June 2010
Volume 51, Issue 6
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Retinal Cell Biology  |   June 2010
Retinoic Acid Receptor (RAR)-α Is Not Critically Required for Mediating Retinoic Acid Effects in the Developing Mouse Retina
Author Affiliations & Notes
  • Laura Cammas
    From the IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), BP 10142, Illkirch, France;
    CNRS (Centre National de la Recherche Scientifique), UMR (Unité Mixte de Recherche) 7104 Illkirch, France;
    INSERM (Institut National de la Santé et de la Recherche Médicale), Unité 964, Illkirch, France;
    Faculté de Médecine, Université de Strasbourg, Strasbourg, France; and
  • Frédéric Trensz
    From the IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), BP 10142, Illkirch, France;
    CNRS (Centre National de la Recherche Scientifique), UMR (Unité Mixte de Recherche) 7104 Illkirch, France;
    INSERM (Institut National de la Santé et de la Recherche Médicale), Unité 964, Illkirch, France;
    Faculté de Médecine, Université de Strasbourg, Strasbourg, France; and
  • Abdeljalil Jellali
    Institut Clinique de la Souris, Illkirch, France.
  • Norbert B. Ghyselinck
    From the IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), BP 10142, Illkirch, France;
    CNRS (Centre National de la Recherche Scientifique), UMR (Unité Mixte de Recherche) 7104 Illkirch, France;
    INSERM (Institut National de la Santé et de la Recherche Médicale), Unité 964, Illkirch, France;
    Faculté de Médecine, Université de Strasbourg, Strasbourg, France; and
  • Michel J. Roux
    From the IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), BP 10142, Illkirch, France;
    CNRS (Centre National de la Recherche Scientifique), UMR (Unité Mixte de Recherche) 7104 Illkirch, France;
    INSERM (Institut National de la Santé et de la Recherche Médicale), Unité 964, Illkirch, France;
    Faculté de Médecine, Université de Strasbourg, Strasbourg, France; and
    Institut Clinique de la Souris, Illkirch, France.
  • Pascal Dollé
    From the IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), BP 10142, Illkirch, France;
    CNRS (Centre National de la Recherche Scientifique), UMR (Unité Mixte de Recherche) 7104 Illkirch, France;
    INSERM (Institut National de la Santé et de la Recherche Médicale), Unité 964, Illkirch, France;
    Faculté de Médecine, Université de Strasbourg, Strasbourg, France; and
  • Corresponding author: Pascal Dollé, 1 rue Laurent Fries, Illkirch, France 67400; dolle@igbmc.fr
Investigative Ophthalmology & Visual Science June 2010, Vol.51, 3281-3290. doi:10.1167/iovs.09-3769
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      Laura Cammas, Frédéric Trensz, Abdeljalil Jellali, Norbert B. Ghyselinck, Michel J. Roux, Pascal Dollé; Retinoic Acid Receptor (RAR)-α Is Not Critically Required for Mediating Retinoic Acid Effects in the Developing Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(6):3281-3290. doi: 10.1167/iovs.09-3769.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To determine the functional contribution of retinoic acid receptor (RAR)-α in the developing murine neural retina, through a phenotypic analysis of the corresponding null mutants.

Methods.: RARα mutant (Rara −/−) mice were compared with wild-type littermates at several stages of pre- and postnatal development. An RA-response element (RARE)–containing reporter transgene was used to assess the contribution of RARα to retinoid signaling in the retina. In situ hybridization was performed on serial eye sections to investigate the expression of main developmental regulators. Immunofluorescence was used to detect differentiated cell types in the adult retina. Mutants were also subjected to clinical observation and visual function evaluation with the optomotor test and electroretinography.

Results.: Both isoform transcripts of RARα were expressed throughout the neural retina at various stages of pre- and postnatal development. In the Rara −/− mice the RARE-reporter transgene consistently failed to activate in the developing neural retina. However, they did not exhibit any alteration of the expression patterns of molecular determinants and had a normal organization of retinal cell types at postnatal stages. Their performance in visual tests was indistinguishable from that of control littermates.

Conclusions.: Although RARα mediates RARE reporter transgene activity in the neural retina, its function is not necessary for the retina to develop and function normally. These data suggest that retinoic acid regulates neural retinal development through other, possibly RAR-independent, pathways.

Retinoids (active vitamin A or retinol derivatives) play complex roles during eye development and physiology. Whereas retinaldehyde is used as a prosthetic group or chromophore by the various opsins to initiate the phototransduction process (for a review, see Ref. 1), its acidic derivative retinoic acid (RA) plays multiple roles as a developmental signaling molecule throughout vertebrate species (for reviews, see Refs. 25). This molecule is the ligand for a subtype of nuclear receptors, the RA receptor (RAR)-α, -β, and -γ, all three existing in the form of two or more N-terminal isoform variants and acting as heterodimers with RXRs to regulate gene expression after binding to specific DNA motifs (RA-response elements or RAREs; for reviews, see Refs. 68). 
The developing retina is one of the tissues containing the highest amount of endogenous RA. 9 Its distribution is the result of dynamic, stage- and region-specific expression of enzymes involved in its synthesis or metabolism. The murine retinol dehydrogenase 10 (Rdh10), as well as three retinaldehyde dehydrogenase (Raldh1, -2, -3 or Aldh1a1, -2, -3) genes, are all specifically expressed in the developing eye. 1015 RALDH2 acts transiently in the eye field of the forebrain and the early optic vesicle, 16,17 and its function is relayed by RALDH1 and -3, which are induced in specific territories of the dorsal and ventral retina. Their expression persists at least until birth, and their combined lack of function abolishes RA activity in the retina and the surrounding tissues, from midgestation onward. 18,19 Two enzymes involved in RA catabolism, CYP26A1 and -C1, are sequentially expressed in a circumferential retinal domain located between the RALDH1- and -3-positive territories. 13,20  
The three Rar genes display complex expression patterns in developing eye tissues, 11,2123 and targeted gene disruption studies have revealed that they act in a redundant manner—mainly as heterodimers with RXRα—to control murine eye morphogenesis. 2326 Indeed, compound mutation of two receptors is necessary to generate a spectrum of eye abnormalities replicating those observed long ago in rodents fed a vitamin A-deficient diet. 27 As the Rara −/−;Rarb −/− and Rara −/−;Rarg −/− double-null mutants are not viable and display rather severe eye defects (including a shortening of the ventral retina and coloboma), they have not been explored with respect to retinal differentiation or visual function. Rarb2;Rarg2 double mutants are viable and do exhibit a thinner and disorganized neural retina. These defects correlate with anomalies of the overlying pigmented epithelium, which is thought to be the target tissue of RARβ2;RARγ2 signaling. 28 Of note, a recent study has reported a Cre-mediated, tissue-specific inactivation of all three RARs in the neural crest–derived perioptic mesenchyme, and the investigators found that this loss of function replicated all the prenatal eye defects observed in the compound-null RAR mutants. 29 Thus, RAR function is required specifically in perioptic cells to control eye morphogenesis. A related conclusion was obtained through the study of Raldh1 −/−;Raldh3 −/− double-null mutants, in which RA synthesis was abolished within the developing retina, although gene expression changes were observed in the perioptic mesenchyme. 18,19  
From the current state of the art, it remains unclear whether RA and the RARs have any intrinsic function within the developing neural retina, and the work just mentioned actually concluded that RA is not required cell-autonomously in the retinal neuroepithelium. 18,19,29 Although this conclusion certainly stands true for events relating to growth or morphogenesis of the embryonic eye and its surrounding tissues, it has not been genuinely tested with respect to gene regulation and cell differentiation events (one limitation of the mouse models used in these studies being that they are lethal before or at birth). Furthermore, this conclusion is not entirely consistent with data obtained in other species through nongenetic approaches—for instance, in the chick embryo where blocking RA signaling by overexpressing a dominant-negative RAR or an RA-catabolizing enzyme does alter gene expression within the retina. 30 Also, several sets of data clearly show that RA treatment (both in vivo and in retinal culture systems) affects the differentiation of retinal cell types in zebrafish, 31,32 Xenopus, 33 chick, 34 and rat. 35 At least in the zebrafish, interfering with endogenous RA synthesis by using the aldehyde dehydrogenase inhibitor citral impeded photoreceptor differentiation. 31  
In an attempt to clarify the relevance of RA signaling in the developing murine neural retina (the layer that includes photoreceptors, excluding the pigmented epithelium), we have undertaken a phenotypic analysis of Rara null (−/−) mutant mice. 36 RARα and -β expression has been detected at the transcript level in the prenatal and adult neural retina. 11,37 On immunohistochemistry, RARα was highly expressed in all layers of the developing and adult neural retina, whereas RARβ was expressed transiently in the inner nuclear layer from embryonic day (E)14.5 to postnatal day (P)7, and in the ganglion cell layer from P4 to P7. 22,23 A previous study has reported a normal retinal histology in adult RARα1 isoform-specific knockout mutants. 38 However, a sensitive lacZ reporter transgene for RARE-mediated transcriptional activity 13,18,19,39 showed that reporter activity was abolished in the neural retina (but persisted in surface ectoderm or perioptic mesenchyme) of Rara −/− embryos at E10.5 to E13.5. 29 By using the same RARE-lacZ reporter transgene, we extended these observations and showed that Rara −/− mutants lack any detectable reporter activity in the neural retina, during both fetal and postnatal differentiation. We concluded that RARα is necessary to transduce RA activity on the RARE-lacZ reporter transgene at all phases of neural retinal development. We then analyzed the expression patterns of various molecular determinants of cell fate within the retina. No abnormality of these mRNA distributions was observed in Rara −/− mutants, in comparison to those in wild-type littermates, at pre- and postnatal stages. We assessed the distribution of differentiated cell types in the postnatal and adult Rara −/− retina using double immunofluorescence labeling and again found no detectable abnormality in the retina of the Rara −/− mutants. Finally, these mutants were subjected to clinical eye observation and functional visual evaluation by the optomotor test and electroretinography, in which they behaved like their control littermates. From this work we conclude that, although RARα transduces RARE-mediated retinoid activity at various stages of neural retina development, it is not critically necessary for proper cell-type differentiation or visual function. These data are discussed with respect to a possible noncanonical (non RAR-mediated) role of RA within the retina. 
Methods
Mice
Mice with a targeted Rara gene disruption after Cre-mediated exon deletion have been previously described. 40 The RARE-lacZ transgenic line 39 was kindly provided by Janet Rossant (University of Toronto, Canada) and was backcrossed with Rara −/+ mutants. All procedures were approved by the local institutional animal care and use committee and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
In Situ Hybridization
In situ hybridization was performed on 10-μm cryosections of whole embryonic heads or postnatal dissected eyes, according to the protocol described in Chotteau-Lelievre et al. 41 and available at http://empress.har.mrc.ac.uk/ (gene expression section; Medical Research Council Mammalian Genome Unit, Harwell, UK). The cDNA templates used to synthesize the Rara total, Rara1, and Rara2 riboprobes were the same as those used in other studies. 21,42 Other ISH template plasmids were kindly provided by Siew-Lan Ang (Otx2. 43 ; MRC National Institute for Medical Research, London, UK), Peter Gruss (Pax6 44 , Six3 45 ; Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany), Gérard Gradwohl (Neurod1 46 ; University of Strasbourg, Illkirch, France), Chi-chung Hui (University of Toronto) (Isl1; EST; GenBank accession no. AA198791; http://www.ncbi.nlm.nih.gov/Genbank/ National Center for Biotechnology Information, Bethesda, MD), Thierry Léveillard (Vsx2 47 ; Institut de la Vision, Paris, France), and Donald Zack (Crx 48 ; Johns Hopkins University, Baltimore, MD). 
Immunohistochemistry
Eyes were dissected and fixed for 30 minutes in 4% paraformaldehyde, impregnated with 10% and 20% sucrose, and cryosectioned at 10 μm. All antibodies were incubated in a solution of DMEM, 10% fetal calf serum, 2% NGS, and 0.1% Triton. Anti-calbindin (AB1778; Chemicon, Temecula, CA), anti-Brn3a (MAB1585; Chemicon), anti-S-opsin (AbCys VPA5407), anti-PKCα (sc-208; Santa Cruz Biotechnology, Santa Cruz, CA), anti-syntaxin1A (HPC-1 clone, S0664; Sigma-Aldrich, St. Quentin Fallavier, France), and anti-rhodopsin (MAB5356; Chemicon) were diluted at 1:3000, 1:400, 1:200, 1:500, 1:400, and 1:500, respectively, and were incubated overnight at 4°C, and goat anti-mouse AlexA488- or AlexA594-conjugated (Molecular Probes, Eugene, OR) were incubated for 1 hour at room temperature with 1 μg/mL DAPI. Sections were analyzed with an epifluorescence microscope (DM4000B; Leica Microsystems GmbH, Wetzlar, Germany). 
Visual Tests
Clinical Observations.
The anterior segment of the eye of unanesthetized mice was examined with a slit lamp (model 990 5X; CSO, Luneau, France) before and after pupil dilatation with a drop of 1% atropine (CibaVision, Blangnac, France). The posterior segment was then evaluated by indirect ophthalmoscopy (Kowa Genesis; Tokyo, Japan) small-animal fundus camera and a condensing 90-D lens (Volk, Mentor, OH). Fundus photographs were taken by topical endoscopy fundus imaging, as described. 49 For angiography, the mice were anesthetized with an intramuscular injection of a mixture of ketamine (120 mg/kg) and xylazine (14 mg/kg) in saline solution and then injected intraperitoneally with 50 μL of a 10% sodium fluorescein solution (CibaVision). The fundus was examined with appropriate excitation and emission filters. Eye weight and size were determined after death, with a balance and precision Vernier caliper under a dissecting microscope. The age difference at the time of death was less than 1 week for all animals (8 months old). 
Optomotor Test.
Optomotor response was scored as previously described. 50 Mice were placed on a grid platform surrounded by a motorized drum, the internal surface of which was covered by alternating black and white stripes at a frequency of 0.064 cyc/deg. The apparatus was enclosed in a light-tight box. After a 5-minute adaptation to the apparatus with a 400-lux light background measured with a luxmeter (LX105; Fisher Bioblock Scientific, Illkirch, France), the stripes were rotated alternately clockwise and counterclockwise for 2 minutes in each direction at an interval of 30 seconds. Experiments were videotaped with a digital video camera (DCR-TRV24E; Sony, Tokyo, Japan) for subsequent scoring of head-tracking movements. 
Electroretinograms.
Mice (3–7 months old) were dark adapted for at least 12 hours and subsequently handled under dim red light. Anesthesia was obtained through intramuscular injection of a mixture of ketamine (120 mg/kg) and xylazine (14 mg/kg) in saline solution. The left pupil was dilated with a drop of 1% atropine on the cornea and the vibrissae were trimmed. The animals were placed on a heating plate to prevent hypothermia due to anesthesia. The reference and ground electrodes were placed in the cheek and in the tail, respectively. The measuring gold electrode was positioned onto the cornea after a drop of methylcellulose gel was added. Flashes were delivered through a Ganzfeld equipped with light-emitting diodes with a maximum output of 318 cd/m2 (Siem Biomédicale, Nîmes, France). The flash duration varied from 3 to 5 ms, with final flash ouput ranging from 0.001 to 1 cd · s/m2. A flicker ERG was performed after a 10-minute adaptation to a 24-cd/m2 light background, with 5-ms flashes of 1.6 cd · s/m2 at frequencies from 2 to 20 Hz. Responses were amplified, filtered (1–300 Hz bandpass), and digitized (Visiosystem; Siem Biomédicale). 
Results
Expression of Rara1 and Rara2 Isoforms in the Developing Neural Retina
As a first step in deciphering the possible contribution of RARα in mediating RA signaling in the developing retina, we comparatively analyzed the expression pattern of its two main isoforms, RARα1 and -α2, 51 at various stages of pre- and postnatal development. As a reference, a probe encompassing the cDNA common to all isoforms (Rara total) was also used. In agreement with the immunolocalization data of Mori et al., 22 Rara total transcripts were detected throughout the developing neural retina, at levels relatively higher than in surrounding periocular tissues (Figs. 1A, 1D), and they continued to be expressed at postnatal (Fig. 1G) and adult stages (Fig. 1J). Analysis of isoform-specific probes revealed similar expression patterns at prenatal (Figs. 1B, 1C, 1E, 1F) and early postnatal stages (Figs. 1H, 1I), with both isoform transcripts being detected throughout the neural retina, at higher levels than in the surrounding tissues. Only Rara1 expression remained detectable in the adult retina (Figs. 1K, 1L). 
Figure 1.
 
(A–L) Expression of RARα isoform transcripts in pre- and postnatal mouse retinas. In situ hybridization was performed with riboprobes recognizing all Rara transcripts (A, D, G, J), or specific for the Rara1 (B, E, H, K) or Rara2 (C, F, I, L) isoforms, on serial sets of sections from E14.5 (AF), P4 (GI), or P70 (JL) eyes. (MP) Analysis of RARE-lacZ reporter transgene activity in the neural retina of WT (M, O) and Rara −/− (N, P) mice. In situ hybridization was performed with a lacZ probe at E14.5 (M, N) and P4 (O, P). Consistent with published data at earlier stages, 29 lack of RARE-lacZ activity is seen in the neural retina of Rara −/− mice. (AC) Overall views of the eye; (DP) details of the ventral retina, with the pigmented epithelium oriented toward the top. Insets: hybridizations with negative control (sense RNA) probes. dr, dorsal retina; gcl, ganglion cell layer; inl, inner nuclear layer; le, lens; on, optic nerve; onl, outer nuclear layer; vr, ventral retina.
Figure 1.
 
(A–L) Expression of RARα isoform transcripts in pre- and postnatal mouse retinas. In situ hybridization was performed with riboprobes recognizing all Rara transcripts (A, D, G, J), or specific for the Rara1 (B, E, H, K) or Rara2 (C, F, I, L) isoforms, on serial sets of sections from E14.5 (AF), P4 (GI), or P70 (JL) eyes. (MP) Analysis of RARE-lacZ reporter transgene activity in the neural retina of WT (M, O) and Rara −/− (N, P) mice. In situ hybridization was performed with a lacZ probe at E14.5 (M, N) and P4 (O, P). Consistent with published data at earlier stages, 29 lack of RARE-lacZ activity is seen in the neural retina of Rara −/− mice. (AC) Overall views of the eye; (DP) details of the ventral retina, with the pigmented epithelium oriented toward the top. Insets: hybridizations with negative control (sense RNA) probes. dr, dorsal retina; gcl, ganglion cell layer; inl, inner nuclear layer; le, lens; on, optic nerve; onl, outer nuclear layer; vr, ventral retina.
RARα-Mediated, RARE-Driven Gene Expression in the Pre- and Postnatal Differentiating Neural Retina
We next investigated to what extent RARα is involved in mediating RA transcriptional activity in the differentiating retina. For this, we used the RARE-lacZ transgene 39 as a reporter system. Activity of this transgene in the retinal neuroepithelium matches the postulated distribution of RA, as inferred by the spatial patterns of expression of its synthesizing and metabolizing enzymes. 10,12,13 Furthermore, a previous study reported a lack of activity of this transgene in the optic neuroepithelium of Rara −/− embryos at midgestation. 29 We compared the patterns of RARE-lacZ transcriptional activity in Rara −/− mutants and wild-type control littermates, at various stages of pre- and postnatal retinal development. As expected, RARE-lacZ mRNA was observed in dorsal and ventral domains of the retinal neuroepithelium in E14.5 control mice (Fig. 1M). RARE-lacZ mRNA was undetectable in the retinas of Rara −/− littermates (Fig. 1N). In the wild-type retina, reporter transgene expression persisted, albeit at a lower level, at late gestational and early postnatal stages (P4; Fig. 1O). At both stages, there was no detectable mRNA from the RARE-lacZ transgene in the retina of Rara −/− mutants (Fig. 1P). This analysis clearly shows that, among the RARs, RARα is the critical receptor in mediating RA transcriptional activity throughout retinal differentiation. 
Effect of RARα Lack of Function on Expression of Main Determinants of Retinal Cell Fate
Retinal cell fate determination depends on a combinatorial transcription factor code involving several basic helix-loop-helix (bHLH) and homeodomain proteins. 52 To investigate the possible phenotypic consequences of RARα loss of function during retinal development, we analyzed the distribution of cells expressing transcription factor genes characteristic of progenitor populations or known cell lineages, on serial sections of Rara −/− or wild-type littermate eyes at two stages of prenatal development (E14.5 and E18.5, Fig. 2; Supplementary Fig. S1). 
Figure 2.
 
In situ analysis of the expression of several developmental determinants in the retina of E14.5 WT and Rara −/− mice. In situ hybridization was performed with riboprobes for Six3 (AD), Pax6 (EH), Isl1 (IL), Neurod1 (MP), Otx2 (QT), or Crx (UX), on serial sets of head sections. For each probe an overall view of the eye is shown (left), with a higher magnification of the ventral retina (right: pigmented epithelium oriented toward the top). Top: genotypes. Insets: hybridizations with sense RNA probes.
Figure 2.
 
In situ analysis of the expression of several developmental determinants in the retina of E14.5 WT and Rara −/− mice. In situ hybridization was performed with riboprobes for Six3 (AD), Pax6 (EH), Isl1 (IL), Neurod1 (MP), Otx2 (QT), or Crx (UX), on serial sets of head sections. For each probe an overall view of the eye is shown (left), with a higher magnification of the ventral retina (right: pigmented epithelium oriented toward the top). Top: genotypes. Insets: hybridizations with sense RNA probes.
Among the genes expressed in retinal progenitor cells (RPCs), Six3 has been shown to be involved in the control of the balance between proliferation and differentiation during retina formation in the medaka, 53 Vsx2 (Chx10) is important for RPCs proliferative ability, 54 and Pax6 mediates the full retinogenic potential of murine retinal progenitor cells. 55 Two of these genes (Six3 and Pax6) were partly downregulated in a mouse mutant deficient in RA synthesis at the optic vesicle stage. 17 However, the distributions of Six3 (Figs. 2A–D)-, Pax6 (Figs. 2E–H)-, or Vsx2 (Supplementary Fig. S1)-expressing cells were comparable in wild-type and Rara −/− eyes. 
Among genes involved in neurogenesis, we chose Islet1 (Isl1), which is mainly expressed in the inner neuroblastic layer of the retina 56,57 (Figs. 2I, 2J). Comparable Isl1 distributions were observed in wild-type and Rara −/− retinas (Figs. 2I–L). As additional cell fate determinants, we analyzed Neurod1, Otx2, and Crx, which mark cells engaged in the photoreceptor lineages. 5860 Again, these genes showed indistinguishable expression patterns between wild-type and Rara −/− neural retinas (Figs. 2M–X; see also Supplementary Fig. S1). From this analysis, we conclude that the correct developmental expression of these determinants of neurogenesis or of retinal cell fate does not necessitate RARα function. 
We reasoned that subtle, RARα-dependent changes in gene expression may not be detectable at prenatal stages, but could become more conspicuous at postnatal stages when the laminar organization of the retina is being established and various cell populations acquire more refined distributions. Indeed, as progenitor cells differentiate, Pax6 expression becomes more restricted to the amacrine and horizontal cells and Vsx2 to bipolar cells. 61 Neurod1 specifies both photoreceptor and amacrine cell fate 59 ; Isl1 is expressed in the ganglion, bipolar, and cholinergic amacrine cells 57 ; and Otx2 is expressed in bipolar and photoreceptor postmitotic cells. 60,62 Crx remains specific to photoreceptors, where it is necessary for their terminal differentiation. 58 Our analysis of the Pax6, Isl1, Vsx2, Otx2, Neurod1, and Crx gene transcripts was pursued at 4 and 10 days postpartum (P4 and P10), and in the adult (4- and 10-week-old: P28 and P70) retina (Fig. 3; Supplementary Fig. S2). All these genes showed indistinguishable expression patterns in the retinas of wild-type (Figs. 3A, 3C, 3E, 3G, 3I, 3K) and Rara −/− mice (Figs. 3B, 3D, 3F, 3H, 3J, 3L), at all stages analyzed (see also Supplementary Fig. S2). Thus, it appears that RARα function is not mandatory for the maintenance of sufficient expression of major retinal cell-type determinants during postnatal life. 
Figure 3.
 
RARα was not necessary for maintenance of the optimal level of Isl1 (A, B), Crx (C, D), Pax6 (E, F, I, J), or Vsx2 (G, H, K, L) expression in the postnatal retina. Expression was studied by in situ hybridization. Stages shown are P4 (Isl1: A, B; Crx: C, D; Pax6: E, F; and Vsx2: G, H), P28 (Pax6: I, J) and P70 (Vsx2: K, L). Top: genotypes.
Figure 3.
 
RARα was not necessary for maintenance of the optimal level of Isl1 (A, B), Crx (C, D), Pax6 (E, F, I, J), or Vsx2 (G, H, K, L) expression in the postnatal retina. Expression was studied by in situ hybridization. Stages shown are P4 (Isl1: A, B; Crx: C, D; Pax6: E, F; and Vsx2: G, H), P28 (Pax6: I, J) and P70 (Vsx2: K, L). Top: genotypes.
Differentiated Cell Types in the Adult Rara−/− Retina
To complete our analysis of the retinal phenotype of Rara −/− mutants, we analyzed the distribution of differentiated cell types using double immunofluorescence with a combination of markers for rod and cone photoreceptors (rhodopsin and short-wave opsin), horizontal and amacrine cells (calbindin and syntaxin1A), ganglion cells implicated in cortical vision (Brn3a), and rod bipolar cells (PKCα). This analysis was performed at 4- and 10-weeks of age (P28 and P70), with results shown for the latter stage (Fig. 4). No detectable difference was observed with these various differentiation markers, between wild-type control and Rara −/− mutant mice. The retina of Rara −/− mice also displayed normal histology on analysis of hematoxylin-eosin–stained sections (Fig. 5). 
Figure 4.
 
RARα function was dispensable for a normal organization of adult retinal cell types. Double fluorescence immunohistochemistry was performed on serial sections of adult (P70: 10 week-old) eyes, with antibodies against rhodopsin (rods; A, B), S-opsin (short-wave cones; C, D), Brn3a (ganglion cells; E, F), calbindin (horizontal and some amacrine cells; G, H), syntaxin1A (amacrine cells and horizontal cells; I, J), and PKCα (rod bipolar cells; K, L). (MR) DAPI staining (marking the cell nuclei). (SX) Merged images. Top: genotypes.
Figure 4.
 
RARα function was dispensable for a normal organization of adult retinal cell types. Double fluorescence immunohistochemistry was performed on serial sections of adult (P70: 10 week-old) eyes, with antibodies against rhodopsin (rods; A, B), S-opsin (short-wave cones; C, D), Brn3a (ganglion cells; E, F), calbindin (horizontal and some amacrine cells; G, H), syntaxin1A (amacrine cells and horizontal cells; I, J), and PKCα (rod bipolar cells; K, L). (MR) DAPI staining (marking the cell nuclei). (SX) Merged images. Top: genotypes.
Figure 5.
 
Histology (hematoxylin-eosin staining) of adult (AD), P4 (E, F), and E14.5 (G, H) wild-type (WT) and Rara −/− retinas. (C, D) The ciliary body. Top: genotypes.
Figure 5.
 
Histology (hematoxylin-eosin staining) of adult (AD), P4 (E, F), and E14.5 (G, H) wild-type (WT) and Rara −/− retinas. (C, D) The ciliary body. Top: genotypes.
Effect of RARα Inactivation on Eye Morphology and Electroretinography
We performed a clinical examination of the eyes of Rara −/− mice and control littermates. No cornea or lens defect was detected by slit lamp examination (data not shown). Fundus photographs of mutant and wild-type animals were comparable, with no detectable anomaly in pigmentation, blood vessel distribution, and optic nerve aspect (Figs. 6A, 6B). The vascularization pattern and blood–retina barrier integrity were not affected, as indicated by fluorescein angiography (Figs. 6C, 6D). Eye weight and size were determined after death. No significant difference was observed between WT and Rara −/− mice for either parameter, with diameters of 3.52 ± 0.02 mm versus 3.54 ± 0.03 mm (P = 0.59, Student's t-test), and weights of 24.6 ± 0.2 mg versus 25.1 ± 0.4 mg (P = 0.25, Student's t-test) for WT (14 eyes of seven animals, four males and three females) and Rara −/− (10 eyes of five animals, three males and two females) mice, respectively. 
Figure 6.
 
Indirect ophthalmoscopy and angiography did not reveal any defects in eyes from Rara −/− mice. Fundus (A, B) and fundus fluorescein angiography (C, D) photographs from control (left) and mutant mice (right) are shown.
Figure 6.
 
Indirect ophthalmoscopy and angiography did not reveal any defects in eyes from Rara −/− mice. Fundus (A, B) and fundus fluorescein angiography (C, D) photographs from control (left) and mutant mice (right) are shown.
In the optomotor test, the two genotypes responded similarly. When the drum was rotating clockwise or counterclockwise, the control animals scored 11.8 ± 0.9 and 12 ± 0.7 head movements (n = 4), versus 11.5 ± 0.9 and 12.3 ± 0.7 for the Rara −/− mice (n = 3). 
Figure 7A shows representative scotopic ERG responses from 3-month-old Rara −/− and control littermates for four flash intensities. On average, the two genotypes had indistinguishable scotopic ERG phenotypes in both a- and b-wave amplitudes (Fig. 7B; P = 0.55 and P = 0.92, respectively; repeated-measures ANOVA) and implicit times (Fig. 7C; P = 0.62 and P = 0.97, respectively; repeated-measures ANOVA; WT: n = 12; Rara −/−: n = 7). Flicker ERG was also seemingly unaffected by inactivation of Rara, with similar responses for frequencies ranging from 2 to 20 Hz (Fig. 7D; P = 0.47, repeated-measures ANOVA when considering all frequencies; when considering each frequency individually: P = 0.22 at 2 Hz, P = 0.57 at 5 Hz, P = 0.91 at 10 Hz, P = 0.56 at 15 Hz, and P = 0.21 at 20 Hz; Student's t-test; WT: n = 12 and Rara −/−: n = 7). 
Figure 7.
 
Absence of RARα did not affect scotopic and photopic ERG responses. (A) Records from scotopic ERGs from WT and Rara −/− mice, for four flash intensities. Neither the a- and b-wave amplitudes (B) nor the implicit times (C) were affected in the Rara −/− mice (n = 7) compared with WT mice (filled symbols, n = 12). In photopic conditions, flicker ERGs for frequencies between 2 and 20 Hz (D) were similarly unaffected (Rara −/−; n = 7; WT; n = 12).
Figure 7.
 
Absence of RARα did not affect scotopic and photopic ERG responses. (A) Records from scotopic ERGs from WT and Rara −/− mice, for four flash intensities. Neither the a- and b-wave amplitudes (B) nor the implicit times (C) were affected in the Rara −/− mice (n = 7) compared with WT mice (filled symbols, n = 12). In photopic conditions, flicker ERGs for frequencies between 2 and 20 Hz (D) were similarly unaffected (Rara −/−; n = 7; WT; n = 12).
Discussion
The Rara −/− knockout mouse is a viable animal model in which, according to the RARE-lacZ reporter system, all RARE-mediated signaling is abolished in the developing neural retina from E10.5 29 to postnatal stages (Fig. 1). We have performed molecular and functional analyses on this mutant and report that Rara −/− mice have a normal organization of retinal cell types (Fig. 4), with no alteration of the distribution of major developmental molecular determinants (Figs. 2, 3). Although previous studies reported a slightly reduced eye size in RARα1 isoform-specific mouse mutants 38 and a more pronounced effect on eye size in chick embryos expressing a dominant-negative RAR, 30 eye size was not affected in our Rara −/− mice. Visual phenotyping of these mice using slit lamp biomicroscopy and indirect ophthalmoscopy, angiography, optomotor test, and ERG analysis, did not reveal any deficit or sign of retinal degeneration. Altogether, these results lead to the conclusion that RARα is dispensable for proper retinal differentiation and visual function. 
During development, RA is present in precisely delineated dorsal and ventral territories of the neural retina. This specific location is due to the regional distribution of two synthesizing enzymes (RALDH1 and -3) and the sequential expression of two RA-metabolizing enzymes (CYP26A1 and -C1) in a horizontal stripe abutting the dorsal and ventral RALDH territories. 10,12,13,20 The CYP26-expressing, RA-poor stripe has been suggested to correspond to the region of the retina that develops the highest visual acuity—a region called the visual streak in such species as rabbit and is functionally similar to the fovea in the human retina. 13,20 It is intriguing that the precise distribution of RA-synthesizing and metabolizing enzymes is the only molecular cue presently known to demarcate this functional specialization. One cannot exclude that a subtle rearrangement of the topology of photoreceptors may occur in the Rara −/− mice, that may affect visual performance in a manner not detectable by our functional tests. Another viable murine model in the RA pathway is the Raldh1 −/− mutant, 63 in which no abnormal retinal phenotype was found; however, these mice were not analyzed by immunohistochemistry for differentiated cell types, as we did for the Rara −/− mutants. 
In our molecular studies, we analyzed the main known developmental regulators acting on retinal progenitor populations (Pax6, Six3, Otx2, Neurod1, Isl1, Vsx2, and Crx) and found them unchanged in the retina of the Rara −/− mouse. This finding does not exclude that other genes could be misregulated in the retina in the absence of RARα. Few genes have been demonstrated to be genuine targets of RA regulation within the embryo, 6466 and in general these are not expressed in the developing neural retina. Pax6 and Six3 were shown to be downregulated in the optic vesicle of Raldh2 −/− embryos 17 ; we did not observe such a downregulation in the Rara −/− mutants (Figs. 2, 3). Global approaches such as whole-transcriptome analysis may be required to clarify whether RARα loss of function affects gene regulation within the retina. It should be noted that RARβ is transiently expressed in the inner nuclear layer and the ganglion cell layer at pre- and perinatal stages, 22 and other studies detected RARβ transcripts in the prenatal 11 and adult retina, using Northern blot analysis and/or PCR analysis. 37 Hence, one cannot exclude that RARβ may to some extent compensate for the lack of RARα in the retina. Further work on knockout mice for RARs and/or RA-synthesizing enzymes is also needed to investigate whether these mutations may affect expression of Eph/Ephrin genes, as reported in the chick using a dominant-negative approach. 30  
Our data on Rara −/− mutants are consistent with results obtained on other murine models deficient for RA synthesis within the retina, 18,19 or with a tissue-specific inactivation of the RARs in perioptic cells, 18,29 indicating that RARα is not functionally essential in the retinal neuroepithelium during eye development and morphogenesis (Fig. 8A). Although RA is produced in the optic vesicle by RALDH2, 13 and eventually by RALDH1 and -3 in specific dorsal and ventral territories (Fig. 8A), during early eye development, it acts as a paracrine signal on neural crest–derived perioptic cells, in which all three RARs function in an essentially redundant manner to regulate gene expression and control cellular events such as apoptosis. 18,29 RXRα is their critical partner, as its sole disruption leads to severe ocular morphogenetic defects. 25,26  
Figure 8.
 
Proposed functions of RA during eye morphogenesis and differentiation. (A) At early stages of eye development, RA is synthesized by RALDH1 and -3 in specific dorsal and ventral domains of the neural retina. Although it can activate RARα within the retina (this study), its function is mainly (if not exclusively) related to diffusion and activation of RARs in the perioptic mesenchyme. 18,19,29 (B) At later stages of retinal differentiation, although RA can still activate RARα, its presence in the neural retina may be relevant for negative regulation of the orphan receptor RORβ, 72 expressed in the entire retina.
Figure 8.
 
Proposed functions of RA during eye morphogenesis and differentiation. (A) At early stages of eye development, RA is synthesized by RALDH1 and -3 in specific dorsal and ventral domains of the neural retina. Although it can activate RARα within the retina (this study), its function is mainly (if not exclusively) related to diffusion and activation of RARs in the perioptic mesenchyme. 18,19,29 (B) At later stages of retinal differentiation, although RA can still activate RARα, its presence in the neural retina may be relevant for negative regulation of the orphan receptor RORβ, 72 expressed in the entire retina.
Does this imply that the presence of RA is not necessary within the differentiating neural retina? As discussed, it is possible that RARβ, 11,37 in combination with RXRα or -β, 67 transduces the RA signal at critical stages of retinal differentiation. Another attractive hypothesis is that RA acts in a noncanonical (non–RAR-RARE–mediated) pathway in the neural retina (Fig. 8B). Such a hypothesis is supported by the following lines of evidence. The lack of RARα has no detectable phenotypic consequence (this study). On the other hand, mice entirely deficient for RA synthesis (the Raldh1 −/−;Raldh3 −/− double mutants) exhibit abnormalities in photoreceptor gene expression (Cammas L, Dollé P, unpublished data, 2009), which suggests that RA could exert some function independently of the RARs. A candidate for noncanonical signaling is the retinoid-related orphan receptor (ROR)-β. This receptor is specifically expressed in the neural retina both during development and adult life, 68 and both loss of function 69 and gain of function 70 studies have demonstrated its implication in the control of retinal differentiation. RORβ was first characterized as an orphan nuclear receptor. 71 However, a subsequent study identified all-trans-RA as a bona fide ligand, which in cotransfection assays had an inhibitory effect on RORβ transactivating properties. 72 Considering the high expression of this receptor throughout the developing neural retina, it is a good candidate for translating the spatial distribution of RA into a regional control of transcriptional activity (Fig. 8B). This hypothesis will be tested in future work. 
Supplementary Materials
Additional in situ hybridization analysis of retinal molecular determinants at pre- or early post-natal stages. Genes, stages and genotypes (WT, Rara-/-) as indicated. 
Additional in situ hybridization analysis of retinal determinants at post-natal and adult stages. Genes, stages and genotypes (WT, Rara-/-) as indicated. Hybridizations with sense RNA probes are shown in insets. 
Footnotes
 Supported by European Union Grant EVI-GENORET LSHG-CT-2005-512036; a RAinBRAIN grant from Agence Nationale de la Recherche; Equipe FRM 2007 Grant from the Fondation pour la Recherche Médicale; CNRS; INSERM; and Hôpitaux Universitaires de Strasbourg.
Footnotes
 Disclosure: L. Cammas, None; F. Trensz, None; A. Jellali, None; N.B. Ghyselinck, None; M.J. Roux, None; P. Dollé, None
The authors thank Valérie Fraulob and Brigitte Schuhbaur for expert technical assistance and Sandra Bour for artwork. 
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Figure 1.
 
(A–L) Expression of RARα isoform transcripts in pre- and postnatal mouse retinas. In situ hybridization was performed with riboprobes recognizing all Rara transcripts (A, D, G, J), or specific for the Rara1 (B, E, H, K) or Rara2 (C, F, I, L) isoforms, on serial sets of sections from E14.5 (AF), P4 (GI), or P70 (JL) eyes. (MP) Analysis of RARE-lacZ reporter transgene activity in the neural retina of WT (M, O) and Rara −/− (N, P) mice. In situ hybridization was performed with a lacZ probe at E14.5 (M, N) and P4 (O, P). Consistent with published data at earlier stages, 29 lack of RARE-lacZ activity is seen in the neural retina of Rara −/− mice. (AC) Overall views of the eye; (DP) details of the ventral retina, with the pigmented epithelium oriented toward the top. Insets: hybridizations with negative control (sense RNA) probes. dr, dorsal retina; gcl, ganglion cell layer; inl, inner nuclear layer; le, lens; on, optic nerve; onl, outer nuclear layer; vr, ventral retina.
Figure 1.
 
(A–L) Expression of RARα isoform transcripts in pre- and postnatal mouse retinas. In situ hybridization was performed with riboprobes recognizing all Rara transcripts (A, D, G, J), or specific for the Rara1 (B, E, H, K) or Rara2 (C, F, I, L) isoforms, on serial sets of sections from E14.5 (AF), P4 (GI), or P70 (JL) eyes. (MP) Analysis of RARE-lacZ reporter transgene activity in the neural retina of WT (M, O) and Rara −/− (N, P) mice. In situ hybridization was performed with a lacZ probe at E14.5 (M, N) and P4 (O, P). Consistent with published data at earlier stages, 29 lack of RARE-lacZ activity is seen in the neural retina of Rara −/− mice. (AC) Overall views of the eye; (DP) details of the ventral retina, with the pigmented epithelium oriented toward the top. Insets: hybridizations with negative control (sense RNA) probes. dr, dorsal retina; gcl, ganglion cell layer; inl, inner nuclear layer; le, lens; on, optic nerve; onl, outer nuclear layer; vr, ventral retina.
Figure 2.
 
In situ analysis of the expression of several developmental determinants in the retina of E14.5 WT and Rara −/− mice. In situ hybridization was performed with riboprobes for Six3 (AD), Pax6 (EH), Isl1 (IL), Neurod1 (MP), Otx2 (QT), or Crx (UX), on serial sets of head sections. For each probe an overall view of the eye is shown (left), with a higher magnification of the ventral retina (right: pigmented epithelium oriented toward the top). Top: genotypes. Insets: hybridizations with sense RNA probes.
Figure 2.
 
In situ analysis of the expression of several developmental determinants in the retina of E14.5 WT and Rara −/− mice. In situ hybridization was performed with riboprobes for Six3 (AD), Pax6 (EH), Isl1 (IL), Neurod1 (MP), Otx2 (QT), or Crx (UX), on serial sets of head sections. For each probe an overall view of the eye is shown (left), with a higher magnification of the ventral retina (right: pigmented epithelium oriented toward the top). Top: genotypes. Insets: hybridizations with sense RNA probes.
Figure 3.
 
RARα was not necessary for maintenance of the optimal level of Isl1 (A, B), Crx (C, D), Pax6 (E, F, I, J), or Vsx2 (G, H, K, L) expression in the postnatal retina. Expression was studied by in situ hybridization. Stages shown are P4 (Isl1: A, B; Crx: C, D; Pax6: E, F; and Vsx2: G, H), P28 (Pax6: I, J) and P70 (Vsx2: K, L). Top: genotypes.
Figure 3.
 
RARα was not necessary for maintenance of the optimal level of Isl1 (A, B), Crx (C, D), Pax6 (E, F, I, J), or Vsx2 (G, H, K, L) expression in the postnatal retina. Expression was studied by in situ hybridization. Stages shown are P4 (Isl1: A, B; Crx: C, D; Pax6: E, F; and Vsx2: G, H), P28 (Pax6: I, J) and P70 (Vsx2: K, L). Top: genotypes.
Figure 4.
 
RARα function was dispensable for a normal organization of adult retinal cell types. Double fluorescence immunohistochemistry was performed on serial sections of adult (P70: 10 week-old) eyes, with antibodies against rhodopsin (rods; A, B), S-opsin (short-wave cones; C, D), Brn3a (ganglion cells; E, F), calbindin (horizontal and some amacrine cells; G, H), syntaxin1A (amacrine cells and horizontal cells; I, J), and PKCα (rod bipolar cells; K, L). (MR) DAPI staining (marking the cell nuclei). (SX) Merged images. Top: genotypes.
Figure 4.
 
RARα function was dispensable for a normal organization of adult retinal cell types. Double fluorescence immunohistochemistry was performed on serial sections of adult (P70: 10 week-old) eyes, with antibodies against rhodopsin (rods; A, B), S-opsin (short-wave cones; C, D), Brn3a (ganglion cells; E, F), calbindin (horizontal and some amacrine cells; G, H), syntaxin1A (amacrine cells and horizontal cells; I, J), and PKCα (rod bipolar cells; K, L). (MR) DAPI staining (marking the cell nuclei). (SX) Merged images. Top: genotypes.
Figure 5.
 
Histology (hematoxylin-eosin staining) of adult (AD), P4 (E, F), and E14.5 (G, H) wild-type (WT) and Rara −/− retinas. (C, D) The ciliary body. Top: genotypes.
Figure 5.
 
Histology (hematoxylin-eosin staining) of adult (AD), P4 (E, F), and E14.5 (G, H) wild-type (WT) and Rara −/− retinas. (C, D) The ciliary body. Top: genotypes.
Figure 6.
 
Indirect ophthalmoscopy and angiography did not reveal any defects in eyes from Rara −/− mice. Fundus (A, B) and fundus fluorescein angiography (C, D) photographs from control (left) and mutant mice (right) are shown.
Figure 6.
 
Indirect ophthalmoscopy and angiography did not reveal any defects in eyes from Rara −/− mice. Fundus (A, B) and fundus fluorescein angiography (C, D) photographs from control (left) and mutant mice (right) are shown.
Figure 7.
 
Absence of RARα did not affect scotopic and photopic ERG responses. (A) Records from scotopic ERGs from WT and Rara −/− mice, for four flash intensities. Neither the a- and b-wave amplitudes (B) nor the implicit times (C) were affected in the Rara −/− mice (n = 7) compared with WT mice (filled symbols, n = 12). In photopic conditions, flicker ERGs for frequencies between 2 and 20 Hz (D) were similarly unaffected (Rara −/−; n = 7; WT; n = 12).
Figure 7.
 
Absence of RARα did not affect scotopic and photopic ERG responses. (A) Records from scotopic ERGs from WT and Rara −/− mice, for four flash intensities. Neither the a- and b-wave amplitudes (B) nor the implicit times (C) were affected in the Rara −/− mice (n = 7) compared with WT mice (filled symbols, n = 12). In photopic conditions, flicker ERGs for frequencies between 2 and 20 Hz (D) were similarly unaffected (Rara −/−; n = 7; WT; n = 12).
Figure 8.
 
Proposed functions of RA during eye morphogenesis and differentiation. (A) At early stages of eye development, RA is synthesized by RALDH1 and -3 in specific dorsal and ventral domains of the neural retina. Although it can activate RARα within the retina (this study), its function is mainly (if not exclusively) related to diffusion and activation of RARs in the perioptic mesenchyme. 18,19,29 (B) At later stages of retinal differentiation, although RA can still activate RARα, its presence in the neural retina may be relevant for negative regulation of the orphan receptor RORβ, 72 expressed in the entire retina.
Figure 8.
 
Proposed functions of RA during eye morphogenesis and differentiation. (A) At early stages of eye development, RA is synthesized by RALDH1 and -3 in specific dorsal and ventral domains of the neural retina. Although it can activate RARα within the retina (this study), its function is mainly (if not exclusively) related to diffusion and activation of RARs in the perioptic mesenchyme. 18,19,29 (B) At later stages of retinal differentiation, although RA can still activate RARα, its presence in the neural retina may be relevant for negative regulation of the orphan receptor RORβ, 72 expressed in the entire retina.
Supplementary Figure S1
Supplementary Figure S2
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