated embryos cultured for 12, 24 and 48 hours exhibited high levels
of Pax1 expression in the walls of the forming first and second
pouches, but not in their most lateral region, which is close to the surface
ectoderm (P1 and P2, Fig.
3A,C,E,G,I,K) (Müller et
al., 1996; Dupé et al.,
1999
). In BMS453-treated embryos, Pax1 was weakly
expressed in the first and second pouches, and sometimes expression was
undetectable in the first pouch (P1 and P2,
Fig. 3B,D,F,H,J,L). Moreover,
these two pouches often appeared to have fused together in embryos cultured
for 48 hours (compare P1 and P2 in Fig.
3E with P1-2 in Fig.
3F). As expression of Pax1 correlates with high levels of
cell proliferation in the pharyngeal endoderm
(Müller et al., 1996
),
our data suggest that 1-2BAFH and fusion of the corresponding aortic arches
may result from the growth failure of the first pharyngeal pouch.
|
BMS453 and TTNPB at concentrations that consistently induced 1-2BAFH in
wild-type embryos (107 M and 5x109
M, respectively, see above), did not yield any abnormality in
Rarb-null embryos (n=10 in each group;
Fig. 5B,D; compare with
Fig. 5A,C; data not shown).
Altogether, these data indicated that the teratogenic effects of retinoids on
BAs were specifically mediated by RARß, provided that BMS453 actually
acted as a bona fide retinoid in the embryo. To check this latter point, we
used transgenic embryos carrying a lacZ gene controlled by the
RA-inducible Rarb2 promoter sequence (Rarb2-lacZ gene),
which is active in some, but not all, embryonic tissues expressing the
endogenous Rarb gene (Mendelsohn
et al., 1991; Mendelsohn et
al., 1994
). In vehicle-treated control embryos (n=11),
expression of the Rarb2-lacZ reporter was detected in the neural tube
from the caudal neuropore up to the seventh rhombomere (R7,
Fig. 6A). In embryos treated
with 107 M BMS453 (n=9), this expression domain was
shifted anteriorly, reaching the boundary between the third and the fourth
rhombomeres (R3/R4 boundary; R3-4; Fig.
6B) (Mendelsohn et al.,
1994
). Adjunction to the culture medium of the panRAR-selective
antagonist BMS493 at 106 M prevented not only the
BMS453-induced ectopic expression of the Rarb2-lacZ transgene in the
R4 to R6 territory (n=6; Fig.
6C), but also the generation of the 1-2BAFH (compare B1-2 in
Fig. 6B with B1 and B2 in
Fig. 6C). As expected, BMS493
also caused a general decrease of Rarb2-lacZ expression (compare
Fig. 6A with
Fig. 6C)
(Wendling et al., 2000
). These
data indicate that the BMS453-induced 1-2BAFH is causally related to the
activation of an RA signalling pathway.
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DISCUSSION |
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RARß/RXR heterodimers mediate teratogenic effects of RA in the
branchial region of the head
We show that the fusion and hypoplasia of the first two BAs (1-2BAFH),
which results from RA administration at a developmental stage equivalent to
E8.0 in the mouse (Webster et al.,
1986; Goulding and Pratt,
1986
; Lee et al.,
1995
; Wei et al.,
1999
), can also be generated by treatment with the
panRAR-selective agonist TTNPB. Furthermore, TTNPB-induced 1-2BAFH is not
observed in Rarb-null embryos, and can be mimicked in wild-type
embryos by treatment with the RARß-selective agonist BMS453 but not with
an RAR
-selective or RAR
-selective agonist. These findings
strongly support the conclusion that abnormal activation of RARß
signalling is both necessary and sufficient to induce the typical early
teratogenic effect induced by RA administration at E8.0. External and middle
ears derive from the first branchial cleft ectoderm, the first pharyngeal
pouch endoderm and the mesenchyme located at the interface between the first
and second BAs, whereas mandibular bone differentiates from the first BA
mesenchyme (Larsen, 1993
).
Thus, ear and mandible deficiencies that, in human newborns, are hallmarks of
RAE (Lammer et al., 1985
;
Coberly et al., 1996
), could
be accounted for by an abnormal activation of RARß signalling during the
fourth week of pregnancy (equivalent to E8.0 in the mouse).
In cultured cells, RAR- and RXR-selective agonists act synergistically to
promote cell proliferation, differentiation or apoptosis through
transactivation of RA-responsive genes. Moreover, the ligand-bound RXR is
transcriptionally active only if its RAR partner is also ligand-bound (the
so-called RXR subordination) (Lotan et
al., 1995; Roy et al.,
1995
; Horn et al.,
1996
; Taneja et al.,
1996
; Botling et al.,
1997
; Chiba et al.,
1997
; Minucci et al.,
1997
; Altucci and Gronemeyer,
2001
). Along the same lines, RXR-selective ligands, which are
inactive on their own, synergize in vivo with RAR
- and
RAR
-selective ligands to generate congenital malformations
(Kochhar et al., 1996
;
Lu et al., 1997
;
Elmazar et al., 1996
;
Elmazar et al., 1997
;
Elmazar et al., 2001
). In our
experiments, the panRXR agonist BMS649 (SR11237) had no effect on its own,
indicating that neither RXR homodimers, nor other heterodimers in which the
RXR partner is transcriptionally active on its own, mediate the teratogenic
effects of retinoids on the branchial region. However, the RXR-selective
ligand can reveal the dysmorphogenetic effects of the RARß agonist at a
concentration that, on its own, is not teratogenic. Thus, retinoid-induced
teratogenesis of BAs is mediated through ectopic activation of RARß/RXR
heterodimers, in which the ligand-dependent activity of RXR is subordinated to
that of its ligand-bound RARß partner.
The pharyngeal endoderm is a primary target tissue of RA-induced
teratogenesis
Because of the multiplicity and complexity of malformations induced upon
exposure to RA, target tissues of RA-induced teratogenicity are difficult to
identify. Several observations have suggested that the cranial neural crest
represents such a target tissue. First, almost all the structures that are
malformed in human RAE are derived from neural crest cells (NCCs) migrating
through BAs (Larsen, 1993).
Second, analyses of animal models indicate that excess RA can alter NCC
survival and migration. For example, NCC apoptosis that occurs as a result of
RA administration to mouse embryos is a probable cause of the craniofacial
defects observed in newborns (Sulik et
al., 1988
; Alles and Sulik,
1992
). Along the same lines, aberrant NCC migration after RA
treatment has been correlated with hypoplasia of the first BA in rat embryos
(Lee et al., 1995
).
Thus, RAE apparently meets the criteria for a neurocristopathy, i.e. `a
condition arising from aberrations of the early migration, growth and
differentiation of NCCs' (Bolande,
1997). However, retinoid-induced fusion of the first and second BA
occurs without alterations of NCC migration or apoptosis
(Lee et al., 1995
). Moreover,
we show that NCCs contributing to the first two BAs do not express a
retinoid-responsive transgene upon treatment with BMS453. Therefore, under our
experimental conditions, NCCs are not the primary targets of retinoid-induced
teratogenicity. Interestingly, recent evidence also indicates that NCCs may
not respond directly to RA under physiological conditions
(Dupé et al., 1999
;
Wendling et al., 2000
;
Iulianella and Lohnes, 2002
;
Jiang et al., 2002
).
By contrast, treatment with BMS453 disrupts the development of the first
two pharyngeal pouches and triggers ectopic RARß-dependent RA signalling
in the endoderm lining the first two BAs, manifested by the rostral shift of
the expression domains of RA-responsive genes (i.e. the
Rare-hsp68-lacZ reporter, the Rarb gene, the Hoxa1
and Hoxb1 genes). Hox gene expression probably plays an important
role in anteroposterior regionalization of the pharyngeal endoderm
(Mulder et al., 1998;
Manley and Capecchi, 1998
;
Wendling et al., 2000
), which
can be set up even in the absence of NCCs
(Veitch et al., 1999
;
Gavalas et al., 2001
;
Escriva et al., 2002
;
Graham and Smith, 2001
).
Conversely, pharyngeal endoderm plays a seminal role in the formation of BAs,
through imparting patterning information to NCCs
(Couly et al., 2002
), as well
as to ectodermal cells (Begbie et al.,
1999
). In our BMS453-treated embryos, impaired development of the
first pharyngeal pouch probably occurs as a direct consequence of improper
regionalization of the pharyngeal endoderm. As BA segmentation cannot proceed
without pharyngeal pouches (Piotrowski and
Nusslein-Volhard, 2000
), the growth failure of the first pouch
might account for the fusion of the first two BAs in embryos exposed to
retinoids at a developmental stage equivalent to mouse E8.0. Interestingly,
treating E9.0 mouse embryos with RA leads to disruption of the third
pharyngeal pouch and branchial cleft, along with a fusion of the third and
fourth BAs, which is accompanied by a marked increase of Hoxa3
expression in the pharyngeal endoderm
(Mulder et al., 1998
). These
data and our present findings support the view that craniofacial, thymic and
cardiovascular defects observed in RAE, as well as in genetically determined
neurocristopathies (Jerome and
Papaioannou, 2001
), can all result from an abnormal function of
the pharyngeal endoderm.
BMS453 acts in vivo as an RARß-selective agonist displaying
RAR- and RAR
-selective antagonistic properties
Retinoids (notably RA) are widely used in cancer chemoprevention, as well
as for treating oncological and dermatological diseases
(Lotan, 1996;
Nason-Burchenal and Dmitrovsky,
1999
). However, undesirable side-effects, including toxicity and
teratogenicity, are observed upon treatment with RA, most probably because of
the panRAR and panRXR agonistic activity of RA
(Orfanos et al., 1987
). The
use of synthetic agonists that selectively interact with given receptor
isotypes is expected to reduce such side-effects. In situ hybridization
analyses (Ruberte et al.,
1991
) have established that the three RARs display distinct
expression profiles at E8.5 (i.e. just a few hours earlier than the
developmental stage illustrated in Figs
7 and
11). During this time period,
both neurectoderm and mesenchyme of the head region strongly express
RAR
, whereas the tail tissues strongly express RAR
. RARß
transcripts are present in trunk tissues and in mesonephric duct, but are not
detectable in the forebrain and tail regions. The overlap between (1) the
expression domains of RAR
, ß and
during normal embryonic
development, and (2) the distribution of responsive cells in
RAR
-selective agonist BMS753-, RARß-selective agonist BMS453- and
RAR
-selective agonist BMS961-treated embryos indicates that RA signals
are transduced preferentially by RAR
in tissues of the embryonic head,
by RARß in tissues of the embryonic neck, and by RAR
in tissues of
the embryonic tail. Interestingly, BMS453-induced ectopic activation of
retinoid signalling in the hindbrain and BA endoderm is accompanied by a
decrease of signalling in the forebrain and tail regions
(Fig. 11). A similar decrease
is observed in the forebrain and tail regions upon treatment with a panRAR
antagonist (Wendling et al.,
2000
), as well as in embryos carrying a null mutation of the
RA-generating enzyme RALDH2 (Niederreither
et al., 1999
). Therefore, this decrease results from a block in RA
signalling and not from a toxic effect of BMS453, which definitely acts, in
vivo, as a bona fide RARß-selective agonist displaying an RAR
and
RAR
antagonistic activity. This is in keeping with data obtained in
vitro in stably transfected cells (Chen et
al., 1995
).
Interestingly, anteriorization of RARß expression triggered by BMS453
has to be mediated by RARß itself and not by other RARs as: (1) BMS453
antagonizes RAR and RAR
signalling in the embryo; and (2)
treatment of RARß-null embryos carrying the
Rare-hsp68-lacZ transgene with BMS453 does not affect the
pattern of lacZ expression in the pharyngeal endoderm
(Fig. 7). Thus, even though the
levels of RARß transcripts are below the threshold for detection in the
anterior pharyngeal endoderm (Fig.
9), RARß has to be present in this region where it can induce
its own promoter if activated by a ligand. This observation is in keeping with
data obtained from cultured cells (Roy et
al., 1995
; Taneja et al.,
1996
; Chiba et al.,
1997
). Many pre-malignant and malignant cells exhibit a reduced
RARß expression (Sun et al.,
2000
), whereas forced-recovered expression of RARß in breast
cancer cells restores RA-induced growth arrest and apoptosis
(Seewaldt et al., 1995
). As
the RARß-selective agonist BMS453 does not activate RAR
and
RAR
, it could be less toxic than RA, and therefore of therapeutic value
in the treatment of cancers characterized by reduced RARß expression.
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ACKNOWLEDGMENTS |
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