Ecdysone receptor

Evolutionary homologs

Table of contents

Retinoic acid receptor RAR: a model for Ecdysone receptor in vertebrates - Roles of RAR in development

Classes of both retinoic acid receptors (RARs) and the retinoic X receptors (RXRs) have (respectively) three different subtypes (RARalpha, RARbeta, and RARgamma, and RXRalpha, RXRbeta, and RXRgamma) that act as ligand-dependent transcription factors. To examine the involvement of the different receptor classes and their subtypes in the biological responses of neuroblastoma cells to retinoids, the effects of a panel of receptor-selective retinoids were analyzed for cell growth, differentiation, and gene expression, using in vitro cultured KCNR cells. Activation of three distinct RXR/RAR heterodimers induces growth arrest and differentiation of neuroblastoma cells. Any association of per se inactive RXR-selective with RAR-selective ligands efficiently regulates growth inhibition, differentiation (neurite extension), and expression of RARbeta, TrkB, and N-myc. SR11383 alone, a very potent retinoid, entirely reproduces the pattern of biological responses induced by naturally occurring retinoids. In contrast to other tumor cell lines, the growth of neuroblastoma cell lines is not altered using AP1-antagonistic retinoids. These studies raise the possibility that three distinct RXR/RAR heterodimers mediate the effects of retinoids on neuroblastoma cells through an AP-1 antagonism-independent mechanism (Giannini, 1997).

The RARgamma gene generates two major isoforms: RARgamma1 and RARgamma2; they originate from two distinct promoters. Mice have been engineered that lack RARgamma1, but RARgamma2 is normally expressed. The effect of this null mutation has been compared with those previously described for RARgamma2 and all RARgamma isoforms (total RARgamma gene inactivation), both in single mutants and in double mutants bearing additional null mutations in their RAR alpha, RARbeta or RXR alpha genes. RARgamma1 mutants (but not RARgamma2 mutants) display a subset of the abnormalities exhibited by total RARgamma null mutants, including growth deficiency, abnormal cricoid cartilage and occasional cervical vertebra defects. This suggests that RARgamma1 is the main isoform mediating the corresponding RARgamma functions. Interestingly, cricoid cartilage defects are also found in a fraction of heterozygote animals for the RARgamma1, RARgamma or RAR alpha mutations, indicating that wild type levels of RARs are required for the normal morphogenesis of this structure. Compound RAR alpha/RARgamma1 and RAR alpha/RARgamma2 double null mutants exhibit only a small fraction of the defects found in RAR alpha/RARgamma double null mutants. Moreover, these defects are often partially penetrant, or correspond to a less severe form. However, they occur preferentially in certain compound mutants, demonstrating that given isoforms mediate specific functions of RARgamma in the context of an RAR alpha null background. In a RXR alpha null background, both RARgamma1 and gamma2 isoform mutations result in increased severity of the RXR alpha null ocular phenotype. Together, the present observations indicate that while the functions of the two RARgamma isoforms overlap to a large extent, each of these isoforms also exhibits a limited functional specificity. The occurrence of morphological defects in heterozygote mutants for a single RAR isoform provides a basis for explaining the strong conservation of these isoforms during vertebrate evolution (Subbarayan, 1997).

RARalpha has two transcriptional activation functions. One of these, known as AF-2, is ligand-dependent, while another, in the amino-terminal region, contains a ligand-independent function. The activity of the N-terminal activation function AF-1 of RAR alpha1 is abrogated upon mutation of a phosphorylatable serine residue (Ser-77). Recombinant RAR alpha is phosphorylated by a variety of proline-directed protein kinases in vitro. However, only the coexpression of cdk7 stimulates Ser-77 phosphorylation in vivo and enhances transactivation by RAR alpha, but not by a S77A RAR mutant. Both free CAK (cdk7, cyclin H, MAT1) and the CAK-containing general transcription factor TFIIH phosphorylate Ser-77 in vitro (see Drosophila Cyclin-dependent kinase 7). RAR alpha binds free CAK and purified TFIIH in vitro, and RAR alpha-TFIIH complexes can be isolated from HeLa nuclear extracts. These findings represent the first example of activation of a transactivator through binding to and phosphorylation by a general transcription factor (Rochette-Egly, 1997).

A motif essential for the transcriptional activation function (AF) present in the E region of retinoic acid receptor and 9-cis retinoic acid receptor has been characterized as an amphipathic alpha-helix whose main features are conserved between transcriptionally active members of the nuclear receptor superfamily. The role of RARalpha1 and RARgamma2 AF-1 and AF-2 activation functions and their phosphorylation was investigated during RA-induced primitive and parietal differentiation of F9 cells. Primitive endodermal differentiation requires RARgamma2, whereas parietal endodermal differentiation requires both RARgamma2 and RARalpha1; in all cases AF-1 and AF-2 must synergize. Primitive endodermal differentiation requires the proline-directed kinase site of RARgamma2-AF-1, whereas parietal endodermal differentiation additionally requires the kinase site of RARalpha1-AF-1. The cAMP-induced parietal endodermal differentiation also requires the protein kinase A site of RARalpha-AF-2, but not that of RARgamma. The AF-1-AF-2 synergism and AF-1 phosphorylation site requirements for RA-responsive gene induction are promoter context-dependent. Thus, AF-1 and AF-2 of distinct RARs exert specific cellular and molecular functions in a cell-autonomous system mimicking physiological situations; their phosphorylation by kinases belonging to two main signaling pathways is required to enable RARs to transduce the RA signal during F9 cell differentiation (Taneja, 1997).

Determination of the dorso-ventral dimension of the vertebrate retina is known to involve retinoic acid (RA): high RA activates expression of a ventral retinaldehyde dehydrogenase and low RA of a dorsal dehydrogenase. In the early eye vesicle of the mouse embryo, expression of the dorsal dehydrogenase is preceded by, and transiently overlaps with, the RA-degrading oxidase CYP26. Subsequently in the embryonic retina, CYP26 forms a narrow horizontal boundary between the dorsal and ventral dehydrogenases, creating a trough between very high ventral and moderately high dorsal RA levels. Most of the RA receptors are expressed uniformly throughout the retina except for the RA-sensitive RARbeta, which is down-regulated in the CYP26 stripe. The orphan receptor COUP-TFII, which modulates RA responses, colocalizes with the dorsal dehydrogenase. The organization of the embryonic vertebrate retina into dorsal and ventral territories divided by a horizontal boundary has parallels to the division of the Drosophila eye disc into dorsal, equatorial and ventral zones, indicating that the similarities in eye morphogenesis extend beyond single molecules to topographical patterns (McCaffery, 1999).

Retinoids regulate gene expression via nuclear retinoic acid receptors, the RARs and RXRs. To investigate the functions of retinoid receptors during early neural development, a dominant negative RARß was expressed in early Xenopus embryos. Dominant negative RARß specifically inhibits RAR/RXR heterodimer-mediated, but not RXR homodimer-mediated, transactivation. Both all-trans- and 9-cis-RA-induced teratogenesis are, however, efficiently opposed by ectopic expression of dominant negative RARß, indicating that only RAR/RXR transactivation is required for retinoid teratogenesis by each of these ligands. Experiments with two RXR-selective ligands confirms that activation of RXR homodimers does not cause retinoid teratogenesis. Dominant negative RARß thus specifically interferes with the retinoid signalling pathway that is responsible for retinoid teratogenesis. Dominant negative RARß-expressing embryos have a specific developmental phenotype leading to disorganization of the hindbrain. Mauthner cell multiplications in the posterior hindbrain, and (both anteriorly and posteriorly) expanded Krox-20 expression domains indicate (partial) transformation of a large part of the hindbrain into (at least partial) rhombomere 3, 4 and/or 5 identity. In contrast, the fore- and mid-brain and spinal cord appeared to be less affected. These data indicate that RARs play a role in patterning the hindbrain (van der Wees, 1998).

All-trans retinoic acid (RA) reduces human neuroblastoma growth by inducing either differentiation or apoptosis. The apoptotic program in these cells is regulated by RA and is paralleled by the transcriptional induction of "tissue" transglutaminase (tTG). tTG is a protein cross-linking enzyme, which specifically accumulates in cells undergoing apoptosis in various in vivo and in vitro systems. In neuroblastoma cells, tTG is detected exclusively in the cells expressing the flat substrate-adherent 'S-type' phenotype (as opposed to the neural 'N-type); these cells show an increase in apoptosis. The present study was undertaken to identify the retinoid receptors involved in the regulation of tTG and apoptosis as well as in the in vitro neuronal differentiation of the human SK-N-BE(2) neuroblastoma cell line. While RARalpha- and RARgamma-selective retinoids alone are able to induce tTG activity, only the combined stimulation of both RARalpha and RARgamma induces apoptosis. Conversely, several combinations of RAR/RXR closely mimic the differentiation effects observed with all-trans retinoic acid. These results indicate that, at variance with differentiation, the induction of apoptosis in human SK-N-BE(2) neuroblastoma cells is under the specific control of RARalpha and RARgamma. These data seem relevant for the reported ability of RARgamma to suppress the clinically malignant tumor phenotype in patients (Melino, 1997).

Retinaldehyde dehydrogenase type 2 (RALDH-2) was identified as a major retinoic acid generating enzyme in the early mouse embryo. The expression domains of the RALDH-2 are likely to indicate regions of endogenous retinoic acid (RA) synthesis. During early gastrulation, RALDH-2 is expressed in the mesoderm adjacent to the node and primitive streak. This suggests that RALDH-2 gene expression may be switched on during the process of cell ingression through the primitive streak. Expression extends rostrally along each side of the node, whereas the node itself is unlabelled. At the headfold stage, mesodermal expression is restricted to posterior regions up to the base of the headfolds. No expression is detected in mesoderm underneath the rhomboencephalon. Later, RALDH-2 is transiently expressed in the undifferentiated somites and the optic vesicles, and more persistently along the lateral walls of the intraembryonic coelom and around the hindgut diverticulum. The RALDH-2 expression domains in differentiating limbs, which include presumptive interdigital regions, coincide with, but slightly precede, those of the RA-inducible RAR beta gene. The RALDH-2 gene is also expressed in specific regions of the developing head, including the tooth buds, inner ear, meninges and pituitary gland, and in several viscera. Administration of a teratogenic dose of RA at embryonic day 8.5 results in downregulation of RALDH-2 transcript levels in caudal regions of the embryo, and may reflect a mechanism of negative feedback regulation of RA synthesis (Niederreither, 1997).

Retinoids have long been known to influence skeletogenesis but the specific roles played by these effectors and their nuclear receptors remain unclear. Thus, it is not known whether endogenous retinoids are present in developing skeletal elements; whether expression of the retinoic acid receptor (RAR) genes alpha, beta, and gamma changes during chondrocyte maturation, or how interference with retinoid signaling affects skeletogenesis. Immature chondrocytes present in stage 27 (Day 5.5) chick embryo humerus exhibit low and diffuse expression of RARalpha and gamma, while RARbeta expression is strong in perichondrium. Emergence of hypertrophic chondrocytes in day 8-10 embryo limbs is accompanied by a marked and selective up-regulation of RARgamma gene expression. The RARgamma-rich type X collagen-expressing hypertrophic chondrocytes lay below metaphyseal prehypertrophic chondrocytes expressing Indian hedgehog (Ihh) and are followed by mineralizing chondrocytes undergoing endochondral ossification. Bioassays reveal that cartilaginous elements in Day 5.5, 8.5, and 10 chick embryo limbs all contain endogenous retinoids; strikingly, the perichondrial tissues surrounding the cartilages contain very large amounts of retinoids. Implantation of beads filled with retinoid antagonists near the humeral anlagens in stage 21 (Day 3.5) or stage 27 chick embryos severely affect humerus development. In comparison to their normal counterparts, antagonist-treated humeri in day 8.5-10 chick embryos are significantly shorter and abnormally bent; their diaphyseal chondrocytes show the continued presence of prehypertrophic Ihh-expressing cells, do not express RARgamma, and do not undergo endochondral ossification. Interestingly, formation of an intramembranous bony collar around the diaphysis is not affected by antagonist treatment. Using chondrocyte cultures, the antagonists have been found to effectively interfere with the ability of all-trans-retinoic acid to induce terminal cell maturation. The results provide clear evidence that retinoid-dependent and RAR-mediated mechanisms are required for completion of the chondrocyte maturation process and endochondral ossification in the developing limb. These mechanisms may be positively influenced by cooperative interactions between the chondrocytes and their retinoid-rich perichondrial tissues (Koyama, 1999).

Both retinoid receptor null mutants and classic nutritional deficiency studies have demonstrated that retinoids are essential for the normal development of diverse embryonic structures (e.g. eye, heart, nervous system, urogenital tract). Detailed analysis of retinoid-modulated events is hampered by several limitations inthese models, including that deficiency or null mutation is present throughout gestation, making it difficult to isolate primary effects, and preventing analysis beyond embryolethality. A mammalian model has been developed in which retinoid-dependent events are documented during distinct targeted windows of embryogenesis. This is accomplished through the production of vitamin A-depleted (VAD) female rats maintained on sufficient oral retinoic acid (RA) for growth and fertility. After mating to normal males, these RA-sufficient/VAD females were given oral RA doses that allowed for gestation in an RA-sufficient state; embryogenesis proceeds normally until retinoids are withdrawn dietarily to produce a sudden, acute retinoid deficiency during a selected gestational window. In this trial, final RA doses were administered on E11.5, vehicle at E12.5, and embryos analyzed on E13.5; during this 48 hour window, the last RA dose is metabolized and embryos progress in a retinoid-deficient state. RA-sufficient embryos are normal. Retinoid-depleted embryos exhibit specific malformations of the face, neural crest, eyes, heart, and nervous system. Some defects were phenocopies of those seen in null mutant mice for RXR alpha(-/-), RXR alpha(-/-)/RAR alpha(-/-), and RAR alpha(-/-)/RAR gamma(-/-), confirming that RA transactivation of its nuclear receptors is essential for normal embryogenesis. Other defects are unique to this deficiency model, showing that complete ligand 'knock-out' is required to see those retinoid-dependent events previously concealed by receptor functional redundancy, and reinforcing that retinoid receptors have separate yet overlapping contributions in the embryo. This model allows for precise targeting of retinoid form and deficiency to specific developmental windows, and will facilitate studies of distinct temporal events (Dickman, 1997).

Vitamin A requirement for early embryonic development is clearly evident in the gross cardiovascular and central nervous system abnormalities that lead to an early death for the vitamin A-deficient quail embryo. This retinoid knockout model system was used to examine the biological activity of various natural retinoids in early cardiovascular development. All-trans-, 9-cis-, 4-oxo-, and didehydroretinoic acids, and didehydroretinol and all-trans-retinol induce and maintain normal cardiovascular development as well as induce expression of the retinoic acid receptor beta2 in the vitamin A-deficient quail embryo. The expression of RARbeta2 is at the same level and at the same sites where it is expressed in the normal embryo. Until the 5-somite stage of development, but not later, retinoids provided to the vitamin A-deficient embryo completely rescue embryonic development, suggesting the 5-somite stage as a critical retinoid-sensitive time point during early avian embryogenesis. Retinoid receptors RARalpha, RARgamma, and RXRalpha are expressed in both the precardiac endoderm and mesoderm in the normal and the vitamin A-deficient quail embryo, while the expression of RXRgamma is restricted to precardiac endoderm. Vitamin A deficiency downregulates the expression of RARalpha and RARbeta. These studies provide strong evidence for a narrow, retinoid-requiring, developmental window during early embryogenesis, in which the presence of bioactive retinoids and their receptors are essential for a subsequent normal embryonic development (Kostetskii, 1998).

At the cellular level, retinoic acid (RA) regulates gene expression through nuclear receptors, which act as ligand-dependent transcription factors. There are two families of retinoid nuclear receptors, the Retinoic Acid Receptors (RARs), which are activated by all-trans and 9-cis retinoic acid, and the Retinoid X Receptors (RXRs), which are activated by 9-cis retinoic acid only. Each family is represented by three genes, Rara, Rarb, and Rarg, and Rxra, Rxrb and Rxrg. Single and compound null mutants for all of these receptors have revealed both unique and redundant functions during development. All combined, the congenital malformations presented by the Rar and Rxr single or compound mutants recapitulate the vitamin A phenotypes induced in rats deprived of vitamin A in utero. Relative to single null mutants, mice bearing mutations in both Hoxd4 and Rarg display malformations of the basioccipital bone, and first (C1) and second cervical vertebrae (C2) at increased penetrance and expressivity, demonstrating synergy between Hoxd4 and Rarg in the specification of the cervical skeleton. In contrast to Rarg mutants, retinoic acid (RA) treatment on embryonic day 10.5 of Hoxd4 single or Hoxd4;Rarg double mutants does not rescue normal development of C2. Somitic expression of Hoxd4 is not altered in wild-type or Rarg mutant animals before or after RA treatment on day 10.5, suggesting that Hoxd4 and Rarg act in parallel to regulate the expression of target genes directing skeletogenesis (Folberg, 1999).

Mutants mice carrying targeted inactivations of both retinoic acid receptor (RAR) alpha and RARgamma (Aalpha/Agamma mutants) were analyzed at different embryonic stages, in order to establish the timing of appearance of defects that are observed during the fetal period. Embryonic day (E)9.5 Aalpha/Agamma embryos display severe malformations, similar to those of retinaldehyde dehydrogenase 2 null mutants. These malformations reflect early roles of retinoic acid signaling in axial rotation, segmentation and closure of the hindbrain; formation of otocysts, pharyngeal arches and forelimb buds, and in the closure of the primitive gut. The hindbrain of E8.5 Aalpha/Agamma embryos shows a posterior expansion of rhombomere 3 and 4 (R3 and R4) markers, but fails to express kreisler, a normal marker of R5 and R6. This abnormal hindbrain phenotype is strikingly different from that of embryos lacking RARalpha and RARß (Aalpha/Aßmutants), in which the territory corresponding to R5 and R6 is markedly enlarged. Administration of a pan-RAR antagonist at E8.0 to wild-type embryos cultured in vitro results in an Aalpha/Aß-like hindbrain phenotype, whereas an earlier treatment at E7.0 yields an Aalpha/Agamma-like phenotype. Altogether, these data suggest that RARalpha and/or RARgamma transduce the RA signal that is required first to specify the prospective R5/R6 territory, whereas RARß is subsequently involved in setting up the caudal boundary of this territory (Wendling, 2001).

The retinoic acid receptors (RARs) recruit coactivator and corepressor proteins to activate or repress the transcription of target genes depending on the presence of retinoic acid (RA). Despite a detailed molecular understanding of how corepressor complexes function, there is no in vivo evidence to support a necessary function for RAR-mediated repression. Signaling through RARs is required for patterning along the anteroposterior (A-P) axis, particularly in the hindbrain and posterior, although the absence of RA is required for correct anterior patterning. Because RARs and corepressors are present in regions in which RA is absent, it is hypothesized that repression mediated through unliganded RARs might be important for anterior patterning. To test this hypothesis, specific reagents were used that either reduce or augment RAR-mediated repression. Derepression of RAR signaling by expressing a dominant-negative corepressor results in embryos that exhibit phenotypes similar to those treated by RA. Anterior structures such as forebrain and cement gland are greatly reduced, as is the expression of molecular markers. Enhancement of target gene repression using an RAR inverse agonist results in up-regulation of anterior neural markers and expansion of anterior structures. Morpholino antisense oligonucleotide-mediated RARalpha loss-of-function phenocopies the effects of RA treatment and dominant-negative corepressor expression. Microinjection of wild-type or dominant-negative RARalpha rescues the morpholino phenotype, confirming that RAR is functioning anteriorly as a transcriptional repressor. Increasing RAR-mediated repression potentiates head-inducing activity of the growth factor inhibitor cerberus, whereas releasing RAR-mediated repression blocks cerberus from inducing ectopic heads. It is concluded that RAR-mediated repression of target genes is critical for head formation. This requirement establishes an important biological role for active repression of target genes by nuclear hormone receptors and illustrates a novel function for RARs during vertebrate development (Koide, 2001).

Early neural patterning in vertebrates involves signals that inhibit anterior (A) and promote posterior (P) positional values within the nascent neural plate. In this study, the contributions of, and interactions between, retinoic acid (RA), Fgf and Wnt signals have been investigated in the promotion of posterior fates in the ectoderm. Expression and function of cyp26/P450RAI, a gene that encodes retinoic acid 4-hydroxylase, has been examined as a tool for investigating these events. Cyp26 is first expressed in the presumptive anterior neural ectoderm and the blastoderm margin at the late blastula. When the posterior neural gene hoxb1b is expressed during gastrulation, it shows a strikingly complementary pattern to cyp26. Using these two genes, as well as otx2 and meis3 as anterior and posterior markers, it has been shown that Fgf and Wnt signals suppress expression of anterior genes, including cyp26. Overexpression of cyp26 suppresses posterior genes, suggesting that the anterior expression of cyp26 is important for restricting the expression of posterior genes. Consistent with this, knock-down of cyp26 by morpholino oligonucleotides leads to the anterior expansion of posterior genes. Fgf- and Wnt-dependent activation of posterior genes is mediated by RA, whereas suppression of anterior genes does not depend on RA signaling. Fgf and Wnt signals suppress cyp26 expression, while Cyp26, an enzyme that degrades RA, limits the range of RA-mediated posteriorization in the embryo by suppressing the RA signal. Thus, cyp26 has an important role in linking the Fgf, Wnt and RA signals to regulate AP patterning of the neural ectoderm in the late blastula to gastrula embryo in zebrafish (Kudoh, 2002).

Exogenous retinoic acid (RA) can evoke vertebral homeosis when administered during late gastrulation. These vertebral transformations correlate with alterations of the rostral limit of Hox gene expression in the prevertebrae, suggesting that retinoid signaling regulates the combinatorial expression of Hox genes dictating vertebral identity. Conversely, loss of certain RA receptors (RARs) results in anterior homeotic transformations principally affecting the cervical region. Despite these observations, the relationship between retinoid signaling, somitic Hox expression, and vertebral patterning is poorly understood. Cdx1 homozygous null mutants exhibit anterior homeotic transformations, some of which are reminiscent of those in RARgamma null offspring. In Cdx1 mutants, these transformations occur concomitant with posteriorized prevertebral expression of certain Hox genes. Cdx1 is a direct RA target, suggesting an indirect means by which retinoid signaling may impact vertebral patterning. To further investigate this relationship, a complete allelic series of Cdx1-RARgamma mutants was generated and the skeletal phenotype assessed either following normal gestation or after administration of RA. Synergistic interactions between these null alleles were observed in compound mutants, and the full effects of exogenous RA on vertebral morphogenesis requires Cdx1. These findings are consistent with a role for RA upstream of Cdx1 as regards axial patterning. However, exogenous RA attenuates several defects inherent to Cdx1 null mutants. This finding, together with the increased phenotypic severity of RARgamma-Cdx1 double null mutants relative to single nulls, suggests that these pathways also function in parallel, likely by converging on common targets (Allan, 2001).

Fusion and hypoplasia of the first two branchial arches, a defect typically observed in retinoic acid (RA) embryopathy, is generated in cultured mouse embryos upon treatment with BMS453, a synthetic compound that exhibits retinoic acid receptor ß (RARß) agonistic properties in transfected cells. By contrast, no branchial arch defects are observed following treatment with synthetic retinoids that exhibit RARalpha or RARgamma agonistic properties. The BMS453-induced branchial arch defects are mediated through RAR activation, since they are similar to those generated by a selective pan-RAR agonist -- they are prevented by a selective pan-RAR antagonist and cannot be mimicked by exposure to a pan-RXR agonist alone. They are enhanced in the presence of a pan-RXR agonist, and cannot be generated in Rarß-null embryos. Furthermore, they are accompanied, in the morphologically altered region, by ectopic expression of Rarß and of several other direct RA target genes. Therefore, craniofacial abnormalities characteristic of the RA embryopathy are mediated through ectopic activation of RARß/RXR heterodimers, in which the ligand-dependent activity of RXR is subordinated to that of RARß. Endodermal cells lining the first two branchial arches respond to treatment with the RARß agonist, in contrast to neural crest cells and ectoderm, which suggests that a faulty endodermal regionalization is directly responsible for RA-induced branchial arch dysmorphologies. Additionally, the first in vivo evidence is provided that the synthetic RARß agonist BMS453 exhibits an antagonistic activity on the two other RAR isotypes (Matt, 2003).

The functional links of specific retinoid receptors to early developmental events in the avian embryo are not known. Before such studies are undertaken, knowledge is required of the spatiotemporal expression patterns of the receptor genes and their regulation by endogenous retinoic acid levels during the early stages of development. The expression patterns are reported of mRNAs for RARalpha, RARalpha2, RAß2, RARgamma, RARgamma2, RXRalpha, and RARgamma from neurulation to HH10 in the normal and vitamin A-deficient (VAD) quail embryo. The transcripts for all retinoid receptors are detectable at HH5, except for RXRgamma, which is detected at the beginning of HH6. At the 4/5 somite stage of HH8, when retinoid signaling is initiated in the avian embryo, mRNAs of all receptors are present, with very strong and ubiquitous expression patterns for RARalpha, RARalpha2, RARgamma, RARgamma2, and RXRalpha, a persistent expression of RARgamma in the neural tissues, a strong expression of RAß2 in lateral plate mesoderm and somites, and an anterior expression of RXRgamma. All retinoid receptors are expressed in the heart primordia. In the VAD quail embryo, the general pattern of retinoid receptor transcript localization is similar to that of the normal, except that the expression of RARalpha2 and RAß2 is severely diminished. Administration of retinol or retinoic acid to VAD embryos at or before the 4/5 somite stage rescues the expression of RARalpha2 and RAß2 within approximately 45 min and restores normal development. RAß2 expression requires the expression of RARalpha2. After neurulation, the expression of all retinoid receptors in the VAD quail embryo becomes independent of vitamin A status and is similar to that of the normal. The mRNA levels and sites of expression of the key enzyme for retinoic acid biosynthesis, Raldh-2, are not affected by vitamin A status; the expression pattern is restricted and does not correspond to that of retinoid receptors at all sites. The general patterns and intensity of retinoid receptor gene expression during early quail development are comparable to those of the mammalian and thus validate the application of results from retinoid-regulated avian development studies to those of the mammalian (Cui, 2003).

Vertebrate body axis extension involves progressive generation and subsequent differentiation of new cells derived from a caudal stem zone; however, molecular mechanisms that preserve caudal progenitors and coordinate differentiation are poorly understood. FGF maintains caudal progenitors and its attenuation is required for neuronal and mesodermal differentiation and to position segment boundaries. Furthermore, somitic mesoderm promotes neuronal differentiation in part by downregulating Fgf8. retinoic acid (RA) has been identified as this somitic signal; retinoid and FGF pathways have opposing actions. FGF is a general repressor of differentiation, including ventral neural patterning, while RA attenuates Fgf8 in neuroepithelium and paraxial mesoderm, where it controls somite boundary position. RA is further required for neuronal differentiation and expression of key ventral neural patterning genes. These data demonstrate that FGF and RA pathways are mutually inhibitory and suggest that their opposing actions provide a global mechanism that controls differentiation during axis extension (Diez del Dorral, 2003).

FGF can maintain an undifferentiated cell state, and retinoids can drive differentiation in many different contexts; for example, mouse ES cells form neural precursors in vitro under the influence of FGF signaling, while exposure to RA promotes neuronal differentiation. In the mouse embryo, excess RA, due to mutation of the RA-metabolizing enzyme CYP26, has been shown to repress Fgf8 expression in the tail bud, and RA downregulates Fgf8 in both neural and mesodermal tissues in vitro. Although Fgf8 expression perdures in caudal regions of Vitamin A-deficient (VAD) quails, it is still eventually lost from the presomitic mesoderm and neuroepithelium. This suggests either that somites provide another signal that can repress Fgf8 or that Fgf8 transcripts normally decay and that RA acts to accelerate this process. In the presomitic mesoderm, Fgf8 can be induced by FGF8 and so RA could effect Fgf8 reduction by interfering with the FGF signaling pathway. Conversely, FGF8 controls RA synthesis by inhibiting onset of Raldh2 in the paraxial mesoderm. Furthermore, exposure to FGF also blocks neuronal differentiation in explants of neural tube that do not express Raldh2, suggesting that FGF can also oppose RA activity in the neuroepithelium. It is proposed that during normal extension of the axis, a slight decline in Fgf8 transcripts (facilitated by regression of the primitive streak that expresses FGFs able to induce Fgf8) is sufficient for Raldh2 onset. As presomitic mesoderm begins to synthesize RA, retinoid signaling then accelerates Fgf8 downregulation in both the paraxial mesoderm and adjacent preneural tube. This mutual opposition of FGF and RA pathways thus helps to ensure the coordinated differentiation of mesodermal and neural tissues (Diez del Corral, 2003).

The level of FGF signaling in the presomitic mesoderm controls where a somite boundary will form, and the ability of RA to attenuate Fgf8 in the paraxial mesoderm identifies a role for the retinoid pathway in this process. According to the current model, somite size is determined by two components: the period of oscillation of transiently expressed mRNAs associated with Notch signaling such as Hairy1 (the segmentation clock) and the speed of FGF decline in the presomitic mesoderm (the maturation wavefront). Changes in FGF signaling do not alter the period of oscillation: resulting segmentation defects are due to alteration in the speed at which FGF levels fall below a threshold. Since RA downregulates Fgf8 in the presomitic mesoderm, it must set the rate of maturation wavefront progression and thereby influence somite size. Further, since FGF and RA pathways are mutually inhibitory, this could create a sharp transition in cell signaling in the presomitic mesoderm, and one possibility is that this change precisely defines the future somite border. Consistent with this, in VAD embryos where Fgf8 expression is prolonged and wavefront progression is slowed, initial somite size is smaller (Diez del Corral, 2003).

Finally, opposition of FGF and RA pathways may be a conserved mechanism for controlling differentiation and maintaining progenitor pools in the developing embryo. A striking analogy can be drawn with the proximo-distal extension of the limb in which distal FGF signaling provided by the apical ridge restricts RA synthesis and RARß receptor expression to the proximal limb. FGF signaling also opposes RA in the forming hindbrain, preserving rhombomere1 as a site of FGF activity that undergoes extensive proliferation to generate the cerebellum. The data suggest that mutual inhibition and opposing activities of FGF and RA pathways act to maintain a critical balance between preservation of the progenitor pool/stem zone and the progressive differentiation of neural and mesodermal tissues during the extension of the embryonic axis (Diez del Corral, 2003).

The identity of motor neurons diverges markedly at different rostrocaudal levels of the spinal cord, but the signals that specify their fate remain poorly defined. Retinoid receptor activation in newly generated spinal motor neurons has a crucial role in specifying motor neuron columnar subtypes. Blockade of retinoid receptor signaling in brachial motor neurons inhibits lateral motor column differentiation and converts many of these neurons to thoracic columnar subtypes. Conversely, expression of a constitutively active retinoid receptor derivative impairs the differentiation of thoracic motor neuron columnar subtypes. These findings provide evidence for a regionally restricted role for retinoid signaling in the postmitotic specification of motor neuron columnar identity (Sockanathan, 2003).

The involvement of retinoid signaling in many aspects of caudal neural patterning has led to a consideration of whether retinoids might also participate in the specification of motor neuron columnar identity along the rostrocaudal axis of the spinal cord. Two lines of evidence suggest such a role. (1) At the time of motor neuron generation, Raldh2 is expressed at a high level by paraxial mesodermal cells that flank brachial (forelimb) levels of the spinal cord, but at a much lower level by paraxial mesoderm at thoracic levels and lumbar levels. (2) Transgenic mice that serve as in vivo reporters of retinoid signaling have revealed a high level of retinoid signaling activity in the brachial spinal cord but a low level of signaling at thoracic levels. Collectively, these findings suggest that newly generated brachial motor neurons are exposed to higher levels of retinoid signaling than are thoracic motor neurons. Thus, a differential in retinoid signals provided by the paraxial mesoderm could contribute to the specification of motor neuron columnar identity along the rostrocaudal axis of the spinal cord (Sockanathan, 2003).

To test this possibility, retinoid receptor signaling was manipulated in postmitotic spinal motor neurons through expression of dominant-negative or constitutively active retinoid receptor derivatives. Blockade of retinoid receptor signaling in newly generated brachial motor neurons prevents them from acquiring an lateral motor column identity, as assessed by gene expression profile, neuronal settling position, and axonal projection pattern. Moreover, many brachial motor neurons now acquire molecular and anatomical characteristics of thoracic level Column of Terni (CT) and lateral median motor column (MMC) neurons despite their preserved rostrocaudal position. Conversely, the expression of a constitutively active retinoid receptor derivative in thoracic motor neurons impairs the differentiation of CT and lateral MMC subtypes and leads ultimately to motor neuron death. Thus, the status of retinoid receptor signaling in postmitotic motor neurons appears to regulate motor neuron columnar subtype identity along the rostrocaudal axis of the spinal cord (Sockanathan, 2003).

Anteroposterior (AP) patterning of the developing neural tube is crucial for both regional specification and the timing of neurogenesis. Several important factors are involved in AP patterning, including members of the WNT and FGF growth factor families, retinoic acid receptors, and HOX genes. The interactions between FGF and retinoic signaling pathways have been studied. Blockade of FGF signaling downregulates the expression of members of the RAR signaling pathway, RARalpha, RALDH2 and CYP26. Overexpression of a constitutively active RARalpha2 rescues the effects of FGF blockade on the expression of XCAD3 and HOXB9. This suggests that RARalpha2 is required as a downstream target of FGF signaling for the posterior expression of XCAD3 and HOXB9. Surprisingly, it was found that posterior expression of FGFR1 and FGFR4 is dependent on the expression of RARalpha2. Anterior expression is also altered with FGFR1 expression being lost, whereas FGFR4 expression is expanded beyond its normal expression domain. RARalpha2 is required for the expression of XCAD3 and HOXB9, and for the ability of XCAD3 to induce HOXB9 expression. It is concluded that RARalpha2 is required at multiple points in the posteriorization pathway, suggesting that correct AP neural patterning depends on a series of mutually interactive feedback loops among FGFs, RARs and HOX genes (Shiotsugu, 2004).

Endogenous retinoids are important for patterning many aspects of the embryo including the branchial arches and frontonasal region of the embryonic face. The nasal placodes express retinaldehyde dehydrogenase-3 (RALDH3) and thus retinoids from the placode are a potential patterning influence on the developing face. Experiments have been carried out that have used Citral, a RALDH antagonist, to address the function of retinoid signaling from the nasal pit in a whole embryo model. When Citral-soaked beads are implanted into the nasal pit of stage 20 chicken embryos, the result is a specific loss of derivatives from the lateral nasal prominences. Providing exogenous retinoic acid rescues development of the beak demonstrating that most Citral-induced defects are produced by the specific blocking of RA synthesis. The mechanism of Citral effects is a specific increase in programmed cell death on the lateral (lateral nasal prominence) but not the medial side (frontonasal mass) of the nasal pit. Gene expression studies were focused on the Bone Morphogenetic Protein (BMP) pathway, which has a well-established role in programmed cell death. Unexpectedly, blocking RA synthesis decreased rather than increased Msx1, Msx2, and Bmp4 expression. Cell survival genes were examined, the most relevant of which was Fgf8, which is expressed around the nasal pit and in the frontonasal mass. Fgf8 was not initially expressed along the lateral side of the nasal pit at the start of the experiments, whereas it was expressed on the medial side. Citral prevented upregulation of Fgf8 along the lateral edge and this may have contributed to the specific increase in programmed cell death in the lateral nasal prominence. Consistent with this idea, exogenous FGF8 was able to prevent cell death, rescue most of the morphological defects and was able to prevent a decrease in retinoic acid receptorβ (Rarβ) expression caused by Citral. Together, these results demonstrate that endogenous retinoids act upstream of FGF8 and the balance of these two factors is critical for regulating programmed cell death and morphogenesis in the face. In addition, these data suggest a novel role for endogenous retinoids from the nasal pit in controlling the precise downregulation of FGF in the center of the frontonasal mass observed during normal vertebrate development (Song, 2004).

Retinoic acid (RA) activity plays sequential roles during the development of the ventral spinal cord. The functions of local RA synthesis in the process of motoneuron specification and early differentiation have been investigated using a conditional knockout strategy that ablates the function of the retinaldehyde dehydrogenase 2 (Raldh2) synthesizing enzyme essentially in brachial motoneurons, and later in mesenchymal cells at the base of the forelimb. Mutant (Raldh2^L–/–) embryos display an early embryonic loss of a subset of Lim1+ brachial motoneurons, a mispositioning of Islet1+ neurons and inappropriate axonal projections of one of the nerves innervating extensor limb muscles, which lead to an adult forepaw neuromuscular defect. The molecular basis of the Raldh2^L–/– phenotype relies in part on the deregulation of Hoxc8, which in turn regulates the RA receptor RARß. Hoxc8 mutant mice, which exhibit a similar congenital forepaw defect, display at embryonic stages molecular defects that phenocopy the Raldh2^L–/– motoneuron abnormalities. Thus, interdependent RA signaling and Hox gene functions are required for the specification of brachial motoneurons in the mouse (Vermot, 2005).

During anteroposterior (AP) patterning of the developing hindbrain, the expression borders of many transcription factors are aligned at interfaces between neural segments called rhombomeres (r). Mechanisms regulating segmental expression have been identified for Hox genes, but for other classes of AP patterning genes there is only limited information. The murine retinoic acid receptor ß gene (Rarb) was analyzed and shown to be induced prior to segmentation, by retinoic-acid (RA) signalling from the mesoderm. Induction establishes a diffuse expression border that regresses until, at later stages, it is stably maintained at the r6/r7 boundary by inputs from Hoxb4 and Hoxd4. Separate RA- and Hox-responsive enhancers mediate the two phases of Rarb expression: a regulatory mechanism remarkably similar to that of Hoxb4. By showing that Rarb is a direct transcriptional target of Hoxb4, this study identifies a new molecular link, completing a feedback circuit between Rarb, Hoxb4 and Hoxd4. It is proposed that the function of this circuit is to align the initially incongruent expression of multiple RA-induced genes at a single segment boundary (Serpente, 2005).

The mechanism regulating Rarb in the presegmented hindbrain is similar to that of Hoxb4. Both genes are transcriptionally induced by a Raldh2-dependent RA source and both possess RARE-containing enhancers [for Hoxb4, this is termed the early neural enhancer (ENE) that directs neural expression with borders that recede after E8.5. These caudal shifts presumably reflect regression of the inducing ability of the paraxial mesoderm with increasing embryonic age. Although there are strong parallels between the RARE enhancers of Rarb and Hoxb4, there are also some differences. For example, proximal enhancer activity begins at around the two-somite stage, whereas ENE activity begins at the nine-somite stage. In addition, at E8.5, the anterior border of the Rarb proximal enhancer is at presumptive r5/6 but that of the Hoxb4 ENE is at presumptive r6/r7. Although the DNA element responsible for these expression differences is undefined, it may be relevant that the DR5 class of RARE, present in both enhancers, differs at 3/12 nucleotide positions (Serpente, 2005).

The regulatory parallels between Rarb and Hoxb4 also extend to the later phase of segmental expression. Like Rarb, Hoxb4 uses a two-step regulatory strategy of establishment and maintenance within the hindbrain, involving two enhancer elements that are mechanistically and physically separable. For Hoxb4, the late hindbrain element is termed the late neural enhancer (LNE). Both the Rarb distal enhancer and the Hoxb4 LNE drive expression with a sharp r6/r7 border and respond to stabilizing inputs from group 4 Hox genes. In both cases, these late Hox inputs serve to halt the caudal regression of diffuse borders that were established by RARE-containing enhancers. However, when the functions of Hoxb4 and Hoxd4 are completely removed, Hoxb4 LNE activity is lost only from r7, whereas Rarb distal enhancer activity is abolished within the entire neural tube. This suggests that, although group 4-6 Hox paralogues activate the Hoxb4 LNE, only some or all of the group 4 Hox genes may be capable of activating the Rarb distal enhancer (Serpente, 2005).

Retinoic acid (RA) generated in the mesoderm of vertebrate embryos controls body axis extension by downregulating Fgf8 expression in cells exiting the caudal progenitor zone. RA activates transcription by binding to nuclear RA receptors (RARs) at RA response elements (RAREs), but it is unknown whether RA can directly repress transcription. This study analyzed a conserved RARE upstream of Fgf8 that binds RAR isoforms in mouse embryos. Transgenic embryos carrying Fgf8 fused to lacZ exhibited expression similar to caudal Fgf8, but deletion of the RARE resulted in ectopic trunk expression extending into somites and neuroectoderm. Epigenetic analysis using chromatin immunoprecipitation of trunk tissues from E8.25 wild-type and Raldh2(-/-) embryos lacking RA synthesis revealed RA-dependent recruitment of the repressive histone marker H3K27me3 and polycomb repressive complex 2 (PRC2) near the Fgf8 RARE. The co-regulator RERE, the loss of which results in ectopic Fgf8 expression and somite defects, was recruited near the RARb RARE by RA, but was released from the Fgf8 RARE by RA. These findings demonstrate that RA directly represses Fgf8 through a RARE-mediated mechanism that promotes repressive chromatin, thus providing valuable insight into the mechanism of RA-FGF antagonism during progenitor cell differentiation (Kumar, 2014a).

RAR and cell proliferation

NB4, a human acute promyelocytic leukemia cell line expressing the promyelocyte-retinoic acid receptor alpha (PML-RAR alpha) hybrid protein was treated with RAR- and retinoid X receptor (RXR)-selective analogs to determine their effects on cell proliferation, retinoblastoma (RB) tumor-suppressor protein phosphorylation, and differentiation. An RAR- or just RAR alpha-selective analog alone induces similar cell population growth arrest, cell cycle arrest without restriction to G1, hypophosphorylation of RB, and myelomonocytic cell surface differentiation marker expression (CD11b). An RAR alpha antagonist can inhibit the effects of the RAR alpha agonist completely. The RAR alpha-selective analog-elicited response is attenuated by simultaneous addition of various RXR-selective analogs. In contrast, each of the RXR-selective analogs is unable to induce any of the cellular responses analyzed. The growth arrest of NB4 cells is not G1-restricted and occurs at all points in the cell cycle. Cells growth arrested by treatment with an RAR alpha-selective analog shows primarily hypophosphorylated RB. When these cells are sorted into G1 or S + G2/M subpopulations by flow cytometry, hypophosphorylated RB protein is found in G1 as well as S + G2/M cells. This suggests that the hypophosphorylated RB protein may be mediating the growth arrest of NB4 cells at all points in the cell cycle. These results are consistent with an involvement of PML-RAR alpha and/or RAR alpha in the transduction of the retinoid signal in NB4 cells (Brooks, 1997).

Myeloproliferative syndromes (MPS) are a heterogeneous subclass of nonlymphoid hematopoietic neoplasms which are considered to be intrinsic to hematopoietic cells. The causes of MPS are largely unknown. This study demonstrates that mice deficient for retinoic acid receptor γ (RARγ), develop MPS induced solely by the RARγ-deficient microenvironment. RARγ^−/− mice have significantly increased granulocyte/macrophage progenitors and granulocytes in bone marrow (BM), peripheral blood, and spleen. The MPS phenotype continues for the lifespan of the mice and is more pronounced in older mice. Unexpectedly, transplant studies revealed this disease was not intrinsic to the hematopoietic cells. BM from wild-type mice transplanted into mice with an RARγ^−/− microenvironment rapidly developed the MPS, which was partially caused by significantly elevated TNFα in RARγ^−/− mice. These data show that loss of RARγ results in a nonhematopoietic cell-intrinsic MPS, revealing the capability of the microenvironment to be the sole cause of hematopoietic disorders (Walkley, 2007).

Transcriptional activation of the nuclear receptor RAR by retinoic acid (RA) often leads to inhibition of cell growth. However, in some tissues, RA promotes cell survival and hyperplasia, activities that are unlikely to be mediated by RAR. This study shows that, in addition to functioning through RAR, RA activates the 'orphan' nuclear receptor PPARβ/δ, which, in turn, induces the expression of prosurvival genes. Partitioning of RA between the two receptors is regulated by the intracellular lipid binding proteins CRABP-II and FABP5. These proteins specifically deliver RA from the cytosol to nuclear RAR and PPARβ/δ, respectively, thereby selectively enhancing the transcriptional activity of their cognate receptors. Consequently, RA functions through RAR and is a proapoptotic agent in cells with high CRABP-II/FABP5 ratio, but it signals through PPARβ/δ and promotes survival in cells that highly express FABP5. Opposing effects of RA on cell growth thus emanate from alternate activation of two different nuclear receptors (Schug, 2007).

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