ReviewVolume 7 | 2009
Cite this Article: Nuclear Receptor Signaling (2009) 7, e002.
Function of retinoic acid receptors during embryonic development
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Département de Biologie Cellulaire and Développement (MM and NG) and Département de Génomique Fonctionnelle (PC), and Collège de France (PC), Strasbourg, France
Retinoids, the active metabolites of vitamin A, regulate complex gene networks involved in vertebrate morphogenesis, growth, cellular differentiation and homeostasis. Studies performed in vitro, using either acellular systems or transfected cells, have shown that retinoid actions are mediated through heterodimers between the RAR and RXR nuclear receptors. However, in vitro studies indicate what is possible, but not necessarily what is actually occurring in vivo, because they are performed under non-physiological conditions. Therefore, genetic approaches in the animal have been be used to determine the physiological functions of retinoid receptors. Homologous recombination in embryonic stem cells has been used to generate germline null mutations of the RAR- and RXR-coding genes in the mouse. As reviewed here, the generation of such germline mutations, combined with pharmacological approaches to block the RA signalling pathway, has provided genetic evidence that RAR/RXR heterodimers are indeed the functional units transducing the RA signal during prenatal development. However, due to (i) the complexity in “hormonal” signalling through transduction by the multiple RARs and RXRs, (ii) the functional redundancies (possibly artefactually generated by the mutations) within receptor isotypes belonging to a given family, and (iii) in utero or early postnatal lethality of certain germline null mutations, these genetic studies have failed to reveal all the physiological functions of RARs and RXRs, notably in adults. Spatio-temporally-controlled somatic mutations generated in given cell types/tissues and at chosen times during postnatal life, will be required to reveal all the functions of RAR and RXR throughout the lifetime of the mouse.
AD: activating domain; AF: activation function; BA: branchial arche; CYP: cytochrome P450 hydroxylase; DORV: double outlet right ventricle; E: embryonic day; INZ: interdigital necrotic zone; NCC: neural crest cells; NR: nuclear receptor; PHPV: persistent and hyperplastic vitreous body; POM: periocular mesenchyme; R1 to R7: rhombomeres 1 to 7; RA: retinoic acid; RALDH: retinaldehyde dehydrogenase; RAR: retinoic acid receptor; RBP: retinol-binding protein; RDH: retinol dehydrogenase; RXR: retinoid X receptor; STRA: stimulated by retinoic acid; VAD: vitamin A-deficiency
Both clinical findings and experimental approaches have revealed that vitamin A (retinol) and its active derivatives (i.e., the retinoids) exert a wide variety of effects on vertebrate embryonic body shaping and organogenesis, tissue homeostasis, cell proliferation, differentiation and apoptosis (reviewed in [Blomhoff, 1994; Kastner et al., 1995; Mark et al., 2006; Morriss-Kay and Ward, 1999; Sporn et al., 1994]). Following Hale’s initial demonstration that vitamin A-deficiency (VAD) induces congenital ocular malformations [Hale, 1933], Warkany and his collaborators showed that a large array of congenital malformations affecting the ocular, cardiac, respiratory and urogenital systems (collectively referred as to the fetal VAD syndrome) occurred in fetuses from vitamin A-deficient (VAD) rats (reviewed in [Wilson et al., 1953]). It was shown, much later, that retinoic acid (RA) could replace vitamin A during embryogenesis, at least at certain stages and in certain organs [Dickman et al., 1997; White et al., 1998].
How RA can exert such pleiotropic effects was a long-standing question, which found its solution with the discovery of two classes of RA-binding transcriptional regulators, the retinoic acid receptors (the multiple isoforms of the RARα, β and γ isotypes), and the retinoid X receptors (the multiple isoforms of the RXRα, β and γ isotypes) (reviewed in [Chambon, 1996; Leid et al., 1992].
The present review focuses on three main questions: (i) are RARs and RXRs involved in the transduction of RA signals in vivo? (ii) to what extent do the observations made in vivo support the molecular mechanisms deduced from in vitro studies? (iii) what are the developmental events controlled by RARs and RXRs? We summarize and discuss in this review the developmental phenotypes induced by mutations that have been introduced at loci encoding RARs and RXRs, and compare them to the phenotypes resulting either from VAD or from administration of pharmacological doses of antagonistic ligands for RARs in vertebrates, placing emphasis on results obtained in mammals.
Rara, Rarb, and Rarg null mutant mice are viable. They display some aspects of the fetal (and postnatal) VAD syndromes, as well as a few additional congenital malformations (Table 1). However, their abnormalities are restricted to a subset of tissues normally expressing these receptors, probably reflecting the existence of functional redundancies between RARs (discussed in [Kastner et al., 1995; Kastner et al., 1997a; Mark and Chambon, 2003; Mark et al., 1995; Mascrez et al., 1998]). To test this hypothesis, mutants lacking two RAR isotypes (Rara/b-, Rara/g- and Rarb/g-null mutants), or several isoforms belonging to distinct isotypes were generated. For the sake of clarity, only abnormalities displayed by double null mutants lacking a couple of RAR isotypes (all isoforms deleted) are listed in Table 2 and Table 3. Similar abnormalities, albeit often less penetrant, which are displayed by "isoform-specific" double null mutants are listed in [Kastner et al., 1995] (mutants lacking RARα1/RARβ2/4, RARα1/RARγ, RARα1α2+/-/RARγ and RARβ2/4/RARγ), in [Subbarayan et al., 1997] (mutants lacking RARα/RARγ1 and RARα/RARγ2), in [Luo et al., 1996] (mutants lacking RARα1/RARβ), in [Ghyselinck et al., 1998] (mutants lacking RARα/RARβ1/3 and RARβ1/3/RARγ), and in [Grondona et al., 1996] (mutants lacking RARβ2/RARγ2).
Rara/b-, Rara/g- and Rarb/g-null mutants die in utero or at birth because of severe developmental defects that altogether include the complete spectrum of malformations belonging to the fetal VAD-induced syndrome reported by Warkany’s group 55 years ago [Wilson et al., 1953] (Table 2). As with Rar-single-null mutants (see Table 1), Rar-double-null mutants (Table 3) also exhibit congenital abnormalities that were not described in Hale’s and Warkany’s pioneering fetal VAD studies [Hale, 1933; Wilson et al., 1953], encompassing ageneses of the Harderian glands, skeletal defects of the skull, face, vertebrae, limbs and forebrain [Ghyselinck et al., 1997; Lohnes et al., 1993; Lohnes et al., 1994; Luo et al., 1996; Mendelsohn et al., 1994; Subbarayan et al., 1997]. The occurrence of these “non-VAD” defects in Rar- single and double-null mutant mice is most probably accounted for by the difficulty to achieve, by dietary deprivation, a state of profound VAD compatible with pregnancy. In fact, almost all these “non-VAD” defects have been subsequently produced in rodent embryos (i) deficient in vitamin A, but supplemented with RA [Dickman et al., 1997; White et al., 2000; White et al., 1998]; (ii) deficient in both RBP (retinol-binding protein) and vitamin A [Quadro et al., 2005]; (iii) lacking the retinaldehyde synthesising enzyme RDH10 (retinol dehydrogenase 10) [Sandell et al., 2007]; (iv) lacking the RA synthesising enzymes RALDH2 (retinaldehyde dehydrogenase 2) [Halilagic et al., 2007; Molotkova et al., 2007; Niederreither et al., 2002a; Niederreither et al., 1999; Niederreither et al., 2001; Niederreither et al., 2000; Niederreither et al., 2002b; Ribes et al., 2006] or RALDH3 [Dupe et al., 2003; Halilagic et al., 2007] or (v) treated with synthetic retinoids possessing panRAR antagonistic activities [Kochhar et al., 1998; Wendling et al., 2000; Wendling et al., 2001]. In addition, it was recently found that the Matthew-Wood syndrome, which consists of a spectrum of congenital abnormalities also observed in Rara/b, Rara/g and Rarb/g-null mutants, is caused by mutations in the RBP receptor gene, STRA6 [Golzio et al., 2007; Pasutto et al., 2007]. As STRA6 loss-of-function mutations most probably yield a state of RA insufficiency in embryonic tissues [Kawaguchi et al., 2007], this discovery provides evidence that RAR signalling performs similar functions during embryonic development in mice and humans.
The comparison of the Rar-null mutant phenotypes with those of rodents and humans carrying the aforementioned blocks in RA signal transduction, demonstrate that liganded RARs play crucial roles at many distinct stages of the development of numerous organs ([Kastner et al., 1995] and references therein). For example, the severe malformations found in Rara/g-null embryos [Wendling et al., 2001] are similar to those of Aldh1a2 (formerly Raldh2)-null embryos [Niederreither et al., 1999], and reflect early roles of RAR signalling in axial rotation, segmentation and closure of the hindbrain, formation of otocysts, pharyngeal arches and forelimb buds, as well as in closure of the primitive gut. RARs are also indispensable for the ontogenesis of (almost) all the anatomical structures that are derived from mesectodermal cells, i.e. the cranial neural crest cells (NCC) that give rise to mesenchymal derivatives (reviewed in [Kastner et al., 1995; Mark et al., 1998; Mark et al., 1995]). RARs are involved in antero-posterior patterning of the somitic mesoderm and hindbrain neurectoderm [Dupe et al., 1999b; Ghyselinck et al., 1997; Lohnes et al., 1993; Lohnes et al., 1994; Wendling et al., 2001], notably through controlling expression of homeobox genes [Allan et al., 2001; Dupe et al., 1997; Dupe et al., 1999b; Houle et al., 2000; Oosterveen et al., 2003; Serpente et al., 2005]. RARs are also involved in the establishment of the antero-posterior axis of the limbs [Dupe et al., 1999a; Lohnes et al., 1994; Mascrez et al., 1998]. RARs are required for the development of a large number of eye structures (Table 2 and Table 3) and for histogenesis of the retina [Ghyselinck et al., 1997; Grondona et al., 1996; Lohnes et al., 1994], cardiomyocyte differentiation [Kastner et al., 1994; Kastner et al., 1997b], as well as for the control of physiological apoptosis in the retina [Grondona et al., 1996], the frontonasal and interdigital mesenchymes [Crocoll et al., 2002; Dupe et al., 1999a; Ghyselinck et al., 1997; Lohnes et al., 1994], the conotruncal segment of the embryonic heart [Ghyselinck et al., 1998] and the embryonic nephric duct [Batourina et al., 2005]. In the embryonic urogenital tract, RARs control epithelial-mesenchymal interactions in the kidney through expression of the receptor tyrosine kinase Ret [Batourina et al., 2002; Batourina et al., 2001; Mendelsohn et al., 1999], as well as the formation of the genital ducts and ureters [Batourina et al., 2001; Batourina et al., 2005; Ghyselinck et al., 1997; Mendelsohn et al., 1994]. In the developing respiratory tract, RA-liganded RARs are necessary for the morphogenesis of the nasal cavities and for their communication with the more caudal airways [Dupe et al., 2003; Ghyselinck et al., 1997] (Table 2 and Table 3). RA-liganded RARs are also required for the partitioning of the primitive foregut into oesophagus and trachea, and regulate lung branching morphogenesis [Chen et al., 2007; Desai et al., 2006; Malpel et al., 2000; Mollard et al., 2000; Wang et al., 2006], as well as lung alveoli septation [Massaro and Massaro, 2003; Massaro et al., 2003; Massaro et al., 2000; McGowan et al., 2000] (Table 1, Table 2, and Table 3).
Altogether, the developmental abnormalities of Rar-null mutant mice faithfully recapitulate those of rodents lacking the RAR ligand either through dietary vitamin A deprivation or genetically-impaired RA synthesis. There are, however, two notable exceptions to this rule which are related to the control, through RA signalling, of (i) the symmetry of somite patterning and (ii) germ cell differentiation in the embryonic ovary. Concerning somitogenesis, it has been firmly established, in several vertebrate species, that RA is instrumental to the generation of bilaterally symmetrical pairs of somites [Echeverri and Oates, 2007; Kawakami et al., 2005; Sirbu and Duester, 2006; Vermot and Pourquie, 2005]. It was also recently demonstrated that signalling by RA is required for expression of Stra8 (stimulated by retinoic acid 8) which, in turn, triggers meiosis in the embryonic ovary [Baltus et al., 2006; Bowles et al., 2006; Koubova et al., 2006]. However, defects in the symmetry of somite derivatives, such as vertebrae and muscles, or in the histology of the ovaries, were never observed in Rara/b-, Rara/g- and Rarb/g-null mutant mice. As mentioned above, the apparent lack of phenotypic convergence between the animal models lacking RA and those lacking RARs is probably a consequence of the artificial compensation of the functions of the RARs that are missing in the knockout mice by the remaining one.
In addition to the dramatic craniofacial skeletal deficiencies affecting Rara/g-null mutants [Lohnes et al., 1994], subtle defects which often alter the shape of a single skeletal piece are observed in several Rar-null mice, including: a cartilaginous or osseous connection between the incus middle ear bone and the alisphenoid bone (the pterygoquadrate element), a cartilage separating the trigeminal ganglion from the brain (the pila antotica) and an agenesis of the rostral ethmoturbinate and maxillary sinus (Table 3) [Ghyselinck et al., 1997; Lohnes et al., 1994; Mark et al., 1998]. The pterygoquadrate element and the pila antotica, which were lost during evolution from reptiles to mammals, represent atavistic features (discussed in [Mark et al., 1998; Mark et al., 1995]). Along the same lines, ethmoturbinate bones and paranasal sinuses (such as the maxillary sinus) are typical mammalian features not present in reptiles [Novacek, 1993]. It is thus conceivable that agenesis of these nasal structures in Rar-null mutants also mimics an atavistic condition. The presence of atavistic characteristics in Rar-null mutants supports the possibility that changes in the temporal or spatial patterns of expression of Rar genes has provided a general mechanism for modifying the number and shape of individual cranial skeletal elements during vertebrate evolution. Interestingly, recent teratogenic experiments using RA in excess have led to similar conclusions [Vieux-Rochas et al., 2007].
The persistent and hyperplastic vitreous body (PHPV) seen in Rarb-null mutants is comparable to the pecten oculi, a normal vascular and pigmented projection from the optic disk found in some reptiles, which is thought to function in the nutrition of the retina [Mann, 1937]. The shortening of the ventral retina observed in Rarb/g-null mutants might also be interpreted as a modification towards an ancestral condition, since comparative embryology of the retina shows that the ventral retinal field increases in size as one ascends the vertebrate scale from fishes to amphibia, reptiles and mammals [Mann, 1937]. Thus, RARβ and RARγ might have been instrumental in the expansion of the ventral retinal field during vertebrate evolution.
Rxra-null mutants all display a hypoplasia of the compact layer of the ventricular myocardium (Table 2), which appears to be the main cause of mutant death occurring by cardiac failure around E14.5 [Kastner et al., 1994; Kastner et al., 1997b; Ruiz-Lozano et al., 1998; Sucov et al., 1994]. That a similar myocardium defect is observed in VAD and Rar-null fetuses [Mendelsohn et al., 1994; Wilson et al., 1953], suggests that RXRα is involved in the transduction of the RA signal required for myocardial growth. This requirement is unlikely to be cell-autonomous, as over-expressing RXRα in cardiomyocytes by means of trangenesis does not prevent the Rxra-null mutation-induced hypoplasia of the ventricular myocardium [Subbarayan et al., 2000]. Other data further indicate that Rxra expression in the epicardium is required for triggering a paracrine signal necessary for myocardial growth [Chen et al., 2002; Kang and Sucov, 2005; Merki et al., 2005].
About one third of the Rxra-null mutants lack the conotruncal septum, which normally divides the embryonic heart outflow tract (or conotruncus) into the intracardiac portions of the aorta and the pulmonary trunk [Kastner et al., 1994]. Interestingly, deficiencies of this septum represent both a classical VAD defect in rodents and a leading cause of human congenital heart defects, ranging from high interventricular septal defects to double outlet right ventricle (DORV). In Rxra-null mutants, the agenesis of the conotruncal septum appears secondary to an enhanced rate of cell death in both the mesenchymal cells of the conotruncal ridges and the parietal conotruncal cardiomyocytes, therefore indicating that RXRα is required for the transduction of the RA signal that controls apoptosis in the conotruncal segment of the embryonic heart [Ghyselinck et al., 1998].
In addition to heart defects, all Rxra-null fetuses display a characteristic ocular syndrome characterized by a PHPV, closer eyelid folds, a thickened ventral portion of the corneal stroma, a ventral rotation of the lens, an agenesis of the sclera and a shortening of the ventral retina (Table 2) [Kastner et al., 1994]. As similar defects are present in VAD fetuses and in Rarb/g-null mutants (Table 2) [Ghyselinck et al., 1997; Wilson et al., 1953], RXRα appears to play an essential role in the transduction of the RA signals required for several ocular morphogenetic processes, notably the formation of the ventral retinal field.
Importantly, the fact that mice lacking both RXRβ and RXRγ (Rxrb/g-null mutants) do not display any obvious morphogenetic defects, even when additionally lacking one allele of Rxra, clearly indicates that RXRα is functionally the most important RXR during morphogenesis of the embryo proper [Krezel et al., 1996].
The AF-1-containing A/B domain and the ligand-dependent AF-2 of RXRα are differentially involved in development
The role played by RXRs as either “active” or “silent” heterodimerization partners in the transcription of target genes, inferred from in vitro studies, has been a controversial issue. To determine the transcriptional role of RXRα in vivo, mouse mutants were engineered that express truncated RXRα proteins lacking (i) the N-terminal activation function (AF)-1-containing A/B region (Rxraaf1o mutants) [Mascrez et al., 2001], (ii) the AF-2 activating domain (AD) core-containing helix 12 located at the C-terminus of the E region (Rxraaf2o mutants) [Mascrez et al., 1998] and (iii) both AF-1 and AF-2 (Rxraafo mutants) [Mascrez et al., 2009].
The Rxraaf2o mutants display the myocardium hypoplasia and the ocular syndrome that are hallmarks of the Rxra-null phenotype [Kastner et al., 1994], but at low frequency [Mascrez et al., 1998]. This may reflect a functional compensation by RXRβ, as (i) the frequency of the myocardium hypoplasia increases from 5% in Rxraaf2o mutants to 50% upon the additional inactivation of Rxrb (which, on its own, has no effect) [Kastner et al., 1996], and (ii) the frequency of the ocular syndrome increases from about 15% in Rxraaf2o mutants to 100% upon further inactivation of Rxrb [Mascrez et al., 1998]. The full penetrance of the Rxra-null ocular syndrome observed in Rxraaf2o/Rxrb-null mutants [Mascrez et al., 1998], as well as in Rxraafo mutants [Mascrez et al., 2009], supports the view that the AF-2 of RXRα (and thus possibly a RXR agonistic ligand) is indispensable for ocular morphogenesis. On the other hand, the rare or modest penetrance of the myocardium hypoplasia in Rxraaf2o (5%), Rxraaf2o/Rxrb-null (50%) and Rxraaf2o/Rxrb/g-null mutants (50%) suggests that the transcriptional activity of RXRα becomes necessary for myocardial growth only in “unfavourable” genetic backgrounds. Accordingly, we recently found that the heart histology is normal in 80% of Rxraafo fetuses [Mascrez et al., 2009], indicating that a transcriptionally “silent” RXRα can promote myocardial growth.
Involution of the primary vitreous body represents the developmental process which likely requires the highest concentration of RA-liganded retinoid receptors, since both VAD [Wilson et al., 1953], Rarb ablation [Ghyselinck et al., 1997], Rxra ablation [Kastner et al., 1994], deletion of RXRα AF-2 [Mascrez et al., 1998] and deletion of RXRα AF-1 and AF-2 [Mascrez et al., 2009] frequently yield a PHPV. Apart from an occasional PHPV, the Rxraaf1o mutants never display any of the Rxra-null developmental defects [Mascrez et al., 2001]. This near-absence of defects does not reflect a functional compensation by RXRβ or RXRγ, as Rxraaf1o/Rxrb-null and Rxraaf1o/Rxrb/g-null mutants display no other developmental defect than a PHPV. However, the frequency of this PHPV increases from 10% in Rxraaf1o mutants to 100% in Rxraaf1o/Rxrb/g-null mutants [Mascrez et al., 2001]. Altogether, these observations indicate that involution of the primary vitreous body requires both RXRα AF-1 and AF-2, while the other RA-dependent ocular morphogenetic events require RXRα AF-2 only, as they normally take place in the absence of the RXRα AF-1-containing A/B domain.
Thus, these data support the view that the activation functions of RXRα are differentially required for eye morphogenesis and that they can be dispensable for heart development. They also indicate that, due to functional redundancy, the role played by each activation function can be revealed only in genetic backgrounds impaired for RA-signalling (or under RA-insufficiency conditions). Assuming that the frequent requirement of RXRα AF-2 for developmental events reflects the binding of an agonistic ligand, this raises the question of the possible existence and nature of a physiological RXR ligand(s) in vivo. The fact that 9-cis RA is undetectable in rodent embryos [Horton and Maden, 1995; Matt et al., 2005b] makes it doubtful that this RXR physiological ligand could be 9-cis RA (reviewed in [Wolf, 2006]; see also [Calleja et al., 2006]).
The RXRα AF-1-containing A/B domain has a specific function in the involution of the interdigital mesenchyme
The AF-2 of RXRα appears functionally more important during development than its AF-1, as (i) Rxraaf2o/Rxrb/g-null fetuses all display a large array of congenital defects and die in utero, while Rxraaf1o/Rxrb/g-null fetuses display only a few congenital defects and are often viable; and (ii) transcription of a RA-responsive lacZ reporter transgene in the mouse requires RXRα AF-2, but not AF-1 [Mascrez et al., 1998; Mascrez et al., 2001]. However, the RXRα AF-1-containing A/B region has a unique role in the RA-dependent disappearance of the interdigital mesenchyme.
The first evidence implicating RA in the involution of the interdigital mesenchyme was provided by organ culture experiments using whole limb in a RA-deprived medium [Lussier et al., 1993]. Subsequently, it was shown that mice lacking both alleles of either Rara or Rarg, as well as mice heterozygous for the Rxra-null mutation occasionally exhibit mild forms of interdigital webbing (i.e., soft tissue syndactyly) (Table 1) [Ghyselinck et al., 1997; Kastner et al., 1994; Lohnes et al., 1993; Lufkin et al., 1993]. Surprisingly, this defect was absent in Rarb-null mutants, even though Rarb is strongly and specifically expressed in the interdigital necrotic zones (INZs) [Ghyselinck et al., 1997]. However, disruption of one (or both) allele(s) of Rarb in a Rarg-null genetic background consistently yields interdigital webbing [Ghyselinck et al., 1997]. The persistence of the fetal interdigital mesenchyme responsible for digit webbing is caused by a marked decrease in programmed cell death, as well as by an increase of cell proliferation in the mutant INZs [Dupe et al., 1999a]. As Rarb and Rarg are not co-expressed in the INZs, interdigital mesenchyme involution must involve paracrine interactions between this mesenchyme, which expresses Rarb, and either the cartilaginous blastema of the digits or the surface epidermis, which both express Rarg (discussed in [Dupe et al., 1999a]).
The RXRα AF-1-containing A/B region is indispensable for the function of RXRα/RARβ and/or RXRα/RARγ heterodimers involved in interdigital mesenchyme involution, since the majority of Rxraaf1o mutants and all Rxraaf1o/Rxrb/g-null mutants display a soft tissue syndactyly [Mascrez et al., 2001]. In contrast, Rxraaf2o and Rxraaf2o/Rxrb/g-null mutants never display this defect [Mascrez et al., 1998], indicating a specific requirement of the RXRα AF-1-containing A/B region in the involution of the interdigital mesenchyme. Interestingly, phosphorylation of RXRα at a specific serine residue located in the A domain is necessary for the anti-proliferative response of F9 teratocarcinoma cells to RA [Bastien et al., 2002; Rochette-Egly and Chambon, 2001]. Phosphorylation of the RXRα A domain may therefore play an important function in the cascade of molecular events that, in vivo, leads to the normal disappearance of the interdigital mesenchyme.
Compound mutants, in which a null mutation of a given RAR isotype is associated either with a Rxra-null, a Rxraaf1o or a Rxraaf2o mutation, altogether recapitulate the abnormalities exhibited by Rar-null mutants (Table 4) [Kastner et al., 1994; Kastner et al., 1997a; Mascrez et al., 1998; Mascrez et al., 2001]. This synergism between Rar and Rxra loss-of-function mutations supports the conclusion that RXRα/RARα, RXRα/RARβ and RXRα/RARγ heterodimers are the fun