Interactive Fly, Drosophila

Retinoic acid receptor RAR: a model for Ecdysone receptor in vertebrates - Transcriptional targets of RAR

Bmp2, a highly conserved member of the transforming growth factor-beta gene family, is crucial for normal development. Retinoic acid, combined with cAMP analogs, sharply induces the Bmp2 mRNA during the differentiation of F9 embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA) also induces the Bmp2 gene in chick limb buds. Since normal Bmp2 expression may require an endogenous retinoid signal and aberrant Bmp2 expression may cause some aspects of RA-induced teratogenesis, the mechanism underlying the induction of Bmp2 was investigated. Measurements of the Bmp2 mRNA half-life and nuclear run-on assays indicate that RA stimulates the transcription rate of the Bmp2 gene. The results of ribonuclease protection and primer extension assays indicate that Bmp2 transcription starts 2,127 nucleotides upstream of the translation start site in F9 cells. To identify genetic elements controlling this transcription rate increase, upstream and downstream genomic sequences flanking the Bmp2 gene were screened using chloramphenicol acetyltransferase reporter genes in F9 cells and beta-galactosidase reporter genes in Saccharomyces cerevisiae that were cotransformed with retinoic acid receptor and retinoid X receptor expression plasmids. RA-dependent transcriptional activation is detected between base pairs -2,373 and -2,316 relative to the translation start site. A required Sp1 binding site was identified between -2,308 and -2,298. The data indicate that Bmp2 is directly regulated by retinoic acid-bound receptors and Sp1 (Heller, 1999).

The role of retinoic acid receptors (RARs) in intercellular regulation of cell growth was assessed by targeting a dominant-negative RARalpha mutant (dnRARalpha) to differentiated suprabasal cells of mouse epidermis. dnRARalpha lacks transcriptional activation but not DNA-binding and receptor dimerization functions. Analysis of transgenic mice reveals that dnRARalpha dose-dependently impairs induction of basal cell proliferation and epidermal hyperplasia by all-trans RA (tRA). dnRARalpha forms heterodimers with endogenous retinoid X receptor-alpha (RXRalpha) over RA response elements in competition with remaining endogenous RARgamma-RXRalpha heterodimers, and dose-dependently impairs retinoid-dependent gene transcription. To identify genes regulated by retinoid receptors and involved in cell growth control, the retinoid effects were analyzed on expression of the following: epidermal growth factor (EGF) receptor; EGF; transforming growth factor-alpha; heparin-binding EGF-like growth factor (HB-EGF), and amphiregulin genes. In normal epidermis, tRA rapidly and selectively induces expression of HB-EGF but not the others. This induction occurs exclusively in suprabasal cells. In transgenic epidermis, dnRARalpha dose-dependently inhibits tRA induction of suprabasal HB-EGF and subsequent basal cell hyperproliferation. Together, these observations suggest that retinoid receptor heterodimers located in differentiated suprabasal cells mediate retinoid induction of HB-EGF, which in turn stimulates basal cell growth via intercellular signaling. These events may underlie retinoid action in epidermal regeneration during wound healing (Xiao, 1999).

All-trans-retinoic acid (RA) is a potent inducer of tissue transglutaminase (TGase II) and apoptosis in the rat tracheobronchial epithelial cell line SPOC-1. These cells express the retinoid receptors RAR alpha, RAR gamma, and RXR beta. To identify which of these receptors are involved in regulating these processes, the effects of several receptor-selective agonists, an antagonist, and a dominant-negative RAR alpha were analyzed. The RAR-selective retinoid SRI-6751-84 strongly increases TGase II expression at both the protein and mRNA levels, whereas the RXR-selective retinoid SR11217 has little effect. The RAR alpha-selective retinoid Ro40-6055 is also able to induce TGase II, whereas the RAR gamma-selective retinoid CD437 is inactive. The induction of TGase II by the RAR-selective retinoid is completely inhibited by the RAR alpha-antagonist Ro41-5253. Overexpression of a truncated RAR alpha gene with dominant-negative activity also inhibits the induction of TGase II expression. The increase in TGase II is associated with an induction of apoptosis as revealed by DNA fragmentation and the generation of apoptotic cells. Apoptosis is affected by retinoids in a manner similar to TGase II. These results suggest that the induction of TGase II expression and apoptosis in SPOC-1 cells are mediated through an RAR alpha-dependent signaling pathway (Zhang, 1995).

Mice lacking the RARgamma gene and one or both alleles of the RARbeta gene (i.e., RARbeta+/-/RARgamma-/- and RARbeta-/-/RARgamma-/- mutants) display a severe and fully penetrant interdigital webbing (soft tissue syndactyly), caused by the persistence of the fetal interdigital mesenchyme. These compound mutants were used to investigate the cellular and molecular mechanisms involved in retinoic acid (RA)-dependent formation of the interdigital necrotic zones (INZs). The mutant INZs show a marked decrease in the number of apoptotic cells accompanied by an increase of cell proliferation. This marked decrease is not paralleled by a reduction of the number of macrophages, indicating that the chemotactic cues that normally attract these cells into the INZs are not affected. The expression of a number of genes known to be involved in the establishment of the INZs, the patterning of the autopod, and/or the initiation of apoptosis is also unaffected. These genes included BMP-2, BMP-4, Msx-1, Msx-2, 5' members of Hox complexes, Bcl2, Bax, and p53. In contrast, the mutant INZs display a specific, graded, down-regulation of tissue transglutaminase (tTG) promoter activity and of stromelysin-3 expression upon the removal of one or both alleles of the RARbeta gene from the RARgamma null genetic background. Because retinoic acid response elements are present in the promoter regions of both tTG and stromelysin-3 genes, it is proposed that RA might increase the amount of cell death in the INZs through a direct modulation of tTG expression and that it also contributes to the process of tissue remodeling, which accompanies cell death, through an up-regulation of stromelysin-3 expression in the INZs. Expression of tTG is known to be a precocious feature of cells that are committed to apoptosis: tTG catalyzes the irreversible cross-linking of intracellular proteins. Stromolysin-3 is a matrix metalloproteinase whose expression has been associated with tissue remodeling processes characterized by extensive extracellular matrix turnover during embryonic development, wound healing, or tumor invasion. Approximately 10% of the RARbeta-/-/RARgamma-/- mutants display a supernumerary preaxial digit on hindfeet, which is also a feature of the BMP-7 null phenotype. BMP-7 is globally down-regulated at an early stage in the autopods of these RAR double null mutants, prior to the appearance of the digital rays. Therefore, RA may exert some of its effects on anteroposterior autopod patterning through controlling BMP-7 expression (Dupe, 1999).

The posteriorizing agent retinoic acid can accelerate anterior neuronal differentiation in Xenopus laevis embryos. To elucidate the role of retinoic acid in the primary neurogenesis cascade, an investigation was carried out to see whether retinoic acid treatment of whole embryos can change the spatial expression of a set of genes known to be involved in neurogenesis. Retinoic acid expands the N-tubulin, X-ngnr-1, X-MyT1, X-Delta-1 and Gli3 domains and inhibits the expression of Zic2 and sonic hedgehog in the neural ectoderm, whereas a retinoid antagonist produces the opposite changes. In contrast, sonic and banded hedgehog overexpression reduce the N-tubulin stripes, enlarge the neural plate at the expense of the neural crest, downregulate Gli3 and upregulate Zic2. Thus, retinoic acid and hedgehog signaling have opposite effects on the prepattern genes Gli3 and Zic2 and on other genes acting downstream in the neurogenesis cascade. In addition, retinoic acid cannot rescue the inhibitory effect of Notch^ICD, Zic2 or sonic hedgehog on primary neurogenesis. These results suggest that retinoic acid acts very early, upstream of sonic hedgehog, and a model is proposed for regulation of differentiation and proliferation in the neural plate, showing that retinoic acid might be activating primary neurogenesis by repressing sonic hedgehog expression (Franco, 1999).

RA treatment can accelerate neuronal differentiation in the anterior neural plate of whole embryos. Could RA also alter neuronal differentiation in the posterior neural plate where endogenous RA might mainly play its role and where primary neurogenesis occurs? It has been shown that RA exposure during gastrulation greatly expands the normal domains of N-tubulin expression at the neural plate stage. In contrast, retinoic acid antagonist Ro treatments decrease N-tubulin expression, in agreement with the loss of primary neurons produced by the microinjection of dominant negative forms of retinoic acid receptors. RA treatment increases the domains of genes previously shown to promote neuronal differentiation, such as X-ngnr-1, X-MyT1 and Gli3. The deletion of spacing between the stripes of X-ngnr-1 and X-MyT1 suggests that RA changes the activity of prepattern genes, thus directing the neural plate toward a uniform proneural territory. Indeed, RA produces a widespread Gli3 expansion in the posterior neural plate and a dramatic downregulation of Zic2, a gene proposed to inhibit neuronal differentiation. The involvement of endogenous retinoids in this regulatory hierarchy was confirmed by blocking RA signaling with Ro, which produced opposite changes in the expression patterns of these genes (Franco, 1999).

Evidence is presented that endogenous retinoids downregulate the expression of genes that inhibit neurogenesis, like Zic2 and X-shh. While RA treatment reduces their expression, after blocking RA signaling, X-shh expression is increased along the dorsal midline and Zic2 expression becomes dispersed over the mediolateral axis of the neural plate, accounting for the inhibition of primary neurogenesis by Ro. Because previous work in chicken limb and zebrafish fin buds has demonstrated an induction of Shh expression in response to RA, it was surprising that X-shh expression is downregulated by RA at neurula stage both in the notochord and floor plate. These results agree with the very early transient downregulation observed in developing and regenerating axolotl limbs. Furthermore, the upstream region of zebrafish shh contains a retinoic acid responsive element (RARE), implying a direct regulation of the shh gene by RA. Thus there is solid evidence for a link between X-shh and RA at the molecular level (Franco, 1999).

The suppression of primary neurogenesis produced by the overexpression of X-shh and X-bhh is not due to inhibition of neural development, because the neural plate is expanded on the injected side, as shown with the general neural marker nrp-1. When compared to RA treatments, X-shh and X-bhh overexpression produce opposite changes in the expression patterns of different members of the neurogenesis cascade that resemble Ro effects, suggesting that a counterbalance exists between retinoid and hedgehog signaling to restrict primary neurogenesis to the normal sites. Precursors of the primary and secondary neurons arise from different layers of the neural plate. The superficial layer contains predominantly secondary precursors, whereas the deep layer contains both types of precursors at a similar density. Although the fate of the cells inhibited to differentiate by X-shh and X-bhh was not followed, they probably participate in subsequent waves of neurogenesis, as suggested by the fact that both hedgehog members later expande the number of cells expressing Xsal-1, a marker of ventral motor and intermediate neurons in the neural tube of tadpoles (Franco, 1999).

The evident expansion of the neural ectoderm and the paraxial mesoderm together with the increase in cell number are consistent with X-shh and X-bhh playing a proliferative role in both germ layers, but an inhibition of cell death cannot be excluded. Indeed, Shh promotes proliferation in the sclerotome and prevents differentiation, and induces a proliferative response in cerebellar cells. Therefore, it is proposed that both hedgehog members produce a differential effect on primary and secondary neuronal precursors, perhaps withdrawing cells from premature differentiation, holding their proliferative state and precluding them from subsequent waves of neuron formation (Franco, 1999).

RA downregulates X-shh expression whereas Ro produces the opposite change. It is proposed that, in the normal embryo, X-shh expression in the dorsal midline should be controlled by positive and negative regulators. When negative regulation of X-shh is impaired by Ro, the equilibrium is displaced toward a gain-of-function of shh that correlates with decreased primary neuron differentiation (Franco, 1999).

Because RA treatments could not rescue the inhibitory effect of X-shh on neuronal differentiation, while X-shh overexpression produces a widespread expansion of Zic2 and suppresses Gli3, it is suggested that a cascade of interactions occurs, wherein endogenous retinoids act far upstream, promoting primary neurogenesis by inhibiting X-shh expression in the dorsal midline. This in turn changes the balance of prepattern genes (activation of Gli3 and reduction of Zic2), thus altering the expression of other intermediary genes, ultimately leading to N-tubulin activation. Because in the normal embryo X-shh is expressed along the dorsal midline, it is evident that endogenous retinoids do not completely block shh signaling. This fact suggests that a precise balance between retinoid and hedgehog signaling must be established, resulting in the normal primary neurogenesis pattern. While endogenous retinoids constitute an early signal that promotes primary neuron formation by inclining the entire neural plate towards a uniform proneural territory, shh signaling is necessarily required at the same time and at an accurate level, limited at least by endogenous retinoids, to save a pool of neuronal precursors from premature differentiation by retinoid signaling, keeping them in a mitotic, undifferentiated state for subsequent waves of neurogenesis (Franco, 1999).

By culturing neural progenitor cells in the presence of retinoid receptor agonists, the components of the retinoid-signalling pathway that are important for the birth and maintenance of neuronal cells have been defined. Evidence is provided that depending on the order and combination of retinoid receptors activated, different neuronal cells are obtained. Astrocytes and oligodendrocytes are predominantly formed in the presence of activated retinoic acid receptor (RAR) alpha, whereas motoneurons are formed when RARß is activated. The regulation of islet-1 and islet-2, which are involved in neuronal development, was examined. Activated RARß up-regulates islet-1 expression, whereas activation of RARalpha can either act in combination with RARß signalling to maintain islet-1 expression or induce islet-2 expression in the absence of activated RARß. RARgamma cannot directly regulate islet-1/2 but can down-regulate RARbeta expression, which results in loss of islet-1 expression. Activated RARalpha is one of the final steps required for a mature motoneuron phenotype (Goncalves, 2005).

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