2173Development 122, 2173-2188 (1996) Printed in Great Britain ©
The Company of Biologists Limited 1996 DEV3430
Retinal dysplasia and degeneration in RARβ2/RARγ2 compound mutant
Jesús M. Grondona†,‡, Philippe Kastner‡, Anne Gansmuller, Didier
Décimo, Pierre Chambon and Manuel Mark*
Institut de Génétique et de Biologie Moléculaire et Cellulaire,
CNRS / INSERM / ULP / Collège de France, BP 163, 67404 Illkirch
Cedex, C.U. de Strasbourg, France
*Author for correspondence (e-mail: [email protected]
†Present address: Laboratorio de Fisiologia Animal, Departamento de
Biologia Animal, Facultad de Ciencias, Universidad de Malaga, 29071
Malaga, Spain ‡These should be considered as equal first
The eye is the organ whose development is the most fre- quently
altered in response to maternal vitamin A defi- ciency [VAD;
Warkany, J. and Schraffenberger, S. (1946). Archs Ophthalmol. 35,
150-169]. With the exception of prenatal retinal dysplasia, all the
ocular abnormalities of the fetal VAD syndrome are recapitulated in
mouse mutants lacking either RARα and RARβ2, RARα and RARγ, RARγ
and RARβ2, or RXRα [Lohnes, D., Mark, M., Mendelsohn, C., Dollé,
P., Dierich, A., Gorry, P., Gans- muller, A. and Chambon, P. (1994)
Development 120, 2723- 2748; Mendelsohn, C., Lohnes, D. Décimo, D.,
Lufkin, T., LeMeur, M., Chambon, P. and Mark, M. (1994) Develop-
ment 120, 2749-2771; Kastner, P., Grondona, J. Mark, M.,
Gansmuller, A., LeMeur, M., Décimo, D., Vonesch, J.L., Dollé, P.
and Chambon, P. (1994) Cell 78, 987-1003], thus demonstrating that
retinoic acid (RA) is the active vitamin A metabolite during
prenatal eye morphogenesis. Whether retinoids are also involved in
postnatal eye development could not be investigated, as VAD
newborns are not viable and the above RAR double null mutants and
RXRα null mutants died in utero or at birth.
We report here the generation of viable RARβ2/RARγ2 double null
mutant mice, which exhibit several eye defects. The neural retina
of newborn RARβ2γ2 mutants is thinner than normal due to a reduced
rate of cell proliferation, and from day 4 shows multiple foci of
disorganization of its layers. These RARβ2γ2 mutants represent the
cally characterized model of retinal dysplasia and their phenotype
demonstrates that RARs, and therefore RA, are required for retinal
histogenesis. The RARβ2γ2 retinal pigment epithelium (RPE) cells
display histological and/or ultrastructural alterations and/or fail
to express cellular retinol binding protein I (CRBPI). Taken
altogether, the early onset of the RPE histological defects and
their striking colocalisation with areas of the neural retina dis-
playing a faulty laminar organization, a reduced neuro- blastic
proliferation, and a lack of photoreceptor differen- tiation and/or
increased apoptosis, make the RPE a likely target tissue of the
RARβ2γ2 double null mutation. A degeneration of the adult neural
retina, which may similarly be secondary to a defective RPE, is
also observed in these mutants, thus demonstrating an essential
role of RA in the survival of retinal cells. Moreover, all RARβ2γ2
mutants display defects in structures derived from the peri- ocular
mesenchyme including local agenesis of the choroid and of the
sclera, small eyelids, and a persistence of the primary mesenchymal
vitreous body. A majority of the RARβ2 single null mutants also
exhibit this latter defect, thus demonstrating that the RARβ2
isoform plays a unique role in the formation of the definitive
Key words: vitamin A, retinoic acid, nuclear receptors, retinal
histogenesis, retinal pigment epithelium, mouse, dysplasia
Vitamin A deficiency (VAD) studies have shown that vitamin A
(retinol) is required during prenatal and postnatal develop- ment,
and during adult life. After birth, retinol is indispensable for
survival, growth, reproduction and vision and also for the
maintenance of numerous tissues. Widespread squamous meta- plasia
of the conjunctival, corneal, respiratory, urinary and various
glandular epithelia (i.e. olfactory, salivary, Harderian, seminal
vesicle and prostate glands and/or their associated excretory
ducts), together with degeneration of the seminifer- ous tubules
and of the neural retina, are hallmarks of the postnatal VAD
syndrome (Wolbach and Howe, 1925; Johnson,
1939). Interestingly, retinoic acid (RA) can prevent or reverse the
deleterious effects of a postnatal VAD diet, with the exception of
night blindness and photoreceptor degeneration (Dowling and
Gibbons, 1961; Dowling, 1964; Howell et al., 1963; Thompson et al.,
1964; Van Pelt and de Rooij, 1991). Furthermore, conceptuses of VAD
dams exhibit a large number of congenital malformations (i.e., the
fetal VAD syndrome) affecting the eye, the kidney and genitourinary
tract, the heart and aortic arch-derived great arteries, the lung
and the diaphragm. Retinol can prevent these malformations provided
that it is supplied to the dams at specific times of gestation,
thus demonstrating that it is required at several stages during
ontogenesis (reviewed in Wilson et al., 1953).
2174 J. M. Grondona and others
Two families of nuclear receptors for retinoids have been
characterized. Members of the RAR family (types α, β and γ, and
their isoforms α1, α2, β1 to β4, and γ1 and γ2) are activated by
most physiologically occurring retinoids (all-trans RA, 9-cis RA,
4-oxo RA and 3,4 dihydro RA). In contrast, members of the RXR
family (types α, β and γ, and their isoforms) are activated by
9-cis RA only. In addition to the multiplicity of receptors, the
complexity of retinoid signalling is further increased by the fact
that, at least in vitro, RARs bind to their cognate response
elements as heterodimers with RXRs. Moreover, RXRs can also bind in
vitro to some DNA elements as homodimers and are heterodimeric
partners for a number of nuclear receptors other than RARs
(reviewed in Chambon, 1994; Giguère, 1994; Mangelsdorf and Evans,
Null mutations of the RAR genes (either α, β or γ), as well as
isoform-specific knock-outs for RARα1, RARβ2/β4 and RARγ2 have been
generated (reviewed in Kastner et al., 1995). RARβ single null
mutant were apparently normal (Luo et al., 1995), whereas RARα, and
RARγ single null mutants were viable and displayed abnormalities
which, however, were confined to a small subset of the tissues that
express these receptors (Lufkin et al., 1993; Lohnes et al., 1993).
These findings suggested that there could be some functional redun-
dancy in the RAR family. To test this hypothesis, double null
mutants lacking either RARα1 and RARβ2, RARα and RARβ2, RARα1 and
RARγ, RARα and RARγ, or RARβ2 and RARγ, were generated (Lohnes et
al., 1994; Mendelsohn et al., 1994a). In contrast to RAR single
mutants, these double mutants exhibited a dramatically reduced
viability, as about half of the RARαγ mutants died in utero, and
the remaining half as well as the other RAR double null mutants
survived for 12 hours at most following delivery by Caesarean
section at full term (Lohnes et al., 1994). Furthermore, almost all
of the malformations of the fetal VAD syndrome were recapitulated
in the different RAR double mutants, with the exception of a
shortening of the ventral retina, which was, however, found in RXRα
single null mutants (Kastner et al., 1994) and of a prenatal
retinal dysplasia (Warkany and Schraffenberger, 1946). These
findings demon- strated that RA is the vitamin A derivative that is
active during ontogenesis, and that its effects are mediated by the
We have recently generated double null mutant mice lacking the
RARβ2 and RARγ2 isoforms. These RARβ2−/−/RARγ2−/−
mutants (hereafter referred to as RARβ2γ2 mutants) were normally
viable and fertile. However, they displayed severe ocular defects.
Their analysis demonstrates that RARs, and therefore RA, play a
crucial role in histogenesis and mainte- nance of the neural
retina, and that the retinal pigment epithe- lium (RPE) most
probably represents the primary target tissue of the RARβ2γ2
MATERIALS AND METHODS
Generation of RARβ2γ2 double null mutants RARγ2 (Lohnes et al.,
1993) and RARβ2 (Mendelsohn et al., 1994b) mutant mice were bred to
generate double heterozygote mice, which were intercrossed to
produce RARβ2−/−/RARγ2−/−, RARβ2−/−/ RARγ2+/− and RARβ2+/−/RARγ2−/−
mice, from which most of the mutants used in this study were
Histology and immunohistochemistry Mice were killed by cervical
dislocation. The eyes were enucleated and
fixed by immersion in Bouin’s fluid for 2 days, transferred to 70%
ethanol overnight, and then bisected with a razor blade along a
plane defined by the superior-inferior axis and the optic nerve.
Lenses were removed and the eyes embedded in paraffin.
Occasionally, skinned skulls from newborn, 4 days and 1-week-old
mice were fixed in toto in Bouin’s fluid to prevent any damage that
might have occurred during enucleation. 7 µm serial sections were
mounted on slides coated with 0.01% poly-L-lysine (Mr 350,000;
Sigma). The sections were stained with Groat’s hematoxylin and
Mallory’s trichrome (Mark et al., 1993) or employed for
immunohistochemistry. The following antibodies were used : a mouse
monoclonal IgG directed against rod-specific opsin (4D2, in the
form of a hybridoma supernatant) (a gift from R. Molday, University
British Columbia, Vancouver and D. Hicks, Uni- versity Louis
Pasteur, Strasbourg; Hicks and Barnstable 1987); a rabbit
polyclonal antibody directed against CRBPI (in the form of an
antiserum; a gift of U. Eriksson, Ludwig Institute for Cancer
Research, Stockholm; Gustafson et al., 1993); a polyclonal antibody
against glial fibrillary acidic protein (GFAP; Sigma); and an
anti-BrdU monoclonal antibody (Boehringer, Mannheim). These
antibodies were used at dilutions of 1:40, 1:100, 1:400 and 1:100,
respectively. Biotinylated anti-mouse IgG, biotinylated anti-rabbit
IgG (Vectastain Elite ABC Kit, Vector) were used according to the
manufacturer’s instructions. The reagents of the Vectastain ABC
Elite system and diaminobenzi- dine (Sigma) were employed for
immunoperoxidase labelling. Cy3- conjugated streptavidin (Jackson
ImmunoResearch) was used for immunofluorescence labelling. All
primary antibodies, anti-IgG anti- bodies and components of the ABC
system were diluted in PBS pH 7.3, containing 0.05% Tween 20 and
0.5% normal goat serum. Control sections were incubated with either
mouse or rabbit preimmune serum instead of the primary
Fixation by perfusion was employed in one histological experiment
(illustrated in Fig. 2) in order to visualize better the capillary
network of the periocular mesenchyme. Under general anaesthesia,
adult mice were perfused with 1% glutaraldehyde–4% paraformaldehyde
in PBS (pH 7.2) at an outflow rate of 5 ml/minute for 5 minutes.
The eyes were enucleated, then postfixed in Bouin’s fluid for 24
Electron microscopy Eyes from 7-day-old and 1-month-old animals
were fixed by immersion in 2% glutaraldehyde in 0.1 M cacodylate
buffer (pH 7.4) for 16 hours at 4°C, and the lenses removed. The
eyes were then rinsed in cacodylate buffer, postfixed in 1% osmium
tetroxide in the same buffer for 2 hours at 4°C, dehydrated with
graded alcohols series and embedded in Epon. 1 µm sections were
stained with toluidine blue. Ultrathin sections from selected areas
were contrasted with uranyl acetate and lead citrate and examined
with a Philips 208 electron microscope operating at 80 kV.
Labeling of S-phase nuclei Bromodeoxyuridine (BrdU) (Sigma)
dissolved in PBS was injected intraperitoneally at a dose of 50 mg
per kg of body weight. The mice were killed 2 hours later, the eyes
fixed in Bouin’s fluid for 2 days, then embedded in paraffin. BrdU
incorporation was detected by using an anti-BrdU monoclonal
antibody (Boehringer) and immunoperoxi- dase labelling.
End-labeling of DNA nicks in tissue sections (TUNEL staining) In
situ detection of fragmented DNA was performed as described by
Gavrieli et al. (1992) with some modifications. 7 µm sections from
Bouin-fixed, paraffin-embedded eyes were collected on poly-L-lysine
coated slides. The sections were dewaxed and then hydrated. After
rinsing in distilled water (3× 5 minutes) the sections were
digested for 15 minutes at 24°C with 20 µg/ml proteinase K in 50 mM
Tris-HCl pH 7.5 containing 50 µM EDTA, rinsed in distilled water
(3×5 minutes), and then incubated for 1 hour at 37°C with
biotinylated dUTP in terminal transferase buffer (all from
2175Retinoic acid in retinal development
reaction was terminated by transferring the slides to distilled
water. The sections were permeabilized with 0.05% Tween 20 in PBS
(3×5 minutes) and biotin incorporation was revealed with
Cy3-conjugated streptavidin (Jackson ImmunoResearch) diluted 1:400
(30 minutes at 24°C). Negative controls were obtained by omitting
terminal trans- ferase in the incubation mixture.
Detection of RARs by in situ hybridization The RARβ and γ probes
used for in situ hybridization were synthe- sised from cDNA
fragments covering the entire open reading frame (Dollé et al.,
1990). In situ hybridization was performed on 10 µm frozen sections
from albino mice, as described (Décimo et al., 1995).
RARβ2γ2 double null mutant mice are viable In contrast to the RAR
compound mutants analysed previously (Lohnes et al., 1994;
Mendelsohn et al., 1994a), which all died within 12 hours after
birth, RARβ2γ2 homozygotes were normally viable and fertile. From
external inspection, however, their eyes appeared abnormal (e.g.
compare Fig. 1a and b).
Retinal degeneration and dysplasia in adult RARβ2γ2 mutants In the
neural retina of the normal (wild-type, WT) adult mouse (R, Fig.
1c,e,h), there are 8 major cell types located in charac- teristic
positions in the outer nuclear layer (ONL: rod pho- toreceptors),
the inner nuclear layer (INL: horizontal, bipolar, amacrine and
Müller cells) and the ganglion cell layer (GCL: ganglion cells and
astrocytes). These layers of cell bodies are separated by two
layers of synaptic interplay, namely the outer plexiform layer
(OPL) between the ONL and INL and the inner plexiform layer (IPL)
between the INL and GCL (Fawcett,
Table 1. Eye abnormalities in
Retinal abnormalities •Generalized thinning of the neural retina
16/22 •Multifocal agenesis of the RPE 3/22 •Retinal dysplasia
(presence of rosettes or folds) 0/22 •Patches of neural retina
degeneration with : - ONL (only) atrophic or missing NA* - ONL and
INL atrophic or missing ; normal GCL NA* - ONL and INL atrophic or
missing, GCL missing NA*
Non-retinal abnormalities •Sclera locally thinner or missing #
•Choroid locally thinner or missing NA‡ •Retrolenticular membrane #
•Harderian gland agenesis 6/22 •Blepharophimosis NA† •Cataracts
# these abnormalities are fully penetrant. NA, not applicable. *The
ONL and INL are not defined at birth. †The eyes open at 12 to 14
days post-partum (Theiler, 1972). ‡The choroid becomes visible only
at P4 when melanocytes start to differentia §It is no longer
possible to score this abnormality because of the extensive deg Nb,
newborn; P, post-natal day; m, month; ONL, INL and GCL, outer
1986; Huxlin et al., 1992; Sarthy et al., 1991 and refs therein;
The neural retina of adult RARβ2γ2 mutants (i.e. 1-month old or
older) exhibited two types of abnormal phenotypes, namely a marked
atrophy (degeneration) and a disorganisation (dysplasia) of the
Retinal degeneration Macroscopic examination of 1-month-old eyes
revealed a marked reduction in the thickness of some portions of
the neural retina (compare R, Fig. 1c and d). In older mutants (6
to 12 months), this reduction in thickness was observed over larger
patches (compare R, Fig. 1e and f). In the most severely affected
eyes (Fig. 1f and i), only 2 to 3 rows of nuclei persisted in the
central retina, whereas in the peripheral retina, close to the
iris, all nuclear and the synaptic retinal layers remained
identifiable although the ONL, INL, OPL and IPL were con-
spicuously thinner and the GCL contained fewer cells, as compared
to their wild-type counterparts (compare Fig. 1h and k with i and
l). The patches of neural retinal atrophy always involved the ONL
(Table 1, results not shown), whereas the INL and GCL were affected
only in place where the ONL had almost disappeared (e.g. Figs 1l
In WT mice, the glial fibrillary acidic protein (GFAP) gene is
specifically expressed in the astrocytes present in the GCL (Sarthy
et al., 1991; Huxlin et al., 1992). In contrast, accumu- lation of
GFAP in Müller cells represents an unspecific response to eye
injuries and retinal degeneration or detachment (references in
Sarthy et al., 1991). Immunostaining with anti- GFAP of the mutant
retina showed, in the thin areas, numerous positive cellular
processes, consistent with a degenerative process (data not
Even though the penetrance of the retinal degeneration was complete
(i.e. every eye displayed the defect; Table 1), its extent
RARβ2/RARγ2 null mutants Number of RARβ2γ2 eyes analysed
Retinal differentiation period Adult retina
P4 P7 P14 1-3m 6-12m 16 13 14 14 18
10/16 11/13 9/14 NA§ NA§ 14/16 10/13 9/14 # # 12/16 9/13 8/14 6/14
# # # # # # # # # # # # # # #
0 0 0 4/14 9/18
te. eneration. , inner nuclear and ganglion cell layers,
respectively; RPE, retinal pigment
2176 J. M. Grondona and others
Fig. 1. Comparison of the palpebral fissure, eyeball and retina
between WT and RARβ2γ2 (β2γ2) mutant adults, at one year (a,b,e-m)
and 1 month (c,d). h, i and j correspond to histological sections
of e, f and g, respectively; note that the lenses were removed
prior to paraffin embedding. k, l and m correspond to high
magnification of regions boxed in h, i and j respectively. The open
arrow in j points to a focus of degeneration. The arrowheads in l
and m indicate the absence of retinal pigmented epithelium (RPE).
The detachment of the neural retina from the RPE (asterisk in l) is
artifactual. Abbreviations: CH, choroid; F, retinal fold; GCL,
ganglion cell layer; HG, Harderian gland; I, iris; IC, inner
canthus; INL, inner nuclear layer; IPL, inner plexiform layer; L,
lens; OC, outer canthus; ONL, outer nuclear layer; OPL, outer
plexiform layer; PS, photoreceptor segments; R, retina; RL,
retrolenticular membrane; Ro, rosettes; RPE, retinal pigment
epithelium; SC, sclera; V, vitreous body. Mallory’s
trichrome-hematoxylin (h-m). Magnifications: ×13 (c and d); ×9
(e-j); ×155 (k-m).
was highly variable among mutants from the same age, partic- ularly
among young adults (i.e. 1-month-old); note in this respect that
Fig. 1d shows an example of one of the most affected eyes at
1-month. Moreover, in a given mutant, the two eyes could be
affected to very different degrees. For instance, Fig. 2 shows the
two eyes from the same mutant: the left neural
retina is essentially normal (e.g. compare PS, ONL, OPL and INL in
Fig. 2a and b), exhibiting only rare foci of degeneration, whereas
the right retina (Fig. 2c) is essentially degenerated.
Retinal dysplasia The second abnormal phenotype displayed by about
2177Retinoic acid in retinal development
Fig. 2. Scleral and choroidal defects in RARβ2γ2 mutant eyes.
Histological sections from perfusion-fixed, 6 months old, WT (a)
and RARβ2γ2 eyes (left and right eyes from the same mutant, as
indicated in b and c). Note the paucity (c) or the absence (b) of
the choroidal melanocytes (CH) and the thinning of the sclera (SC).
Also note that b and c represent typical aspect of the left and the
right neural retinas in this mutant. Abbreviations: CH, choroid;
GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner
plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform
layer; RPE, retinal pigment epithelium. The arrowheads point to
periocular capillaries. Magnification: ×380 (a-c).
of the adult mutant eyes (Table 1) corresponds to a disorgan-
isation of the layers of the neural retina manifested by the
presence of retinal folds and rosettes (F and Ro in Fig. 1g,j,m).
Note that each of these dysplastic neural retinas also exhibited
occasional foci of degeneration (open arrow in Fig. 1j, and data
not shown) and large areas with a normal laminar pattern (e.g., R
in Fig. 1j).
It is noteworthy that these retinal defects were never observed in
the RARβ2 and in RARγ2 single null mutants.
Developmental abnormalities of RARβ2γ2 mutant neural retinas In
order to gain insights into the pathogenesis of the above retinal
abnormalities in adult RARβ2γ2 mutants, we studied the processes of
lamination, rod functional differentiation, cell proliferation and
apoptosis in the neural retina.
Abnormal retinal lamination During normal mouse retinal
development, the IPL begins to form by embryonic day 17.5 (E17.5)
and separates the future GCL from the outer neuroblastic layer (Pei
and Rhodin, 1970). Likewise, by postnatal day 4 (P4), the OPL
appears in the central retina, thus dividing the outer neuroblastic
layer into a
ONL and a INL (Young, 1984). By P7, the retinal layers (i.e. ONL,
INL and GCL) are distinctly present in the entire neural retina
In ~70% of the RARβ2γ2 newborn mutant eyes (Table 1), the entire
outer neuroblastic layer appeared thinner than its WT counterpart
(compare NBL, in Figs 3a, 6a and 6c with 3b, 6b and 6d; the overall
thickness of these mutant retinas was 25% lower than normal).
Additionally, in a few mutant eyes (3 out of 22; Table 1), focal
areas of RPE agenesis were observed in the central retina
(arrowheads in Figs 3b, 6b and d, and see below). However, even in
these areas devoid of RPE, the lam- ination of the mutant neural
retinas was normal (e.g. NBL, IPL and GCL, in Figs 3b and
In P4, P7 and P14 mutant eyes, the generalized thinning of the
neural retina, already noticed at birth, was conspicuous. It
affected the photoreceptor segments (PS), the ONL and the INL. In
contrast, at the same developmental stages, the mutant GCL was
indistinguishable from its WT counterpart (Table 1 and compare PS,
ONL and INL in Fig. 3f and g). Additionally, multiple foci of
dysplasia were observed in 80% of the mutant neural retina that
displayed rosettes (Ro1 and Ro2 in Fig. 3c-e; Ro in Figs 3g, 4b,
d), folds (F, Fig. 4c) and/or local retinal detachments (RD, Fig.
4e). The rosettes comprised tubular arrangements of photoreceptors
displaying well defined segments (e.g. Ro, Fig. 3g). Interestingly,
the earliest-formed (i.e. P4) rosettes were often connected with
the periocular mes- enchyme by bridges of cells ‘escaping’ from the
ONL (Fig. 3c- e). Folds are probably generated at sites of retinal
detachment (RD, Fig. 4e) and involve all the layers of the neural
retina (F, Fig. 4c). Extreme thinning of the ONL (unlabelled arrows
in Fig. 3g) and of the INL (not shown) were frequently observed in
these dysplastic areas at P14. At P4, P7 and P14 the dys- plastic
areas of neural retina were always contiguous with patches of
abnormal or absent RPE, and always commenced abruptly at the point
where the morphologically normal RPE terminated (open arrows in
Figs 3c,d,g and 4b-e; arrowheads in Figs 3g, 4b,d, 6f; see below
for a description of RPE defects).
Thus, on the basis of histological criteria, the RARβ2γ2 mutant
neural retinas display two distinct abnormal develop- mental
phenotypes: (1) a congenital thinning of the neural retina, which
is ubiquitous and thus does not correlate with the patchy
distribution of the RPE defects, and (2) multiple foci of retinal
dysplasia, which are spatially correlated with defects in the
Abnormal differentiation of mutant photoreceptors Functional
differentiation of the rods, the major photoreceptor type in the
mouse retina, occurs during the first days after birth and can be
followed by the expression of the rod-specific opsin in the
presumptive ONL (Hicks and Barnstable, 1987). Rod differentiation
is achieved at P14 which corresponds to the completion of segment
formation (Obata and Usubura, 1992; Theiler, 1972).
The 4D2 monoclonal antibody, which specifically recog- nises opsin
in rod photoreceptors (Hicks and Barnstable, 1987), was employed as
a marker of the differentiated state of the ONL in newborn and P4
retinas (Fig. 4). In both WT mice and RARβ2γ2 mutants, the first
opsin-immunoreactive cells were detected in the central retina at
the time of birth (not shown). In P4 WT eyes, almost all cells of
the ONL expressed opsin (Fig. 4a), and the P4 RARβ2γ2 mutant ONL
2178 J. M. Grondona and others
stogenesis in WT and RARβ2γ2 mutants at birth (a,b), P4 (c-e) and
P14 e successive sections through the same portion of a mutant
retina. The f missing RPE and the open arrows to scattered RPE
cells within the e arrows in g indicate an area where the ONL is
atrophic. Asterisk ent of the RPE and neural retina during tissue
processing. Abbreviations: n cell layer; INL, inner nuclear layer;
IPL, inner plexiform layer; NBL, L, outer plexiform layer; ONL,
outer nuclear layer; PS, photoreceptor , rosettes; RPE, retinal
pigment epithelium; SC, sclera; V, vitreous body.
oxylin. Magnifications: ×155 (a,b,f,g) and ×195 (c-e).
guishable from its WT counterpart in areas facing a normal RPE
(Fig. 4b,c,e). In contrast, the ONL cells adjacent to areas lacking
an histologically normal RPE did not express opsin (unlabelled
small arrows in Fig. 4b-e), with the notable exception of some
rosettes (Ro in Fig. 4d), nor did they form segments (PS in Fig. 5,
compare a and c with b and d). These results strengthen the
conclusion that the presence of RPE is essential for proper
photoreceptor differentiation (Stiemke et al., 1994, and references
therein), and further suggest that the abnormalities in the RARβ2γ2
RPE could be, at least in part, responsible for the defects
observed in the neural retina (see below for further analysis of
the mutant RPE).
Reduced cell proliferation in RARβ2γ2 mutant neural retinas Cell
proliferation in the retina was examined by bromodeoxyuridine
(BrdU) incorporation (Fig. 6). The fraction of cells incorporating
BrdU during a short exposure reflects the fraction of cells in S
phase during this period, therefore permitting an assessment of the
proliferation rate of a population of cells.
In RARβ2γ2 newborns, the labelling index in the outer neurob-
lastic layer was markedly reduced (between 18 and 28% of the total
cell number, depending of the animal) when compared with WT (40% of
the total cell number) (compare Fig. 6a and c with 6b and d). This
reduced labelling index affected both the central and peripheral
retinas (Fig. 6b), and was apparently identical whether the RPE was
missing (arrow- heads in Fig. 6b,d) or present. At P4, the rate of
cell proliferation had decreased in both WT and mutant neural
retinas, which displayed similar labelling indices in places where
the RPE was present (data not shown). In contrast, in portions of
the mutant neural retina facing large areas devoid of RPE, the
percentage of proliferating cells was markedly reduced (12% in
peripheral retina) compared to WT (26% in peripheral retina)
(compare Fig. 6e and f).
Thus, a reduced cell proliferation rate during the perinatal period
of retinal development may account for the generalized thinning of
the RARβ2γ2 neural retina. However, this decrease in cell
proliferation cannot explain the loss of the retinal cells in adult
mutants, since cell pro- liferation already ceases by P6 and by P11
in the central and peripheral portions of the WT retina, respec-
tively (Young, 1985a,b).
Fig. 3. Aspects of retinal hi (f,g). c-e correspond to thre
arrowheads point to areas o periocular mesenchyme. Th indicate
artifactual detachm CH, choroid; GCL, ganglio outer neuroblastic
layer; OP segments; Ro, Ro1 and Ro2 Mallory’s trichrome-hemat
Increased apoptosis in RARβ2γ2 mutant neural retinas. In the normal
mouse retina, programmed cell death occurs primarily during the
first 2 weeks after birth and is essentially completed by the end
of the third week (Young, 1984). The in situ end-labelling method
of DNA nicks (TUNEL method; Gavrieli et al., 1992) was employed to
compare cell death between normal and mutant mouse retinas (Fig.
At P4 and P14, the majority of apoptotic cells of WT retinas were
located in the INL and the GCL in agreement with Young’s data
(Young, 1984); apoptotic cells were scarce in WT ONL (Fig. 7a,d).
In dysplastic areas of mutant retinas at the same ages (i.e. P4 and
P14), the number of apoptotic cells in the INL was markedly
increased (compare Fig. 7a and d with
2179Retinoic acid in retinal development
b and e), and dying cells were also often observed in the ONL
(arrowheads in Fig. 7e). This suggests that, in addition to the
reduced rate of cell proliferation mentioned above, an increase of
apoptosis also contributes to the extreme thinning of the INL
Fig. 4. Expression of the rod-specific opsin evaluated by
immunohistochemistry in P4 WT (a) and RARβ2γ2 (b-e) mutants. The
open arrows point to scattered RPE cells in the periocular
mesenchyme, the arrowheads to portions lacking RPE, and the small
arrows to areas of the ONL lacking expression of opsin. Note that
these latter areas are always juxtaposed to an abnormal or absent
RPE. The detachments between the RPE and neural retinal such as the
one illustrated in e likely occur in vivo; they have regular
outlines which are maintained on more than 20 serial sections;
their size is small and the tissues bordering them are not damaged
in any fashion. These features distinguish them from artifactual
retinal detachments (asterisk in b and c) generated during tissue
processing. Abbreviations: CH, choroid; GCL, ganglion cell layer;
INL, inner nuclear layer; ONL, outer nuclear layer; Ro, rosettes;
RD, retinal detachment; RPE, retinal pigment epithelium; V,
vitreous body. Immunoperoxidase with hematoxylin counterstain.
Magnifications: ×77 (a-c), ×155 (d) and ×90 (e).
and/or ONL which is frequently observed in the dysplastic areas of
P14 mutants (e.g. unlabelled arrows in Fig. 3g).
In WT animals older than 1 month, retinal cell death was no longer
detectable (Young, 1984, and data not shown). In contrast, retinas
from adult mutant mice displayed an average of 4-6 apoptotic cells
per section that were preferentially located in areas of thin or
folded retina (data not shown). Thus, apoptosis occurring after the
period of physiological retinal cell death is likely to account for
the progression of degeneration in the retina of adult
Retinal pigment epithelium defects in RARβ2γ2 mutants As mentioned
above, foci of dysplastic neural retina were always located
adjacent to an absent or morphologically abnormal RPE. Moreover,
the RPE defects preceded the neural retina dysplasia in mutant
eyes. Since the RPE is known to play an important trophic influence
in the development and main- tenance of the neural retina
(Campochiaro, 1993; Bok, 1993), these observations suggested to us
that a defective RPE could be instrumental in the generation of the
RARβ2γ2 retinal defects. Therefore, the morphology of the mutant
RPE was analysed by electron microscopy, and its functional state
was investigated by examining the distribution of cellular retinol
binding protein I (CRBPI) which is normally expressed uniformly in
RPE cells (Fig. 9a) and is believed to play a crucial role in the
delivery of retinol to the neural retina (reviewed in Saari, 1994).
From an analysis during the period of retinal histogenesis (i.e. at
P4 and P7) and in adult retina (i.e. at 1 month), mutant RPEs could
be classified within three different categories: disorganised RPE,
abnormal RPE without loss of epithelial organisation and apparently
Disorganised RPE During the period of retinal histogenesis,
scattered pigmented cells were often detected within the periocular
mesenchyme adjacent to the areas of dysplastic neural retina (open
arrows in Figs 3c,d,g, 4b-e, 7c). Semithin sections and electron
microscopy of P7 retina (Fig. 5b,d) showed flattened RPE cells that
had lost their epithelial arrangement and, in some cases, their
contact with the neural retina (compare Fig. 5a and c with b and
d). Note that these flattened pigmented cells represent altered RPE
cells, not choroidal melanocytes, since they were usually lying
within large portions of the periocular mes- enchyme that were
totally devoid of choroid (see Figs 4a-e and 5b).
Abnormal ‘epithelial’ RPE Electron microscopic analysis of mutant
RPE at P7 (results not shown) and 1 month (Fig. 8b,d) revealed
abnormalities in areas where its epithelial organization was
preserved: the cytoplasm of these RPE cells was highly vacuolated
(V in Fig. 8d) and their apical microvilli (Mi, Fig. 8c), which
normally inter- digitate with the photoreceptor outer segments,
were absent. The photoreceptor outer segments facing these altered
RPE cells were reduced in number and/or disorganized, showing
improper piling of their disks (compare POS in Fig. 8a and c with b
and d). Immunostaining on P7 (not shown) and 1-month- old mutant
RPEs, revealed weak (with respect to wild type) or absent CRBPI
expression in several areas where this tissue appeared
histologically normal (compare Fig. 9a with b and c).
2180 J. M. Grondona and others
d electron microscopy of P7 RPE from WT and RARβ2γ2 mutants. Note
mutant RPE cells in b and d. Abbreviations: C, capillaries; CH,
layer; N, RPE cell nuclei; PS, photoreceptor segments; RPE, retinal
era. Magnifications: ×380 (a,b); ×2250 (c,d).
Apparently normal RPE In some areas of the mutant retina, the RPE
was indistin- guishable from its WT counterpart, based on
ultrastructural characters or levels of CRBPI expression.
It is noteworthy that, during the period of histogenesis, the
neural retina in contact with unaffected RPE always appeared
normal. However, in the neural retina of adult mutants,
degenerating, dysplastic and normal areas could be observed
adjacent to both normal or abnormal RPE (based on the same
ultrastructural and immunohistochemical criteria; data not shown).
In any event, these data suggest that (i) large portions of the
mutant RPE are morphologically and/or functionally abnormal, (ii)
during retinal differentiation, but not in the adult retina, there
is a tight spatial correspondence between the morphological defects
in the RPE and the abnormalities of the neural retina (i.e.
rosettes, folds, foci of degeneration, reduced cell proliferation,
absence of opsin expression by photoreceptors and/or lack of the
photoreceptor segment formation).
Apoptotic bodies were occasionally observed in RPE of both
differentiating (small arrows in Fig. 7e) and mature mutant retinas
(results not shown), but never in the RPE-derived cells scattered
in the periocular mesenchyme (open arrows in Fig. 7b and c). These
observations suggest that the loss of mutant RPE cells occurs
either by migration away from the neural retina or by cell
Distribution of RARs in ocular tissues The distribution of the
transcripts of the 3 RARs was investigated by in situ hybridization
at several prenatal and postnatal stages of eye ontogenesis as well
as in the adult eye (Table 2 and Fig. 10). Both RARβ and RARγ were
strongly expressed in the periocular mes- enchyme from E15.5 to P7
(e.g. POM, Fig. 10b-d). Both RARγ and RARβ transcripts were also
detected in the RPE at the same developmental stages (Fig. 10d,e).
Previous studies in E9.5 to E14.5 mouse embryos and fetuses (Dollé
et al., 1990; Ruberte et al., 1990) showed expression of RARγ in
periocular mesenchyme and expression of RARβ in the peri- ocular
mesenchyme, the primary vitreous body and the RPE. Alto- gether,
these observations indicate that the periocular mesenchyme and the
RPE express RARγ and/or RARβ throughout the period of eye
development. RARβ and RARγ transcripts were occasion- ally present
in the INL and GCL, whereas RARα transcripts were detected in all
nuclear layers; RARα appears to be the main RAR
Fig. 5. Semithin sections an the fibroblastic aspect of the
choroid; ONL, outer nuclear pigment epithelium; SC, scl
expressed in the prenatal and the postnatal neural retina (Table
Thus, even though our probes did not specifically identify RARβ2 or
RARγ2 transcripts, the in situ hybridization data strongly suggest
that the periocular mesenchyme and/or the RPE are the target
tissues of the double mutation.
Non-retinal abnormalities in RARβ2γ2 mutants RARβ2γ2 adult mutant
eyes exhibited several abnormalities in addition to retinal
dysplasia and degeneration (Table 1): (i) a reduction of the
palpebral aperture (i.e. blepharophimosis) was observed in all
RARβ2γ2 mutants (Table 1). Its degree, evaluated by measuring the
intercanthal distance (e.g. compare the distance between IC and OC
in Fig. 1a and b), ranged from small decreases to near absence of
the palpebral fissure. (ii) a posterior persistent hyperplastic
primary vitreous (retrolenticu- lar membrane: RL in Fig. 1d,f,g)
was observed in all RARβ2γ2 mutants. However, this defect was not
specific to these double mutants, since it was also seen in ~70%
(11 out of 16) of the RARβ2 single null mutant eyes examined, in
~90% (16 out of 18 eyes) of RARβ2−/−/RARγ2+/− compound mutants
(data not shown) and in ~90% (62 out of 72 eyes) of the RARβ single
null mutant eyes (N. Ghyselinck, M. M. and P. C., unpublished
results). (iii) colobomas of the sclera and/or of the choroid
2181Retinoic acid in retinal development
ation evidenced by BrdU incorporation in newborn (a-d) and P4 (e,f)
RARβ2γ2 (b,d,f) mutants. The arrowheads indicate the absence of the
s of newborn mutants, the cell proliferation is unaffected in areas
of al retinas the areas lacking RPE show a lower proportion of
labelled n cell layer; L, lens; NBL, outer neuroblastic layer; R,
neural retina; C, sclera; V, vitreous body. Immunoperoxidase with
hematoxylin a,b); ×77 (c,d); ×155 (e,f).
large portions of the eyeball completely lacking the sclera and/or
the choroid) were present in all RARβ2γ2 eyes; these defects were
often readily visible in the form of distended and translu- cent
regions at external inspection of enucleated eyes (Fig. 1d,f).
Where present, the sclera (SC) and the choroid (CH) usually showed
a marked thinning (compare Fig. 2a with b and c). It is noteworthy
that the choroid is made up of two main cell types, which have
distinct embryological origins, namely neural crest- derived
melanocytes and capillary endothelial cells originating from the
head mesoderm (Johnston et al., 1979). At the histo- logical level,
fewer capillaries were observed adjacent to the retina in areas
completely lacking melanocytes (arrow- heads in Fig. 2b, compare
with 2a). (4) Unilateral or bilateral absence of the Harderian
gland (i.e. the main periocular gland in the mouse) was observed in
approximately one fourth of the mutant eyes. Similar agenesis of
the Harderian gland occurs occa- sionally in RARγ single null
mutants and is fully penetrant in RARα1γ and RARβ2γ double null
mutants (Lohnes et al., 1993, 1994). (5) Lens cataracts were
observed in about one third of the RARβ2γ2 eyes (Table 1), and were
characterized macro- scopically by the presence of large vacuoles
between the lens fibers and/or by the presence of a posterior
lenti- conus (which consists of a bowing of the posterior pole of
the lens capsule; data not shown).
The retrolenticular mem- brane, the local agenesis of the sclera
and choroid and the agenesis of the Harderian glands were also
observed in newborn RARβ2γ2 mutants, and thus correspond to con-
genital defects (Table 1). The local absence of the RARβ2γ2 choroid
was con- spicuous as early as P4, when ocular melanocytes start to
differentiate. In contrast, the lens abnormalities were only
detected in adult mutants and their penetrance appeared to increase
with aging (Table 1). Since, in the mouse, the palpebral fissure
starts to form only at P12-P14 (Theiler, 1972), the ble-
pharophimosis could only be
Fig. 6. Comparison of cell prolifer neural retinas from WT (a,c,e)
and RPE. Note that, in the neural retina RPE agenesis; however, in
P4 neur cells. Abbreviations: GCL, ganglio RPE, retinal pigment
epithelium; S counterstain. Magnifications: ×40 (
diagnosed in adult RARβ2γ2 mutants; note, however, that in all
E14.5 RARβ2γ2 mutants the palpebral fissure was con- spicuously
smaller than in wild-type fetuses of the same age and weight (Fig.
11a,b, and N. Ghyselinck, M. M. and P. C., unpublished results)
implying that eyelid hypoplasia, which is the underlying cause of
the blepharophimosis, is determined early, in any event before
eyelid closure at E15-E16 (Theiler, 1972; Juriloff and Harris, 1993
and references therein). It is also noteworthy that the defects
observed in the periocular con- nective tissues were not spatially
correlated with the neural retina defects in newborn, young or
2182 J. M. Grondona and others
The phenotypic analysis of the RARβ2γ2 mutant mice provides the
first evidence of the indispensability of retinoic acid (RA), both
for the postnatal stages of neural retina histo- genesis and the
survival of differentiated neural retina cells, in vivo. At least
some of the effects of RA on the neural retina which are revealed
by the RARβ2γ2 compound mutation are likely to be mediated by the
RPE, as discussed below.
Pathogenesis of the retinal dysplasia in RARβ2γ2 null mutants The
RARβ2γ2 null mutant mice analysed in this report represent the
first genetically characterized model of retinal dysplasia. Retinal
dysplasia usually refers to a disorganization of the laminar
pattern of the developing neural retina and is defined
histologically by foldings of the neural retina or by the presence
of rosettes composed of neurons and glial cells (Sil- verstein et
al., 1971; Lahav and Albert, 1973). Retinal dysplasia in humans
(Lahav and Albert, 1973; Godel et al., 1981; Potter and Traboulsi,
1993) and animals (Randall et al., 1983; Fite et al., 1982; Cook et
al., 1991; Whiteley, 1991;
Table 2. Detection of RAR trans
Periocular mesenchyme Retinal pigment epithelium Outer neuroblastic
layer Outer nuclear layer Inner nuclear layer Ganglion cell
E, embryonic day; P, post-natal day; Nb, newborn; m, months. NA,
not applica neuroblastic layer at P4.
*Internal portion of the outer neuroblastic layer (presumptive
inner nuclear laye respectively.
+ + + + + + + ++ + + + + + + + + + + + + + + NA NA NA − −
NA NA NA NA + + + NA N NA NA NA NA ++ ++ ++ NA N ++ ++ ++ ++ ++ ++
++ − +/
Caffé et al., 1993; Toole, 1983) can be caused by a variety of
genetic and environmental factors and are frequently associ- ated
with multiple eye defects.
The RARβ2γ2 dysplastic retina is associated with a persis- tent
primary vitreous body, an absent or abnormal RPE, a partial
agenesis of the choroid, and sclera and lens degenera- tion,
raising the question as to which ocular tissue(s) is (are)
primarily affected by the compound mutation. The earliest ocular
defect observed in RARβ2γ2 mutants at E14.5 (data not shown) is the
persistence of the primary mesenchymal vitreous body, resulting in
the presence of a retrolenticular membrane in all postnatal
mutants. A persistent hyperplastic vitreous body is often
associated with retinal dysplasia in human patients (Lahav and
Albert, 1973; Godel et al., 1981; Potter and Traboulsi 1993; and
references therein). However, the two defects appear to be
unrelated in the present mouse model, since ~70% of the RARβ2
single null mutants display a retro- lenticular membrane which, in
this case, coexists with a normal retina.
Both embryonic and adult neural retinas contain high levels of RA
as well as the enzymatic machinery required for its synthesis
(McCaffery et al., 1993, and references therein). RA
Fig. 7. Comparison of apoptosis evidenced by the TUNEL method in P4
(a,b) and P14 (d,e) retinas from WT (a,d) and RARβ2γ2 mutant mice
(b,e). c and f are bright- field views of b and e, respectively.
The open arrows in b and c indicate the absence of apoptosis in the
pigmented cells scattered within the periocular mesenchyme. The
small arrows and the arrowheads in e point to apoptotic bodies in
the RPE and in the ONL respectively. Abbreviations: GCL, ganglion
cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; Ro,
rosettes; V, vitreous body. Magnifications: ×77 (a-f).
cripts by in situ hybridisation
ble: the inner and the outer nuclear layers are formed from the
r). +/−, + and ++, weak (close-to-background), moderate and strong
.5 Nb P3 P7 P21 2m E15.5 E17.5 Nb P3 P7 P21 2m
+ ++ ++ ++ ++ − ++ ++ ++ ++ ++ + + + + + + − + + + + + + + − +* NA
NA NA − − − − NA NA NA
A NA NA − − − NA NA NA NA − − − A NA NA + + − NA NA NA NA − + + −
+/− +/− + + − − − − − − + +
2183Retinoic acid in retinal development
copy of 1 month-old retinas from WT (a,c) and RARβ2γ2 (b,d) mutant
a higher magnification of the area boxed in b. The mutant RPE cells
lack ir cytoplasm is highly vacuolated. Abbreviations: BM, Bruch’s
basement ndria; Mi, microvilli; N, nuclei of RPE cells; PH,
phagosomes; POS, ments; V, vacuoles. Magnifications: ×4500 (a,b);
was shown to promote the differentiation and survival of isolated
embryonic retina photoreceptors (Kelley et al., 1994; Stenkamp et
al., 1993). However, the neural retina is unlikely to represent a
primary target tissue of the compound mutation since RARβ and RARγ
do not appear to be expressed in the outer neuroblastic layer
(which expresses RARα) during the period of retinal lamination.
Moreover, the differentiation of the ganglion cells, in which RARβ
transcripts are detected from E17.5 onwards, seems to occur
normally in RARβ2γ2 mutants as, in newborn mutants, these cells
have withdrawn from the mitotic cycle and possess well-developed
axons which have reached the diencephalon, and both the thickness
of the ganglion cell layer and calibre of the optic nerve appear
normal at this stage.
In contrast, the wild-type RPE expresses both RARβ and RARγ tran-
scripts before and during the period of retinal lamination. In
RARβ2γ2 mutants, focal histological abnormalities of the RPE
precede the retinal dysplasia and their spatial distribution
strikingly corre- sponds to that of the dysplastic areas. A spatial
correlation between RPE struc- tural defects and retinal dysplastic
areas has also been reported in heritable forms of retinal
dysplasia in mice (Cook et al., 1991) and chicks (Randall et al.,
1983; Fite et al., 1983), as well as in a variety of human retinal
dysplasia (reviewed in Silverstein et al., 1971). Contact between
the RPE and the neural retina is required for morphological and
functional pho- toreceptor differentiation in cultures of Rana
pipiens ocular rudiments (Holly- field and Witkowsky, 1974) and co-
culture of RPE cells is necessary for laminar organisation within
the neural retina of chick (Vollmer and Layers, 1986). Fetal RPE
cells secrete a protein that induces a neuronal phenotype in
cultured retinoblastoma cells, suggesting that RPE-derived
paracrine factors play a role in the differentiation of the neural
retina (Steele et al., 1990). The impor- tance of the RPE in the
maintenance of the structural integrity of the retina, in vivo, has
been elegantly demonstrated by Raymond and Jackson (1995) in a
trans- genic mouse line expressing the attenu- ated diptheria toxin
A gene under the control of the tyrosinase-related protein- 1
promoter which is specifically active in RPE cells and melanocytes.
Perinatal tox- icogenic ablation of the RPE results in a retinal
dysplasia strikingly resembling that of the RARβ2γ2 mutants,
notably with respect to the possible origin of rosette formation:
the rosettes observed in both Raymond and Jackson’s RPE- deficient
mice and in our mutants appear to arise from the migration of ONL
Fig. 8. Electron micros mice. d corresponds to microvilli (Mi) and
the membrane; M, mitocho photoreceptor outer seg
between the neural retina and the locally disrupted RPE. Since, in
the adjacent areas, the RPE is attached to the neural retina (note
that the detachment indicated by asterisks in Fig. 3c-e are typical
artifacts due to tissue processing), mechanical con- straints are
generated, which cause an inward bending of the ONL. This bending,
in turn, might provoke an inversion of the polarity of the
photoreceptors located in the hinge region. Interestingly, this
pathological cell rearrangement permits the photoreceptor to
express differentiated features (i.e. segment formation and
expression of opsin) in the absence of contact with the RPE (Figs
1m, 4d). Taken together, these data strongly suggest that a
defective RPE might be responsible for the retinal dysplasia in the
RARβ2γ2 mutants, either through a
2184 J. M. Grondona and others
Fig. 9. Immunodetection of CRBPI in 1 month old retinas from WT and
RARβ2γ2 mutant. Note the near absence of CRBPI expression in the
lower right portion of the mutant’s RPE in panel b. Abbreviations:
R, neural retina; RPE, retinal pigment epithelium; SC, sclera; V,
vitreous body. Magnifications: ×40 (a) and ×45 (b).
local disruption of the blood-retinal barrier (whose major
component is the RPE) subjecting the neural retina to systemic
disrupting influences, or through the lack of a RPE-derived
positive signal, normally inducing and maintaining the neural
The periocular mesenchyme is another candidate as a primary target
tissue of the double null mutation, as it is the ocular tissue that
expresses the highest levels of RARβ and RARγ: it is conceivable
that, under the influence of RA, some periocular mesenchymal cells
might synthesize paracrine factors required for the laminar
organisation of the retina. Such factors might act either
indirectly, e.g. on differentiation, prolif-
Fig. 10. Detection RARβ and RARγ transcripts in a E17.5 WT eye by
in situ hybridization. a is a bright field of b. The large arrows
in d and e point to RPE cell nuclei. Note that the silver grains
located over the neural retina (R) correspond to background
labelling. Abbreviations: C, cornea; GCL, ganglion cell layer; EY,
eyelids; HG, Harderian gland; I, iris; L, lens; POM, periocular
mesenchyme; R, neural retina; RPE, retinal pigment epithelium; V,
vitreous body. Magnifications: ×20 (a-c); ×310 (d,e).
eration or survival of RPE cells, or directly after having crossed
the blood-ocular barrier. Recent transplantation experiments
demonstrating the absence of specificity of the peri-ocular mes-
enchyme in supporting the differentiation of the RPE cells (Buse et
al., 1993) argue against the first of these two possi-
Interestingly, Johnson (1939, 1943) also reported the occur- rence
of rosettes in the retinas of VAD rats. However, contrary to the
rosettes seen in our RARβ2γ2 mutants, these VAD- induced rosettes
were determined after the period of retinal lamination and were
interpreted as being the result of the reor- ganisation of the
remaining photoreceptors in the most severely degenerated areas
Pathogenesis of the retinal degeneration in RARβ2γ2 null mutants
Retinal degeneration is characterized histologically by an atrophy
of one or more layers of the neural retina. Several lines of
evidence suggest that retinal degeneration might be a direct
consequence of retinal dysplasia. Firstly, massive apoptosis is
observed within the rosettes and folds at P4, suggesting that these
sites will evolve toward degeneration. Accordingly, the frequency
of the mutant retinas displaying signs of dysplasia apparently
decreases as degeneration proceeds (Table 1). Secondly, the first
histological signs of retinal degeneration that are manifested by
focal atrophy of the ONL, are observed at the dysplastic sites at
P14. Thirdly, several similar cases of retinal atrophy accompanying
or preceded by retinal dysplasia have been described in mice and
chicks (Randall et al., 1983; Cook et al., 1991; Caffé et al.,
1992). Therefore retinal degen- eration in RARβ2γ2 mutants might be
initiated at sites of retinal dysplasia, and then spread toward the
retinal periphery either because the cells of the dysplastic areas
2185Retinoic acid in retinal development
Fig. 11. External aspect of the eye of E14.5 WT (a) and RARβ2γ2 (b)
mutant mice. Note the striking reduction of the palpebral aperture
in the mutant.
‘death’ factors or because they fail to produce a trophic factor
normally required for the survival of neural retinal cells (see
Huang et al., 1993 for further discussion).
Alternatively, or additionally, degeneration might occur de novo,
at least in some portions of the mutant retina. In contrast to the
‘dysplastic phenotype’, the ‘degenerative phenotype’ is fully
penetrant, suggesting that degeneration may not be solely a
consequence of dysplasia. Moreover, areas with a normal laminar
pattern, but displaying both a marked atrophy of all 3 nuclear
layers (ONL, INL and GCL) and apoptotic cells in the ONL and INL,
were observed in adult mutant retinas. Such areas of retinal
degeneration were not always contiguous to areas of absent or
histologically abnormal RPE. However, it is noteworthy that many
more RPE cells were found to be altered on the basis of
ultrastructural and immunocytochemical criteria than on
histological criteria alone. The adult RPE has a critical role in
the homeostasis of the neural retina through the regen- eration of
the 11-cis retinaldehyde (the chromophore of the visual pigment) in
the visual cycle, the selective transport of retinoid and nutrients
to the photoreceptors (forming part of the blood-retinal barrier),
the phagocytosis of the distal portion of the rod outer segments,
the production of interphotorecep- tor matrix material and of
trophic factors for the neural retina (Bok, 1993; Campochiaro,
1993; Saari, 1994). Culture media conditioned by normal RPE cells
promote photoreceptor survival, indicating that cytokines and other
growth factors produced by RPE cells may exert a trophic influence
on the maintenance of the neural retina (reviewed in Campochiaro et
al., 1993 and Sheedlo et al., 1993). A defective RPE appears to be
the direct cause of neural retina degeneration in the Royal College
of Surgeons strain of rats (RCS rats; Bok and Hall, 1971; Malecaze
et al., 1993; and references therein) and in human choroideremia
(reviewed in Bird and Jay, 1994). Recent immunohistochemical
studies indicate that RPE cells syn- thesize bFGF, which is an
important photoreceptor survival factor as it can rescue
photoreceptor degeneration in RCS rats and in light-damaged rats
(Steinberg, 1994, and references therein). Moreover, the RPE
appears to be a target tissue of RA action since (i) RA can prevent
dedifferentiation and loss of density-dependent growth control of
human RPE cells in culture (Campochiaro et al., 1991), and (ii) the
VAD-induced RPE defects in adult rats (i.e. flattening and
RPE cells; Johnson, 1939, 1943; Dowling and Wald, 1958) can
apparently be prevented by supplementing their diet with RA
(Dowling, 1964; Dowling and Gibbons, 1961; Carter-Dawson et al.,
1979). Taken together, all of these data raise the possi- bility
that a defective RPE could also be instrumental in the genesis of
the RARβ2γ2 retinal degeneration through events that are not
secondary to the retinal dysplasia.
The choroid is thought to represent the main source of blood supply
for the ONL (reviewed in Bernstein, 1961) and large portions of
this tissue are lacking in RARβ2γ2 mutants. However, we did not
find any correlation between the regions displaying choroidal
agenesis and the presence of lesions in the neural retina, e.g.
RARβ2γ2 mutants often show portions lacking choroid juxtaposed with
areas of normal retina (see Fig. 2b). Moreover, mutant mice
deficient in melanocyte pre- cursors, such as Dominant white
spotting (w) and Steel (Sl), do not develop retinal defects
(Jackson, 1994). These data suggest that the choroidal defects are
not the cause of retinal degener- ation.
In any event, the phenotype of the RARβ2γ2 mutants strongly
suggests that RA is most likely required for the survival of the
rod photoreceptors, the bipolar neurons, (which represent the major
INL cell type), and the ganglion cells. Experiments aimed at
rescuing the RARβ2γ2 mutant phenotype through specific reexpression
of RARβ or RARγ in the RPE should demonstrate whether these trophic
effects of RA on the neural retina are mediated by the RPE, as
Pleiotropic role of RARs in retinal maintenance and eye development
Retinoids have trophic effects on the neural retina as first
demonstrated by Johnson (1939, 1943) and later by Dowling and
colleagues (Dowling and Wald, 1958; Dowling and Gibbons, 1961;
Dowling, 1964) in studies of degenerative changes in retina of rats
deprived of vitamin A. According to Johnson’s data (1939, 1943),
the degeneration of the neural retina induced by avitaminosis A is
progressive. It begins with the loss of the photoreceptor outer
segment, then involves suc- cessively ONL and the INL, and is
always more pronounced in the central than in the peripheral region
of the retina. The identity of the retinoids exerting these trophic
effects is unclear (discussed in Stenkamp et al., 1993). Systemic
administration of RA to VAD rats apparently prevents the death of
the cells of the INL, but not the night blindness, the
deterioration of the photoreceptor outer segment and death of the
photoreceptor cells, whereas administration of retinol can prevent
the appear- ance of all of these defects, (Dowling and Gibbons
1961; Dowling, 1964). However, as it is the case for the
blood-testis barrier (Van Pelt and de Rooij, 1991), RA is probably
not trans- ported across the blood-retinal barrier (Bridges et al.,
1983) and RA synthesized within the neural retina (or the RPE)
through metabolic conversion of retinol could be involved in the
trophic effect of vitamin A.
In this context, it is noteworthy that there are at least two
points of convergence between the neural retina degeneration in VAD
animals and RARβ2γ2 mutants. The first is purely morphological: the
neural retina degeneration in RARβ2γ2 mutants progresses from the
center towards the periphery of the retina and from the ONL towards
the internal retinal layers, thus resembling the progression
observed in VAD animals (see
2186 J. M. Grondona and others
above). The second deals with the physiopathology of the lesions:
in RARβ2γ2 mutants, CRBPI expression by RPE cells is impaired and,
since this protein likely plays an important role in the delivery
of retinoids to the photoreceptors (Saari et al., 1994), this
condition could create a state of VAD in the neural retina.
Interestingly, CRBPI mRNA levels are increased by RA treatment of
whole animals (Haq and Chytil, 1988) and the CRBPI gene contains a
RA response element in its promoter (Smith et al., 1991) which
further supports the proposal that the RPE could be a primary
target of the double null mutation (see above).
Warkany and Schraffenberger reported half a century ago (1946) that
the developing rat eye is the organ that is most sensitive to
vitamin A deprivation since, in less severely affected VAD fetuses,
it represented the only site of malfor- mations. We show here that
more than two thirds of the RARβ2 single null mutants display a
retrolenticular membrane that actually corresponds to the commonest
abnormality of the fetal VAD syndrome. It arises by persistence and
hyperplasia of the primary vitreous body, a structure that starts
to develop at E10.5 from periocular mesectodermal cells that enter
the optic cup, and has regressed at E14.0 by mechanisms that are
still unknown. In adult RARβ2 or RARβ2γ2 mutants, the per- sistence
of the primary vitreous is manifested by the presence of a plaque
of pigmented fibrovascular tissue connecting the posterior pole of
the lens with the optic papilla (the optic nerve exit point and
point of entry of retinal blood vessels). This abnormality, which
must result in poor vision, was previously overlooked in RARβ2
mutants (Mendelsohn et al., 1994b) due to its lack of behavioural
manifestation in the laboratory (note, in this respect, that
blindness does not overtly affect the behaviour of the laboratory
mouse; Grüneberg, 1952). RARβ2+/−/RARα−/− mice (Lohnes et al.,
1994) and RARβ2+/−/RARγ2−/− mice (our present data) never displayed
a retrolenticular membrane. Thus, one functional copy of the RARβ2
gene is sufficient to ensure the involution of the primary
mesenchymal vitreous. The penetrance of the persis- tent
retrolenticular membrane phenotype increased in a graded manner
upon removal of one, and then of both alleles of the RARγ2 gene
from the RARβ2 null genetic background and, in RARβ2γ2 double null
mutants, it was fully penetrant. These results suggest that RARγ2
can functionally compensate for the lack of RARβ2 in some RARβ2γ2
Aside from the neural retina, the RPE and the vitreous body, the
sclera, the choroid, the eyelids, and the lens were affected in
RARβ2γ2 mutants. Eyelids start to develop at E13.5 as mes- enchymal
outgrowths of the neural crest-derived periocular mesenchyme
covered by the ectoderm, whereas the sclera arises from the
compaction of the peripheral layers of the peri- ocular mesenchyme
at E16.5 This compaction event also indi- vidualizes the choroid, a
loose, highly vascularized mesenchy- mal tissue located between the
sclera and the RPE. The blepharophimosis and the local agenesis or
thinning of the choroid and sclera in RARβ2γ2 mutants might reflect
a direct effect of the double mutation in the periocular mesenchyme
which normally expresses high levels of both RARβ and RARγ in
embryos, fetuses and young mice. However, the cataracts could be
secondary to vascular invasion of the lens by blood vessels coming
from the retrolenticular membrane (discussed in Traboulsi,
Our previous analysis of RARαβ2, RARαγ, RARβ2γ and
RXRα mutants (Lohnes et al., 1994; Kastner et al., 1994), together
with the classical VAD studies of Warkany and Schraffenberger
(1946), have implicated retinoid signaling at almost every step of
prenatal eye morphogenesis. These include lens formation,
separation of the lens from the ectoderm, development of the outer
layer of the optic cup as RPE (demonstrated by the development of
neural retina in the place of RPE on the dorsal aspect of RARαγ
mutant eyes; Lohnes et al., 1994; P. Gorry, M. M. and P. C.,
unpublished data), development of the ventral retina, closure of
the optic fissure, involution of the primary vitreous body,
development of the eye’s anterior segment (cornea, conjunctival
sac, anterior chamber), development of periocular structures
(sclera, choroid and Harderian gland) and formation and fusion of
the eyelids). In addition, RA may be implicated in the formation of
the optic cup, as the development of the eye anlage is arrested at
the optic cup stage in cultured mouse embryos deprived of RA by
inhibition of yolk-sac retinol binding protein (RBP) synthesis
(Båvik et al., 1996). Thus, our present demonstration that RARs are
also required for retinal histo- genesis and survival of retinal
cells, further establishes the pleiotropic role of RA in eye
development. Interestingly, in the retina of VAD rat fetuses, the
formation of the inner neuro- blastic layer (the future GCL) and of
the IPL apparently fails to occur (Warkany and Schraffenberger,
1946). This absence of retinal laminar organisation is the only eye
defect of the fetal VAD syndrome which was not recapitulated in the
RAR and RXR single and double null mutants studied so far (Lohnes
et al., 1994; Kastner et al., 1994). It remains therefore to be
seen whether RA is also involved in fetal retinal
The problems of penetrance and expressivity of the retinal defects
The retinas (i.e. neural retina and RPE) of young RARβ2γ2 mutants
usually displayed only focal lesions (dysplasia and abnormal RPE)
coexisting with large, apparently intact areas. Thus, removal of
RARβ2 and RARγ2 does not completely abolish retinoid responsiveness
in target cells, but rather seems to bring this responsiveness
close to a threshold level below which the realization of
RA-dependent cellular events is impaired. Stochastic variation of
this residual retinoid respon- siveness among the target cells may
account for the observa- tion that defects are confined to limited
portions of the neural retina and of the RPE, and also for the
incomplete penetrance and expressivity of the retinal dysplastic
phenotype (Table 1). This possibility is further supported by the
observations that additional RAR inactivations in the RARβ2γ2
mutant back- ground (e.g. removal of one allele of RARγ1 or RARα1)
result in a marked increase in the number of dysplastic foci within
a given retina (our unpublished data). These observations also
suggest that the incomplete penetrance and expressivity of the
RARβ2γ2 retinal dysplastic phenotype does not result from
functional redundancy with RA-independent regulators.
The stochastic variation of retinoid responsiveness within retinal
cells may occur both at the spatial (i.e. between different cells
at a given time) and temporal (i.e. in a given cell at different
times) levels. For instance in a given RPE cell, this
responsiveness may be adequate at the time of birth and fall below
the critical threshold only after the completion of retinal
histogenesis, which would then impair the function of retinoids in
retinal maintenance. This may account for the fact that, in
2187Retinoic acid in retinal development
old animals, retinal degeneration is fully penetrant and affects
extensive portions of the retina. Thus, the degenerative phenotype
may be the consequence of either the retinal dysplasia and/or a
further impairment of RA function in retinas (or regions of the
retina) that had escaped defects during his- togenesis. Even though
we cannot exclude that differences in genetic background may
account for some of the phenotypic variations seen among different
animals, the considerable vari- ations in expressivity often
observed between the two eyes of a given animal cannot be explained
on that basis, and thus must be related to the stochastic processes
The present study demonstrates an essential role for RARs, and
therefore for retinoic acid, in retinal histogenesis and survival
of retinal cells. The present RARβ2γ2 mutant mice represent the
first genetically characterized animal model for retinal dysplasia.
Even though homozygous null compound mutations for both RARβ2 and
RARγ2 are unlikely to occur at a signif- icant rate in humans, our
data raise the possibility that genetic lesions affecting the
retinoid signalling pathway could underlie some cases of human
retinal dysplasia and/or degenerations, for which the genetic basis
is currently unknown. RAR- and/or RXR-deficient mice may also
provide interesting models to investigate the mechanism underlying
the therapeutic effects of vitamin A in some retinal degenerations
(Jacobson et al. 1995; Acott and Weleber, 1995).
We are grateful to Drs R. Molday and D. Hicks for the gift of anti-
opsin antibody, Dr Eriksson for the gift of anti-CRBPI antibody and
C. Mendelsohn for the RARβ2 mutant mice. We thank B. Weber, C.
Fisher, V. Giroult and S. Heyberger for excellent technical
assistance. We also thank Drs D. Hicks and J. Sahel for advice, B.
Boulay, J. M. Lafontaine and the secretariat staff for their help
in the preparation of this manuscript, and S. Ward for critically
reading the manuscript. This work was supported by funds from the
Centre National de la Recherche Scientifique, the Institut National
de la Santé et de la Recherche Médicale, the Centre Hospitalier
Universitaire Régional, the Association pour la Recherche sur le
Cancer, the Human Frontier Science Program, the Collège de France
and the Bristol-Myers Squibb Pharmaceutical Research Institute. J.
M. G. was supported by a long- term fellowship from the European
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