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(Investigative Ophthalmology and Visual Science. 2001;42:1312-1318.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Systematic Immunolocalization of Retinoid Receptors in Developing and Adult Mouse Eyes

Mikiro Mori, Norbert B. Ghyselinck, Pierre Chambon and Manuel Mark

From the Institut de Génétique et de Biologie Moléculaire et Cellulaire, Collège de France, Illkirch-CU de Strasbourg, France.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Conclusions
 References
 
PURPOSE. To determine the localization of retinoic acid receptors (RAR) {alpha}, ß, and {gamma} and retinoid X receptors (RXR) {alpha}, ß, and {gamma} in developing and adult mouse eyes at the level of single cells.

METHODS. Immunohistochemistry was performed on paraformaldehyde-lysine-periodate–fixed cryosections of mouse eyes, from embryonic day 10.5 to adulthood, with polyclonal antibodies directed against each receptor isoform. Histologic sections from null mutant mice for each receptor served as negative controls.

RESULTS. RAR{alpha} was present ubiquitously in the prenatal eye and preferentially located in the posnatal retina and ciliary body. RARß was detected predominantly in the periocular mesenchyme and ciliary body. RAR{gamma} was distributed in the periocular mesenchyme, choroid, sclera, cornea, conjunctiva, and lids. RXR{alpha} was found preferentially in the prenatal periocular mesenchyme and retina and in the postnatal ciliary body, cornea, and conjunctiva. RXRß was ubiquitous at all the stages. RXR{gamma} was detected mainly in subsets of prenatal retinal cells and in postnatal ganglion cells as well as a subset of photoreceptor cells that were characterized as cones in adults.

CONCLUSIONS. RAR{alpha}, ß, and {gamma} and RXR{alpha} and {gamma} exhibit specific and dynamic patterns of distribution in ocular tissues throughout the course of development. The abundance of RARß, RAR{gamma}, and RXR{alpha} in the periocular mesenchyme suggests that this tissue represents an important site of retinoid actions during eye development and in adulthood.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Conclusions
 References
 
Early studies on the effects of dietary vitamin A deficiency (VAD) in adult and embryonic rats have demonstrated that this molecule is required for vision, maintenance of the adult retinal structure,1 2 3 and eye morphogenesis.4 Vitamin A acts through its metabolites: retinaldehyde, which forms the visual chromophore5 ; and retinoic acid (RA), which regulates gene expression.6 RA has been shown to be a key molecule for eye development.7 It is indispensable for eye morphogenesis in fish8 and mouse9 embryos and can increase the number of cells expressing rod opsin in fish10 and rats11 in vivo as well as in cultures from dissociated embryonic rat retinal cells.12 13 RA also induces rod-specific apoptosis in mouse retinal explants.14 15 In the adult mouse retina, RA is produced in response to light stimuli16 and regulates expression of vision-related genes such as arrestin.17

The actions of RA are mediated by nuclear receptors, which are ligand-inducible transcriptional regulators and belong to two distinct families: RARs activated by all-trans RA and 9-cis RA and RXRs activated by 9-cis RA only. Each family consists of three genetic isoforms (RAR or RXR {alpha}, ß, and {gamma}). Retinoid signaling pathways are complex because (i) RARs bind to their cognate response elements as heterodimers with RXRs, (ii) RXRs can also bind to certain DNA response elements as homodimers, and (iii) RXRs heterodimerize with a number of nuclear receptors, such as thyroid hormone receptors (TR{alpha} and ß), the vitamin D3 receptor (VDR), peroxisome proliferator–activated receptors (PPAR{alpha}, ß, and {gamma}), and various orphan nuclear receptors (i.e., whose ligands, if any, remain to be discovered).18 19 20

Gene targeting studies in the mouse have demonstrated that null mutants for RXR{alpha}21 and double mutants for RAR{alpha} and RAR{gamma} (RAR{alpha}/RAR{gamma} mutants22 ) and for RARß and RAR{gamma} (RARß/RAR{gamma} mutants23 ) display a large spectrum of ocular malformations recapitulating that caused by embryonic VAD. Furthermore, congenital eye defects observed in the RXR{alpha} null mutants become severe upon inactivation of RARß or RAR{gamma} alleles in the RXR{alpha} null genetic background, demonstrating that RXR{alpha}/RARß and RXR{alpha}/RAR{gamma} heterodimers are critically involved in eye development.21 24 25 Mice lacking both RARß2 and RAR{gamma}2 isoforms (RARß2/RAR{gamma}2 mutants26 ) exhibit postnatal retinal dysplasia and degeneration, indicating that expression of RARß and RAR{gamma} is required for retinal histogenesis and for the survival of retinal cells. Despite the accumulated evidence for retinoid functions in eye development, the localization of RAR and RXR isoforms is partially documented only in the adult mouse eye.27 To provide a comprehensive map of RAR and RXR distribution at the level of single cells in ocular tissues, we have systematically analyzed the localization of RAR{alpha}, ß, and {gamma} and RXR{alpha}, ß, and {gamma} proteins in the developing and adult mouse eye.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Conclusions
 References
 
Immunohistochemistry
Animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Eyes or embryo heads were collected from CD1 mice at embryonic (E) days 10.5, E12.5, E14.5; and E17.5, postnatal (P) days 1, P4, P7, P10, and P14; and at adulthood (3 weeks and 2 months), fixed with 2% paraformaldehyde-lysine-periodate28 for 4 hours at 4°C, and cryoprotected in a series of 5%, 10%, 15%, and 20% sucrose in phosphate-buffered saline for 2, 2, 6, and 16 hours, respectively. Frozen sections (10-µm-thick) were collected on poly-L-lysine–coated slides (Mentzel-Glaser, Braunschweig, Germany) and incubated with affinity-purified polyclonal antibody for RAR{alpha}29 (diluted 1:2000), RARß30 (1:400), RAR{gamma}31 (1:1000), RXR{alpha}32 (1:2000), RXRß33 (1:1000), or RXR{gamma} (1:1000, sc555; Santa Cruz, CA) for 16 hours at 4°C. The immunoreactivity was visualized by incubation with CY3-labeled anti-rabbit IgG (Chemicon International, Temecula, CA) diluted 1:500 for 1 hour at room temperature. Counterstaining was done with DAPI in the mounting medium (Vectashield; Vector Laboratories, Burlingame, CA). Histologic sections from mutant mice carrying targeted inactivation of RAR{alpha},34 RARß,23 RAR{gamma},35 RXR{alpha},21 RXRß,36 and RXR{gamma}37 were used as negative controls of the immunostaining procedure as previously described.38

Double Detection of RXR{gamma} Protein and Cone Photoreceptors
Adult eye sections reacted with the anti-RXR{gamma} antibody, as described above, were then incubated with fluorescein-labeled anti-rabbit IgG (Chemicon International) diluted 1:500 in the presence of TRITC-labeled peanut lectin (Sigma, St. Louis, MO) diluted 1:50 for 2 hours at room temperature.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Conclusions
 References
 
RXRß showed ubiquitous distribution in developing and adult eyes (Fig. 1P and data not shown) without significant spatiotemporal variations. In contrast, RAR{alpha}, ß, {gamma}, and RXR{alpha} and {gamma} were immunolocalized to specific tissues or cells throughout the course of prenatal and postnatal stages of eye development (Table 1) . Mice at 3 weeks and at 2 months after birth showed indistinguishable patterns of immunostaining for all the receptors and were collectively referred to as "adults." The specificity of the immunoreactivity was confirmed by the absence of signals on eye sections from null mutants for each receptor at E14.5 and, except for the embryonically lethal RXR{alpha} mutants, at adulthood (Figs. 1C 1F 1I 1L 1O 1Q and 2E 2J 20 ). It should be noted that cells staining weakly for a given receptor (Table 1) are not necessarily visible on micrographs taken at low magnifications.



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  		Figure 1. Immunolocalization of retinoid receptors in the prenatal mouse eye. The cryosections were incubated with an antibody specific for RAR{alpha} (A{alpha}), RARß (Aß), RAR{gamma} (A{gamma}), RXR{alpha} (X{alpha}), RXRß (Xß), or RXR{gamma} (X{gamma}). The signal was visualized with a secondary antibody conjugated to CY3, and cell nuclei were counterstained with DAPI. Control sections were prepared from null mutant mice (-/-) for the corresponding receptor. See text for details. (A through Q) Left: immunohistochemistry (IHC); right: DAPI staining. INBL, inner neuroblastic layer; ONBL, outer neuroblastic layer; RPE, retinal pigment epithelium; PM, periocular mesenchyme; L, lens; C, cornea; R, neural retina. Bars, 100 µm.

 

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  		Table 1. Distribution of Retinoic Acid Receptors {alpha}, ß, and {gamma} and Retinoid X Receptors {alpha} and {gamma} in the Developing and Adult Mouse Eye

 


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  		Figure 2. Immunolocalization of retinoid receptors in the postnatal mouse eye. See the legend of Figure 1 . (E, J, and O) Left: immunoreactivity; right: DAPI staining. (H and I) Arrows, scattered immunoreactive cell nuclei. INBL, inner neuroblastic layer; ONBL, outer neuroblastic layer; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; CH, choroid; SC, sclera; L, lens; C, cornea; CB, ciliary body. Bars, 50 µm.

 
Neuroretina
During mouse development, the retina begins to differentiate and form the inner neuroblastic layer (INBL) and the outer neuroblastic layer (ONBL) by E14. The INBL is destined to give rise principally to the ganglion cell layer (GCL). By E15, the inner plexiform layer (IPL) forms between the INBL and the ONBL. By postnatal day 4 (P4), the outer plexiform layer (OPL) forms, dividing the ONBL into the outer nuclear layer (ONL) and inner nuclear layer (INL). By P7, these layers are distinctly present throughout the entire retina.39

RAR{alpha} was detected at all the stages from E10.5 to adulthood (Figs. 1A 1B and 2A 2B 2C 2D ; Table 1 ). At E10.5, the immunoreactive cell nuclei were uniformly distributed (Figs. 1A 1B and 2A) and at P4 relatively concentrated in the inner region of the ONBL (Fig. 2B) . At P7, nuclei in the middle rows of the INL were strongly immunoreactive (Fig. 2C) . By P14, the signals decreased to low levels in the INL and ONL and remained stronger in some nuclei in the GCL and in the innermost rows of the INL (Fig. 2D) , which presumably correspond to amacrine cell nuclei. The RARß signal was weak and transient (from E14.5 to P7) and absent in the ONL (Figs. 1D 1E ; Table 1 ). RAR{gamma} signal was never detected in the neural retina (Figs. 1G 1H and 2N ; Table 1 ). The RXR{alpha} signal was strong and uniformly distributed in the embryonic retina before the onset of retinal lamination (i.e., at E10.5 and E12.5; Fig. 1J ; Table 1 ), decreased thereafter, and was not detected in the adult retina (Table 1) . The RXR{gamma} signal was restricted to the peripheral border of the optic cup where the neural retina is continuous with the retinal pigment epithelium (RPE) at E10.5 (Fig. 1M) and then was abundant in the inner and outer portions of the neural retina at E12.5 (data not shown). From E14.5 to P7, immunoreactive nuclei were essentially present in the outer portion of the ONBL (or in the ONL) and in the INBL (or in the GCL; Figs. 1N and 2F 2G 2H ). The immunoreactive nuclei in the ONL became confined to the outermost rows in adults (Figs. 2I) . This later distribution pattern suggested that the immunoreactive nuclei in the ONL are those of cone photoreceptor cells. A few immunoreactive nuclei were also detected in the innermost rows of the INL, which presumably correspond to amacrine cells (Fig. 2I , arrow).

To identify the cell type of the immunoreactive nuclei in the ONL, double staining was performed with the RXR{gamma} antibody and the peanut lectin. Almost all the nuclei immunoreactive for RXR{gamma} corresponded to the outer segments stained with this lectin (Figs. 3A 3B 3C) , confirming that the photoreceptors expressing RXR{gamma} are mostly, if not exclusively, cone photoreceptors.



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  		Figure 3. Double detection of RXR{gamma} protein and cone photoreceptor cells in the adult mouse eye. RXR{gamma} immunostaining (A) and lectin staining (B) are superimposed in (C). Note that the positive cell nuclei (A) correspond to the positive outer segments (B). Bars, 50 µm.

 
Retinal Pigment Epithelium
In the RPE, RAR{alpha} was detected at all the stages from E10.5 with decrease at postnatal stages (Figs. 1A 1B and 2A 2B 2C 2D ; Table 1 ). RARß was weakly detected from E14.5 to P7 (Fig. 1E ; Table 1 ). RAR{gamma} was not detected at any stages (Figs. 1G 1H and 2N ; Table 1 ). RXR{alpha} was detected weakly from E10.5 to E17.5 but not at later stages (Figs. 1J 1K ; Table 1 ). RXR{gamma} was detected only at E10.5 (Fig. 1M ; Table 1 ).

Lens
RAR{alpha} was detected at all the stages in the lens (Figs. 1A and 4C ; Table 1 ). RXR{alpha} and RXR{gamma} were detected only at E10.5 and E12.5 (Figs. 1J 1M ; Table 1 ). RARß and {gamma} and RXR{alpha} and {gamma} were not detected at any stages.



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  		Figure 4. Immunolocalization of retinoid receptors in the mouse eye anterior segments and primary vitreous (PV) at E14.5. See the legend of Figure 1 . L, lens; C, cornea; LI, lid; R, neural retina; CB, ciliary body. Bars, 50 µm.

 
Periocular Mesenchyme and Its Derivative Tissues
The periocular mesenchyme (PM) begins to differentiate into sclera, choroid, ciliary body, and iris by E16. RARß and {gamma} were strongly and specifically detected in the PM or in the choroid and sclera from E10.5 to P4 (Figs. 1D 1E 1G 1H ; Table 1 ). The RARß signal in the choroid and sclera decreased thereafter (Table 1) , whereas the RAR{gamma} signal remained strong in these tissues in the adult (Fig. 2N) . RAR{alpha} and RXR{alpha} signals were more broadly distributed prenatally (Figs. 1A 1B 1J 1K ; Table 1 ) and diminished postnatally (Figs. 2A 2B 2C 2D ; Table 1 ). RARß and RXR{alpha} were also present in the primary vitreous body (Figs. 4A 4B ), which is a transient embryonic structure consisting of fibroblastic cells that stem from the PM and a capillary network around the hyaroid artery. In the iris and ciliary body (and in their precursor PM), RAR{alpha}, RARß, and RXR{alpha} were present from E14.5 to adult (Figs. 2K 2L 2M and 4C 4D 4F ; Table 1 ), whereas RAR{gamma} and RXR{gamma} were not detected at any stages (Fig. 4E ; Table 1 ).

Cornea, Conjunctiva, and Lids
RAR{gamma} and RXR{alpha} were detected in these tissues from E14.5 to adulthood both in the epithelium and stroma. (Figs. 2M 4E 4F and 5A 5B 5C 5D ; Table 1 ). RAR{alpha} was also detected from E14.5 with a decrease in signal intensity at postnatal stages (Figs. 2K and 4C ; Table 1 ). RARß and RXR{gamma} were not detected in these tissues at any stages (Figs. 2L and 4D and data not shown).



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  		Figure 5. Immunolocalization of RAR{gamma} and RXR{alpha} in the cornea (A and B) and conjunctiva (C and D) in the adult mouse. See the legend of Figure 1 . EP, corneal epithelium; EN, corneal endothelium; ST, corneal stroma; CS, conjunctival sac. Bars, 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Conclusions
 References
 
The present study describes the localization of RAR and RXR proteins in the developing and adult mouse eye. The distribution patterns of the receptor proteins are in general accordance with those of the corresponding mRNAs reported in in situ hybridization studies of prenatal mice.23 26 40 41 Discrepancies between our results and the distributions of mRNA signals reported in the previous studies (e.g., detection of RARß mRNA in the cornea,42 retina,27 43 and sclera44 ) could be at least partly explained by possible posttranscriptional control of receptor gene expression. Similar apparent discrepancies between distributions of retinoid receptor mRNAs and proteins have also been reported in the central nervous system.38 The immunoreactivity for the antibodies, whose specificity was demonstrated by the absence of any signals in cognate RAR and RXR mutant mice, was confined to cell nuclei in accordance with the view that RARs and RXRs are essentially if not exclusively nuclear proteins. In this respect, we want to stress that the previously reported immunoreactivity of extranuclear components27 45 could be artifactual, because cross-reactivity of the antibodies used in these studies to proteins other than the retinoid receptors cannot be ruled out simply from the loss of positive signals after preadsorption of the antibodies on the corresponding immunization peptides. Note also that our results cannot be readily compared with those obtained in the chick with non–isoform-specific antibodies for RARs and RXRs.45 The ubiquitous RXRß immunoreactivity is in agreement with reverse transcription-PCR data, which demonstrate that RXRß mRNA is the most abundant of all the six receptor mRNAs in the cornea, iris, ciliary body, neural retina, and RPE isolated from P7 mice (our unpublished results). Because no apparent eye abnormalities are found in the RXRß single null mutants,36 the physiological relevance of its ubiquitous distribution in the eye is currently unknown.

Neuroretina and RPE
In the developing neuroretina, RAR{alpha} and RXR{gamma} show dynamic and rapidly changing patterns of distribution and are eventually localized to specific subsets of cells of the mature retina. RAR{alpha} is present at high levels in INL cells that correspond to differentiating bipolar cells. At later stages it is detected in a subset of retinal ganglion cells as well as in cells located in the inner rows of the INL, most probably amacrine cells. Possible roles for RAR{alpha} in differentiating bipolar cells and developed amacrine cells are currently not documented. The presence of RAR{alpha} in the retinal ganglion cells could underlie the effects of RA on regeneration of ganglion cells in vitro.46 Therefore, although the retinas of RAR{alpha} null mutants show no light microscopic abnormalities (our unpublished data), it will be interesting to analyze them in terms of cell differentiation, electrophysiological functions, and regeneration properties.

RXR{gamma} is present in small subsets of cells largely comprising photoreceptor cells, which are identified to be cone photoreceptor cells in adults. This finding is compatible with the previous report showing that RXR{gamma} mRNA is expressed in the cells that eventually differentiate into photoreceptors in the chick.47 A recent report using the same antibody as in the present study showed strong immunoreactivity in the outer segments and weaker signals in the nuclei of the cone photoreceptor cells.27 In wild-type eyes, we detected RXR{gamma} signals exclusively in cell nuclei even when a higher concentration (1:200) of the antibody was used. This discrepancy might reflect differences in the immunohistochemical procedures. Interestingly, in single null mutants for RXR{gamma} the histologic aspects of the retina and the number of cone photoreceptor cells are normal, and there are no significant abnormalities in both photopic and scotopic electroretinograms (Mori M, Porto F, Picaud S, unpublished data, 2000). Likewise, double null mutants for RAR{alpha} and RXR{gamma}, which theoretically lack the major RAR/RXR heterodimer in cone photoreceptor cells, show no apparent retinal abnormalities at least at histologic levels (our unpublished data). Further functional studies are required to elucidate the specific roles of RXR{gamma} in cone photoreceptors. RXRs can function as heterodimerization partners not only for RARs but also for TRs, VDR, PPARs, and several other orphan receptors.18 20 Although TR{alpha} and ß transcripts,48 VDR protein,49 and PPAR{alpha}, ß, and {gamma} transcripts50 are reported to be present in the neural retina, no retinal abnormalities have been reported in single null mutants for these nuclear receptors. However, there is strong support for involvement of RXR/TR heterodimers in the differentiation of rat retinal progenitor cells into cone photoreceptor cells, because this process is induced by thyroid hormone and modulated by 9-cis RA in cell cultures.51

In the RPE, RAR{alpha} is present throughout eye development including adulthood, whereas RARß, RXR{alpha}, and RXR{gamma} are detected at some embryonic and perinatal stages (see Table 1 ). RA inhibits proliferation of RPE cells and promote their differentiation in vitro.52 In retinal explants, the RPE regulates the stimulatory effects of excess RA on photoreceptor differentiation and mediates RA-induced rod-specific apoptosis.14 15 These findings suggest that some effects of endogenous RA on postnatal neural retina could be modulated through the RPE.

Periocular Mesenchyme and Its Derivative Tissues
RAR{alpha}, RARß, RAR{gamma}, and RXR{alpha} proteins are abundant in the PM. Gene targeting of either one or two of these receptor genes results in eye abnormalities:21 23 24 26 Persistent hyperplastic primary vitreous (PHPV) is present in null mutants for RARß; those for RXR{alpha} as well as RAR{alpha}/RAR{gamma} and RARß/RAR{gamma} compound mutants show severe ocular malformations. The PM could be a major site of RA production, because retinaldehyde dehydrogenase 2 (RALDH2) is intensely expressed in this tissue in mouse fetuses.53 Thus, retinoids probably have autocrine actions in the PM. The presence of RARß and RXR{alpha} in the primary vitreous combined with the findings that the vast majority of RARß and RXR{alpha} single null mutants exhibit a PHPV21 23 indicates that these two receptors are required for the disappearance of the primary vitreous body in a cell-autonomous manner, probably in the form of RXR{alpha}/RARß heterodimers. Altogether, these findings suggest that retinoids play crucial roles in the PM during eye morphogenesis. It will be interesting to investigate whether diffusible factors regulated by RA in the PM may have paracrine effects on the RPE and neural retina.

RAR{alpha}, RARß, and RXR{alpha} are present in the iris and ciliary body. Because severe malformations of the anterior segments are found in null mutants for RXR{alpha} and double null mutants for RAR{alpha}/RAR{gamma} and RARß/RAR{gamma},21 23 24 normal development of iris and ciliary body appears to require RAR{gamma} (which is present in the precursor mesenchyme and absent in the differentiated iris and ciliary body) as well as RAR{alpha}, RARß, and RXR{alpha}. The detection of RAR{alpha}, RARß, and RXR{alpha} in the iris and ciliary body of adults suggests possible roles of retinoids in the functions of retinoids in these tissues, for example, accommodation, pupil responses, and aqueous humor production.

RAR{gamma} is the major retinoid receptor present in adult mouse sclera. Recent studies have shown that endogenous RA synthesis in the choroid and/or retina is involved in the growth of sclera in form-deprivation myopia in the chick.54 55 It is probable that signaling through RAR{gamma} underlies this pathology. In this respect, the RAR{gamma} null mutant mice might provide a useful model to gain insights into the molecular mechanisms of the form-deprivation myopia.

Ocular Surface and Lids
VAD causes xerophthalmia, indicating that vitamin A is indispensable for the structural integrity of the cornea and conjunctiva.56 Moreover, topically applied retinoids have therapeutic effects on these tissues.57 58 59 RAR{gamma} and RXR{alpha} are constantly and uniformly present in the corneal and conjunctival epithelia and lids from E14.5 onward, whereas RAR{alpha} immunoreactivity decreases postnatally. This finding indicates that RAR{gamma}/RXR{alpha} is the major heterodimer functioning in these tissues.


   Conclusions
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
References
 
The present study demonstrates that all the retinoid receptors, RXRß excepted, show dynamic patterns of distribution in the course of eye development and that some of these receptors are localized to specific cell types in the adult retina. These results provide a basis for further studies on retinoid actions in the development and functions of the eye.


   Acknowledgements
 
The authors thank Cécile Rochette-Egly for providing antibodies for RAR{alpha}, RARß, RAR{gamma}, and RXR{alpha} and Bénédicte Mascrez for providing RXR{alpha} null mutant mice.


   Footnotes
 
Supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Collège de France, the Institut Universtitaire de France, the Hôpital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale, and the Bristol-Myers Squibb Pharmaceutical Research Institute. MMo was supported by fellowships from the Institut National de la Santé et de la Recherche Médicale (France) and from the Ministry of Education, Science, Sports, and Culture (Japan).

Submitted for publication October 18, 2000; revised January 3, 2001; accepted January 18, 2001.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Manuel Mark, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Parc d’Innovation 1, rue Laurent Fries, 67404 Illkirch-CU de Strasbourg, France. marek{at}igbmc.u-strasbg.fr


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 References
 

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