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Page 1: Retinal dysplasia and degeneration in RAR²2/RAR 2 compound mutant

2173Development 122, 2173-2188 (1996)Printed in Great Britain © The Company of Biologists Limited 1996DEV3430

Retinal dysplasia and degeneration in RARβ2/RARγ2 compound mutant mice

Jesús M. Grondona†,‡, Philippe Kastner‡, Anne Gansmuller, Didier Décimo, Pierre Chambonand Manuel Mark*

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS / INSERM / ULP / Collège de France, BP 163, 67404 IllkirchCedex, 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 authors

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 ofprenatal retinal dysplasia, all the ocular abnormalities ofthe fetal VAD syndrome are recapitulated in mousemutants lacking either RARα and RARβ2, RARα andRARγ, 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], thusdemonstrating that retinoic acid (RA) is the active vitaminA metabolite during prenatal eye morphogenesis. Whetherretinoids are also involved in postnatal eye developmentcould not be investigated, as VAD newborns are not viableand the above RAR double null mutants and RXRα nullmutants died in utero or at birth.

We report here the generation of viable RARβ2/RARγ2double null mutant mice, which exhibit several eye defects.The neural retina of newborn RARβ2γ2 mutants is thinnerthan normal due to a reduced rate of cell proliferation, andfrom day 4 shows multiple foci of disorganization of itslayers. These RARβ2γ2 mutants represent the first geneti-

cally characterized model of retinal dysplasia and theirphenotype demonstrates that RARs, and therefore RA, arerequired for retinal histogenesis. The RARβ2γ2 retinalpigment epithelium (RPE) cells display histological and/orultrastructural alterations and/or fail to express cellularretinol binding protein I (CRBPI). Taken altogether, theearly onset of the RPE histological defects and theirstriking 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 likelytarget tissue of the RARβ2γ2 double null mutation. Adegeneration of the adult neural retina, which maysimilarly be secondary to a defective RPE, is also observedin these mutants, thus demonstrating an essential role ofRA in the survival of retinal cells. Moreover, all RARβ2γ2mutants display defects in structures derived from the peri-ocular mesenchyme including local agenesis of the choroidand of the sclera, small eyelids, and a persistence of theprimary mesenchymal vitreous body. A majority of theRARβ2 single null mutants also exhibit this latter defect,thus demonstrating that the RARβ2 isoform plays a uniquerole in the formation of the definitive vitreous body.

Key words: vitamin A, retinoic acid, nuclear receptors, retinalhistogenesis, retinal pigment epithelium, mouse, dysplasia

SUMMARY

INTRODUCTION

Vitamin A deficiency (VAD) studies have shown that vitaminA (retinol) is required during prenatal and postnatal develop-ment, and during adult life. After birth, retinol is indispensablefor survival, growth, reproduction and vision and also for themaintenance of numerous tissues. Widespread squamous meta-plasia of the conjunctival, corneal, respiratory, urinary andvarious glandular epithelia (i.e. olfactory, salivary, Harderian,seminal vesicle and prostate glands and/or their associatedexcretory ducts), together with degeneration of the seminifer-ous tubules and of the neural retina, are hallmarks of thepostnatal VAD syndrome (Wolbach and Howe, 1925; Johnson,

1939). Interestingly, retinoic acid (RA) can prevent or reversethe deleterious effects of a postnatal VAD diet, with theexception 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 largenumber of congenital malformations (i.e., the fetal VADsyndrome) affecting the eye, the kidney and genitourinarytract, the heart and aortic arch-derived great arteries, the lungand the diaphragm. Retinol can prevent these malformationsprovided that it is supplied to the dams at specific times ofgestation, thus demonstrating that it is required at severalstages during ontogenesis (reviewed in Wilson et al., 1953).

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Two families of nuclear receptors for retinoids have beencharacterized. Members of the RAR family (types α, β and γ,and their isoforms α1, α2, β1 to β4, and γ1 and γ2) areactivated by most physiologically occurring retinoids (all-transRA, 9-cis RA, 4-oxo RA and 3,4 dihydro RA). In contrast,members of the RXR family (types α, β and γ, and theirisoforms) are activated by 9-cis RA only. In addition to themultiplicity of receptors, the complexity of retinoid signallingis further increased by the fact that, at least in vitro, RARs bindto their cognate response elements as heterodimers with RXRs.Moreover, RXRs can also bind in vitro to some DNA elementsas homodimers and are heterodimeric partners for a number ofnuclear receptors other than RARs (reviewed in Chambon,1994; Giguère, 1994; Mangelsdorf and Evans, 1995).

Null mutations of the RAR genes (either α, β or γ), as wellas isoform-specific knock-outs for RARα1, RARβ2/β4 andRARγ2 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 wereviable and displayed abnormalities which, however, wereconfined to a small subset of the tissues that express thesereceptors (Lufkin et al., 1993; Lohnes et al., 1993). Thesefindings suggested that there could be some functional redun-dancy in the RAR family. To test this hypothesis, double nullmutants 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 mutantsexhibited a dramatically reduced viability, as about half of theRARαγ mutants died in utero, and the remaining half as well asthe other RAR double null mutants survived for 12 hours at mostfollowing delivery by Caesarean section at full term (Lohnes etal., 1994). Furthermore, almost all of the malformations of thefetal VAD syndrome were recapitulated in the different RARdouble mutants, with the exception of a shortening of the ventralretina, 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 duringontogenesis, and that its effects are mediated by the RARs.

We have recently generated double null mutant mice lackingthe RARβ2 and RARγ2 isoforms. These RARβ2−/−/RARγ2−/−

mutants (hereafter referred to as RARβ2γ2 mutants) werenormally viable and fertile. However, they displayed severeocular defects. Their analysis demonstrates that RARs, andtherefore 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 tissueof the RARβ2γ2 compound mutation.

MATERIALS AND METHODS

Generation of RARβ2γ2 double null mutantsRARγ2 (Lohnes et al., 1993) and RARβ2 (Mendelsohn et al., 1994b)mutant mice were bred to generate double heterozygote mice, whichwere intercrossed to produce RARβ2−/−/RARγ2−/−, RARβ2−/−/RARγ2+/− and RARβ2+/−/RARγ2−/− mice, from which most of themutants used in this study were generated.

Histology and immunohistochemistryMice 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 planedefined by the superior-inferior axis and the optic nerve. Lenses wereremoved and the eyes embedded in paraffin. Occasionally, skinnedskulls from newborn, 4 days and 1-week-old mice were fixed in totoin Bouin’s fluid to prevent any damage that might have occurred duringenucleation. 7 µm serial sections were mounted on slides coated with0.01% poly-L-lysine (Mr 350,000; Sigma). The sections were stainedwith Groat’s hematoxylin and Mallory’s trichrome (Mark et al., 1993)or employed for immunohistochemistry. The following antibodieswere used : a mouse monoclonal IgG directed against rod-specificopsin (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 rabbitpolyclonal antibody directed against CRBPI (in the form of anantiserum; a gift of U. Eriksson, Ludwig Institute for Cancer Research,Stockholm; Gustafson et al., 1993); a polyclonal antibody against glialfibrillary acidic protein (GFAP; Sigma); and an anti-BrdU monoclonalantibody (Boehringer, Mannheim). These antibodies were used atdilutions of 1:40, 1:100, 1:400 and 1:100, respectively. Biotinylatedanti-mouse IgG, biotinylated anti-rabbit IgG (Vectastain Elite ABCKit, 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 forimmunofluorescence labelling. All primary antibodies, anti-IgG anti-bodies and components of the ABC system were diluted in PBS pH7.3, containing 0.05% Tween 20 and 0.5% normal goat serum. Controlsections were incubated with either mouse or rabbit preimmune seruminstead of the primary antibody.

Fixation by perfusion was employed in one histological experiment(illustrated in Fig. 2) in order to visualize better the capillary networkof the periocular mesenchyme. Under general anaesthesia, adult micewere perfused with 1% glutaraldehyde–4% paraformaldehyde in PBS(pH 7.2) at an outflow rate of 5 ml/minute for 5 minutes. The eyeswere enucleated, then postfixed in Bouin’s fluid for 24 hours.

Electron microscopyEyes from 7-day-old and 1-month-old animals were fixed byimmersion 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 thenrinsed in cacodylate buffer, postfixed in 1% osmium tetroxide in thesame buffer for 2 hours at 4°C, dehydrated with graded alcohols seriesand embedded in Epon. 1 µm sections were stained with toluidineblue. Ultrathin sections from selected areas were contrasted withuranyl acetate and lead citrate and examined with a Philips 208electron microscope operating at 80 kV.

Labeling of S-phase nucleiBromodeoxyuridine (BrdU) (Sigma) dissolved in PBS was injectedintraperitoneally at a dose of 50 mg per kg of body weight. The micewere killed 2 hours later, the eyes fixed in Bouin’s fluid for 2 days,then embedded in paraffin. BrdU incorporation was detected by usingan anti-BrdU monoclonal antibody (Boehringer) and immunoperoxi-dase labelling.

End-labeling of DNA nicks in tissue sections (TUNELstaining)In situ detection of fragmented DNA was performed as described byGavrieli et al. (1992) with some modifications. 7 µm sections fromBouin-fixed, paraffin-embedded eyes were collected on poly-L-lysinecoated slides. The sections were dewaxed and then hydrated. Afterrinsing in distilled water (3× 5 minutes) the sections were digested for15 minutes at 24°C with 20 µg/ml proteinase K in 50 mM Tris-HClpH 7.5 containing 50 µM EDTA, rinsed in distilled water (3×5minutes), and then incubated for 1 hour at 37°C with biotinylateddUTP in terminal transferase buffer (all from Boehringer). The

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reaction was terminated by transferring the slides to distilled water.The sections were permeabilized with 0.05% Tween 20 in PBS (3×5minutes) and biotin incorporation was revealed with Cy3-conjugatedstreptavidin (Jackson ImmunoResearch) diluted 1:400 (30 minutes at24°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 µmfrozen sections from albino mice, as described (Décimo et al., 1995).

RESULTS

RARβ2γ2 double null mutant mice are viableIn contrast to the RAR compound mutants analysed previously(Lohnes et al., 1994; Mendelsohn et al., 1994a), which all diedwithin 12 hours after birth, RARβ2γ2 homozygotes werenormally viable and fertile. From external inspection, however,their eyes appeared abnormal (e.g. compare Fig. 1a and b).

Retinal degeneration and dysplasia in adultRARβ2γ2 mutantsIn 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 areseparated by two layers of synaptic interplay, namely the outerplexiform layer (OPL) between the ONL and INL and the innerplexiform layer (IPL) between the INL and GCL (Fawcett,

Table 1. Eye abnormalities in

Nb22

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 0

# 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 degNb, newborn; P, post-natal day; m, month; ONL, INL and GCL, outer nuclear

epithelium.

1986; Huxlin et al., 1992; Sarthy et al., 1991 and refs therein;Fig. 1k).

The neural retina of adult RARβ2γ2 mutants (i.e. 1-monthold or older) exhibited two types of abnormal phenotypes,namely a marked atrophy (degeneration) and a disorganisation(dysplasia) of the retinal layers

Retinal degenerationMacroscopic examination of 1-month-old eyes revealed amarked reduction in the thickness of some portions of theneural retina (compare R, Fig. 1c and d). In older mutants (6to 12 months), this reduction in thickness was observed overlarger patches (compare R, Fig. 1e and f). In the most severelyaffected eyes (Fig. 1f and i), only 2 to 3 rows of nuclei persistedin the central retina, whereas in the peripheral retina, close tothe iris, all nuclear and the synaptic retinal layers remainedidentifiable although the ONL, INL, OPL and IPL were con-spicuously thinner and the GCL contained fewer cells, ascompared to their wild-type counterparts (compare Fig. 1h andk with i and l). The patches of neural retinal atrophy alwaysinvolved the ONL (Table 1, results not shown), whereas theINL and GCL were affected only in place where the ONL hadalmost disappeared (e.g. Figs 1l and 2c).

In WT mice, the glial fibrillary acidic protein (GFAP) geneis 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 unspecificresponse 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, numerouspositive cellular processes, consistent with a degenerativeprocess (data not shown).

Even though the penetrance of the retinal degeneration wascomplete (i.e. every eye displayed the defect; Table 1), its extent

RARβ2/RARγ2 null mutantsNumber of RARβ2γ2 eyes analysed

Retinal differentiation period Adult retina

P4 P7 P14 1-3m 6-12m16 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 5/18

0 0 9/14 1/14 0/180 0 3/14 5/14 2/180 0 0 8/14 16/18

# # # # ## # # # ## # # # #

6/16 3/13 3/14 3/14 6/18NA† NA† NA† # #

0 0 0 4/14 9/18

te.eneration., inner nuclear and ganglion cell layers, respectively; RPE, retinal pigment

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Fig. 1. Comparison of thepalpebral fissure, eyeball andretina between WT and RARβ2γ2(β2γ2) mutant adults, at one year(a,b,e-m) and 1 month (c,d). h, iand j correspond to histologicalsections of e, f and g,respectively; note that the lenseswere removed prior to paraffinembedding. k, l and m correspondto high magnification of regionsboxed in h, i and j respectively.The open arrow in j points to afocus of degeneration. Thearrowheads in l and m indicatethe absence of retinal pigmentedepithelium (RPE). Thedetachment of the neural retinafrom the RPE (asterisk in l) isartifactual. Abbreviations: CH,choroid; F, retinal fold; GCL,ganglion cell layer; HG,Harderian gland; I, iris; IC, innercanthus; INL, inner nuclear layer;IPL, inner plexiform layer; L,lens; OC, outer canthus; ONL,outer nuclear layer; OPL, outerplexiform layer; PS,photoreceptor segments; R,retina; RL, retrolenticularmembrane; Ro, rosettes; RPE,retinal pigment epithelium; SC,sclera; V, vitreous body.Mallory’s trichrome-hematoxylin(h-m). Magnifications: ×13 (c andd); ×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 thisrespect that Fig. 1d shows an example of one of the mostaffected eyes at 1-month. Moreover, in a given mutant, the twoeyes 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 andINL in Fig. 2a and b), exhibiting only rare foci of degeneration,whereas the right retina (Fig. 2c) is essentially degenerated.

Retinal dysplasiaThe second abnormal phenotype displayed by about one third

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Fig. 2. Scleral and choroidal defects in RARβ2γ2 mutant eyes.Histological sections from perfusion-fixed, 6 months old, WT (a) andRARβ2γ2 eyes (left and right eyes from the same mutant, asindicated in b and c). Note the paucity (c) or the absence (b) of thechoroidal melanocytes (CH) and the thinning of the sclera (SC). Alsonote that b and c represent typical aspect of the left and the rightneural retinas in this mutant. Abbreviations: CH, choroid; GCL,ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiformlayer; ONL, outer nuclear layer; OPL, outer plexiform layer; RPE,retinal pigment epithelium. The arrowheads point to periocularcapillaries. 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 thepresence of retinal folds and rosettes (F and Ro in Fig. 1g,j,m).Note that each of these dysplastic neural retinas also exhibitedoccasional foci of degeneration (open arrow in Fig. 1j, and datanot shown) and large areas with a normal laminar pattern (e.g.,R in Fig. 1j).

It is noteworthy that these retinal defects were neverobserved in the RARβ2 and in RARγ2 single null mutants.

Developmental abnormalities of RARβ2γ2 mutantneural retinasIn order to gain insights into the pathogenesis of the aboveretinal abnormalities in adult RARβ2γ2 mutants, we studiedthe processes of lamination, rod functional differentiation, cellproliferation and apoptosis in the neural retina.

Abnormal retinal laminationDuring normal mouse retinal development, the IPL begins toform by embryonic day 17.5 (E17.5) and separates the futureGCL from the outer neuroblastic layer (Pei and Rhodin, 1970).Likewise, by postnatal day 4 (P4), the OPL appears in thecentral 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 neuralretina (Young, 1984).

In ~70% of the RARβ2γ2 newborn mutant eyes (Table 1),the entire outer neuroblastic layer appeared thinner than its WTcounterpart (compare NBL, in Figs 3a, 6a and 6c with 3b, 6band 6d; the overall thickness of these mutant retinas was 25%lower than normal). Additionally, in a few mutant eyes (3 outof 22; Table 1), focal areas of RPE agenesis were observed inthe central retina (arrowheads in Figs 3b, 6b and d, and seebelow). However, even in these areas devoid of RPE, the lam-ination of the mutant neural retinas was normal (e.g. NBL, IPLand GCL, in Figs 3b and 6d).

In P4, P7 and P14 mutant eyes, the generalized thinning ofthe neural retina, already noticed at birth, was conspicuous. Itaffected the photoreceptor segments (PS), the ONL and theINL. In contrast, at the same developmental stages, the mutantGCL was indistinguishable from its WT counterpart (Table 1and compare PS, ONL and INL in Fig. 3f and g). Additionally,multiple foci of dysplasia were observed in 80% of the mutantneural retina that displayed rosettes (Ro1 and Ro2 in Fig. 3c-e;Ro in Figs 3g, 4b, d), folds (F, Fig. 4c) and/or local retinaldetachments (RD, Fig. 4e). The rosettes comprised tubulararrangements of photoreceptors displaying well definedsegments (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 inFig. 3g) and of the INL (not shown) were frequently observedin these dysplastic areas at P14. At P4, P7 and P14 the dys-plastic areas of neural retina were always contiguous withpatches of abnormal or absent RPE, and always commencedabruptly at the point where the morphologically normal RPEterminated (open arrows in Figs 3c,d,g and 4b-e; arrowheads inFigs 3g, 4b,d, 6f; see below for a description of RPE defects).

Thus, on the basis of histological criteria, the RARβ2γ2mutant neural retinas display two distinct abnormal develop-mental phenotypes: (1) a congenital thinning of the neuralretina, which is ubiquitous and thus does not correlate with thepatchy distribution of the RPE defects, and (2) multiple foci ofretinal dysplasia, which are spatially correlated with defects inthe RPE.

Abnormal differentiation of mutant photoreceptorsFunctional differentiation of the rods, the major photoreceptortype in the mouse retina, occurs during the first days after birthand can be followed by the expression of the rod-specific opsinin the presumptive ONL (Hicks and Barnstable, 1987). Roddifferentiation is achieved at P14 which corresponds to thecompletion 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 theONL in newborn and P4 retinas (Fig. 4). In both WT mice andRARβ2γ2 mutants, the first opsin-immunoreactive cells weredetected 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 was indistin-

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stogenesis in WT and RARβ2γ2 mutants at birth (a,b), P4 (c-e) and P14e successive sections through the same portion of a mutant retina. Thef missing RPE and the open arrows to scattered RPE cells within thee arrows in g indicate an area where the ONL is atrophic. Asteriskent 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 normalRPE (Fig. 4b,c,e). In contrast, the ONL cells adjacent to areaslacking an histologically normal RPE did not express opsin(unlabelled small arrows in Fig. 4b-e), with the notableexception of some rosettes (Ro in Fig. 4d), nor did they formsegments (PS in Fig. 5, compare a and c with b and d). Theseresults strengthen the conclusion that the presence of RPE isessential for proper photoreceptor differentiation (Stiemke etal., 1994, and references therein), and further suggest that theabnormalities in the RARβ2γ2 RPE could be, at least in part,responsible for the defects observed in the neural retina (seebelow for further analysis of the mutant RPE).

Reduced cell proliferation inRARβ2γ2 mutant neural retinasCell proliferation in the retina wasexamined by bromodeoxyuridine(BrdU) incorporation (Fig. 6). Thefraction of cells incorporating BrdUduring a short exposure reflects thefraction of cells in S phase duringthis period, therefore permitting anassessment of the proliferation rateof a population of cells.

In RARβ2γ2 newborns, thelabelling index in the outer neurob-lastic layer was markedly reduced(between 18 and 28% of the total cellnumber, depending of the animal)when compared with WT (40% of thetotal cell number) (compare Fig. 6aand c with 6b and d). This reducedlabelling index affected both thecentral and peripheral retinas (Fig.6b), and was apparently identicalwhether the RPE was missing (arrow-heads in Fig. 6b,d) or present. At P4,the rate of cell proliferation haddecreased in both WT and mutantneural retinas, which displayedsimilar labelling indices in placeswhere the RPE was present (data notshown). In contrast, in portions of themutant neural retina facing largeareas devoid of RPE, the percentageof proliferating cells was markedlyreduced (12% in peripheral retina)compared to WT (26% in peripheralretina) (compare Fig. 6e and f).

Thus, a reduced cell proliferationrate during the perinatal period ofretinal development may account forthe generalized thinning of theRARβ2γ2 neural retina. However,this decrease in cell proliferationcannot explain the loss of the retinalcells in adult mutants, since cell pro-liferation already ceases by P6 andby P11 in the central and peripheralportions of the WT retina, respec-tively (Young, 1985a,b).

Fig. 3. Aspects of retinal hi(f,g). c-e correspond to threarrowheads point to areas operiocular mesenchyme. Thindicate artifactual detachmCH, choroid; GCL, ganglioouter neuroblastic layer; OPsegments; Ro, Ro1 and Ro2Mallory’s trichrome-hemat

Increased apoptosis in RARβ2γ2 mutant neural retinas.In the normal mouse retina, programmed cell death occursprimarily during the first 2 weeks after birth and is essentiallycompleted by the end of the third week (Young, 1984). The insitu end-labelling method of DNA nicks (TUNEL method;Gavrieli et al., 1992) was employed to compare cell deathbetween normal and mutant mouse retinas (Fig. 7).

At P4 and P14, the majority of apoptotic cells of WT retinaswere located in the INL and the GCL in agreement withYoung’s data (Young, 1984); apoptotic cells were scarce inWT ONL (Fig. 7a,d). In dysplastic areas of mutant retinas atthe same ages (i.e. P4 and P14), the number of apoptotic cellsin the INL was markedly increased (compare Fig. 7a and d with

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b and e), and dying cells were also often observed in the ONL(arrowheads in Fig. 7e). This suggests that, in addition to thereduced rate of cell proliferation mentioned above, an increaseof apoptosis also contributes to the extreme thinning of the INL

Fig. 4. Expression of the rod-specific opsin evaluated byimmunohistochemistry in P4 WT (a) and RARβ2γ2 (b-e) mutants.The open arrows point to scattered RPE cells in the periocularmesenchyme, the arrowheads to portions lacking RPE, and the smallarrows to areas of the ONL lacking expression of opsin. Note thatthese latter areas are always juxtaposed to an abnormal or absentRPE. The detachments between the RPE and neural retinal such asthe one illustrated in e likely occur in vivo; they have regularoutlines which are maintained on more than 20 serial sections; theirsize is small and the tissues bordering them are not damaged in anyfashion. These features distinguish them from artifactual retinaldetachments (asterisk in b and c) generated during tissue processing.Abbreviations: CH, choroid; GCL, ganglion cell layer; INL, innernuclear layer; ONL, outer nuclear layer; Ro, rosettes; RD, retinaldetachment; 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 dysplasticareas of P14 mutants (e.g. unlabelled arrows in Fig. 3g).

In WT animals older than 1 month, retinal cell death was nolonger detectable (Young, 1984, and data not shown). Incontrast, retinas from adult mutant mice displayed an averageof 4-6 apoptotic cells per section that were preferentiallylocated in areas of thin or folded retina (data not shown). Thus,apoptosis occurring after the period of physiological retinal celldeath is likely to account for the progression of degenerationin the retina of adult mutants.

Retinal pigment epithelium defects in RARβ2γ2mutantsAs mentioned above, foci of dysplastic neural retina werealways located adjacent to an absent or morphologicallyabnormal RPE. Moreover, the RPE defects preceded the neuralretina dysplasia in mutant eyes. Since the RPE is known to playan 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 couldbe instrumental in the generation of the RARβ2γ2 retinaldefects. Therefore, the morphology of the mutant RPE wasanalysed by electron microscopy, and its functional state wasinvestigated by examining the distribution of cellular retinolbinding protein I (CRBPI) which is normally expresseduniformly in RPE cells (Fig. 9a) and is believed to play acrucial role in the delivery of retinol to the neural retina(reviewed in Saari, 1994). From an analysis during the periodof retinal histogenesis (i.e. at P4 and P7) and in adult retina(i.e. at 1 month), mutant RPEs could be classified within threedifferent categories: disorganised RPE, abnormal RPE withoutloss of epithelial organisation and apparently normal RPE.

Disorganised RPEDuring the period of retinal histogenesis, scattered pigmentedcells were often detected within the periocular mesenchymeadjacent to the areas of dysplastic neural retina (open arrowsin Figs 3c,d,g, 4b-e, 7c). Semithin sections and electronmicroscopy of P7 retina (Fig. 5b,d) showed flattened RPE cellsthat had lost their epithelial arrangement and, in some cases,their contact with the neural retina (compare Fig. 5a and c withb and d). Note that these flattened pigmented cells representaltered RPE cells, not choroidal melanocytes, since they wereusually lying within large portions of the periocular mes-enchyme that were totally devoid of choroid (see Figs 4a-e and5b).

Abnormal ‘epithelial’ RPEElectron microscopic analysis of mutant RPE at P7 (results notshown) and 1 month (Fig. 8b,d) revealed abnormalities in areaswhere its epithelial organization was preserved: the cytoplasmof these RPE cells was highly vacuolated (V in Fig. 8d) andtheir apical microvilli (Mi, Fig. 8c), which normally inter-digitate with the photoreceptor outer segments, were absent.The photoreceptor outer segments facing these altered RPEcells were reduced in number and/or disorganized, showingimproper piling of their disks (compare POS in Fig. 8a and cwith b and d). Immunostaining on P7 (not shown) and 1-month-old mutant RPEs, revealed weak (with respect to wild type) orabsent CRBPI expression in several areas where this tissueappeared histologically normal (compare Fig. 9a with b and c).

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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, retinalera. Magnifications: ×380 (a,b); ×2250 (c,d).

Apparently normal RPEIn some areas of the mutant retina, the RPE was indistin-guishable from its WT counterpart, based on ultrastructuralcharacters or levels of CRBPI expression.

It is noteworthy that, during the period of histogenesis, theneural retina in contact with unaffected RPE always appearednormal. However, in the neural retina of adult mutants,degenerating, dysplastic and normal areas could be observedadjacent to both normal or abnormal RPE (based on the sameultrastructural and immunohistochemical criteria; data notshown). In any event, these data suggest that (i) large portionsof the mutant RPE are morphologically and/or functionallyabnormal, (ii) during retinal differentiation, but not in theadult retina, there is a tight spatial correspondence betweenthe morphological defects in the RPE and the abnormalitiesof the neural retina (i.e. rosettes, folds, foci of degeneration,reduced cell proliferation, absence of opsin expression byphotoreceptors and/or lack of the photoreceptor segmentformation).

Apoptotic bodies were occasionally observed in RPE of bothdifferentiating (small arrows in Fig. 7e) and mature mutantretinas (results not shown), but never in the RPE-derived cellsscattered in the periocular mesenchyme (open arrows in Fig.7b and c). These observations suggest that the loss of mutantRPE cells occurs either bymigration away from the neuralretina or by cell death.

Distribution of RARs in oculartissuesThe distribution of the transcriptsof the 3 RARs was investigated byin situ hybridization at severalprenatal and postnatal stages of eyeontogenesis as well as in the adulteye (Table 2 and Fig. 10). BothRARβ and RARγ were stronglyexpressed in the periocular mes-enchyme from E15.5 to P7 (e.g.POM, Fig. 10b-d). Both RARγ andRARβ transcripts were alsodetected in the RPE at the samedevelopmental stages (Fig. 10d,e).Previous studies in E9.5 to E14.5mouse embryos and fetuses (Dolléet al., 1990; Ruberte et al., 1990)showed expression of RARγ inperiocular mesenchyme andexpression of RARβ in the peri-ocular mesenchyme, the primaryvitreous body and the RPE. Alto-gether, these observations indicatethat the periocular mesenchymeand the RPE express RARγ and/orRARβ throughout the period ofeye development. RARβ andRARγ transcripts were occasion-ally present in the INL and GCL,whereas RARα transcripts weredetected in all nuclear layers;RARα appears to be the main RAR

Fig. 5. Semithin sections anthe fibroblastic aspect of thechoroid; ONL, outer nuclearpigment epithelium; SC, scl

expressed in the prenatal and the postnatal neural retina(Table 2).

Thus, even though our probes did not specifically identifyRARβ2 or RARγ2 transcripts, the in situ hybridization datastrongly suggest that the periocular mesenchyme and/or theRPE are the target tissues of the double mutation.

Non-retinal abnormalities in RARβ2γ2 mutantsRARβ2γ2 adult mutant eyes exhibited several abnormalities inaddition to retinal dysplasia and degeneration (Table 1): (i) areduction of the palpebral aperture (i.e. blepharophimosis) wasobserved in all RARβ2γ2 mutants (Table 1). Its degree,evaluated by measuring the intercanthal distance (e.g. comparethe distance between IC and OC in Fig. 1a and b), ranged fromsmall decreases to near absence of the palpebral fissure. (ii) aposterior persistent hyperplastic primary vitreous (retrolenticu-lar membrane: RL in Fig. 1d,f,g) was observed in all RARβ2γ2mutants. However, this defect was not specific to these doublemutants, since it was also seen in ~70% (11 out of 16) of theRARβ2 single null mutant eyes examined, in ~90% (16 out of18 eyes) of RARβ2−/−/RARγ2+/− compound mutants (data notshown) and in ~90% (62 out of 72 eyes) of the RARβ singlenull mutant eyes (N. Ghyselinck, M. M. and P. C., unpublishedresults). (iii) colobomas of the sclera and/or of the choroid (i.e.,

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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 thes of newborn mutants, the cell proliferation is unaffected in areas ofal retinas the areas lacking RPE show a lower proportion of labelledn cell layer; L, lens; NBL, outer neuroblastic layer; R, neural retina;C, sclera; V, vitreous body. Immunoperoxidase with hematoxylina,b); ×77 (c,d); ×155 (e,f).

β2γ2

large portions of the eyeball completely lacking the sclera and/orthe choroid) were present in all RARβ2γ2 eyes; these defectswere 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) usuallyshowed a marked thinning (compare Fig. 2a with b and c). It isnoteworthy 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 originatingfrom the head mesoderm (Johnston et al., 1979). At the histo-logical level, fewer capillaries were observed adjacent to theretina in areas completelylacking melanocytes (arrow-heads in Fig. 2b, compare with2a). (4) Unilateral or bilateralabsence of the Harderiangland (i.e. the main perioculargland in the mouse) wasobserved in approximatelyone fourth of the mutant eyes.Similar agenesis of theHarderian gland occurs occa-sionally in RARγ single nullmutants and is fully penetrantin RARα1γ and RARβ2γdouble null mutants (Lohneset al., 1993, 1994). (5) Lenscataracts were observed inabout one third of theRARβ2γ2 eyes (Table 1), andwere characterized macro-scopically by the presence oflarge vacuoles between thelens fibers and/or by thepresence of a posterior lenti-conus (which consists of abowing of the posterior pole ofthe lens capsule; data notshown).

The retrolenticular mem-brane, the local agenesis ofthe sclera and choroid and theagenesis of the Harderianglands were also observed innewborn RARβ2γ2 mutants,and thus correspond to con-genital defects (Table 1). Thelocal absence of theRARβ2γ2 choroid was con-spicuous as early as P4, whenocular melanocytes start todifferentiate. In contrast, thelens abnormalities were onlydetected in adult mutants andtheir penetrance appeared toincrease with aging (Table 1).Since, in the mouse, thepalpebral fissure starts toform only at P12-P14(Theiler, 1972), the ble-pharophimosis could only be

Fig. 6. Comparison of cell proliferneural retinas from WT (a,c,e) andRPE. Note that, in the neural retinaRPE agenesis; however, in P4 neurcells. Abbreviations: GCL, ganglioRPE, retinal pigment epithelium; Scounterstain. Magnifications: ×40 (

WT

diagnosed in adult RARβ2γ2 mutants; note, however, that inall E14.5 RARβ2γ2 mutants the palpebral fissure was con-spicuously smaller than in wild-type fetuses of the same ageand weight (Fig. 11a,b, and N. Ghyselinck, M. M. and P. C.,unpublished results) implying that eyelid hypoplasia, which isthe underlying cause of the blepharophimosis, is determinedearly, in any event before eyelid closure at E15-E16 (Theiler,1972; Juriloff and Harris, 1993 and references therein). It isalso noteworthy that the defects observed in the periocular con-nective tissues were not spatially correlated with the neuralretina defects in newborn, young or adult mutants.

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2182 J. M. Grondona and others

DISCUSSION

The phenotypic analysis of the RARβ2γ2 mutant miceprovides the first evidence of the indispensability of retinoicacid (RA), both for the postnatal stages of neural retina histo-genesis and the survival of differentiated neural retina cells, invivo. At least some of the effects of RA on the neural retinawhich are revealed by the RARβ2γ2 compound mutation arelikely to be mediated by the RPE, as discussed below.

Pathogenesis of the retinal dysplasia in RARβ2γ2null mutantsThe RARβ2γ2 null mutant mice analysed in this reportrepresent the first genetically characterized model of retinaldysplasia. Retinal dysplasia usually refers to a disorganizationof the laminar pattern of the developing neural retina and isdefined histologically by foldings of the neural retina or by thepresence of rosettes composed of neurons and glial cells (Sil-verstein et al., 1971; Lahav and Albert, 1973). Retinaldysplasia 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 mesenchymeRetinal pigment epitheliumOuter neuroblastic layerOuter nuclear layerInner nuclear layerGanglion cell layer

E, embryonic day; P, post-natal day; Nb, newborn; m, months. NA, not applicaneuroblastic layer at P4.

*Internal portion of the outer neuroblastic layer (presumptive inner nuclear layerespectively.

RARα

E15.5 E17.5 Nb P3 P7 P21 2m E15.5 E17

+ + + + + + + ++ ++ + + + + + + + ++ + + + NA NA NA − −

NA NA NA NA + + + NA NNA NA NA NA ++ ++ ++ NA N++ ++ ++ ++ ++ ++ ++ − +/

WT β2γ

Caffé et al., 1993; Toole, 1983) can be caused by a variety ofgenetic 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, apartial 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 earliestocular defect observed in RARβ2γ2 mutants at E14.5 (data notshown) is the persistence of the primary mesenchymal vitreousbody, resulting in the presence of a retrolenticular membranein all postnatal mutants. A persistent hyperplastic vitreousbody is often associated with retinal dysplasia in humanpatients (Lahav and Albert, 1973; Godel et al., 1981; Potterand Traboulsi 1993; and references therein). However, the twodefects 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 normalretina.

Both embryonic and adult neural retinas contain high levelsof RA as well as the enzymatic machinery required for itssynthesis (McCaffery et al., 1993, and references therein). RA

Fig. 7. Comparison ofapoptosis evidenced by theTUNEL method in P4 (a,b)and P14 (d,e) retinas from WT(a,d) and RARβ2γ2 mutantmice (b,e). c and f are bright-field views of b and e,respectively. The open arrowsin b and c indicate the absenceof apoptosis in the pigmentedcells scattered within theperiocular mesenchyme. Thesmall arrows and thearrowheads in e point toapoptotic bodies in the RPEand in the ONL respectively.Abbreviations: GCL, ganglioncell layer; INL, inner nuclearlayer; ONL, outer nuclearlayer; Ro, rosettes; V, vitreousbody. Magnifications:×77 (a-f).

cripts by in situ hybridisation

ble: the inner and the outer nuclear layers are formed from the outer

r). +/−, + and ++, weak (close-to-background), moderate and strong signals

RARβ RARγ

.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 − + +− +/− +/− + + − − − − − − + +

2

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2183Retinoic acid in retinal development

copy of 1 month-old retinas from WT (a,c) and RARβ2γ2 (b,d) mutanta higher magnification of the area boxed in b. The mutant RPE cells lackir cytoplasm is highly vacuolated. Abbreviations: BM, Bruch’s basementndria; Mi, microvilli; N, nuclei of RPE cells; PH, phagosomes; POS,ments; V, vacuoles. Magnifications: ×4500 (a,b); ×7200 (c,d).

T β2γ2

was shown to promote the differentiation and survival ofisolated embryonic retina photoreceptors (Kelley et al., 1994;Stenkamp et al., 1993). However, the neural retina is unlikelyto represent a primary target tissue of the compound mutationsince RARβ and RARγ do not appear to be expressed in theouter neuroblastic layer (which expresses RARα) during theperiod of retinal lamination. Moreover, the differentiation ofthe ganglion cells, in which RARβ transcripts are detectedfrom E17.5 onwards, seems to occur normally in RARβ2γ2mutants as, in newborn mutants, these cells have withdrawnfrom the mitotic cycle and possess well-developed axonswhich have reached the diencephalon, and both the thicknessof the ganglion cell layer and calibre of the optic nerve appearnormal at this stage.

In contrast, the wild-type RPEexpresses both RARβ and RARγ tran-scripts before and during the period ofretinal lamination. In RARβ2γ2 mutants,focal histological abnormalities of theRPE precede the retinal dysplasia andtheir spatial distribution strikingly corre-sponds to that of the dysplastic areas. Aspatial correlation between RPE struc-tural defects and retinal dysplastic areashas also been reported in heritable formsof retinal dysplasia in mice (Cook et al.,1991) and chicks (Randall et al., 1983;Fite et al., 1983), as well as in a varietyof human retinal dysplasia (reviewed inSilverstein et al., 1971). Contact betweenthe RPE and the neural retina is requiredfor morphological and functional pho-toreceptor differentiation in cultures ofRana pipiens ocular rudiments (Holly-field and Witkowsky, 1974) and co-culture of RPE cells is necessary forlaminar organisation within the neuralretina of chick (Vollmer and Layers,1986). Fetal RPE cells secrete a proteinthat induces a neuronal phenotype incultured retinoblastoma cells, suggestingthat RPE-derived paracrine factors play arole in the differentiation of the neuralretina (Steele et al., 1990). The impor-tance of the RPE in the maintenance ofthe structural integrity of the retina, invivo, has been elegantly demonstrated byRaymond and Jackson (1995) in a trans-genic mouse line expressing the attenu-ated diptheria toxin A gene under thecontrol of the tyrosinase-related protein-1 promoter which is specifically active inRPE cells and melanocytes. Perinatal tox-icogenic ablation of the RPE results in aretinal dysplasia strikingly resemblingthat of the RARβ2γ2 mutants, notablywith respect to the possible origin ofrosette formation: the rosettes observed inboth Raymond and Jackson’s RPE-deficient mice and in our mutants appearto arise from the migration of ONL cells

Fig. 8. Electron microsmice. d corresponds to microvilli (Mi) and themembrane; M, mitochophotoreceptor outer seg

W

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 aretypical artifacts due to tissue processing), mechanical con-straints are generated, which cause an inward bending of theONL. This bending, in turn, might provoke an inversion of thepolarity of the photoreceptors located in the hinge region.Interestingly, this pathological cell rearrangement permits thephotoreceptor to express differentiated features (i.e. segmentformation and expression of opsin) in the absence of contactwith the RPE (Figs 1m, 4d). Taken together, these data stronglysuggest that a defective RPE might be responsible for theretinal dysplasia in the RARβ2γ2 mutants, either through a

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2184 J. M. Grondona and others

Fig. 9. Immunodetection of CRBPI in 1 month old retinas from WTand RARβ2γ2 mutant. Note the near absence of CRBPI expression inthe 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 majorcomponent is the RPE) subjecting the neural retina to systemicdisrupting influences, or through thelack of a RPE-derived positive signal,normally inducing and maintaining theneural retina.

The periocular mesenchyme isanother candidate as a primary targettissue of the double null mutation, as itis the ocular tissue that expresses thehighest levels of RARβ and RARγ: it isconceivable that, under the influence ofRA, some periocular mesenchymal cellsmight synthesize paracrine factorsrequired for the laminar organisation ofthe retina. Such factors might act eitherindirectly, e.g. on differentiation, prolif-

Fig. 10. Detection RARβ and RARγtranscripts in a E17.5 WT eye by in situhybridization. a is a bright field of b. Thelarge arrows in d and e point to RPE cellnuclei. Note that the silver grains locatedover the neural retina (R) correspond tobackground labelling. Abbreviations: C,cornea; GCL, ganglion cell layer; EY,eyelids; HG, Harderian gland; I, iris; L, lens;POM, periocular mesenchyme; R, neuralretina; RPE, retinal pigment epithelium; V,vitreous body. Magnifications: ×20 (a-c);×310 (d,e).

eration or survival of RPE cells, or directly after having crossedthe blood-ocular barrier. Recent transplantation experimentsdemonstrating 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-bilities.

Interestingly, Johnson (1939, 1943) also reported the occur-rence of rosettes in the retinas of VAD rats. However, contraryto the rosettes seen in our RARβ2γ2 mutants, these VAD-induced rosettes were determined after the period of retinallamination and were interpreted as being the result of the reor-ganisation of the remaining photoreceptors in the most severelydegenerated areas (Johnson, 1939).

Pathogenesis of the retinal degeneration in RARβ2γ2null mutantsRetinal degeneration is characterized histologically by anatrophy of one or more layers of the neural retina. Several linesof evidence suggest that retinal degeneration might be a directconsequence of retinal dysplasia. Firstly, massive apoptosis isobserved within the rosettes and folds at P4, suggesting thatthese sites will evolve toward degeneration. Accordingly, thefrequency of the mutant retinas displaying signs of dysplasiaapparently decreases as degeneration proceeds (Table 1).Secondly, the first histological signs of retinal degenerationthat are manifested by focal atrophy of the ONL, are observedat the dysplastic sites at P14. Thirdly, several similar cases ofretinal atrophy accompanying or preceded by retinal dysplasiahave 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 ofretinal dysplasia, and then spread toward the retinal peripheryeither because the cells of the dysplastic areas release diffusible

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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 aperturein the mutant.

‘death’ factors or because they fail to produce a trophic factornormally required for the survival of neural retinal cells (seeHuang et al., 1993 for further discussion).

Alternatively, or additionally, degeneration might occur denovo, at least in some portions of the mutant retina. In contrastto the ‘dysplastic phenotype’, the ‘degenerative phenotype’ isfully penetrant, suggesting that degeneration may not be solelya consequence of dysplasia. Moreover, areas with a normallaminar pattern, but displaying both a marked atrophy of all 3nuclear layers (ONL, INL and GCL) and apoptotic cells in theONL and INL, were observed in adult mutant retinas. Suchareas of retinal degeneration were not always contiguous toareas of absent or histologically abnormal RPE. However, it isnoteworthy that many more RPE cells were found to be alteredon the basis of ultrastructural and immunocytochemical criteriathan on histological criteria alone. The adult RPE has a criticalrole in the homeostasis of the neural retina through the regen-eration of the 11-cis retinaldehyde (the chromophore of thevisual pigment) in the visual cycle, the selective transport ofretinoid and nutrients to the photoreceptors (forming part ofthe blood-retinal barrier), the phagocytosis of the distal portionof 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 mediaconditioned by normal RPE cells promote photoreceptorsurvival, indicating that cytokines and other growth factorsproduced by RPE cells may exert a trophic influence on themaintenance of the neural retina (reviewed in Campochiaro etal., 1993 and Sheedlo et al., 1993). A defective RPE appearsto be the direct cause of neural retina degeneration in the RoyalCollege of Surgeons strain of rats (RCS rats; Bok and Hall,1971; Malecaze et al., 1993; and references therein) and inhuman choroideremia (reviewed in Bird and Jay, 1994). Recentimmunohistochemical studies indicate that RPE cells syn-thesize bFGF, which is an important photoreceptor survivalfactor as it can rescue photoreceptor degeneration in RCS ratsand in light-damaged rats (Steinberg, 1994, and referencestherein). Moreover, the RPE appears to be a target tissue of RAaction since (i) RA can prevent dedifferentiation and loss ofdensity-dependent growth control of human RPE cells inculture (Campochiaro et al., 1991), and (ii) the VAD-inducedRPE defects in adult rats (i.e. flattening and degeneration of

RPE cells; Johnson, 1939, 1943; Dowling and Wald, 1958) canapparently be prevented by supplementing their diet with RA(Dowling, 1964; Dowling and Gibbons, 1961; Carter-Dawsonet al., 1979). Taken together, all of these data raise the possi-bility that a defective RPE could also be instrumental in thegenesis of the RARβ2γ2 retinal degeneration through eventsthat are not secondary to the retinal dysplasia.

The choroid is thought to represent the main source of bloodsupply for the ONL (reviewed in Bernstein, 1961) and largeportions of this tissue are lacking in RARβ2γ2 mutants.However, we did not find any correlation between the regionsdisplaying choroidal agenesis and the presence of lesions in theneural retina, e.g. RARβ2γ2 mutants often show portionslacking choroid juxtaposed with areas of normal retina (seeFig. 2b). Moreover, mutant mice deficient in melanocyte pre-cursors, such as Dominant white spotting (w) and Steel (Sl), donot develop retinal defects (Jackson, 1994). These data suggestthat the choroidal defects are not the cause of retinal degener-ation.

In any event, the phenotype of the RARβ2γ2 mutantsstrongly suggests that RA is most likely required for thesurvival of the rod photoreceptors, the bipolar neurons, (whichrepresent the major INL cell type), and the ganglion cells.Experiments aimed at rescuing the RARβ2γ2 mutantphenotype through specific reexpression of RARβ or RARγ inthe RPE should demonstrate whether these trophic effects ofRA on the neural retina are mediated by the RPE, as proposedabove.

Pleiotropic role of RARs in retinal maintenance andeye developmentRetinoids have trophic effects on the neural retina as firstdemonstrated by Johnson (1939, 1943) and later by Dowlingand colleagues (Dowling and Wald, 1958; Dowling andGibbons, 1961; Dowling, 1964) in studies of degenerativechanges in retina of rats deprived of vitamin A. According toJohnson’s data (1939, 1943), the degeneration of the neuralretina induced by avitaminosis A is progressive. It begins withthe loss of the photoreceptor outer segment, then involves suc-cessively ONL and the INL, and is always more pronouncedin the central than in the peripheral region of the retina. Theidentity of the retinoids exerting these trophic effects is unclear(discussed in Stenkamp et al., 1993). Systemic administrationof RA to VAD rats apparently prevents the death of the cellsof the INL, but not the night blindness, the deterioration of thephotoreceptor outer segment and death of the photoreceptorcells, 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-testisbarrier (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 inthe trophic effect of vitamin A.

In this context, it is noteworthy that there are at least twopoints of convergence between the neural retina degenerationin VAD animals and RARβ2γ2 mutants. The first is purelymorphological: the neural retina degeneration in RARβ2γ2mutants progresses from the center towards the periphery ofthe retina and from the ONL towards the internal retinal layers,thus resembling the progression observed in VAD animals (see

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2186 J. M. Grondona and others

above). The second deals with the physiopathology of thelesions: in RARβ2γ2 mutants, CRBPI expression by RPE cellsis impaired and, since this protein likely plays an importantrole in the delivery of retinoids to the photoreceptors (Saari etal., 1994), this condition could create a state of VAD in theneural retina. Interestingly, CRBPI mRNA levels are increasedby RA treatment of whole animals (Haq and Chytil, 1988) andthe CRBPI gene contains a RA response element in itspromoter (Smith et al., 1991) which further supports theproposal that the RPE could be a primary target of the doublenull mutation (see above).

Warkany and Schraffenberger reported half a century ago(1946) that the developing rat eye is the organ that is mostsensitive to vitamin A deprivation since, in less severelyaffected VAD fetuses, it represented the only site of malfor-mations. We show here that more than two thirds of theRARβ2 single null mutants display a retrolenticular membranethat actually corresponds to the commonest abnormality of thefetal VAD syndrome. It arises by persistence and hyperplasiaof the primary vitreous body, a structure that starts to developat E10.5 from periocular mesectodermal cells that enter theoptic cup, and has regressed at E14.0 by mechanisms that arestill unknown. In adult RARβ2 or RARβ2γ2 mutants, the per-sistence of the primary vitreous is manifested by the presenceof a plaque of pigmented fibrovascular tissue connecting theposterior pole of the lens with the optic papilla (the optic nerveexit point and point of entry of retinal blood vessels). Thisabnormality, which must result in poor vision, was previouslyoverlooked in RARβ2 mutants (Mendelsohn et al., 1994b) dueto its lack of behavioural manifestation in the laboratory (note,in this respect, that blindness does not overtly affect thebehaviour of the laboratory mouse; Grüneberg, 1952).RARβ2+/−/RARα−/− mice (Lohnes et al., 1994) andRARβ2+/−/RARγ2−/− mice (our present data) never displayeda retrolenticular membrane. Thus, one functional copy of theRARβ2 gene is sufficient to ensure the involution of theprimary mesenchymal vitreous. The penetrance of the persis-tent retrolenticular membrane phenotype increased in a gradedmanner upon removal of one, and then of both alleles of theRARγ2 gene from the RARβ2 null genetic background and, inRARβ2γ2 double null mutants, it was fully penetrant. Theseresults suggest that RARγ2 can functionally compensate for thelack of RARβ2 in some RARβ2γ2 mutants.

Aside from the neural retina, the RPE and the vitreous body,the sclera, the choroid, the eyelids, and the lens were affectedin RARβ2γ2 mutants. Eyelids start to develop at E13.5 as mes-enchymal outgrowths of the neural crest-derived periocularmesenchyme covered by the ectoderm, whereas the scleraarises 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. Theblepharophimosis and the local agenesis or thinning of thechoroid and sclera in RARβ2γ2 mutants might reflect a directeffect of the double mutation in the periocular mesenchymewhich normally expresses high levels of both RARβ and RARγin embryos, fetuses and young mice. However, the cataractscould be secondary to vascular invasion of the lens by bloodvessels coming from the retrolenticular membrane (discussedin Traboulsi, 1993).

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 andSchraffenberger (1946), have implicated retinoid signaling atalmost every step of prenatal eye morphogenesis. Theseinclude lens formation, separation of the lens from theectoderm, development of the outer layer of the optic cup asRPE (demonstrated by the development of neural retina in theplace of RPE on the dorsal aspect of RARαγ mutant eyes;Lohnes et al., 1994; P. Gorry, M. M. and P. C., unpublisheddata), development of the ventral retina, closure of the opticfissure, involution of the primary vitreous body, developmentof the eye’s anterior segment (cornea, conjunctival sac, anteriorchamber), development of periocular structures (sclera,choroid and Harderian gland) and formation and fusion of theeyelids). In addition, RA may be implicated in the formationof the optic cup, as the development of the eye anlage isarrested at the optic cup stage in cultured mouse embryosdeprived of RA by inhibition of yolk-sac retinol bindingprotein (RBP) synthesis (Båvik et al., 1996). Thus, our presentdemonstration that RARs are also required for retinal histo-genesis and survival of retinal cells, further establishes thepleiotropic role of RA in eye development. Interestingly, in theretina of VAD rat fetuses, the formation of the inner neuro-blastic layer (the future GCL) and of the IPL apparently failsto occur (Warkany and Schraffenberger, 1946). This absenceof retinal laminar organisation is the only eye defect of the fetalVAD syndrome which was not recapitulated in the RAR andRXR single and double null mutants studied so far (Lohnes etal., 1994; Kastner et al., 1994). It remains therefore to be seenwhether RA is also involved in fetal retinal histogenesis.

The problems of penetrance and expressivity of theretinal defectsThe retinas (i.e. neural retina and RPE) of young RARβ2γ2mutants usually displayed only focal lesions (dysplasia andabnormal RPE) coexisting with large, apparently intact areas.Thus, removal of RARβ2 and RARγ2 does not completelyabolish retinoid responsiveness in target cells, but rather seemsto bring this responsiveness close to a threshold level belowwhich the realization of RA-dependent cellular events isimpaired. 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 neuralretina and of the RPE, and also for the incomplete penetranceand expressivity of the retinal dysplastic phenotype (Table 1).This possibility is further supported by the observations thatadditional RAR inactivations in the RARβ2γ2 mutant back-ground (e.g. removal of one allele of RARγ1 or RARα1) resultin a marked increase in the number of dysplastic foci within agiven retina (our unpublished data). These observations alsosuggest that the incomplete penetrance and expressivity of theRARβ2γ2 retinal dysplastic phenotype does not result fromfunctional redundancy with RA-independent regulators.

The stochastic variation of retinoid responsiveness withinretinal cells may occur both at the spatial (i.e. between differentcells at a given time) and temporal (i.e. in a given cell atdifferent times) levels. For instance in a given RPE cell, thisresponsiveness may be adequate at the time of birth and fallbelow the critical threshold only after the completion of retinalhistogenesis, which would then impair the function of retinoidsin retinal maintenance. This may account for the fact that, in

Page 15: Retinal dysplasia and degeneration in RAR²2/RAR 2 compound mutant

2187Retinoic acid in retinal development

old animals, retinal degeneration is fully penetrant and affectsextensive portions of the retina. Thus, the degenerativephenotype may be the consequence of either the retinaldysplasia 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 ingenetic background may account for some of the phenotypicvariations seen among different animals, the considerable vari-ations in expressivity often observed between the two eyes ofa given animal cannot be explained on that basis, and thus mustbe related to the stochastic processes mentioned above.

CONCLUSION

The present study demonstrates an essential role for RARs, andtherefore for retinoic acid, in retinal histogenesis and survivalof retinal cells. The present RARβ2γ2 mutant mice representthe first genetically characterized animal model for retinaldysplasia. Even though homozygous null compound mutationsfor both RARβ2 and RARγ2 are unlikely to occur at a signif-icant rate in humans, our data raise the possibility that geneticlesions affecting the retinoid signalling pathway could underliesome cases of human retinal dysplasia and/or degenerations,for which the genetic basis is currently unknown. RAR- and/orRXR-deficient mice may also provide interesting models toinvestigate the mechanism underlying the therapeutic effectsof 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 andC. 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 ofthis manuscript, and S. Ward for critically reading the manuscript.This work was supported by funds from the Centre National de laRecherche Scientifique, the Institut National de la Santé et de laRecherche Médicale, the Centre Hospitalier Universitaire Régional,the Association pour la Recherche sur le Cancer, the Human FrontierScience Program, the Collège de France and the Bristol-Myers SquibbPharmaceutical Research Institute. J. M. G. was supported by a long-term fellowship from the European Communities (Human Capital andMobility).

REFERENCES

Acott, T. S., Weleber, R. G. (1995). Vitamin A megatherapy for retinalabnormalities. Nature Medicine 1, 884-885.

Båvik, C., Ward, S. J. and Chambon, P. (1996). Developmental abnormalitiesin cultured mouse embryos deprived of retinoic acid by inhibition of yolk-sacretinol binding protein synthesis. Proc. Natl. Acad. Sci., USA 93, 3110-3114.

Bernstein, M. H. (1961). Functional architecture of the retinal epithelium. InThe Structure of the Eye. (ed. G. K. Smelser). pp. 139-150. New York:Academic press.

Bird, A. C. and Jay, B. (1994). Diagnosis in inherited retinal disorders. InMolecular Genetics of Inherited Disorders. (ed. A. F. Wright and B. Jay). pp.53-88. Chur (Switzerland): Harwood Academic Publishers.

Bok, D. (1993). The retinal pigment epithelium: a versatile partner in vision. J.Cell Sci. Supplement 17, 189-195.

Bok, D. and O. Hall, M. (1971). The role of the pigment epithelium in theetiology if inherited retinal distrophy in the rat. J. Cell. Biol. 49, 664-682.

Bridges, C. D. B., Fong, S. L., Alvarez, R. A., Landers, R. A. (1983).

Transport, utilization and metabolism of visual cycle retinoids in the retinaand pigment epithelium. In Progress in Retinal Research. (ed. N. Osborneandg. Chader). pp. 137-162. Oxford: Pergamon.

Buse, E. Eichmann, T., de Groot, H and Leker, A. (1993). Differentiation ofthe mammalian retinal pigment epithelium in vitro: influence of presumptiveretinal neuroepithelium and head mesenchyme. Anat. Embryol. 187, 259-268.

Caffé, A. R., Szél, A., Juliusson, B. and van Veen, T. (1993). Hyperplasticneuroretinopathy and disorder of pigment epithelial cells precede acceleratedretinal degeneration in the SJL/N mouse. Cell Tissue Res. 271, 297-307.

Campochiaro, P. A. (1993). Cytokine production by retinal pigmentedepithelial cells. Int. Rev. Cytol. 146, 75-82.

Campochiaro, P. A., Hackett, S. F. and Conway, B. P. (1991). Retinoic acidpromotes density-dependent growth arrest in human retinal pigmentepithelial cells. Investigative Ophtalmology and Visual Science 32, 65-72

Carter-Dawson, L., Kuwabara, Y., O’Brien, P. and Bieri, J. G. (1979).Structural and biochemical changes in vitamin A-deficient rat retinas.Investigative Ophtalmology and Visual Science 18, 437-446.

Chambon, P. (1994). The retinoid signaling pathway: molecular and geneticanalyses. Sem. in Cell Biol. 5, 115-125.

Cook, C. S., Generoso, W. M., Hester, D. and Peiffer, R. L. (1991). RPEdysplasia with retina duplication in a mutant mouse strain. Exp. Eye. Res. 52,409-415.

Décimo, D., Georges-Labouesse, E., and Dollé, P. (1995). In situhybridization of nucleic acid probes to cellular RNA. In Gene Probes, aPractical Approach Book. (ed. B. D. Hames and S. Higgins) Vol II, in press.

Dollé, P., Ruberte, E., Leroy, P., Morriss-Kay, G. and Chambon, P. (1990).Retinoic acid receptors and cellular retinoid binding proteins. I. A systematicstudy of their differential pattern of transcription during mouseorganogenesis. Development 110, 1133-1151.

Dowling, J. E. (1964). Nutritional and inherited blindness in the rat. Exp. EyeRes. 3, 348-356.

Dowling, J. E. and Gibbons, I. R. (1961). The effect of vitamin A deficiencyon the fine structure of the retina. In The Structure of the Eye. (ed. G. K.Smelser). pp. 85-99. New York: Academic Press.

Dowling, J. E. and Wald, G. (1958). Vitamin A deficiency and nightblindness. Proc. Natl. Acad. Sci. USA 44, 648-661.

Fawcett, D. W. (1986). In A Textbook of Histology. (ed. Bloom and Fawcett).Philadelphia: W. B. Saunders Company,

Fite, K. V., Montgomery, T., Whitney, T,. Boissy, R. and Smyth, JR. (1982).Inherited retinal degeneration and ocular amelanosis in the domestic chicken.Current Eye Research 2, 109-115.

Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. (1992). Identification ofprogrammed cell death in situ via specific labeling of nuclear DNAfragmentation. J. Cell Biol. 119, 493-501.

Giguère, V. (1994). Retinoic acid receptors and cellular retinoid bindingproteins: complex interplay in retinoid signaling. Endocrine Rev. 15, 61-79.

Godel, V., Nemet, P. and Lazar, M. (1981). Retinal dysplasia. DocumentaOphtalmologica. 51, 277-288.

Grüneberg, H. (1952). The Genetics of the Mouse. Second Edition. MartinusNijhoff, The Hague, Holland.

Gustafson, A-L, Dencker, L. and Eriksson, U. (1993). Non-overlappingexpression of CRBPI and CRABPI during pattern formation of limbs andcraniofacial structures in the early mouse embryo. Development 117, 451-460.

Haq, R. and Chytil, F. (1988). Retinoic acid rapidly induces lung cellularretinol-binding protein mRNA levels in retinol deficient rats. Biochem.Biophys. Res. Commun. 156, 712-716.

Hicks, D. and Barnstable, C. J. (1987). Different monoclonal antibodiesreveal different binding patterns on developing and adult retina. J.Histochem. Cytochem. 35, 1317-1328.

Hollyfield, J. E. and Witkovsky, P. (1974). Pigmented retinal epithelium inphotoreceptor development and function. J. Exp. Zool. 189, 357-378.

Howell, J. McG., Thompson, J. N., and Pitt, G. A. J. (1963). Histology of thelesions produced in the reproductive tract of animals fed on a diet deficient invitamin A alcohol, but containing vitamin A acid. I. The male rat. J. Reprod.Fertil. 5, 159-167.

Huang, P. C., Gaitan, A. E., Hao, Y., Petters, R. M. and Wong, F. (1993).Cellular interactions implicated in the mechanism of photoreceptordegeneration in transgenic mice expressing a mutant rhodopsin gene. Proc.Natl. Acad. Sci. USA 90, 8484-8488.

Huxlin, K. R., Sefton A. J. and Furby, J. H. (1992). The origin anddevelopment of retinal astrocytes in the mouse. J. Neurocytology 21, 530-544.

Page 16: Retinal dysplasia and degeneration in RAR²2/RAR 2 compound mutant

2188 J. M. Grondona and others

Jackson, I. J. (1994). Molecular and developmental genetics of mouse coatcolor. Annu. Rev. Genet. 28, 189-217.

Jacobson, S. G., Cideciyan, A. V., Regunath, G., Rodrigez, F. J.,Vandenburgh, K., Sheffield, V. C. and Stone, E. (1995). Night blindness inSorsby’s fundus dystrophy reversed by vitamin A. Nature Genetics 11, 27-32.

Johnson, M. L. (1939). The effect of vitamin A deficiency upon the retina ofthe rat. J. Exp. Zool. 81, 67-89.

Johnson, M. L. (1943). Degeneration and repair of the rat retina inavitaminosis A. Archs Ophtalmol. 29, 793-810.

Johnston, M. C., Noden, D. M., Hazelton, R. D., Coulombre, J. L. andCoulombre, A. J. (1979). Origins of avian ocular and periocular tissues.Exp. Eye Res. 29, 27-43.

Juriloff, D. M. and Harris, M. J. (1993). Retinoic acid, cortisone, or thyroxinesuppresses the mutant phenotype of the eyelid development mutation, IgMl,in mice. J. Exp. Zool. 265, 144-152.

Kastner, P., Grondona, J. Mark, M., Gansmuller, A., LeMeur, M., Décimo,D., Vonesch, J. L., Dollé, P. and Chambon, P. (1994). Genetic analysis ofRXRα developmental function: convergence of RXR and RAR signallingpathways in heart and eye morphogenesis. Cell 78, 987-1003.

Kastner, P., Mark, M. and Chambon, P. (1995). Nonsteroid nuclearreceptors: what are genetic studies telling us about their role in real life ? Cell83, 859-869.

Kelley, M. W., Turner, J. K. and Reh, T. A. (1994). Retinoic acid promotesdifferentiation of photoreceptors in vitro. Development 120, 2091-2102.

Lahav, M. and Albert, D. M. (1973). Clinical and histopathologicclassification of retinal dysplasia. Am. J. Ophthalmol. 75, 648-667.

Lohnes, D. Kastner, P., Dierich, A., Mark, M., Le Meur, M., and Chambon,P. (1993). Function of retinoic acid receptor γ (RARγ) in the mouse. Cell 73,643-658.

Lohnes, D., Mark, M., Mendelsohn, C., Dollé, P., Dierich, A., Gorry, P.,Gansmuller, A., and Chambon, P. (1994). Function of the retinoic acidreceptors (RARs) during development. I. Craniofacial and skeletalabnormalities in RAR double mutants. Development 120, 2723-2748.

Lufkin, T., Lohnes, D., Mark, M., Dierich, A., Gory, P., Gaub, M. P.,LeMeur, M. and Chambon, P. (1993). High postnatal lethality and testisdegeneration in retinoic acid receptor α mutant mice. Proc. Natl. Acad. Sci.USA 90, 7225-7229.

Luo, J., Pasceri, P., Conlon, R. A., Rossant, J., and Giguère, V. (1995). Micelacking all-isoforms of retinoic acid receptor β develop normally and aresusceptible to the teratogenic effects of retinoic acid. Mech. Develop. 53, 61-71.

Malecaze, F., Mascarelli, F., Bugra, K., Fuhrmann, G., Courtois, Y. andHicks, D. (1993). Fibroblast growth factor receptor deficiency in dystrophicretinal pigmented epithelium. J. Cell. Physiol. 154, 631-642.

Mangelsdorf, D. J., and Evans, R. M. (1995). The RXR heterodimers andorphan receptors. Cell 83, 841-850.

Mark, M. Lufkin, T. Vonesch, J. L. Ruberte, E., Olivo, J. C. Dollé, P.Gorry, P., Lumsden, A. and Chambon P. (1993). Two rhombomeres arealtered in Hoxa-1 mutant mice. Development 119, 319-338.

McCaffery, P., Posch, K. C., Napoli, J. L., Gudas, L. and Dräger, U. C.(1993). Changing patterns of the retinoic acid system in the developingretina. Dev. Biol. 158, 390-399.

Mendelsohn, C., Lohnes, D. Décimo, D., Lufkin, T., LeMeur, M.,Chambon, P. and Mark, M. (1994a). Function of the retinoic acid receptors(RARs) during development. (II) Multiple abnormalities at various stage oforganogenesis in RAR double mutants. Development 120, 2749-2771.

Mendelsohn, C., Mark, M., Dollé, P., Dierich, A., Gaub, M. P., Krust, A.,Lampron, C. and Chambon, P. (1994b). Retinoic acid receptor β2(RARβ2) null mutant mice appear normal. Dev. Biol. 166, 246-258.

Obata, S. and Usukura, J. (1992). Morphogenesis of the photoreceptor outersegment during postnatal development in the mouse (BALB/c) retina. CellTissue Res. 269, 39-48.

Pei, Y. F. and Rhodin, J. A. G. (1971). The prenatal development of the mouseeye. Anat. Rec. 168, 105-126.

Potter, M. and Trabouslsi, E. (1993). Retinal Dysplasia. In HumanMalformations and Related Anomalies, vol. 2. (ed. R. E. Stevenson, J. G Halland R. M. Goodman). pp. 184-185. New York: Oxford University Press.

Randall, C. J., Wilson, M. A., Pollock, B. J. Clayton, R. M., Ross, A. S.Bard, J. B. L. and McLachlan, I. (1983). Partial retinal dysplasia andsubsequent degeneration in a mutant strain of domestic fowl (rdd). Exp. EyeRes. 37, 337-347.

Raymond, S. M. and Jackson, I. J. (1995). The retinal pigment epithelium isrequired for development and maintenance of the mouse neural retina.Current Biol. 5, 1286-1295.

Ruberte, E., Dollé, P., Krust, A., Zelent, A., Morriss-Kay, G. and ChambonP. (1990). Specific spatial and temporal distribution of retinoic acid receptorgamma transcripts during mouse embryogenesis. Development 108, 213-222.

Saari, J. C. (1994). Retinoids in photosensitive systems. In The Retinoids:Biology, Chemistry and Medicine, 2nd ed. (ed. M. B. Sporn, A. B. Robertsand D. S. Goodman). pp. 351-385. New York: Raven Press Ltd.

Sarthy, P. V., Fu, M. and Huang, J. (1991). Developmental expression of theglial fibrillary acidic protein (GFAP) gene in the mouse retina. Cell. Mol.Neurobiol. 11, 623-637.

Sheedlo, H. J., Li, L., Fan, W., and Turner, J. E. (1994). Retinal pigmentepithelial cell support of photoreceptor survial in vitro. In Vitro Cell Dev.Biol. 31, 330-333.

Silverstein, A. M., Osburn, B. I. and Prendergast, R. A. (1971). Thepathogenesis of retinal dysplasia. Am. J. Ophthalmol. 72, 13-21.

Smith, W. C., Nakshatri, H., Leroy, P., Rees, J. and Chambon, P. (1991). Aretinoic acid response element is present in the mouse cellular retinol bindingprotein I (mCRBPI) promoter. EMBO J. 10, 2223-2230.

Steele, F. R., Chader, G. J., Johnson, L. V., and Tombran-Tink, J. (1992).Pigment epithelium-derived factor: Neurotrophic activity and identificationas a member of the serine protease inhibitor gene family. Proc. Natl. Acad.Sci. USA 90, 1526-1530.

Steinberg, R. H. (1994). Survival factors in retinal degenerations. CurrentOpin. Neurobiol. 4, 515-524.

Stenkamp, D., Gregory, J. K. and Adler, R. (1993). Retinoid effects inpurified cultures of chick embryo retina neurons and photoreceptors.Investigative Ophthalmology and Visual Science 34, 2425-2436.

Stiemke, M. M., Landers, R. A., Al-Ubaidi, M. R., Rayborn, M. E. andHollyfield, J. G. (1994). Photoreceptor outer segment development inXenopus laevis: influence of the pigmented epithelium. Dev. Biol. 162, 169-180.

Theiler, K. (1972). The House Mouse. Development and Normal Stages fromFertilization to 4 Weeks of Age. Berlin: Springer-Verlag.

Thomson, J. N., Howell, J. McC and Pitt, G. A. J. (1964). Vitamin A andreproduction in rats. Proc. Royal Soc. 159, 510-535.

Toole, D. O., Young, S., Severin G. A. and Neumann, S. (1983). Retinaldysplasia of english springer spaniel dogs: light microscopy of the postnatallesions. Vet. Pathol. 20, 298, 311.

Traboulsi, E. (1993). The eye. In Human Malformations and RelatedAnomalies, vol. 2. (ed. R. E. Stevenson, J. G Hall and R. M. Goodman). pp.163-191. New-York: Oxford University Press.

Van Pelt, H. M. M. and de Rooij, D. G. (1991). Retinoic acid is able toreinitiate spermatogenesis in vitamin A-deficient rats and high replicatedoses support the full development of spermatogenic cells. Endocrinology128, 697-704.

Vollmer, G. and Layer, P. G. (1986). Reaggregation of chick retinal andmixtures of retinal and pigment epithelial cells: the degree of laminarorganization is dependent on age. Neurosci. Lett. 63, 91-95.

Warkany, J. and Schraffenberger, S. (1946). Congenital malformationsinduced in rats by maternal vitamin A deficiency. I. Defects of the eye. Archs.Ophthalmol. 35, 150-169.

Whiteley H. E. (1991). Dysplastic canine retinal morphogenesis. InvestigativeOphthalmology and Visual Science 32, 1492-1498.

Wilson, J. G., Roth, C. B. and Warkany, J. (1953). An analysis of thesyndrome of malformations induced by maternal vitamin A deficiency.Effects of restoration of vitamin A at various times during gestation. Am. J.Anat. 92, 189-217.

Wolbach, S. B. and Howe, P. R. (1925). Tissue changes following deprivationof fat-soluble A vitamin. J. Exp. Med. 42, 753-777.

Young, R. W. (1984). Cell death during differentiation of the retina in themouse. J. Comparative Neurology 229, 362-373.

Young, R. W. (1985a). Cell differentiation in the retina of the mouse. Anat.Rec. 212, 199-205.

Young, R. W. (1985b). Cell proliferation during postnatal development of theretina in the mouse. Dev. Brain Research 21, 229-239.

(Accepted 9 April 1996)


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