Download Function of the retinoic acid receptors (RARs) during development

Development 120, 2723-2748 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
2723
Function of the retinoic acid receptors (RARs) during development
(I) Craniofacial and skeletal abnormalities in RAR double mutants
David Lohnes* ,†, Manuel Mark*, Cathy Mendelsohn*,‡, Pascal Dollé, Andrée Dierich, Philippe Gorry,
Anne Gansmuller and Pierre Chambon§
Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité 184 de Biologie Moléculaire et de Génie Génétique de
l’INSERM, Institut de Chimie Biologique, Faculté de Médecine, 11 rue Humann, 67085 Strasbourg Cedex, France
*Should be considered as equal first authors
address: Institut de recherches cliniques de Montréal, Laboratoire de Biologie Cellulaire et Moléculaire, 110 Avenue des Pins-Ouest, Montréal, Québec H2W
1R7, Canada
‡Present address: Columbia University, Department of Physiology and Biophysics, 630 W 168th Street, New York, NY 10032, USA
§ Author for correspondence
† Present
SUMMARY
Numerous congenital malformations have been observed in
fetuses of vitamin A-deficient (VAD) dams [Wilson, J. G.,
Roth, C. B., Warkany, J., (1953), Am. J. Anat. 92, 189-217].
Previous studies of retinoic acid receptor (RAR) mutant
mice have not revealed any of these malformations [Li, E.,
Sucov, H. M., Lee, K.-F., Evans, R. M., Jaenisch, R. (1993)
Proc. Natl. Acad. Sci. USA 90, 1590-1594; Lohnes, D.,
Kastner, P., Dierich, A., Mark, M., LeMeur, M., Chambon,
P. (1993) Cell 73, 643-658; Lufkin, T., Lohnes, D., Mark,
M., Dierich, A., Gorry, P., Gaub, M. P., LeMeur, M.,
Chambon, P. (1993) Proc. Natl. Acad. Sci. USA 90, 72257229; Mendelsohn, C., Mark, M., Dollé, P., Dierich, A.,
Gaub, M.P., Krust, A., Lampron, C., Chambon, P. (1994a)
Dev. Biol. in press], suggesting either that there is a considerable functional redundancy among members of the
RAR family during ontogenesis or that the RARs are not
essential transducers of the retinoid signal in vivo. In order
to discriminate between these possibilities, we have
generated a series of RAR compound null mutants. These
RAR double mutants invariably died either in utero or
shortly after birth and presented a number of congenital
INTRODUCTION
It has long been known that retinol (vitamin A) is crucial for
normal growth, vision, reproduction, maintenance of numerous
tissues and overall survival (Wolbach and Howe, 1925; see
Sporn et al., 1994 and Blomhoff, 1994, for reviews and references). Retinol is also essential for normal development, as
shown by the appearance of multiple congenital abnormalities
in fetuses from dams fed a vitamin A-deficient (VAD) diet (the
fetal VAD syndrome, see Wilson et al., 1953 and references
therein). Interestingly, with the exception of vision (Wald,
1968), retinoic acid (RA) appears to be the active derivative of
vitamin A, since its administration can prevent or reverse most
of the defects induced by postnatal VAD (Thompson et al.,
1964). Furthermore, RA excess is much more teratogenic than
abnormalities, which are reported in this and in the accompanying study. We describe here multiple eye abnormalities which are found in various RAR double mutant fetuses
and are similar to those previously seen in VAD fetuses.
Interestingly, we found further abnormalities not previously reported in VAD fetuses. These abnormalities affect
ocular glands, salivary glands and their associated ducts,
the axial and limb skeleton, and all skeletal elements
derived from the mesectoderm of the frontonasal mass and
of the second and third pharyngeal arches. RAR double
mutants also exhibit supernumerary cranial skeletal
elements that are present in the ancestral reptilian skull.
The role of retinoic acid (RA) and of the RARs in the ontogenesis of the affected structures, particularly of those that
are derived from mesenchymal neural crest cells, is
discussed.
Key words: retinoic acid, retinoic acid receptors, ontogenesis, neural
crest, evolution, atavisms, mouse, homeotic transformations, eye,
skull, limb
retinol excess, causing many developmental abnormalities, the
precise malformation depending largely on the time of administration (reviewed in Morriss-Kay, 1993; Nau et al., 1994;
Hofman and Eichele, 1994). The spectacular effects of topical
RA application on limb development and regeneration popularized the belief that RA could in fact be a morphogen (for
reviews, see Tabin, 1991; Hofman and Eichele, 1994).
The discovery of a nuclear receptor for RA, acting as a
ligand-inducible transcriptional regulator (RAR; Petkovich et
al., 1987; Giguère et al., 1987), greatly advanced our understanding of the molecular mechanisms underlying the
pleiotropic effects of retinoids (synthetic and natural derivatives
of RA; reviewed in Leid et al., 1992; Kastner et al., 1994; Mangelsdorf et al., 1994; Linney and LaMantia, 1994). Since this
initial finding, it has been shown that the RA signal can be trans-
2724 D. Lohnes and others
duced in cultured cells through two families of retinoid
receptors. The RAR family (RARα, β and γ and their isoforms)
are activated by both all-trans RA and 9-cis RA, whereas the
RXR family (RXR α, β and γ) are activated only by 9-cis RA.
The DNA-binding and ligand-binding regions (regions C and
E, respectively) of the three RAR types are highly similar,
whereas the C-terminal F region and the central D region
exhibit little, if any, conservation. The three RAR types also
diverge in their N-terminal B regions and further diversification
is generated for each receptor type by variant isoforms differing
in their N-terminal-most A regions (RARα1 and α2, β1 to β4,
and γ1 and γ2), which originate from alternate splicing and differential promoter usage (reviewed in Leid et al., 1992). Amino
acid sequence comparisons have revealed that the interspecies
conservation of a given RAR type and of each of its isoforms
is greater than the similarity found between the three RARs
within a given species (see Kastner et al., 1994 for review). Furthermore, the various RAR isoforms contain two transcriptional
activation functions (AFs), located in the N-terminal A/B region
(AF-1) and C-terminal E region (AF-2) which act synergistically, and sometimes differentially, to activate various RAresponsive promoters. Taken together with the distinct spatiotemporal transcript distribution observed for each RAR and
isoforms during mouse embryogenesis and in adult tissues, the
above interspecies sequence conservation and transcriptional
activation specificities suggested that each RAR isoform may
perform unique functions (for refs see Kastner et al., 1994;
Chambon, 1994). Furthermore, the finding that RA-responsive
promoters are likely controlled in cultured cells through RARRXR heterodimers (reviewed in Kastner et al., 1994; Mangelsdorf et al., 1994; Chambon, 1994) suggested that the diverse
effects of retinoids may also reside in the control of various
subsets of retinoid-responsive promoters by different combinations of RAR-RXR types (and isoforms).
To evaluate the function of the various RARs (types and
isoforms) in vivo, we have created mice lacking several of
these receptors. Surprisingly, mice deficient for RARα1 (Li et
al., 1993; Lufkin et al., 1993), RARβ2 (Mendelsohn et al.,
1994a) or RARγ2 (Lohnes et al., 1993) isoforms were apparently unaffected. In contrast, mice deficient for RARα or
RARγ receptors (all α or γ isoforms disrupted) exhibited postpartum lethality and growth deficiency (Lohnes et al., 1993;
Lufkin et al., 1993). Furthermore, RARα null mice presented
a degeneration of the testicular germinal epithelium, which was
similar to that observed in male rats maintained on a VAD diet
(Howell et al., 1963). Male RARγ null mutants also exhibited
VAD-like abnormalities, namely squamous metaplasia of the
seminal vesicles and prostate gland. Agenesis of the ocular
Harderian gland and homeotic transformations of the axial
skeleton were also observed in RARγ null mutants, although
both of these defects occurred with incomplete penetrance and
expressivity. In no case, however, have these RAR null
mutants displayed any of the congenital malformations
observed in fetal VAD studies (Wilson et al., 1953). Furthermore, the phenotype of RARα and RARγ null mice was
confined only to a small subset of tissues expressing these
receptor types. Thus, contrary to our expectations, the RAR
types and isoforms disrupted to date do not apparently possess
the unique functions that were predicted on the basis of their
evolutionary conservation, expression pattern and in vitro transcriptional regulatory characteristics.
Taken together, the above findings suggest either that there
is a high degree of functional redundancy among members of
the RAR family, or that the RARs are not essential transducers of the retinoid signal in vivo. To discriminate between these
possibilities, we have generated and analyzed RAR compound
null mutants. The defects displayed by various double mutants
recapitulate essentially all of the congenital malformations
found in fetal VAD. These double mutants also exhibit a
number of abnormalities not previously described in VAD
experiments (see also the accompanying study). We report here
a detailed analysis of the craniofacial and skeletal defects
found in RAR double null mutants.
MATERIALS AND METHODS
Generation of RAR double mutants
The generation of RARα1, α, β2 and γ single null mutants has been
described (Lohnes et al., 1993; Lufkin et al., 1993; Mendelsohn et al.,
1994a). Initial intercrosses of these single mutants were performed to
derive double heterozygotes. With the exception of RARα+/−/γ+/−
offspring, second generation animals were obtained by mating double
heterozygotes with the appropriate RAR heterozygous or homozygous single mutants to test for viability and fertility of compound
mutants. This was performed in order to optimize subsequent generation of double null mutants. The RAR double mutants and the crosses
used to generate them were as follows: RARα1−/−/β2−/− were derived
from RARα1+/−/β2−/− intercrosses or crosses between RARα1+/−/
β2−/− and RARα1+/−/β2+/− animals; RARα−/−/β2−/− and RARα−/−/
β2+/− mutants were derived from crosses between RARα+/−/β2−/−
males and RARα+/−/β2+/− or RARα+/−/β2−/− females; RARα1−/−/γ−/−
mutants were derived from crossing RARα1−/−/γ+/− males and
RARα1+/−/γ+/− females; RARα1−/−/γ−/−/α2+/− mutants were derived
from crosses between RARα1−/−/γ+/− and RARα+/−/γ+/− animals;
RARα−/−/γ−/− mutants were obtained from intercrosses between
RARα+/−/γ+/− animals; RARβ2−/−/γ−/− mutants were obtained
from crosses between RARβ2−/−/γ+/− males and RARβ2+/−/γ+/− or
RARβ2−/−/γ+/− females.
Matings and genotyping of offspring
Animals were mated overnight and females examined for a vaginal
plug the following morning. Noon of the day of evidence for a vaginal
plug was considered 0.5 dpc. Embryos (10.5-14.5 dpc) or 18.5 dpc
fetuses were obtained by Cesarean section and genotypes determined
by genomic Southern blotting using DNA isolated from the yolk sac
or placenta. Probes, digests and other conditions for southern blotting
have been detailed elsewhere (Lohnes et al., 1993; Lufkin et al., 1993;
Mendelsohn et al., 1994a).
Histological and skeletal analysis
For whole-mount skeletal analysis, fetuses were collected at 18.5 dpc
and stored at −20°C. Skeletons were prepared as described (Lufkin et
al., 1992). For histological analysis, embryos or skinned fetuses were
fixed in Bouin’s solution. Paraffin sections, 7 µm thick, were stained
with hematoxylin and eosin or Groat’s hematoxylin and Mallory’s
trichrome (Mark et al., 1993).
RESULTS
(A) Viability
Animals lacking the RARα1 or β2 isoforms were apparently
normal (Table 1A; see refs also Lufkin et al., 1993; Mendelsohn et al., 1994a). Although exhibiting a high degree of
neonatal lethality, animals lacking all isoforms of either RARα
RARs in ontogenesis 2725
or RARγ survived in isolation for at least 24 hours when
delivered by Cesarean section at 18.5 day postcoitum (dpc),
being in this respect as viable as their control littermates (Table
1A; see Lohnes et al., 1993; Lufkin et al., 1993). With the
exception of αγ double mutants, the distribution of RARα1β2,
αβ2, α1γ, α1γα2+/− and β2γ double mutant offspring at 18.5
dpc indicated that loss of these receptors did not result in
embryonic lethality [Table 1A; for the sake of simplicity
mutants null for RARα1 and RARβ2, RARα (all isoforms) and
RARβ2, etc, are called hereafter and in the accompanying study
α1β2, αβ2, etc, mutants]. However, in contrast to the RAR
single mutants, these double mutants invariably died within at
most 12 hours following Cesarean delivery at 18.5 dpc.
The frequency of RARαγ mutants found at 18.5 dpc was
lower than predicted from Mendelian distribution (Table 1B),
indicating partial embryonic lethality. Although analysis of
earlier stages of development (10.5-13.5 dpc) yielded the
expected frequency, a large fraction were either dead or
partially resorbed (Table 1B). The time of death appeared
variable, as judged by the size and relative proportion of
resorbed and dead mutant embryos (Table 1B, data not shown).
The embryonic and postpartum lethality may be due to one or
more malformations affecting the heart, aortic arches, kidney,
lung or trachea which were observed in RAR double mutant
fetuses (see the accompanying study).
(B) External features
Upon external inspection at 18.5 dpc, α1β2, αβ2 and β2γ
fetuses could not be distinguished from their littermates. In
contrast, αγ mutant fetuses could be readily identified by their
reduced size (compare Fig. 1a and c), the small size or apparent
absence of the eyes (asterisk, compare Fig. 1a with c and g-i)
and the aspect of their mid-facial region (compare Fig. 1d and
f). Their snout was markedly foreshortened and divided by a
sagittal median cleft (large arrow, Fig. 1f). The prolabium
(median third of the upper lip, dashed box in Fig. 1d) was
absent (compare Fig. 1d and f). The maxillary processes which
bear whiskers (double arrow, Fig. 1f) were located farther apart
than in WT animals. Paramedian swellings likely corresponding to the nasomedial processes (single small arrow, Fig. 1f
and see below) were fused to the maxillary processes ventral
to the nostrils, which opened dorsally instead of rostrally
(compare arrowheads, Fig. 1d and f). Additional external
defects occasionally found in these mutants included: exteriorized brain (exencephaly, Fig. 1h and i, and Table 1B),
bilateral agenesis of the auricle (open arrows, compare Fig. 1a
with c and g), umbilical hernia (Fig. 1i, large black arrow), and
abnormal limbs (e.g. Fig. 1c,h and i, and see below).
A persistent opening of the rhombencephalic neural tube
was observed in nine out of twenty living αγ mutant embryos
between 10.5 and 11.5 dpc (e.g. compare Fig. 2b and e) and in
Table 1. (A) Viability of 18.5 dpc RAR double null mutant fetuses
Mutant
genotype
Total
offspring
RARα1
RARα
RARβ2
RARγ
RARα1β2
RARαβ2
RARα1γ
RARα1γα2+/−
RARαγ
218
137
345
280
45
170
243
122
355
RARβ2γ
137
Mutants
Mutant
frequency
%
Expected
frequency
%
58
39
100
65
13
15
29
16
11
27
28
29
23
29
9
12
13
3
25
25
25
25
25
12.5
12.5
12.5
6.25
19
13
12.5
Viability
Viable
Neonatal lethal†
Viable
Neonatal lethal‡
Viability* ≤12 hrs
Viability* ≤1 hr
Viability* ≤12 hrs
Viability* ≤1 hr
Embryonic lethal
Viability* ≤1 hr
Viability* ≤12 hrs
*Fetuses were delivered by Cesarean section at 18.5 dpc and compared to littermates for postpartum (PP) viability in an isolated environment; under these
conditions WT littermates survived for at least 24 hours.
†,‡ Animals delivered by Cesarean section survived as well as WT littermates, but showed a high degree of mortality in the first 4 weeks of life following
natural birth. †See Lufkin et al., 1993; ‡see Lohnes et al., 1993.
(B) Embryonic lethality of RARαγ double mutant offspring from RARα+/−/γ+/− intercrosses
RARαγ double mutants
Mutants with neural tube
defect‡
Days postcoitum
10.5
11.5
13.5
18.5
Total
offspring
Total
mutants*
Mutants
resorbed or
dead†
Mutants
living†
Total
mutants
Relative to
living
mutants
303
190
196
355
20 (6.6%)
11 (5.8%)
11 (5.6%)
13 (3.7%)
6 (30%)
5 (45%)
4 (36%)
2 (15%)
14 (70%)
6 (55%)
7 (64%)
11 (85%)
6
3
1
5
43%
50%
14%
45%
*Percentages are relative to the number of total offspring.
†Percentages are relative to total mutants.
‡Neural tube defect in 10.5 to 13.5 dpc embryos was observed in the rhombencephalic region, whereas in 18.5 dpc fetuses this refers to exposure of the brain
(exencephaly, see text).
2726 D. Lohnes and others
Fig. 1. Comparison of external features between 18.5 dpc (a,d) wild-type
(WT) and (b,c,e-i) RAR double mutant fetuses. The mutant genotype is
indicated in the lower right corner of each photograph. The asterisks indicate
the region of the eye; the open arrows point to the region of the external ear;
the arrowheads point to the nostrils; the large white arrow indicate midfacial
clefts. (i) The large black arrow points to an umbilical hernia; (f) the single
small arrow and the double arrow point to the nasomedian and maxillary
processes of the αγ mutant fetus, respectively. (d) The dashed box indicates
the prolabium. Same magnifications for a-c and for d-f.
RARs in ontogenesis 2727
Fig. 2. Comparison of (a-f) whole mounts and (g-i) histological sections of facial and brain structures between (a,b,d,e,i) 10.5 dpc and (c,f-h)
11.5 dpc wild-type (WT, a-c,g) and αγ exencephalic mutant embryos (d-f,h,i). Histological sections were performed through the forebrain and
frontonasal mesenchyme. Abbreviations: FC, median facial cleft; LV, lateral ventricles of the brain; MS, mesencephalon; NL; nasolateral
process; NM, nasomedial process; NP, nasal placode; OP, olfactory pit; RH, rhombencephalon; TE, telencephalon (cerebral hemispheres). The
arrowheads indicate the eye region; (d,e) the large black arrow indicates the scoliosis; (c,f) the broken line indicates the midline. (e) The
bracket indicates the extent of the rhombencephalic opening;(i) the dashed box indicates an area of cell necrosis in the frontonasal
mesenchyme. Magnifications: ×30 (g,h) and ×150 (i). The same magnifications were used for a,b,d,e and for c,f.
2728 D. Lohnes and others
RARs in ontogenesis 2729
one of seven living αγ embryos at 13.5 dpc (Table 1B). In these
embryos, the telencephalic hemispheres appeared markedly
underdeveloped (TE, compare Fig. 2a,b with Fig. 2d, and Fig.
2c with Fig. 2f). Some αγ mutant embryos also showed a
severe degree of lateral deviation of the vertebral axis
(scoliosis, arrows in Fig. 2d,e). However, the only pathognomonic external feature of αγ mutant embryos between 11.5 and
13.5 dpc was the aspect of the frontonasal segment of the face:
both the nasolateral (NL) and the nasomedial (NM) processes,
located on either side of the ofactory pit (OP), were present
(compare Fig. 2c and f). The nasomedial processes were
normally fused with ipsilateral maxillary processes, but were
never fused at the midline resulting in a median facial cleft
(FC; compare Fig. 2c with f, and Fig. 2g with h). This lack of
fusion may result from cell deficiency caused by excessive cell
death in the frontonasal mesenchyme, which was observed on
serial histological sections in 10.5 dpc αγ embryos (dashed
box, Fig. 2i).
18.5 dpc α1γα2+/− fetuses were brachycephalic (compare
Fig. 1a and b) and occasionally showed a median cleft of the
upper lip (compare Fig. 1d and e). In contrast, α1γ mutants did
not exhibit any abnormal external features.
(C) Craniofacial skeletal abnormalities
(1) Defects of the craniofacial skeleton and teeth
Craniofacial skeletal deficiencies were not observed in 18.5
dpc αβ2, α1β2, or β2γ mutant fetuses. In contrast, most of the
neural crest cell (NCC)-derived craniofacial skeletal elements
were altered in αγ mutants. The most severe skeletal defects
were observed in the midfacial region and rostral cranial base,
consistent with the loss of midfacial structures described
above. The skeletal elements normally derived from the frontonasal mesectoderm are the frontal (F) and nasal (N) bones
(Fig. 3a), the cartilaginous template of the ethmoid bone [comprising the nasal septum (NS, Figs 3k and 4a), the nasal capsule
(NC, Figs 3a,k, 4a) and the lamina cribriform (LC, Fig. 4a)],
Fig. 3. Comparison of the craniofacial skeleton between 18.5 dpc
wild-type (WT) and RAR double mutant fetuses (genotypes as
indicated on the photographs). (a-d) Lateral views of the skull. Note
that c and d represent different fetuses. (e,f) Dorsal views of the
cranial base; the orbitosphenoid bone has been removed in order to
show the pila prooptica (PP) and the pila metoptica (PM) more
clearly. (g-j) Dorsal views of the WT incus (g) or incus plus
alisphenoid bone (h-j). (k-m) Ventral views of the cranial base; the
dentary bone has been removed from these whole mounts.
Abbreviations: AL, alisphenoid bone; BI, body of the incus; BO,
basioccipital bone; BS, basisphenoid bone; D, mandibular (dentary)
bone; E, exoccipital bone; F, frontal bone; IF, incisive foramen; IP
interparietal bone; LI, long process of the incus; N, nasal bone; NC,
nasal capsule; NS, nasal septum; O, otic capsule; OB, orbitosphenoid
bone; OF, optic foramen; P, parietal bone; PA, pila antotica; PL,
palatine bone; PM, pila metoptica; PP, pila prooptica; PS,
presphenoid bone; PX, incisive (premaxillary) bone; Q,
pterygoquadrate rod; S, supraoccipital bone; SI, short process of the
incus; T, tympanic bone; X, maxillary bone; (m) asterisk marks
cartilaginous nodules replacing the nasal capsule; (f) the arrows point
to a deficiency in the presphenoid bone; (c) the small arrows delimit
the median gap between the left and right frontal bones; (a,b) the
broken line delimits the frontoparietal suture; (f,m) the arrowheads
point to a persistent hypophyseal foramen. The same magnifications
were used for a-d and k-m, for e and f, and for g-j.
and the incisive (or premaxillar, PX, Fig. 3a,k) and vomer (not
shown) bones (De Myer, 1975). These elements were grossly
deficient or absent in αγ mutant fetuses. The medial portions
of the frontal (F) and nasal (N) bones were lacking (compare
Fig. 3a with c,d). The nasal capsule was apparently reduced to
laterocaudal rudiments (NC, compare Fig. 3a with c,d, and Fig.
3k with m). The rest of the nasal capsule, the nasal septum, the
lamina cribriform and the vomer and incisive bones could not
be identified (NS and PX, compare Fig. 3a with c,d, and Fig.
3k with m), and were apparently replaced by aggregates of cartilaginous and bony nodules or rods (asterisks in Figs 3m, 4c,
5c,d). In the cranial base, caudal to the ethmoid bone, similar
aggregates replaced the presphenoid bone (data not shown).
The upper incisors, which are largely derived from the nasomedial processes (Lumsden and Buchanan, 1986), were
lacking (not shown). The hypophyseal foramen of the
basisphenoid was never closed (arrowhead in Fig. 3m), thus
the pituitary gland (HY, Fig. 4g) remained in contact with the
pharynx.
Many of the first pharyngeal arch-derived skeletal elements
(Noden, 1988; Le Douarin et al., 1993 and references therein)
were also malformed (e.g. maxillary and palatine bones) or
hypoplastic (e.g. alisphenoid) (compare X, AL and PL in Fig.
3k,m). Surprisingly, however, the mandibular (dentary) bone
(compare D in Fig. 3a,c,d), the temporomandibular joint (not
shown), the malleus middle ear ossicle (not shown) and the
tympanic bone (T, compare Fig. 3k with m) appeared normal,
as did the patterning of the lower dentition and the shape
(cuspal pattern) of the first and second upper and lower molars
and of the lower incisors (compare UM and LM in Fig. 4a,c,
and data not shown). Second and third pharyngeal arch-derived
skeletal elements were either absent (stapes, not shown) or
highly malformed (styloid and hyoid bones, see the accompanying study and data not shown).
In non-exencephalic αγ mutants, the skull vault caudal to the
frontal region was complete, although markedly underossified
[compare the size of the parietal (P) and interparietal (IP) bones
in Fig. 3a,d; also note the absence of interparietal (IP) and
supraoccipital (S) ossification centers in Fig. 3c]. In contrast,
the entire cranial vault was absent in exencephalic mutants
(Fig. 7k).
The cartilaginous otic capsule was always small and incomplete in αγ mutants (O, compare Fig. 3a,c,d), resulting in a
cystic protrusion of the epithelial inner ear within the braincase
(not shown). These malformations are likely to be secondary
to defects of the otocyst (discussed in Mark et al., 1993), which
was consistently hypoplastic in 10.5 dpc αγ mutants (not
shown). That the epithelial inner ear is a RA-target organ
affected early during embryogenesis is supported by the
absence of some of its derivatives (i.e. the spiral organ of Corti
and the spiral ganglion, data not shown) in 18.5 dpc αγ fetuses.
Although less affected than αγ mutants, α1γα2+/− mutants
(but not α1γ mutants) also exhibited several defects of the
cranial skeleton. These included shortening of the frontal bone
(F, compare Fig. 3a and b), duplication of the cartilaginous
nasal septum (NS, compare Fig. 3k and l), cleft palate, aplasia
of the presphenoid (PS, compare Fig. 3e and f), persistence of
the hypophyseal foramen (arrowhead Fig. 3f) and absence of
the incisive foramen (IF, compare Fig. 3k and l). The latter
malformation was closely correlated with the absence or
dysplasia of the upper incisors and may be secondary to this
2730 D. Lohnes and others
RARs in ontogenesis 2731
Table 2. Fusion of the incus with a cartilaginous or osseous
element in RAR double mutant fetuses
Genotype and number
of 18.5 dpc fetuses
examined
WT
RARα1γ
RARα1γα2+/−
RARαγ
RARα1β2
RARαβ2+/−
RARαβ2
RARβ2γ
16
10
8
6
8
9
16
11
Unilateral
fusion
Bilateral
fusion
Fusion
frequency
(%)
0
0
3
0
2
3
5
0
0
0
1
6
1
2
7
0
0
0
31
100
25
39
59
0
defect. Ectopic cartilaginous and bony nodules, formed from
the meninges, were also found in 18.5 dpc αγ and α1γα2+/−
mutant fetuses. In particular, in αγ mutants, the falx cerebri
was completely chondrified (FX, Fig. 4c).
(2) Supernumerary cranial skeletal elements
With the exception of the ectopic cartilaginous and bony
deposits, the above skeletal abnormalities correspond to deficiencies, including the duplicated nasal septum which arises
by failure of coalescence of the nasomedial processes (De
Myer, 1975). Aside from these deficiencies and ectopias, two
supernumerary skeletal elements were frequently detected.
In placental mammals, two cartilaginous pillars, the pila
prooptica and pila metoptica, connect the orbital (optic) region
of the fetal skull to the floor of the braincase (De Beer, 1985).
In normal mice at 18.5 dpc, these pilae (PP and PM in Fig. 3e)
are located on either side of the optic foramen (OF) and are
fused ventrally to the presphenoid bone (PS). All 18.5 dpc αγ,
α1γα2+/− and α1γ mutant fetuses possessed a third, more caudal,
cartilaginous pillar which was fused ventrally to the basisphenoid bone to form a cartilaginous medial wall to the cavum
epiptericum [PA, compare Fig. 3e with f, and Fig. 4e-g with 4d;
the size of this additional pillar was greater in αγ and α1γα2+/−
(not shown) mutants than in α1γ mutants]. The cavum epiptericum (bracketed in Fig. 4d), which corresponds to a normal
extracranial space (i.e. located outside of the dura mater), is so
called as it is limited laterally by the alisphenoid bone (i.e. the
mammalian homologue of the reptilian epipterygoid bone) (AL,
Figs 3e,k, 4d). This cavum lodges the trigeminal ganglion (G5,
Fig. 4d-f) and is crossed by cranial nerves III, IV, V (N5, Fig.
4g) and VI, and the internal jugular vein (JV, Fig. 4g).
Fig. 4. Comparison, on frontal histological sections, of craniofacial
skeletal structures between 18.5 dpc wild-type (WT; a,d) and RAR
double mutant fetuses (b,c,e-g). The genotype of the mutant fetuses
is indicated in the upper-right corner of each micrograph. (a-c)
Sections at the level of the first upper (UM) and lower (LM) molars.
(d-g) Sections through the cavum epiptericum (brackets in d).
Abbreviations: AL, alisphenoid bone; BR, brain; BS, basisphenoid
bone; CP, cleft of the palate; EY, eye : FX, chondrification of the
falx cerebri; G5, ganglion of the trigeminal nerve (5th cranial nerve);
HY, pituitary gland (hypophysis); JV, jugular vein; LC, lamina
cribriform of the ethmoid bone; LM, lower molar; N5, trigeminal
nerve (5th cranial nerve); NC, nasal capsule; NS, nasal septum; OL,
olfactory epithelium; P, secondary palate; PA, pila antotica; PT,
pterygoid bone; TO, tongue; UM, upper molar. (c) Asterisks indicate
cartilaginous rods and nodules replacing the nasal septum and nasal
capsule. Magnifications: ×16 (a,b); ×20 (c-f); ×32 (g).
In mammals, the alisphenoid bone contributes to the base
and lateral walls of the skull between the optic and otic regions
where it forms the lateral limit of the cavum epiptericum,
whereas the incus (Fig. 3g) represents one of the three middle
ear ossicles. In the middle ears of α1β2, αβ2+/−, αβ2, α1γα2+/−
and αγ, but not α1γ or β2γ mutants, the medial aspect of the
body of the incus was continuous with a rostrally oriented cartilaginous or osseous rod (Q, Fig. 3h-j) which was frequently
fused to the alisphenoid bone (Table 2). In a number of these
mutants, the short process of the incus (SI) was conspicuously
larger than its wild-type homologue (compare Fig. 3g and i).
(D) Brain abnormalities
The brains of 18.5 dpc α1β2, αβ2 and β2γ mutants appeared
normal. In contrast, in 10.5 dpc αγ mutants, a wide persistent
opening of the rhombencephalic neural tube was frequently
observed (see above and Fig. 2e). The midbrain and forebrain
of these exencephalic embryos were invariably closed and
covered by ectoderm (Fig. 2d,e). However, the neurectoderm
of the telencephalic vesicles (TE) was abnormally folded and
the lateral ventricles (LV) were collapsed (compare Fig. 2g,h).
Histological analysis of two 18.5 dpc exencephalic fetuses
showed a lack of hindbrain and cerebellar structures; furthermore the cerebral hemispheres were small and displayed
hemorragic foci (not shown). Taken together with the finding
that persistent opening of the rhombencephalon at 10.5-11.5
dpc and exencephaly at 18.5 dpc occurred with similar frequencies (Table 1B), these results suggest that failure of
closure of the rhombencephalic neural tube is the primary
defect leading to exencephaly. This failure, which leaves the
rhombencephalic neurectoderm exposed to the amniotic fluid,
may result in rhombencephalon degeneration and impair the
accumulation of cerebrospinal fluid in the ventricular system.
The absence of hydrostatic pressure would then lead to the
abnormal folding of the cerebral hemispheres (Pexieder and
Jelinek, 1970; Jacobson, 1981), thus altering the relationship
between the neurectoderm and overlying osteogenic cranial
mesectoderm and subsequently impairing the epithelial-mesenchymal skeletogenic interactions required for the formation
of the bones of the skull vault (Hall, 1991). The improper interactions between the folded neuroepithelium and presumptive
dermis might also lead to the absence of skin covering the brain
in 18.5 dpc exencephalic mutants.
The brain of 18.5 dpc non-exencephalic αγ mutant fetuses
appeared considerably distorted (BR, Fig. 4c and data not
shown), probably secondary to increased intracranial pressure
caused by the shortening of the braincase and compression by
intracranial ectopic cartilaginous and bony nodules (e.g. FX in
Fig. 4c). In addition, failure of the rostral interhemispheric
commissures (i.e. corpus callosum, hippocampal commissure
and anterior commissure) to cross the midline was consistently
observed, a condition that is also frequently encountered in
humans with median cleft face syndrome (De Myer, 1975;
Cohen and Sulik, 1992). In the mutant hindbrain, the motor
nucleus of the abducens nerve (derived from rhombomeres 5
and 6, Lumsden et al., 1991) was not identifiable (not shown);
whether this reflects a primary effect on the hindbrain or is
secondary to the abnormalities present in the ocular region (see
below) is unclear, since the target organ of the abducens nerve
is the external rectus muscle of the eye.
Distortions of the brain and absence of the interhemispheric
2732 D. Lohnes and others
RARs in ontogenesis 2733
Table 3. Main abnormalities of the eye of RAR double null mutants
RAR mutant genotype and number of 18.5 dpc fetuses examined
Coloboma of the retina
Coloboma of the optic nerve
Fibrous retrolenticular membrane
Unfused eyelids
Small conjunctival sac
Corneal - lenticular stalk
Abnormal corneal stroma
Absence of the anterior chamber
Abnormal lens fibers
Agenesis of the cornea and conjunctiva
Agenesis of the lens
Shorter ventral retina
α1γ
5
α1γα2+/−
5
αγ
6
αβ2+/−
4
α1β2
3
αβ2
7
β2γ
3
VAD
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4/10
0
4/10
0
0
0
0
0
0
12/12
12/12
12/12
12/12
12/12
4/12
8/12
12/12
4/12
4/12
2/12†
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4/6
0
0
0
0
0
0
0
0
0
0
0
14/14
0
0
0
0
0
0
0
0
0
1/6
6/6
6/6*
0
6/6
0
6/6
6/6
0
0
0
0
+
+
+
+
+
NR
+
+
+
NR
NR
+
The number of eyes showing the abnormality versus eyes analysed on serial sections is given; *chondrified. †Observation made on a 12.5 dpc embryo (see
text). Note that all abnormalities were found bilaterally, with the exception of coloboma of the retina in β2γ mutants.
NR =not reported.
commissures was also found in two out of five 18.5 dpc
α1γα2+/− mutants (BR, Fig. 4b). In these mutants, which
display nearly normal eyes, the abducens nucleus was always
present. The brains of α1γ mutants, which did not exhibit gross
cranial skeletal malformations, appeared normal.
(E) Eye defects
The eye of a normal 18.5 dpc fetus is covered by fused eyelids
(Y, Fig. 5a,e). A wide conjunctival sac (J, Fig. 5a,e) is present
between the lids and the cornea (C). There are two cell-free
spaces, one between the cornea and the lens (the anterior
chamber; A, Fig. 5a,e), and the other between the lens and the
retina (the vitreous body; V, Fig. 5a,e). The retina, comprising
the internal (neural) and external (pigmented) epithelia (IR and
OR, Fig. 5a,e), encloses the lens and the vitreous body and is
continuous with the iris (I, Fig. 5a,e) laterally.
RARαγ, α1γα2+/−, α1β2, αβ2 and β2γ mutants exhibited a
number of ocular defects (Table 3). Microphtalmia, coloboma
of the retina and abnormalities of the cornea, eyelids and conjunctiva, were constant features of 18.5 dpc αγ fetuses. As
shown in Fig. 5c, the eye was conspicuously smaller than
normal (compare Fig. 5a and c). The dorsal margin of the retina
extended well past the equator of the lens (L, Fig. 5c), where
it formed a rudimentary iris (I, Fig. 5c). Ventrally, the retina
barely reached the equator (possibly secondary to the
coloboma), so that the ventral lens region was in direct contact
with periocular mesenchyme (PO, Fig. 5c). Medially, a second
Fig. 5. Comparison of frontal sections through the left eye between
18.5 dpc (a,e) wild-type (WT) and (b-d,f-i) RAR double mutant
fetuses; the genotype of the mutant fetuses is indicated in the upper
right corner of each micrograph. Abbreviations: A, anterior chamber
of the eye; C, corneal stroma; EP, corneal epithelium; ER, everted
retina; F, fibrous retrolenticular membrane; I, iris, IR, internal leaf of
the retina; J, conjunctival sac; L, lens; ON, optic nerve; OR, external
(outer or pigmented) leaf of the retina; PO, periocular mesenchyme;
V, vitreous body; Y, eyelids; asterisks, cartilaginous nodules
replacing the ethmoid bone. (g) The large black arrow points to the
persistent corneal-lenticular stalk. (i) The arrow points to a
cartilaginous nodule developed from the fibrous retrolenticular
membrane. Magnifications: ×46 (a-d,h); ×76 (e,f,i); ×229 (g).
ventral gap permitted communication between periocular mesenchyme and persistent retrolenticular mesenchyme (F, Fig.
5c), which occupied the space normally taken by the vitreous
body (V, Fig. 5a). The two ventral gaps were joined caudally.
In this plane of section, however, they were separated by a
portion of the retina with a duplicated internal leaf. This aspect
is characteristic of an eversion of the retina (ER, Fig. 5c),
which is thought to arise by metaplastic transformation of
pigmented epithelium into neural epithelium (Coulombre and
Coulombre, 1977). The cleft in the ventral portion of the retina,
the penetration of the optic cup by mesenchymal tissue and the
eversion of the retina in the cleft region are characteristic of
the typical complete coloboma of the retina (Mann, 1937). The
developmental fault underlying this defect is a complete persistence of the optic fissure (also called fetal or choroid fissure,
which normally closes completely by 14.0 dpc) through its
entire length from the region of the optic disc (the optic nerve
exit point) to the iris. Additional defects in this eye included:
absence of fusion of the eyelids (compare Y, in Fig. 5a and c),
agenesis of the upper (dorsal) conjunctival sac (compare J, in
Fig. 5a and c), persistent corneal-lenticular stalk (see below),
absence of differentiation of the corneal stroma (C, in Fig. 5a,e)
and absence of the anterior chamber of the eye (A, in Fig. 5a,e).
Another αγ mutant eye is displayed in Fig. 5d showing a cleft
restricted to the medial portion of the ventral retina (i.e. typical
‘partial’ coloboma of the retina). The corneal stroma was
present (C, in Fig. 5d,f), but was fused with that of the iris (I,
Fig. 5e,f), resulting in an absence of the anterior chamber; the
corneal epithelium was locally hyperplastic and keratinized
(EP, compare Fig. 5e and f). Additional ocular abnormalities
in two 18.5 dpc exencephalic αγ mutants included agenesis of
the conjunctiva and cornea, and abnormal lens fibers (data not
shown). Primary aphakia (i.e. failure of lens formation) was
observed bilaterally in one non-exencephalic 12.5 dpc αγ
mutant (data not shown; see also Table 3).
Interestingly, the only ocular malformations found in two of
five α1γα2+/− mutants were unfused eyelids (not shown) and
a corneal-lenticular stalk. This latter abnormality was characterized by a persistent continuity of the corneal and lens
epithelia at the center of the cornea (large arrow, Fig. 5g),
which most probably results from the failure of the lens cup to
2734 D. Lohnes and others
pinch off from its parental surface ectoderm (Coulombre and
Coulombre, 1977). The eyes of α1γ mutants were unaffected
(Fig. 6b; Table 3).
A partially chondrified fibrous retrolenticular membrane (F
and arrow, Fig. 5h,i) was a specific feature of β2γ mutants. It
was always continuous with a thick strand of mesenchyme,
which was embedded in the optic nerve (ON, Fig. 5h) and was
carried along with it into the eye through a coloboma of the
optic nerve. The retina appeared normal, with the exception of
a unilateral coloboma of the ventral retina in one out of three
mutants (see ER, Fig. 5h). All β2γ eyes also exhibited a small
conjunctival sac (J, compare Fig. 5a and h, and Fig. 5e and i),
poorly differentiated corneal stroma and absence of the anterior
chamber of the eye (Fig. 5h,i; see also Table 3).
A fibrous retrolenticular membrane (F, Fig. 5b) was the only
abnormality present in the eyes of all αβ2 and of two (out of
three) α1β2 18.5 dpc fetuses (Table 3). It is noteworthy that,
with the exception of the fibrous retrolenticular membrane, the
ocular defects were confined to animals disrupted for RARγ
plus either α or β2 (Table 3).
(F) Glandular defects
The intraorbital, submandibular and sublingual glands and
their ducts were normal in 18.5 dpc α1β2 and αβ2 mutants
(Table 4). In contrast, in all 18.5 dpc α1γ (Fig. 6b) and β2γ
(not illustrated) mutant fetuses, the epithelial rudiments of all
intraorbital glands [which include the lachrymal and Harderian
glands (HG, Fig. 6a)] were missing bilaterally (Table 4).
However, the neural crest-derived, melanocyte-containing
stroma of the Harderian glands was always present (data not
shown; see Lohnes et al., 1993). The nasolachrymal duct,
which was consistently missing in α1γ fetuses (compare NLD
in Fig. 6c and d), was sometimes present in β2γ fetuses, indicating that its development was independent from that of the
lachrymal glandular epithelium.
The parenchyma of the submandibular and sublingual
salivary glands develop from downgrowth of the buccal
ectoderm and from the adjacent mesenchyme, whereas the
main ducts of these two glands are formed by closure, in a
rostral direction, of gutter-like grooves in the oral ectoderm
(Hamilton et al., 1945). In 18.5 dpc wild-type fetuses, the two
ducts (MAD and LID, Fig. 6e) open in the mouth cavity at the
sublingual caruncle, a median mucosal fold located at the level
of the rostral third of the lower incisors (LI, Fig. 6e). Both ducts
were shortened in all α1γ fetuses (Table 4): the submandibular duct (MAD, Fig. 6f) opened at the caudal end of the lower
incisor (LI, Fig. 6f), whereas the sublingual duct opened even
more caudally at the level of the 2nd lower molar (not shown).
The sublingual caruncle was always absent. In contrast, only
the sublingual duct was shortened in β2γ fetuses and the sublingual caruncle was always present (Table 4). Dysplasia of the
sublingual gland, consisting of cystic epithelial formations
within the parenchyma (compare LIG, Fig. 6g and h), was frequently observed bilaterally in α1γ fetuses (Table 4), whereas
the submandibular glands appeared normal (compare MAG,
Fig. 6g with h; Table 4).
As expected, glandular defects similar those described above
in α1γ mutants were found in all α1γα2+/− and αγ 18.5 dpc
fetuses (Table 4), with agenesis of both sublingual and submandibular glands being occasionally observed (Table 4).
(G) Abnormalities of the axial skeleton
RARγ null mice exhibit various vertebral abnormalities which
include homeotic transformations and affect primarily the
cervical region (Lohnes et al., 1993). These abnormalities
occurred with variable penetrance and bilateral expressivity,
although the majority (89%) of RARγ null offspring exhibited
one or more vertebral malformations. Although vertebral
defects were not initially found in RARα null mutants (Lufkin
et al. 1993), analysis of RARα null offspring inbred in a 129
Table 4. Abnormalities of the Harderian, sublingual and submandibular glands and their associated excretory ducts in
RAR double null mutants
RAR mutant genotype and number of
18.5 dpc fetuses examined†
α1γ
5
α1γα2+/5
αγ
5
β2γ
3
3 (B)
1 (B)
2 (U)
Agenesis of the Harderian glands*
5 (B)
5 (B)
5 (B)
Agenesis of the nasolachrymal duct
5 (B)
5 (B)
5 (B)
Cystic dysplasia of the sublingual gland
4 (B)
1 (B)
3 (U)
NA
0
0
2 (B)
2 (B)
1 (U)
0
Agenesis of the sublingual gland and duct
0
1 (B)
3 (U)
5 (B)
0
Agenesis of the submandibular gland and duct
0
0
2 (B)
1 (U)
0
Shortening of the sublingual duct
5 (B)
1 (B)
3 (U)
NA
3 (B)
Shortening of the submandibular duct
5 (B)
5 (B)
5
5
Cystic dysplasia of the submandibular gland
Absence of the sublingual caruncle
*The lachrymal glands are not individualized at this stage.
†No abnormalities were seen in α1β2 and αβ2 double null mutants.
NA, not applicable; B, bilateral defect; U, unilateral defect.
2 (B)
1 (U)
5
0
0
RARs in ontogenesis 2735
Fig. 6. Comparison of the Harderian glands, nasolachrymal duct, and sublingual and submandibular glands and ducts between (a,c,e,g) day
18.5 wild-type (WT) and (b,d,f,h) α1γ fetuses. (a,b) Frontal sections at the level of the eye and upper molar (UM). (c,d) Frontal sections at the
level of the upper incisors (UI). (e,f) Frontal sections at the level of the rostral third (e) or caudal end (f) of the lower incisors (LI). (g,h)
Sections through the submandibular (MAG) and sublingual (LIG) glands. Abbreviations: CY, epithelial cyst in the sublingual gland; HG,
Harderian gland; L, Lens; LI, lower incisor; LID, main duct of the sublingual gland; LIG, sublingual gland; MAD, main duct of the
submandibular gland; MAG, submandibular gland; N5, maxillary branch of the trigeminal nerve (5th cranial nerve); NLD, nasolachrymal duct;
NS, nasal septum; NT, nasal cavity; PX, incisive (premaxillary) bone; R, retina; TO, tongue; UI, upper incisor; UM, first upper molar. The
large arrows in (c) and (d) point towards the midline. Magnifications: ×30 (a,b,e,f); ×75 (c,d,g,h).
2736 D. Lohnes and others
SV background (third generation of inbreeding) revealed a low
frequency of malformations affecting the second (C2) and third
(C3) cervical vertebrae (Table 5).
(1) Homeotic transformations
With the exception of fusion of the basioccipital bone with the
anterior arch of the atlas, which likely represents a posterior
homeotic transformation (Lohnes et al., 1993), α1γ and
α1γα2+/− mutants showed increases in the frequency of all
homeotic transformations previously observed in RARγ null
fetuses (Table 5). Anterior transformation of C2 to a first
cervical identity, evidenced by an increase in the thickness of
the neural arches and the appearance of an ectopic anterior arch
appeared two to four times more frequently in α1γ and
α1γα2+/− mutants, respectively, when compared to γ null
fetuses (this transformation was, however, incomplete since
the axis dens was unaffected). Although an ectopic anterior
arch of atlas was occasionally observed in αγ skeletons (e.g.
AAA* in Fig. 7i and l), the severe malformations of the
cervical vertebrae in these mutants usually precluded evaluation of homeosis.
Anterior transformation of C6 or C7 to a C5 or C6 identity,
respectively, was found twice as frequently in α1γ null mutants
and approximately five times more frequently in α1γα2+/−
mutants than in RARγ null fetuses (Table 5 and data not
shown). C7 to C6 transformation was evidenced by the appearance of tuberculi anterior (TA) on the ventral aspect and of
foramina transversaria on the lateral processes of C7 (see
Lohnes et al., 1993 and Fig. 7d). C6 to C5 transformation was
inferred from loss of the C6-specific tuberculi anterior. Interestingly, in 50% of the affected α1γα2+/− mutants, the C6 to
C5 and C7 to C6 transformations were bilateral (data not
shown), whereas these transformations were essentially unilateral in RARγ and α1γ null mutants. Additional anterior
transformations of cervical vertebrae may exist in α1γ and
α1γα2+/− offspring that cannot be identified due to the lack of
morphological landmarks (see Kessel and Gruss, 1991). Since
the C2 to C1, C6 to C5 and C7 to C6 anterior transformations
were found simultaneously in two α1γ and in six α1γα2+/−
mutants (data not shown), the entire cervical region may have
undergone an anterior transformation in these fetuses.
RARα1γ, α1γα2+/− and αγ (but not γ) mutants also exhibited
a posterior homeotic transformation characterized by an
extensive rib anlage on C7, which in some cases fused
ventrally with the first thoracic rib (data not shown; Table 5).
This transformation was usually unilateral and the ectopic C7
rib never contacted the sternum. Ectopic cervical ribs (CR)
were found unilaterally on both C7 and C6 in two αγ mutants,
with the C6 rib joining the cervical rib projecting from C7
(compare Fig. 7i with d; Table 5). These additional ribs did not
alter the total number of presacral vertebrae, thus this likely
represents homeotic transformations of C6 and C7 to thoracic
vertebral identities. (‘T1’ and ‘T2’ in Fig. 7i).
Relative to RARγ null offspring, αβ2 mutant skeletons
exhibited an increase only in the frequency of anterior transformations of C6 to C5, and C7 to C6, which were bilateral
transformations in 50% of the cases (Table 5 and data not
shown), whereas RARβ2γ mutants did not exhibit any increase
in the frequency of the homeotic transformations described for
RARγ null mutants (Table 5). No skeletal abnormalities were
found in α1β2 mutant fetuses.
(2) Malformations of the axial skeleton
In RARα1γ, α1γα2+/− and β2γ mutants, bifidus of C1 and/or
fusion with C2 occurred eight to ten times more frequently than
in RARγ null mutants (Table 5 and data not shown). A similar
increase was also observed for fusion of the neural arches of
C2 and C3 (compare Fig. 7d and e, white arrow; Table 5 and
data not shown). RARα1γ, α1γα2+/− and β2γ mutants also
exhibited malformations not previously observed in RARγ null
.
Table 5 Axial skeletal malformations in RAR double mutants
Genotype and number of 18.5 dpc mutant featuses examined
Number of mutants with abnormal skeletons
ABNORMALITIES
Homeotic Transformations
Basioccipital AAA fusion
Anterior transformations of C2 to C1
C6 to C5
C7 to C6
Posterior transformations of C6 to T1
C7 to T1 or T2
Malformations
C1 malformed
C2 malformed
Fusions of cervical neural arches
Agenesis of cervical neural arches
Dyssymphysis of cervical neural arches
Ectopic bone in cervical region
Rib fusions
Basioccipital Exoccipital fusion
Sternum malformations
RARα
21
RARγ
29
RARα1γ
16
RARα1γα2+/−
11
RARαγ
6
RARβ2γ
9
RARαβ2
10
4(19%)
25(86%)
16(100%)
11(100%)
6(100%)
7(78%)
10(100%)
0
1(5%)
0
0
0
0
8(28%)
5(17%)
4(14%)
4(14%)
0
0
3(19%)
5(31%)
4(25%)
4(25%)
0
6(38%)
2(18%)
7(64%)
8(73%)
8(73%)
0
3(27%)
NA
NA
NA
NA
2(33%)
4(67%)
2(22%)
1(11%)
0
0
0
0
0
2(20%)
8(80%)
8(80%)
0
0
0
3(14%)
1(5%)
0
3(14%)
0
0
0
0
2(7%)
3(10%)
5(17%)
0
0
0
4(14%)
0
0
10(63%)
4(25%)
5(31%)
0
10(63%)
0
3(19%)
0
6(38%)
9(82%)
5(45%)
11(100%)
0
10(91%)
0
2(18%)
0
3(27%)
6(100%)
6(100%)
6(100%)
6(100%)
6(100%)
2(33%)
2(33%)
1(17%)
6(100%)
5(56%)
5(56%)
6(66%)
0
2(22%)
0
0
0
0
8(80%)
7(70%)
4(40%)
0
4(40%)
0
0
7(70%)
0
Skeletal malformations were not observed in RARα1 or RARβ2 single mutants, nor in RARα1β2 double mutants.
NA, not applicable; AAA, anterior arch of atlas.
RARs in ontogenesis 2737
Fig. 7. Malformations of the axial skeleton in RAR double mutant mice. (a-c) Dorsal views of the cervical and upper thoracic region of wild-type
(a) and αβ2 mutant (b,c) skeletons. (b) The long arrow indicates bifidis of the neural arch of the first cervical vertebra, the short arrow indicates
fusion between the neural arches of the second and third cervical vertebrae. (c) The asterisk denotes dyssymphysis of the first cervical vertebra. C1
to C7, first to seventh cervical vertebrae. (d,e,f,i) Lateral views of the cervical and upper thoracic region of wild-type (d) α1γα2+/− mutant (e) and
αγ (f,i) mutant skeletons. (e,f) Asterisks indicate dyssymphysis of the first cervical vertebrae. (e) Arrows indicate fusions between the neural
arches of the second and third and third and fourth cervical vertebrae. (f) Arrow indicates fusion of the neural arches of the third, fourth and fifth
cervical vertebrae. (i) AAA* indicates an ectopic anterior arch ventral to the second cervical vertebra, CR indicates cervical ribs and ‘T1’ and ‘T2’
denote posterior transformation of the sixth and seventh cervical vertebrae to a first and second thoracic identity, respectively. (f,i) Note the
agenesis of the neural arch of the second cervical vertebrae. (g,h) Ventral views of the cranial base of wild-type (g) and αβ2 mutant (h) skeletons.
(h) The asterisk indicates an ossified fusion between the basioccipital (BO) and exoccipital (E) bones. (j-l) Lateral views of the cranial to lumbar
regions of a wild-type (j) and αγ mutant (k,l) skeletons. (k) EC indicates an ectopic ossified structure in the cervical region. Note also the complete
lack of the cranial vault of this specimen. (l) Asterisk denotes dyssymphysis of the first cervical vertebra and AAA* indicates an ectopic anterior
arch. Note also the agenesis of the neural arch of the second cervical vertebra. T1, first thoracic vertebra; L1, first lumbar vertebra.
2738 D. Lohnes and others
Fig. 8. Limb defects in αγ double mutants. (a,b) External ventral views of wild-type (a) and αγ mutant (b) right forelimbs; I* indicates a
supernumerary preaxial digit; I-V, first to fifth digits. (c,d) Frontal external views of a wild-type (c) and αγ double mutant (d) left forelimbs;
note the abnormal aspect and fusions of the digits. (e,f) Dorsal external views of the right forefeet of a wild-type (e) and an αγ mutant (f); note
the abnormal aspect of the fifth digit. (g,h) Lateral views of the right hindlimbs of a wild-type (g) and an αγ mutant (h); open arrowhead
indicates delayed ossification and abnormal appearance of the phalanges; F, fibula; T, tibia. (i-l) Whole-mount skeletal stainings of the
forelimbs of wild-type and αγ double mutants. (i) Small arrow indicates supernumerary preaxial digit. Arrows (j-l) indicate missing first digit.
Asterisks (i-l) indicate malformations of the scapulae. Note that the αγ mutant limbs (i,j) were from the same animal. H, humerus; R, radius; S,
scapula; U, ulna.
RARs in ontogenesis 2739
offspring, including dyssymphysis of the neural arch of C1
(asterisk in Fig. 7e compare to 7d) or C2 (not shown), and
fusion of C3 with C4 (white arrow in Fig. 7e compare to 7d).
RARαγ mutant fetuses had severe defects of all cervical
vertebrae, usually making detailed analysis impossible, save
for cervical ribs on C6 or C7 (discussed above). Bifidus (not
shown) and dyssymphysis of C1 (asterisks in Fig. 7f,i,l,
compare to Fig. 7d,j) and agenesis or fusions of the neural
arches of C2 to C5 (large arrow in Fig. 7f, compare to 7d; note
the absence of the neural arch of C2 in Figs. 7f,i,l) were
observed in all specimens. In two cases an ectopic ossified
structure between C1 and C4 or C5 was observed dorsal to the
cervical region (EC in Fig. 7k).
Most interestingly, with the exception of rib fusions, which
occurred in α1γ, α1γα2+/− and αγ mutants with a frequency
similar to that in RARγ null mutants (arrow in Fig. 7l; Table
5), vertebrae caudal to the cervical region appeared unaffected
in all double mutants analyzed (e.g. compare thoracic (T) and
lumber (L) vertebrae in Fig. 7j and k; note the complete loss
of the cranial vault of the specimen in Fig. 7k). The only additional malformations found in these animals involved occipital
bones (see below) and the sternum. In some 18.5 dpc α1γ,
α1γα2+/− and αγ mutant fetuses, the sternum was distorted and,
in one case, appeared incompletely closed (arrowhead, Fig. 7l)
and possessed four instead of five sternebrae.
The cervical region of αβ2 mutants was also malformed,
with a high frequency of dyssymphysis and bifidus of C1, and
fusions between C1 and C2 or C2 and C3 (Fig.7a compare to
b and c; arrow in Fig. 7b denotes C1 bifidus and arrowhead
indicates C2-C3 fusion; asterisk in Fig. 7c denotes dyssymphysis of C1). Furthermore, the majority of αβ2 and 1 (of 6) αγ
mutant fetuses exhibited an osseous fusion of the basioccipital
(BO) and exoccipital (E) bones (asterisk in Fig. 7h, compare
to g; Table 5), a malformation not observed in RARγ mutants.
(H) Malformations of the appendicular skeleton
The limbs of all double mutants were essentially normal with
the exception of RARαγ mutants.
(1) External features
Although forelimbs were affected in all 18.5 dpc αγ mutants
examined, the nature of the limb defects showed considerable
variation. Syndactyly was frequently observed (compare Fig.
8e and f). The precise digits fused varied among animals and
fusion was at the level of soft tissues only (see below). Digits
frequently looked abnormal (compare Fig. 8c and d, e and f)
and polydactyly (with 6 digits; compare Fig. 8a and b), or
ectrodactyly (3 or 4 digits; not illustrated) was often apparent.
RARαγ mutant hindlimbs exhibited a consistent aberrant
external aspect with the hindfoot twisted such that the footplate
faced the lateral abdominal wall (compare Fig. 1a and c).
However, no cases of syndactyly or other digit malformations
were found in the hindlimbs upon external examination.
(2) Skeletal analysis
Whole-mount skeletal preparations from six 18.5 dpc αγ
mutants were analyzed in detail. All six exhibited a number of
malformations affecting diverse skeletal elements of the
forelimb. However, the severity and frequency of most of these
defects varied both among animals and between contralateral
forelimbs from the same animal (Table 6).
The entire forelimb was shorter when compared to 18.5 dpc
control littermates (Fig. 8i-l). This may be related to the
general growth deficiency of these mutants (e.g. compare Fig.
1a and c). The scapula was always malformed in αγ mutants.
In one fetus, the two scapulae were partially agenic with a
greatly reduced shaft diameter and vertebral (medial) region
(asterisk in Fig. 8l). In another fetus, the median portion of one
scapula appeared bifurcated (Fig. 8k). In a third fetus, one
scapula was partially agenic, whereas the central portion of the
contralateral scapula was bifurcated (data not shown). The
other scapula defects corresponded to mild aplasia of the
superior or inferior margins of the vertebral region (e.g. Fig.
8i and j, asterisks).
The humerus, although smaller than controls, appeared
otherwise normal (e.g. Fig. 8i-l). In contrast, the radius and
ulna, which were also reduced in size, were abnormal in all αγ
mutants. In four of six fetuses, the radius of either the left or
right forelimb was missing unilaterally (e.g. Fig. 8l; Table 6).
The ulna was always formed, but exhibited an abnormal
curvature as did the radius when present (e.g. Fig. 8i-l).
With the exception of the pyramidal (PY) and pisiform (PI)
bones [the nomenclature of Milaire (1978) is used for the
carpal bones], which were well defined (e.g. compare Fig. 9e
with f and g with h), all other carpal bones were malformed.
The central bone (C) was missing in all but one case (compare
Fig 9e with f, g with h; asterisks denote the missing central
Table 6. Forelimb malformations in RARαγ double null
mutants
18.5 dpc mutant fetuses
Malformation or partial
agenesis of the scapula
Agenesis of the radius
L
R
L
R
Malformation of the
L
scapholunatum
R
Ectopic distal carpal bone L
R
Agenesis of the D1
L
carpal bone
R
Agenesis of the central
L
carpal bone
R
Prepollex agenic or
L
rudimentary
R
6 Digits
L
R
5 Digits
L
R
4 Digits
L
R
3 Digits
L
R
Hypoplastic first digit
L
R
1
2
3
4
5
6
Total
+
+
+
+
+
ND
ND
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
5
5
2
2
6
6
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+*
+
+
+
+†
+†
+
+†
+§
+†
+†
+‡
+
1
3
2
5
6
5
4
0
1
2
2
3
3
1
0
1
0
ND; not determined; *additional presumptive first digit; †loss of
presumptive first digit; ‡loss of presumptive first and second digit; §loss of
presumptive second digit. L and R, left and right forelimb, respectively. 1 to
6, correspond to the different fetuses which were examined. Total, total
number of limbs that exhibited a given malformation.
2740 D. Lohnes and others
bone). The scapholunatum (SL), which was always present
regardless of the absence or presence of the radius (e.g. see SL
in Fig. 9m), was always misshapen and small (compare SL in
Fig 9e and f, g and h), and in many cases was partially cleft
(arrow in Fig. 9f). All distal carpals (D1-D4/5) were consis-
tently hypoplastic (compare Fig. 9e and f; g and h). The
prepollex (PX) was frequently rudimentary or absent (compare
Fig. 9i and j).
In 6 (of 12) mutant forelimbs, only 4 digits were present, but
this defect was bilateral in only one instance (Table 6 and see
RARs in ontogenesis 2741
below). In five cases, the missing digit was presumably digit 1
(e.g. compare Fig. 9c and d), as determined by phalangeal
count (digit one has two phalanges, whereas digits 2 to 5 have
three) and the position of the remaining digits with respect to
the distal carpal bones (using D4/5 as a landmark). The loss of
digit 1 was correlated with the absence of the carpal D1 in four
cases (Table 6 and data not shown). In one other specimen, the
first digit was present, but markedly smaller (Table 6 and data
not shown). In one fetus, which was missing the first digit on
the right forelimb, the second digit was absent on the contralateral forelimb, with only a small cartilaginous anlage
found in its place (arrow II in Fig. 9h). Interestingly, the first
digit in this forelimb resembled a normal second digit rather
than a thumb, as judged by its length and the advanced ossification of the metacarpal bone, yet only two phalanges were
present (not shown). Unilateral ectrodactyly with three digits
was observed in one animal, where digits 1 and 2 were missing
and carpal D1 was agenic (Table 6 and data not shown).
Polydactyly with six digits was found in one mutant
(compare Fig. 9a and b, e and f); the additional preaxial
element was associated with an ectopic distal carpal bone,
whereas the prepollex was lacking (data not shown). The
diameter of the putative metacarpal bone was reduced and only
one phalange was apparent. Interestingly, in this specimen the
normal first digit may have undergone a transformation to a
second digit, as supported by the increase in its length and
diameter, advanced ossification of the metacarpal bone and
larger size of the phalanges (compare digit I in Fig. 9a and b,
e and f; note that the third phalange was broken during preparation of this specimen). Remarkably, the contralateral limb of
this mutant possessed only four digits (Fig. 9d; Table 6).
Additional malformations observed in αγ mutant forelimbs
included a delay in the ossification of the metacarpals and/or
phalanges (e.g. compare Fig. 9c and d). Although this may
result from the general growth deficiency of these animals,
comparison to 18.5 dpc controls of only slightly larger size
(e.g. compare Fig. 9a and b) indicates that this delay may be a
direct effect of the mutations. In this respect, note a complete
lack of metacarpal ossification centers in the mutant forelimb
shown in Fig. 9m (although hypertrophic chondroblasts are
evident), even though ossification of metacarpals 3 and 4
usually commences as early as 15.5 dpc (Dollé et al., 1993;
Kaufman, 1992). The phalanges of αγ mutants were also
abnormal, appearing bulbous with the outline of the synovial
joints between the phalanges difficult to discern (compare Fig.
9a to b, c to d, k to l). This latter malformation occurred irrespective of other forelimb malformations.
Examination of the hindlimbs revealed (in five of six cases)
a dramatic bilateral reduction in the length and increase in the
diameter of the ossification of the fibula (F), with a concomitant bending of the tibia (T; compare Fig. 8g and h; note that a
connection exists between the head of the tibia and the proximal
end of the fibula in the specimen in Fig. 8h). The disproportionate growth between the fibula and tibia is the likely cause
of the bending of the tibia and of the abnormal external aspect
of the hindlimbs. In one αγ mutant fetus, the ossification of the
fibula was intermediate between controls and severely affected
mutants (data not shown). The only additional defects found in
the hindlimbs of RARαγ skeletons were a slight retardation in
ossification of the metatarsals and phalanges, and bulbous
phalanges with indistinct synovial joints as described above for
the forelimbs (compare Fig. 8h and g). No loss or gain of
hindlimb digits or tarsal elements was observed.
The forelimbs of α1γα2+/− fetuses were unaffected.
However, of 11 RARα1γα2+/− fetuses examined, 1 exhibited a
defect of the fibula identical to that shown in Fig. 8h and two
other specimens exhibited a similar, but milder malformation
(data not shown).
DISCUSSION
Fig. 9. Skeletal analysis of the malformations of the forefeet in αγ
mutants. (a,b) Dorsal views of skeletons of the right forefeet of a
wild-type (a) and an αγ mutant (b); I* indicates supernumerary
preaxial digit; note that the adjacent digit (digit I) of this foot
originally possessed three phalanges; I-V, first to fifth digits; R,
radius; U, ulna. (c,d) Dorsal views of the skeletons of the left forefeet
of a wild-type (c) and an αγ mutant (d); arrow indicates missing first
digit. (e,f) Higher magnification of the carpal regions of the
specimens shown in a and b, respectively; asterisk denotes missing
central (C) carpal bone, arrow indicates split in scapholunatum (SL)
and I* indicates supernumerary preaxial digit. Note also the small
size of the distal carpals (D1-D4/5). PI, pisiform carpal bone; PY,
pyramidal carpal bone. (g,h) Dorsal views of the carpal skeleton of
the left forefeet of a wild-type (g) and an αγ mutant (h); asterisk
indicates missing central carpal bone and arrow indicates
cartilaginous rudiment in place of the second digit. Note also the
increased size and the advanced ossification of the first digit in h
compared to g. (i,j) Lateral views of skeletons of the left forefeet of a
wild-type (i) and an αγ mutant (j). Note the reduction of the
prepollex (PX) in j. (k,l) Dorsal views of the phalanges of the forefeet
of a wild-type (k) and an αγ mutant (l); arrows indicate the bulbous
appearance of the phalanges and the indistinct synovial joint between
the second and third phalanges. (m) Dorsal view of the left forefoot
of an αγ mutant. Short arrow indicates hypertrophic chondroblasts
and absence of ossification of the metacarpals and the long arrow
indicates missing first digit. Note also the severe malformation of the
scapholunatum and the absence of the radius.
The present results show that RARs are essential for normal
development of many structures in the mouse. However, with
the exception of malformations of the axial skeleton and
agenesis of the Harderian glands (which have been previously
described in RARγ null offspring; Lohnes et al., 1993), congenital defects are observed only in RAR double mutants.
Offspring from dams fed a vitamin A-deficient (VAD) diet also
exhibit a number of developmental abnormalities (Wilson et
al., 1953). The recapitulation of most of these VAD-associated
congenital malformations (see also Mendelsohn et al., 1994b)
indicate that retinoic acids, the known ligands of the RARs,
most probably represent the active retinoid developmental
signal. With the exceptions of eye defects (Warkany and
Schraffenberger, 1946) and cleft palate (Hale, 1933), the malformations described here in RAR double mutants have not
been reported in VAD studies, probably because extreme
deprivation of vitamin A results in embryonic lethality (Mason,
1935; Wilson and Barch, 1949). Complete inactivation (by full
dietary deprivation of RA) of all RARs is likely to result in
early embryonic death and resorption, as reflected here by the
embryonic death of approximately 50% of the RARαγ double
mutants. These observations indicate that some RA-dependent
developmental processes are more sensitive than others to RA
deficiency (e.g. eye development; see below) and that only
2742 D. Lohnes and others
abnormalities related to these events are observed in VAD
studies.
RARβ isoform transcripts and amount of RARβ2 protein
were not affected in 13.5 or 14.5 dpc αγ double mutant
embryos, respectively, nor was the pattern of expression of
RARβ transcripts apparently altered in 10.5, 11.5 or 13.5 dpc
αγ mutant embryos as judged by in situ hybridization data (our
unpublished results). Thus the extreme malformations specific
to αγ double mutants are unlikely to reflect an additional defect
in RARβ expression. Moreover, since RARβ2 is believed to
be transcriptionally modulated by RA in vivo, its expression
in RARαγ double null mutants may be controlled by the other
RARβ isoforms (RARβ1, β3 and β4), through an autoregulatory mechanism (i.e. by RARβ2 itself), or by other nuclear
receptors, such as the RXRs.
(A) RA is required at several stages during eye
morphogenesis
The eye is the most sensitive organ to retinol deprivation and
in less severely affected VAD fetuses, it is often the only site
of malformation (Warkany and Schraffenberger, 1946). The
spectrum of VAD-induced ocular malformations is largely
recapitulated in αγ (with the exception of a shorter ventral
retina) and to a lesser extent in β2γ, double mutants (see Table
3).
Eye formation involves the coordinated development of
forebrain neuroectoderm (which gives rise to the retina, optic
nerve and epithelial portion of the iris), surface ectoderm
(which gives rise to the lens and epithelia of the cornea and
conjunctiva) and cranial neural crest-derived mesenchyme
(which forms the choroid, sclera, stroma of the cornea and iris,
anterior chamber, and vitreous body; Pei and Rhodin, 1970;
Johnston et al., 1979; Le Douarin et al., 1993; and references
therein). In WT mice at 9.5 dpc, the optic vesicle, which has
evaginated from the forebrain neuroectoderm, comes into
contact with the lens placode, which immediately invaginates
and, at 11.5 dpc, pinches off from its parental surface ectoderm
to become a hollow lens vesicle, leaving behind the presumptive corneal epithelium at the surface. At 10.5 dpc, the optic
vesicle invaginates from its ventral side to form the optic cup
(i.e. the anlage of the retina and of the epithelial portion of the
iris; Kaufman, 1992; Pei and Rhodin, 1970). The area of
invagination represents the optic fissure. Mesoderm extends
from this fissure into the cup forming the primary vitreous
body. The two lips of the optic fissure come into contact and
fuse at 12.5 dpc, except in the regions of the iris and of the
optic disc, where closure is delayed until 14.0 dpc. By 14.5
dpc, the mesenchymal cells of the vitreous body (i.e. the retrolenticular mesenchyme) disappear.
The ocular defects in RAR double mutants correspond
mostly to structures arrested in ontogenesis and partly to
abnormal embryonic formations. Absence of the lens, cornea,
conjunctiva or anterior chamber, persistence of a corneal lenticular stalk or a fibrous retrolenticular membrane, coloboma of
the retina or optic nerve, poor differentiation of the corneal
stroma or lens fibres, hypoplasia of the conjunctival sac and lack
of fusion of the eyelids are developmental arrests. However, the
chondrification of the persistent retrolenticular tissue in
RARβ2γ mice and the keratinization of the corneal epithelium
have no equivalent at earlier developmental stages.
RARs are clearly required for morphogenetic processes at
distinct stages of eye development. The complete absence of
lens tissue observed in one 12.5 dpc RARαγ mutant embryo
likely results from a fault in the process of lens induction
occurring before 9.5 dpc (reviewed in Grainger et al., 1992).
The presence of a corneal lenticular stalk implies an arrest at
11.5 dpc in the separation of the lens from the parental
ectoderm. These processes depend on interactions between the
lens placode and the optic vesicle. Interestingly, the embryonic
retina is capable of synthesizing RA (McCaffery et al., 1992)
and RA-reporter mice suggest that the eye contains RA as early
as 9.5 dpc, with later synthesis (12.5 dpc) in the neural retina
(Balkan et al., 1992; Rossant et al., 1991). These data suggest
that the lack of a RA-dependent inductive signal from the optic
vesicle may be the underlying basis for these lens defects.
The fusion of the two lips of the optic fissure, which starts
with an inversion of the retinal pigmented epithelium, occurs
first between undifferentiated cells at the junction of the neural
retina and the retinal pigmented epithelium, and involves the
disintegration of the basement membrane between the retina
and the mesodermal tissue (Geeraets, 1976; Suzuki et al., 1988;
Hero, 1989, 1990). Partial or complete persistence of the optic
fissure (coloboma) in 18.5 dpc RARαγ and β2γ mutants might
then be caused by: (i) overgrowth of the inner layer of the optic
cup relative to its external counterpart, thus preventing the
normal inversion of the latter along the line of the optic fissure,
(ii) precocious differentiation of the retinal cells located at the
junction between the two layers or, (iii) maintenance of the
basement membrane preventing the fusion of the two lips of
the optic fissure. Further studies at the electron microscopic
level are required to distinguish between these possibilities.
Although the RA-dependent events leading to coloboma are
unknown, this defect appears to be determined shortly before
the begining of the closure of the optic fissure (12.5 dpc), as
administration of vitamin A to VAD rat embryos can reduce
the incidence of coloboma if given before the equivalent of
11.5 dpc of mouse gestation (Wilson et al., 1953). The persistence of the retrolenticular mesenchyme appears to correspond
to a later arrest, since its occurrence could be likewise
prevented in VAD embryos by vitamin A administration before
13.5 dpc (Wilson et al., 1953). The additional malformations
in the eyes of both VAD offspring and RAR double mutants
(including isolated persistent retrolenticular mesenchyme and
malformations of the eyelids, conjunctival sac, cornea and
anterior chamber) may be due to defects in mesenchymal NCC,
as all of these structures are derived from NCC originating
from the forebrain and/or midbrain of the developing embryo
(see below; Serbedzija et al., 1992; Le Douarin et al., 1993 and
references therein).
The major ocular defects were found in RARαγ and
RARβ2γ double null mutants, suggesting that RARγ is
essential (in the absence of RARα1 and RARα2 or RARβ2)
for normal eye development. Furthermore, in contrast to
RARαγ mutants, RARα1γα2+/− double mutants had near
normal eyes, suggesting that (in the absence of RARγ) one
copy of RARα2 suffices for most of the events needed for eye
development. The eye defects observed in RAR mutants are in
good agreement with the presence of RA in the eye (see above)
and the expression pattern of the RARs during the period of
eye development known to be sensitive to vitamin A deprivation (8.5 to 13.5 dpc in the mouse; Dollé et al., 1990; Ruberte
RARs in ontogenesis 2743
et al., 1990, 1991). RARα is expressed ubiquitously, whereas
RARγ and RARβ are expressed in the periocular mesenchyme
throughout this period of development. Furthermore, RARβ2
(but not RARγ) transcripts are also detected at 12.5 to 13.5 dpc
in the retrolenticular mesenchyme (our unpublished results),
the abnormal persistence of which is the only ocular abnormality found in RARα1β2 and αβ2 double mutants. Thus, in
these mutants, this abnormality may be a primary defect
whereas, in RARαγ and β2γ mutants, it may be secondary to
the coloboma.
(B) RARs and specification of the axial skeleton
The homeotic transformations and other axial malformations
that occur in RAR double mutants are strikingly confined to
cervical vertebrae. The present results indicate that RARs may
be functionally redundant for specification of these vertebrae,
as the penetrance and expressivity (bilateral versus unilateral
defects) of cervical anterior transformations previously
observed in RARγ null offspring (Lohnes et al., 1993)
increased in a graded manner with subsequent loss of RARα1
and RARα2 isoforms from the RARγ−/− background. Furthermore, RARβ2 (in the absence of RARα) also appears to play
a role in axial specification, as RARαβ2 double mutants (but
not RARα mutants) displayed a high frequency of anterior
homeotic transformations, particularly of the sixth and seventh
cervical vertebrae. That the posteriorization of the sixth and/or
seventh cervical vertebrae was observed only in α1γ, α1γα2+/−
and αγ double null mutants suggests that RARα and RARγ are
redundant with regard to events leading to these posteriorizations and that RARβ2 has no role in this particular event.
Gain-of-function (Kessel et al., 1990: Lufkin et al., 1992)
and loss-of-function (Le Mouellic et al., 1992; Ramirez-Solis
et al., 1993; Jeannotte et al., 1993; Condie and Capecchi, 1993)
studies have shown that some Hox genes specify the identity
of somites. Although there are notable exceptions (Pollock et
al., 1992; Jegalian and De Robertis, 1992), Hox gain-offunction mutations usually lead to posteriorization, while Hox
loss-of-function mutations lead to anteriorization of vertebral
identities. RAR double mutants and some Hox null mutants
exhibit similar cervical vertebral transformations, suggesting
that RA may affect vertebral patterning by controlling Hox
gene expression. The most notable similarities are with Hoxb4 null mice, which exhibit anterior transformation of C2 to a
C1 identitiy (Ramirez-Solis et al., 1993), and with Hoxa-5 null
mice, which exhibit anterior transformation of C6 to a C5
identity and posterior transformation of the C7 to a T1 identity
(Jeannotte et al., 1993). That Hox gene expression may be controlled by RA during development has been suggested by the
observation that some Hox gene transcripts accumulate in
cultured embryonal carcinoma (EC) cells exposed to RA
(Simeone et al., 1990, 1991; Mavilio, 1993 and refs therein)
and by the presence of functional RAREs in the promoter
region of some of these RA-responsive genes (e.g. Hoxa-1,
Langston and Gudas, 1992; and Hoxd-4, Pöpperl and Featherstone, 1993). That only cervical vertebrae were transformed in
RAR double mutants may reflect the greater sensitivity (at least
in EC cells) of 3′ Hox paralogues to RA (Mavilio, 1993). Some
of these 3′ genes may be preferentially affected in RAR double
mutants, leading to selective vertebral transformation in the
cervical region. The severe malformations of cervical vertebrae
observed in αγ double mutants may occur when the expression
of several of these Hox genes are concommitantly altered.
In situ hybridization studies have shown that RARγ (and α)
are expressed posterior to the caudal neuropore in the late gastrulating mouse embryo in all three germ layers prior to somite
formation. RARγ expression then apparently disappears concomitant with the appearance of somites (Ruberte et al., 1990),
suggesting that RARγ and α may control Hox gene expression
during somite formation and specification. This also coincides
with the embryonic stages when RA excess affects both Hox
gene expression and vertebral identities (Kessel and Gruss,
1991). However, there is also evidence indicating that vertebral
transformations can occur at later stages (10.5-11.5 dpc)
through mechanisms not involving Hox genes (Kessel, 1992).
At these later stages, RARα and γ transcripts are found in sclerotomes (Ruberte et al., 1990, 1991; Dollé et al., 1990), suggesting that RA could also be involved in the maintenance of
vertebral identities through a Hox gene-independent
mechanism. Thus, the vertebral malformations observed here
could reflect a RA requirement at these later stages. RARβ
transcripts were not detected in presomitic mesoderm, somites
or sclerotomes in mouse embryos, while present in the neural
tube (Ruberte et al., 1991; Dollé et al., 1990; and our unpublished results). This suggests that the defects seen in RARαβ2
and RARβ2γ mutants may reflect an indirect effect of RARβ
in vertebral morphogenesis, involving RA-dependent signals
emanating from the neural tube, since perturbations of this
structure can result in vertebral malformations (Hall, 1977).
However, isolated transformations of C6 and C7 and a restriction of malformations to the cervical region have not been
reported in such studies.
(C) RARs and patterning of the limb
We previously speculated that the lack of limb malformations
in RARα or γ null mutants may be due to functional redundancy between these receptors (Lohnes et al., 1993; Lufkin et
al., 1993), since each is uniformly expressed throughout the
mesenchyme of the limb bud at 9.5-11.5 dpc (Dollé et al.,
1989). This is clearly the case, as the limbs from RARαγ
double mutants consistently exhibited malformations. Strikingly, forelimbs from RARα1γα2+/− mutants were normal,
showing that (as for the eye) a single copy of RARα2 suffices
for limb morphogenesis in the absence of RARγ and RARα1.
The anteroposterior axis of the limb is patterned by the zone
of polarizing activity (ZPA), whereas limb outgrowth requires
a functional apical ectodermal ridge (AER; see Tabin, 1991 for
review). The effect of the ZPA on the specification of the
anteroposterior axis of the limb can be mimicked by topical
application of RA, and both RA and the ZPA can trigger the
expression of some Hox genes believed to be critical for limb
specification (for reviews, see Duboule, 1992; Dollé and
Duboule, 1993). A role for RA in maintaining AER activity is
suggested by the finding that RA, in combination with FGF-4,
can fulfill most AER functions (Niswander et al., 1993).
However, the AER of RARαγ mutants appeared histologically
normal, although its formation may be slightly delayed (our
unpublished results). It is currently believed that RA may indirectly influence limb patterning by generating (or maintaining)
a functional ZPA (Wanek et al., 1993), possibly through the
regulation of a secreted protein, sonic hedgehog (Riddle et al.,
2744 D. Lohnes and others
1993). The malformations in RARαγ mutants do not appear to
result from an early ZPA defect, since the limbs displayed a
clear anteroposterior asymmetry. This does not exclude a role
for RA in anteroposterior limb patterning, since RARβ transcripts (which appear unaffected in the limbs of αγ mutants)
are expressed in the flanking mesenchyme and proximal
regions of the early limb bud in a region that overlaps with the
ZPA (Dollé et al., 1989; Mendelsohn et al., 1991; our unpublished results). In addition, the expression of some molecular
markers of ZPA and AER activity, including Hoxd-9 and
Hoxd-13 (Dollé and Duboule, 1993), MSX-1 (Robert et al.,
1989; Hill et al., 1989), BMP-2 (Lyons et al., 1990), FGF-4
(Niswander et al., 1993) and sonic hedgehog appeared unaffected as judged from in situ hybridization studies from 10.5
and 11.5 dpc RARαγ mutants (P. Dollé and D. Décimo, unpublished data).
Although two RARαγ mutants exhibited putative digit transformations, firm conclusions cannot be drawn concerning the
role of RA in specification of limb axes. It is clear, however,
that RA is essential for the generation of some skeletal
elements of the forelimb. In tetrapods, the limb skeletal
elements are established through a conserved series of
branching and segmentation events from prechondrogenic
blastemas arising during limb outgrowth (reviewed in Shubin
and Alberch, 1986; Shubin, 1991). Branching of the humeral
blastema gives rise to those of the radius and ulna. The
proximal and central carpals, as well as the digital arch, arise
from segmentation and branching events initiating from the
ulnar condensation, while distal carpal bones and subsequent
digit formation generally proceed in a posterior-to-anterior
direction in the mouse. All of the elements consistently
affected in RARαγ mutants are derived from the preaxial
(anterior) portion of the limb bud (i.e. radius, central and D1
carpals, digits 1 and 2 and the prepollex) and arise from the
last branching event occurring in a given region of the limb.
Loss of radius, first digit, central carpal bone and prepollex
may be due to the absence of the final specific branching events
giving rise to these structures (i.e. branching of the radial condensation from the humeral condensation, branching of the
central carpal condensation from the pyramidal condensation
and final branching of the digital arch to yield the prepollex or
first digit). These defects may reflect a requirement of RA to
generate the proper amount of limb mesenchyme, since a
deficit in this mesenchyme leads to a preferential loss of
anterior skeletal elements (Alberch and Gale, 1983). Such a
deficit is also suggested by the generalized size reduction of
the carpals in RARαγ double mutants (note also in this respect
that the entire forelimb of RARαγ mutants was often shorter).
Smaller mesenchymal condensations may also provide the
extra space needed for generating supernumerary condensations, occasionally resulting in polydactyly.
The phalanges of both the forelimbs and hindlimbs of 18.5
dpc RARαγ double mutants were consistently malformed,
appearing bulbous with poorly defined boundaries. This malformation does not appear to correspond to a developmental
delay, since the phalanges of WT 15.5 to 17.5 dpc fetuses are
always well defined (our unpublished observation; Kaufman,
1992). The perichondrial cells, surrounding the emerging
blastemae, are believed to be important in directional growth
of the blastemae and in determining their final shape (Shubin
and Alberch, 1986; and refs therein). Since RARα and RARγ
are both expressed in the perichondrial region of blastemal
condensations (Dollé et al., 1989), the loss of these receptors
could conceivably alter the functional integrity of the phalangeal perichondrium, thus leading to their altered morphology.
It is noteworthy that preaxial malformations of the limbs
exhibited by RARαγ double mutants are restricted to the
forelimbs (with the exception of bending of the tibia, which is
likely secondary to the severe malformation of the fibula).
These observations suggest that either RA plays different roles
in forelimbs and hindlimb development, or that events related
to differences in time of development of the two limbs allow
phenotypic rescue of the preaxial derivatives of the hindlimb.
Note in this respect that several mouse mutants, such as Po
(postaxial; Nakamura et al., 1963) and Px (postaxial hemimelia;
Searle, 1964) also exhibit defects confined largely to the
forelimbs. However malformations affecting the anteroposterior axis of the limb usually affect homologous structures in
both forelimbs and hindlimbs (e.g. Xt, Batchelor et al., 1966).
(D) Neural crest and RARs
Malformations of most of the structures derived from cranial
and cardiac mesenchymal neural crest cells (NCC) were
observed in RAR double mutants (this and the accompanying
study of Mendelsohn et al., 1994b). These structures originate
from osteogenic NCC (i.e. membrane bones of the skull and
face), from chondrogenic NCC [i.e. endochondral bones of the
skull and face and second (hyoid) and third arch skeletal
elements], from odontogenic NCC (i.e. upper incisors), from
smooth muscle NCC precursors (i.e. the tunica media of the
aortic arches and the aorticopulmonary septum), from dermal
NCC precursors (contributing to the pinna, eyelids and
prolabium) and from periocular NCC (i.e. corneal stroma and
retrolenticular mesenchyme). Mesenchymal NCC also contributes to the stroma of the glands whose development and/or
migration are altered in RAR double mutants (i.e. Harderian,
lachrymal, thyroid, thymus and parathyroid glands) (for review
and references see Noden, 1988; Le Douarin et al., 1993).
Since NCC appeared with the emergence of vertebrates (Gans
and Northcutt, 1983) and RARs have been found only in vertebrates (Kastner et al., 1994; Linney and LaMantia, 1994; and
references therein), our observations raise the interesting possibility that these receptors may have evolved to fulfill
functions necessary for the development of mesenchymal
NCC-derived structures.
In αγ (and to a lesser extent in α1γα2+/−) mutant mice, all
the structures derived from mesenchymal NCC originating
from the forebrain and the rostral midbrain (i.e. prolabium,
frontal, nasal, premaxillary, ethmoid, presphenoid and
sphenoid bones, and upper incisors; Le Douarin et al., 1993
and references therein) were either agenic or severely
malformed. Ocular structures derived from fore- and rostral
midbrain mesenchymal NCC were also affected in αγ and
other RAR mutants (see above). All elements derived from
more caudal mesenchymal NCC emanating from the level of
rhombomeres 4 and 6 (R4 and R6) and from the unsegmented
caudal portion of the rhombencephalon, and populating the
second, third, fourth and sixth arches, (Lumsden et al., 1991;
Noden, 1988; Le Douarin et al., 1993; Serbedzija et al., 1992;
Sechrist et al., 1993), were also likely malformed or ectopi-
RARs in ontogenesis 2745
cally localized in RARαγ and other double mutants. These
structures included the hyoid and styloid bones and stapes (i.e.
second and third pharyngeal arch derivatives), aortic arches,
aorticopulmonary septum and thymus, thyroid and parathyroid
glands (Mendelsohn et al., 1994b). In contrast, the first pharyngeal arch skeletal elements, which are derived from caudal
midbrain and rostral hindbrain (i.e. R1 and R2) levels, were all
identifiable in RARαγ double mutant fetuses, although some
were abnormal. Most of the misshapen first pharyngeal archderived skeletal elements (e.g. maxillary and palatine bones)
are derived from the maxillary (rostral) process of this arch,
which is populated mainly by mesenchymal NCC from the
caudal mesencephalon. Note that the alisphenoid and incus
bones, which are also derived from the maxillary process, were
fused, but otherwise not grossly affected (see below). In
contrast, most of the unaffected skeletal elements (e.g. dentary
bone, Meckel’s cartilage, malleus and tympanic bone) are
derived from the mandibular (caudal) process of the first pharyngeal arch, which is essentially populated by mesenchymal
NCC originating from R1 and R2 (Lumsden et al., 1991).
This lack of effect of RARαγ mutation on first pharyngeal
arch mesectoderm is unlikely due to a compensation by RARβ,
since RARβ transcripts are expressed at a much lower level in
the first arch than in the frontonasal region, nor can it be
explained by the absence of expression of RARα and γ, since
their transcripts are equally abundant in the first arch and frontonasal region (Dollé et al., 1990; Ruberte et al., 1990, 1991).
Interestingly, first arch NCC appear to be embodied with a
ground state morphogenetic program, which is also present in
the second pharyngeal arch mesectoderm where it is respecified by expression of Hoxa-2 (Rijli et al., 1993). Frontonasal
mesectodermal cells are also embodied with a similar program,
since they can generate first arch skeletal elements when
grafted to the level of either the presumptive first or second
arch (Noden, 1983). Thus, our data suggest that the realization
of at least part of the ground state program present in the first
arch does not require RA, whereas modification of this
program both in the frontonasal and second arch mesectoderm
involves RA-dependent processes. Since the lack of expression
of Hoxa-2 results in homeotic transformation of second arch
to first arch skeletal elements (Rijli et al.,1993), the second arch
defects seen here cannot simply reflect a requirement of RA
for Hoxa-2 expression.
Substantial evidence suggests that RA excess can affect premigratory NCC (Morriss-Kay, 1993 and references therein).
However, our results suggest a RA requirement for events
occurring during and/or after NCC migration, since RARγ has
not been detected in the presumptive forebrain or midbrain, nor
in presumptive NCC progenitors in the rhombencephalon
(Ruberte et al., 1990). In contrast, all three RARs are highly
expressed in the frontonasal process, whereas RARα and
RARγ, and to a lesser extent RARβ2, are expressed in the pharyngeal arches after (or possibly during) NCC migration into
these structures (Dollé et al., 1990; Ruberte et al., 1990, 1991;
our unpublished results). Furthermore, VAD-induced aortic
arch and aorticopulmonary septal defects can be prevented by
vitamin A administration up to mouse 9.5 or 10.5 dpc, respectively, whereas all NCC contributing to these structures have
migrated by 9.0 dpc (Wilson et al., 1953; Le Douarin et al.,
1993; and references therein; see also Mendelsohn et al.,
1994b). Although the molecular defects underlying the effect
of RAR inactivation on mesenchymal NCC are unknown, two
phenomena have been observed: (i) abnormal cell death in the
mesectoderm of the frontonasal process, which precedes the
aplasia of midfacial structures in RARαγ mutants, and (ii)
abnormal specification of the fate of some NCC populations,
as evidenced by chondrification of the meninges and the persistence and occasional chondrification of the retrolenticular
mesenchyme. Additional cartilaginous ectopias found in the
diaphragm, the peritoneum and the semilunar cusps of the heart
(see Mendelsohn et al., 1994b), may also be of NCC origin,
here reflecting either an abnormal specification of trunk NCC
(which normally have no chondrogenic potential; Hall, 1991
and references therein) or abnormal migration or specification
of cranial NCC.
It is remarkable that, with the exception of the lack of the
spiral ganglion (acoustic portion of the VIIIth cranial nerve) in
RARαγ mutants (which, however, may be secondary to abnormalities of the otocyst; see results section), we have not
observed primary malformations of neurogenic NCC-derived
structures. Thus, whether the effect of RA excess on many of
these structures (reviewed in Armstrong et al., 1994) is a pharmacological phenomenon awaits examination of RARβ (all
isoforms) null mice. In this respect, RA has been proposed to
regulate directly transcription of the Hoxa-1 gene (Langston et
al., 1992; Boylan et al., 1993). However, with the exception of
the lack of structures derived from the otocyst and lack of the
abducens nerve, RAR double mutants do not exhibit any of the
defects found in Hoxa-1 null mice (Lufkin et al., 1991; Chisaka
and Capecchi, 1991; Mark et al., 1993).
Finally, the phenotype of αγ mutants (see also Mendelsohn
et al., 1994b) present some striking similarities with a human
neurocristopathy called the CHARGE syndrome (Pagon et al.,
1981; Siebert et al., 1985): Coloboma of the retina, Heart
disease (consisting of aorticopulmonary septal defects and
abnormalities of aortic arch-derived great arteries), Atresia of
the choana (likely resulting from a defect in the formation of
the ethmoid bone), Retardation of physical and mental development, Genital hypoplasia in males and Ear abnormalities
(i.e. malformation of the pinna) and/or deafness. Additional
defects in these patients include hypoplasia or agenesis of the
thymus and parathyroid glands. Although it is unlikely that the
CHARGE association is due to inactivation of both RARα and
RARγ, it could result from the mutation of a RA-dependent
gene that is mis-expressed in αγ mutant mice (potential candidates are discussed in the conclusion section of Mendelsohn et
al., 1994b).
(E) RAR double mutants exhibit atavistic changes
Two types of supernumerary skeletal structures were often
detected in the skulls of RAR double mutant fetuses, the first
forming a cartilaginous medial wall to the cavum epiptericum,
the second linking the incus and alisphenoid. Comparative
anatomical data strongly suggest that these two skeletal
elements correspond to atavistic structures (Allin, 1975;
Presley, 1989; De Beer, 1985).
In reptiles and monotremes (lower mammals), the orbital
and the basal regions of the skull are connected by three pillars,
the pilae prooptica, metoptica and antotica (cranial to caudal
order) all of which arise by local chondrification of the
primitive cranial wall (i.e. the dura mater). The caudalmost
reptilian pillar (the pila antotica) forms a solid medial wall to
2746 D. Lohnes and others
the cavum epiptericum, thus physically separating it from the
braincase. The ability to form a pila antoptica from the dura
mater has been lost in placental mammals, thus permitting the
expansion of the braincase. The resulting gap caused by this
loss became filled in a more lateral plane by a first pharyngeal
arch-derived skeletal element, the alisphenoid bone. In the
RARγ−/− genetic background, disruption of the RARα1
isoform resulted in the bilateral appearance of a small supernumerary chondrification center at the base of the medial wall
of the cavum epiptericum. With subsequent loss of only one
copy of the α2 isoform, a complete supernumerary pillar was
formed which, based on its anatomical relationships, likely corresponds to the reptilian pila antotica. Thus RA appears to
modify the ancestral reptilian program, suppressing the
formation of this pillar in mammals.
In reptiles, the quadrate bone (the dorsal element of the jawjoint) and the epipterygoid bone (which forms the lateral limit
of the cavum epiptericum) develop from a single element, the
pterygoquadrate (upper jaw) cartilage. During the emergence
of mammal-like reptiles (therapsids), the quadrate bone, while
losing its connection with the epipterygoid bone, underwent
size reduction, with its otic process becoming the short process
of the incus middle ear bone [the long or stapedial process of
the incus likely represents a mammalian neomorph (Presley,
1989)]. It is also widely held that the mammalian pterygoid
and alisphenoid bones evolved from the reptilian epipterygoid
bone which develops from the rostral portion of the pterygoquadrate cartilage. A number of RAR double mutant fetuses
exhibited abnormal skeletal structures in which the body of the
incus was continuous with a rostrally oriented cartilaginous or
osseous rod, which was often fused to the alisphenoid (Table
2), while the short process of the incus was larger than in
normal fetuses. It is likely that this is an atavistic structure corresponding to the therapsid evolutionary state in which the
incus had appeared, but was still linked to the newly derived
alisphenoid bone through quadrate remnants. Interestingly, the
present day ground state morphogenetic program of the first
arch appears to encode a similar atavistic structure (Rijli et al.,
1993). It is tempting to conclude that an RA-dependent process
has been recruited during the course of evolution to modify this
program.
The re-emergence of ancestral skeletal features in RAR
double mutants not only indicates that the underlying mesectodermal developmental programs are still present in
mammals, but also that RA-dependent mechanisms have been
recruited during the reptilian-mammalian transition to modify
some features of the reptilian skull.
We would like to thank Dr T. Pexieder for a critical reading of the
manuscript, Dr M. LeMeur for her collaboration and the members of
the retinoid group for useful discussions; B. Weber, C. Fischer and
V. Giroult and the technical staff of the animal facility for excellent
help; B. Boulay, J. M. Lafontaine and C. Werlé for the illustrations
and the secretarial staff for assembling the manuscript. D. L. was a
recipient of a fellowship from the MRC Canada and C. M. was
supported by a fellowship from the NIH (5F32 GM13597-03) and
from the ARC. This work was supported by funds from the Institut
National de la Santé et de la Recherche Médicale, the Centre National
de la Recherche Scientifique, the Centre Hospitalier Universitaire
Régional, the Association pour la Recherche sur le Cancer, the Human
Frontier Science Program and the Fondation pour la Recherche
Médicale.
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(Accepted 15 July 1994)