Excess Retinoic Acid Research Articles

Retinoic acid, midkine, and defects of secondary neurulation

Background Retinoic acid (RA) is necessary for normal differentiation of the tail bud into the secondary neural tube. Excess RA, however, is teratogenic and causes neural tube defects (NTDs). The way in which RA modulates secondary neurulation is unclear but probably involves RA-regulated downstream genes such midkine (MK), which encodes a growth factor implicated in tail bud mesenchymal-neuroepithelial conversion. Our objective was to determine whether RA-deficiency would produce similar defects and if MK is involved. Methods Citral, a drug that blocks endogenous RA formation, as well as a neutralizing antibody, were used to block RA activity in chick embryos. Immunohistochemistry and in situ hybridization were used to localize RA and MK in the tail bud. Competitive RT-PCR was used to examine the effects of excess RA and RA deficiency due to citral on the expression of MK mRNA. Results Citral-induced NTDs displayed a morphological resemblance to those caused by excess RA. However, citral treatment did not significantly increase embryonic mortality, and RA rescue of citral-treated embryos proved unsuccessful. MK mRNA was detected in the differentiating tail bud by in situ hybridization. Competitive RT-PCR showed that excess RA decreased MK expression by 60%. Doses of citral that caused a comparable incidence of defects, however, caused only a 25% decrease. Conclusions The results show that excess RA and RA deficiency both cause defects of secondary neurulation. While excess RA decreased MK expression, RA deficiency had minimal effects. However, whether or not MK is an intermediary in the developmental phenomena regulated physiologically or pathologically by RA remains to be elucidated. Teratology 62:123–133, 2000. © 2000 Wiley-Liss, Inc.

Characterization of Retinoic Acid Receptor-deficient Keratinocytes

Retinoids are essential for normal epidermal growth and differentiation and show potential for the prevention or treatment of various epithelial neoplasms. The retinoic acid receptors (RARalpha, -beta, and -gamma) are transducers of the retinoid signal. The epidermis expresses RARgamma and RARalpha, both of which are potential mediators of the effects of retinoids in the epidermis. To further investigate the role(s) of these receptors, we derived transformed keratinocyte lines from wild-type, RARalpha, RARgamma, and RARalphagamma null mice and investigated their response to retinoids, including growth inhibition, markers of growth and differentiation, and AP-1 activity. Our results indicate that RARgamma is the principle receptor contributing to all-trans-retinoic acid (RA)-mediated growth arrest in this system. This effect partially correlated with inhibition of AP-1 activity. In the absence of RARs, the synthetic retinoid N-(4-hydroxyphenyl)-retinamide inhibited growth; this was not observed with RA, 9-cis RA, or the synthetic retinoid (E)-4-[2-(5, 5, 8, 8 tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl] benzoic acid. Finally, both RARalpha and RARgamma differently affected the expression of some genes, suggesting both specific and overlapping roles for the RARs in keratinocytes.

Retinoic acid, midkine, and defects of secondary neurulation.

Retinoic acid (RA) is necessary for normal differentiation of the tail bud into the secondary neural tube. Excess RA, however, is teratogenic and causes neural tube defects (NTDs). The way in which RA modulates secondary neurulation is unclear but probably involves RA-regulated downstream genes such midkine (MK), which encodes a growth factor implicated in tail bud mesenchymal-neuroepithelial conversion. Our objective was to determine whether RA-deficiency would produce similar defects and if MK is involved. Citral, a drug that blocks endogenous RA formation, as well as a neutralizing antibody, were used to block RA activity in chick embryos. Immunohistochemistry and in situ hybridization were used to localize RA and MK in the tail bud. Competitive RT-PCR was used to examine the effects of excess RA and RA deficiency due to citral on the expression of MK mRNA. Citral-induced NTDs displayed a morphological resemblance to those caused by excess RA. However, citral treatment did not significantly increase embryonic mortality, and RA rescue of citral-treated embryos proved unsuccessful. MK mRNA was detected in the differentiating tail bud by in situ hybridization. Competitive RT-PCR showed that excess RA decreased MK expression by 60%. Doses of citral that caused a comparable incidence of defects, however, caused only a 25% decrease. The results show that excess RA and RA deficiency both cause defects of secondary neurulation. While excess RA decreased MK expression, RA deficiency had minimal effects. However, whether or not MK is an intermediary in the developmental phenomena regulated physiologically or pathologically by RA remains to be elucidated.

A Role for GATA-4/5/6 in the Regulation of Nkx2.5 Expression with Implications for Patterning of the Precardiac Field

Interactions between the key regulatory genes of the cardiogenic pathway, including those from the GATA and Nkx2 transcription factor families, are not well defined. Treating neurula-stage Xenopus embryos with retinoic acid (RA) causes a specific block in cardiomyocyte development that correlates with a progressive reduction in the region of the presumptive heart-forming region expressing Nkx2.5. In contrast, RA does not block expression of the GATA-4/5/6 genes, which are transcribed normally in an overlapping pattern with Nkx2.5 throughout cardiogenesis. Instead, GATA-4/5/6 transcription levels are increased, including an expansion of the expression domain corresponding to lateral plate mesoderm that is part of the early heart field, but that normally is progressively restricted in its ability to contribute to the myocardium. GATA-dependent regulatory sequences of the Nkx2.5 gene that implicate GATA-4/5/6 as upstream positive regulators were described recently. However, our experiments also indicate that GATA factors might normally antagonize transcription of Nkx2.5. To test this hypothesis we generated a dominant negative isoform of GATA-4 (SRG4) capable of inhibiting transcription of GATA-dependent target genes. Ectopic expression of SRG4 results in a transient expansion of the Nkx2.5 transcript pattern, indicating that a normal function of GATA factors is to limit the boundary of the Nkx2.5 expression domain to the most anterior ventral region of the heart field. Regulatory mechanisms altered by excess RA must function normally to limit GATA-4/5/6 expression levels, to define the region of Nkx2.5 expression and regulate myocardial differentiation.

Retinoic Acid Affects Left–Right Patterning

Our understanding of the means by which the left–right axis is patterned is not fully understood, although a number of key intermediaries have been recently described. We report here that retinoic acid (RA) excess affects heart situs concomitant with alterations in the expression of genes implicated in the establishment of the left–right axis. Specifically, RA exposure during a specific developmental window evoked bilateral expression of lefty-1, lefty-2, nodal, and pitx-2 in the lateral plate mesoderm. Time course experiments, together with analysis of midline markers, suggest that nascent mesoderm constitutes a predominant RA target involved in this process. These events are likely to underlie the perturbations of heart looping provoked by excess RA and suggest a means by which retinoids influence the early steps in establishment of the left–right embryonic axis.

Retinoic acid is required in the mouse embryo for left-right asymmetry determination and heart morphogenesis.

Determination of the left-right position (situs) of visceral organs involves lefty, nodal and Pitx2 genes that are specifically expressed on the left side of the embryo. We demonstrate that the expression of these genes is prevented by the addition of a retinoic acid receptor pan-antagonist to cultured headfold stage mouse embryos, whereas addition of excess retinoic acid leads to their symmetrical expression. Interestingly, both treatments lead to randomization of heart looping and to defects in heart anteroposterior patterning. A time course analysis indicates that only the newly formed mesoderm at the headfold-presomite stage is competent for these retinoid effects. We conclude that retinoic acid, the active derivative of vitamin A, is essential for heart situs determination and morphogenesis.

Postnatal effects of retinoic acid on cerebellar development.

Retinoic acid(RA) is a potent teratogen to which the early CNS is known to be highly sensitive. However, very little is know about the postnatal effects of RA. The cerebellum is a candidate for postnatal RA toxicity, as it develops late and exhibits temporal patterns of RA synthesis that are synchronized with developmental stages. Northern blotting shows that the cerebellum expresses receptors for RA, predominantly of the RXR group activated by 9-cis RA. To determine whether development can be disrupted by RA excess, newborn rats were injected with RA at a time when endogenous RA levels are normally low. Histological examinations at postnatal day 14 revealed loss of a subpopulation of proliferating granule cells, and adult cerebella showed clusters of ectopic granule cells located in the molecular layer. Thus, although the granule cells may normally be regulated by endogenous RA, they are sensitive to abnormally high RA concentrations.

A molecular basis for retinoic acid-induced axial truncation.

Dietary deprivation and gene disruption studies clearly demonstrate that biologically active retinoids, such as retinoic acid (RA), are essential for numerous developmental programs. Similar ontogenic processes are also affected by retinoic acid excess, suggesting that the effects of retinoid administration reflect normal retinoid-dependent events. In the mouse, exogenous retinoic acid can induce both anterior (anencephaly, exencephaly) and posterior (spina bifida) neural tube defects depending on the developmental stage of treatment. Retinoic acid receptor gamma (RARgamma) mediates these effects on the caudal neural tube at 8.5 days postcoitum, as RARgamma-/- mice are completely resistant to spina bifida induced by retinoic acid at this stage. We therefore used this null mouse as a model to examine the molecular nature of retinoid-induced caudal neural tube defects by using a panel of informative markers and comparing their expression between retinoic acid-treated wild-type and RARgamma-/- embryos. Our findings indicate that treatment of wild-type embryos led to a rapid and significant decrease in the caudal expression of all mesodermal markers examined (e.g., brachyury, wnt-3a, cdx-4), whereas somite, neuroepithelial, notochord, floorplate, and hindgut markers were unaffected. RARgamma-/- mutants exhibited normal expression patterns for all markers examined, consistent with the notion that mesodermal defects underlie the etiology of retinoid-induced spina bifida. We also found that posterior somitic, but not caudal presomitic, embryonic tissues contained detectable bioactive retinoids, an observation which correlated with the ability of caudal explants to rapidly clear exogenous RA. Interestingly, transcripts encoding mP450RAI, a cytochrome P450, the product of which is believed to catabolize retinoic acid, were abundant in the retinoid-poor region of the caudal embryo. mP450RAI was rapidly induced by retinoic acid treatment in vivo, consistent with previous studies suggesting that it plays a critical role in retinoid signaling. These data suggest that nascent mesoderm is highly sensitive to retinoic acid and that mP450RAI serves to tightly regulate retinoid levels in the caudal embryo. These findings also raise the possibility that RA may play a role in the generation of posterior mesoderm derivatives in part by affecting brachyury expression.

Retinoic acid disrupts anterior ectodermal and endodermal development in ascidian larvae and postlarvae.

In vertebrates, excess all-trans retinoic acid (RA) applied during axis formation leads to the apparent truncation of anterior structures. In this study we sought to determine the type of defects caused by ectopic RA on the development of the ascidian Herdmania curvata. We demonstrate that H. curvata embryos cultured in the presence of RA develop into larvae whose trunks are shortened and superficially resemble those of early metamorphosing postlarvae. Despite RA-treated larvae lacking papillar structures they respond normally to natural cues that induce metamorphosis, indicating that chemosensory functionality previously mapped to the most anterior region of normal larvae is unaffected by RA. Excess RA applied during postlarval development leads to a graded loss of the juvenile pharynx, apparently by respecifying anterior endoderm to a more posterior fate. This structure is considered homologous to the gill slits of amphioxus, which are also lost upon RA treatment. This suggests that RA may have had a role in the development of the pharynx of the ancestral chordate and that this function has been maintained in ascidians and cephalochordates and lost in vertebrates.

Metabolic inactivation of retinoic acid by a novel P450 differentially expressed in developing mouse embryos.

Retinoic acid (RA) is a physiological agent that has a wide range of biological activity and appears to regulate developmental programs of vertebrates. However, little is known about the molecular basis of its metabolism. Here we have identified a novel cytochrome P450 (P450RA) that specifically metabolizes RA. In vitro, P450RA converts all-trans RA into 5,8-epoxy all-trans RA. P450RA metabolizes other biologically active RAs such as 9-cis RA and 13-cis RA, but fails to metabolize their precursors, retinol and retinal. Overexpression of P450RA in cell culture renders the cells hyposensitive to all-trans RA. These functional tests in vitro and in vivo indicate that P450RA inactivates RA. The P450RA gene is not expressed uniformly but in a stage- and region-specific fashion during mouse development. The major expression domains in developing embryos include the posterior neural plate and neural crest cells for cranial ganglia. The expression of P450RA, however, is not necessarily inducible by excess RA. These results suggest that P450RA regulates the intracellular level of RA and may be involved in setting up the uneven distribution of active RA in mammalian embryos.

Synergistic effect of retinoic acid on Sn-PP mediated suppression of heme oxygenase activity in vivo in rats

The present investigation is designed to probe the influence of excess retinoic acid (50,000 I.U.) and Sn-protoporphyrin (50 μmol/kg bw) along with retinoic acid on the activity patterns of the rate-limiting enzyme, heme oxygenase, of the heme catabolic pathway in the liver, spleen, kidney, brain, heart, and lung of male Wistar rats. Our results are noteworthy as SnPP is being used for the amelioration and management of hyperbilirubinemia, and they emphasize that the combined dosing of retinoic acid and SnPP attenuates the suppression of the activity of HMOX, thereby decreasing plasma bilirubin levels. The features of action of retinoic acid and SnPP together in vivo, i.e., a substantial suppression of the formation of a potentially neurotoxic metabolite, bilirubin, and the enhancement of disposal of the untransformed substrate (heme) of the enzyme that is inhibited, define some of the prerequisites of a therapeutically useful formulation.

Unilateral vitelline vein ligation alters intracardiac blood flow patterns and morphogenesis in the chick embryo.

To study the role of blood flow in normal and abnormal heart development, an embryonic chicken model was developed. The effect of altered venous inflow on normal intracardiac blood flow patterns was studied by visualization of blood flow with India ink. At stage 17, India ink was injected into a capillary or small venule within a specific yolk sac region. After determination of the normal intracardiac flow pattern, the right lateral vitelline vein was ligated, and the new intracardiac flow pattern was studied. Ligation resulted in disturbance of normal intracardiac flow patterns, which was most obvious in the conotruncus. The long-term effect of these abnormal intracardiac flow patterns on the development of the heart and pharyngeal arch arteries was investigated by permanent ligation in ovo with a microclip at stage 17 and subsequent evaluation at stages 34, 37, and 45. These experiments revealed anomalies of the vascular system in 58 of the 91 ligated embryos studied. We observed intracardiac malformations consisting of subaortic ventricular septal defects (n = 52), semilunar valve anomalies (n = 19), atrioventricular anomalies (n = 7), and pharyngeal arch artery malformations (n = 32). It is concluded that abnormal intracardiac blood flow, resulting from hampered venous inflow, may result in serious intracardiac and pharyngeal arch artery malformations comparable to defects observed in embryonic chicken models subjected to neural crest ablation, cervical flexure experiments, and excessive retinoic acid treatment.

In vivo functional analysis of the Hoxa-1 3′ retinoic acid response element (3′ RARE)

Retinoids are essential for normal development and both deficiency and excess of retinoic acid (RA) are teratogenic. Retinoic acid response elements (RAREs) have been identified in Hox gene promoters suggesting that endogenous retinoids may be involved in the direct control of Hox gene patterning functions. In order to test this hypothesis, we have mutated the Hoxa-1 3'RARE using the Cre-loxP targeting strategy, and studied its functional role during mouse development. We find that this enhancer plays an important role in the early establishment of the Hoxa-1 anterior expression boundary in the neural plate. This early disturbance in Hoxa-1 activation results in rhombomere and cranial nerve abnormalities reminiscent of those obtained in the Hoxa-1 total knockout, although their severity and penetrance are lower, thus providing strong evidence for direct control of Hox gene function by retinoids during normal development. Interestingly, we also find that the Hoxa-1 expression response to RA treatment is not entirely controlled by the RARE, suggesting the existence of other retinoid-induced factors mediating the Hoxa-1 response to RA and/or the presence of additional RAREs. Interestingly, although the RARE is not required for the spatiotemporal control of colinear expression of the Hoxa genes, it is absolutely required for correct Hoxa-2 expression in rhombomere 5.

Retinoic acid is required for the initiation of outgrowth in the chick limb bud

Background Retinoic acid (RA) is present in the chick limb bud, and excess RA induces limb duplications. Here, we have investigated the role of endogenous RA during chick limb development by preventing the synthesis of RA and testing the effect on various genes expressed during limb initiation and outgrowth.Results We demonstrate that the stage 20/21 limb bud synthesizes didehydroretinoic acid (ddRA), and that the posterior half of the limb bud synthesizes ddRA at a higher rate than the anterior half. Disulphiram inhibits this synthesis at micromolar concentrations. Administering disulphiram to embryos prior to limb bud outgrowth (stages 12–18) abolishes outgrowth, and no limb develops in the majority of cases. Disulphiram treatment also prevents the expression of Sonic hedgehog (Shh), but the expression of the fibroblast growth factor-8 gene (Fgf-8) appears as normal in the ectoderm over the prospective limb bud. The application of a bead soaked in RA can rescue Shh expression. Disulphiram treatment of later limb buds (stages 20–23) similarly down-regulates Shh, and also Fgf-4, expression, whereas the expression of Fgf-8, as at earlier stages, is initially unaffected. Again, RA can rescue the expression of Shh in these limb buds.Conclusions RA, in conjunction with Fgf-8, may be needed for the induction of the chick limb bud and the induction of Shh and Fgf-4 expression. The expression of Shh and Fgf-4 remains dependent upon the continued synthesis of RA within the limb bud. Didehydroretinoic acid is the major active retinoid in the stage 20 chick limb bud.

Expression of AmphiHox-1 and AmphiPax-1 in amphioxus embryos treated with retinoic acid: insights into evolution and patterning of the chordate nerve cord and pharynx

Excess all-trans retinoic acid (RA) causes severe craniofacial malformations in vertebrate embryos: pharyngeal arches are fused or absent, and a rostrad expansion of Hoxb-1 expression in the hindbrain shows that anterior rhombomeres are homeotically respecified to a more posterior identity. As a corollary, neural crest migration into the pharyngeal arches is abnormal. We administered excess RA to developing amphioxus, the closest invertebrate relative of the vertebrates and thus a key organism for understanding evolution of the vertebrate body plan. In normal amphioxus, the nerve cord has only a slight anterior swelling, the cerebral vesicle, and apparently lacks migratory neural crest. Nevertheless, excess RA similarly affects amphioxus and vertebrates. The expression domain of AmphiHox-1 (homologous to mouse Hoxb-1) in the amphioxus nerve cord is also extended anteriorly. For both the amphioxus and mouse genes, excess RA causes either (1) continuous expression throughout the preotic hindbrain (mouse) and from the level of somite 7 to the anterior end of the nerve cord (amphioxus) or (2) discontinuous expression with a gap in rhombomere 3 (mouse) and a gap at the posterior end of the cerebral vesicle (amphioxus). A comparison of these expression patterns suggests that amphioxus has a homolog of the vertebrate hindbrain, both preotic and postotic. Although RA alters the expression of AmphiHox-1 expression in the amphioxus nerve cord, it does not alter the expression of AmphiHox-1 in presomitic mesoderm or of alkali myosin light chain (AmphiMlc-alk) in somites, and the axial musculature and notochord develop normally. The most striking morphogenetic effect of RA on amphioxus larvae is the failure of mouth and gill slits to form. In vertebrates effects of excess RA on pharyngeal development have been attributed solely to the abnormal migratory patterns of Hox-expressing cranial neural crest cells. This cannot be true for amphioxus because of the lack of migratory neural crest. Furthermore, expression of Hox genes in pharyngeal tissues of amphioxus has not yet been detected. However, the absence of gill slits in RA-treated amphioxus embryos correlates with an RA-induced failure of AmphiPax-1 to become down-regulated in regions of pharyngeal endoderm that would normally fuse with the overlying ectoderm. In vertebrates, RA might similarly act via Pax-1/9, also expressed in pharyngeal endoderm, to impair pharyngeal patterning.

Retinoic Acid Differentially Up-Regulates the Gene Expression of Retinoic Acid Receptor α and γ Isoforms in Embryo and Adult Rats

The diverse biological effects of retinoic acid (RA) are exerted by its nuclear receptor-mediated gene expression. One of the two nuclear retinoic acid receptor subfamilies is composed of three subtypes of the all-trans retinoic acid receptor (RAR-α,RAR-β,and RAR-γ). Furthermore, several isoforms are generated from each of three RARs by differential promoter usage and/or alternative splicing. It is thus thought that the developmental stage-specific actions of RA are modulated through the spatio-temporal expression of the subtype and isoforms of RARs. In this study, the auto-regulation of the RAR subtypes (RAR-α total, RAR-β total and RAR-γ total) and their major isoforms (RAR-α1, α2, RAR-β1, β2 and RAR-γ1, γ2) by RA was examined by means of Northern blotting in the 11.5 day embryo and maternal tissues by administering pregnant rats with an excess of all-trans RA. The expression of RAR-β isoforms as well as the RAR-β total was auto-regulated by RA in all maternal tissues and embryos examined. The gene expression of RAR-α2, which was not affected by RA in the maternal tissues, was up-regulated in embryos, though there were no significant effects of RA on the levels of RAR-α1 and the α total in the maternal tissues and the embryos. Likewise, RA did not affect the levels of RAR-γ1 and γ total. However, unlike RAR-α2, RAR-γ2 expression was up-regulated by RA only in the maternal tissues. Thus, these results indicates that two retinoic acid receptor isoforms (RAR-α2 and RAR-γ2) are differentially auto-regulated in embryo and adult rats.

Gene expression of retinoic acid receptors, retinoid-X receptors, and cellular retinol-binding protein I in bone and its regulation by vitamin A.

We investigated the gene expression of retinoic acid (RA) receptors (RARs) and retinoid-binding proteins, and the effect of vitamin A on gene expression in the rat tibia to understand the actions of vitamin A on bone tissue. The transcripts of all three subtypes of all-trans RAR (alpha, beta, and gamma) and two of three subtypes of retinoid-X receptor (alpha and beta) were detected by Northern blotting. Among cellular retinol-binding protein I (CRBP-I) and CRBP-II and cellular retinoic acid-binding protein I and II, only the CRBP-I gene was expressed. These results indicated that in bone, the actions of vitamin A are exerted through these nuclear receptors by regulating target gene expression, and through CRBP-I by modulating the intracellular transport of vitamin A. Moreover, using rats of various retinoid status, we investigated whether the expression of target genes for vitamin A (RAR beta and CRBP-I) is regulated by retinoic acid (RA) in the adult rat tibia. The messenger RNA levels of these genes in vitamin A-deficient rats decreased to half of those in normal rats and were quickly restored (4 h) by either all-trans-RA or 9-cis-RA. Excess RA given to normal rats doubled the messenger RNA levels of these two genes. These results verified that, like other target tissues, bone is a target for vitamin A in terms of gene expression. In addition, we examined the effect of RAs on the expression of the target genes for vitamin D, because it is possible that 9-cis-RA is involved in the transcriptional control of vitamin D receptor by forming a heterodimer complex with retinoid-X receptor. The vitamin D-regulated osteopontin gene was induced 4 h after the administration of RA regardless of retinoid or vitamin D status. RA also induced osteopontin gene expression in concert with vitamin D in normal rats. Specific inhibitors of transcription showed that gene expression may be regulated by RA at the transcriptional level. Thus, the results presented here clarified at the molecular level that bone is a target organ for vitamin A in terms of gene expression.

Modulation of limb bud chondrogenesis by retinoic acid and retinoic acid receptors.

An excess of retinoic acid (RA) in the mouse embryo in utero produces hypochondrogenesis and severe limb bone deformities. Since one of the RA receptors--RAR-beta 2, is specifically induced in the limb bud cells upon treatment of embryos with teratogenic doses of RA, we investigated if this receptor played a role in teratogenesis by regulating the process of chondrogenesis. In micromass cultures of mouse limb bud mesenchymal cells, we found that a downregulation of RAR-beta 2 as well as several other RAR isoforms by supplementation of the culture medium with specific oligodeoxynucleotides stimulated chondrogenesis: cartilage nodule number, sulfated proteoglycans, and synthesis of collagen type IIB were all enhanced in a dose-dependent manner. However, only the antisense RAR-beta 2 probe efficiently prevented the strong inhibitory effects of exogenous RA on chondrogenesis in these cells. The data suggest that the RAR-RA complexes play a role in position-dependent patterning of the limb skeleton in normal development and that, in particular, RAR-beta 2 serves to prevent the mesenchymal cells from expressing their chondrogenic bias. Our results further strengthen the argument that RA-dependent elevation in RAR-beta 2 levels plays a unique role in RA-induced teratogenesis.

Retinoic acid provokes metaplasia of epithelium formed in vitro by adult human epidermal keratinocytes

A striking effect of retinoids is their ability to alter cell fate during development. The mucous metaplasia produced by treating chick embryo skin in organ culture with retinoic acid is a classical example of this property. It has been impossible so far to demonstrate that retinoids are able to provoke metaplasia of adult keratinocytes grown in vitro, although these agents have been shown to block terminal epidermal differentiation, to induce increased synthesis of mucopolysaccharides, and to induce the ectopic expression of K19 and K13 keratins. Our previous work showed that adult human epidermal keratinocytes grown on dermal equivalents at the surface of a culture medium containing physiological amounts of retinoids form a normal keratinized epidermis, while when excess retinoic acid is added to the culture medium, keratinization is prevented but stratification is not. Here we show that the distribution of tissue- and differentiation-stage-specific markers in retinoic acid-treated epithelium is similar to that of the oral mucosa. Moreover, when the excess retinoic acid is removed, a new epithelium is formed beneath the “old” one and this epithelium displays an epidermal orthokeratinized phenotype, whereas the “old” epithelium remains unchanged. This phenomenon of “partial reversibility”, as well as the mutually exclusive distribution of the markers of the two alternative routes of differentiation, demonstrate that retinoic acid is indeed able to provoke metaplasia of adult epidermal keratinocytes.

Retinoic acid regulates the development of oligodendrocyte precursor cells in vitro.

Cultures of oligodendrocyte precursor cells can be grown from brain hemispheres of newborn rats. These cells, also called O-2A progenitor cells, can differentiate in vitro into oligodendrocytes or type 2 astrocytes. Basic FGF and PDGF are known to stimulate their proliferation and delay their differentiation. Lack or excess of retinoic acid (RA) has been known for a long time to alter brain development suggesting that this compound is involved in normal brain development. Here we report that RA partially inhibits both the proliferation and the differentiation of oligodendrocyte precursor cells. It also down-regulates the mitogenic effect of bFGF on these cells while keeping them in an immature stage. RA is more effective than bFGF in inhibiting myelin basic protein mRNA expression in these cells, and like bFGF, it preserves their bipotential character. RA nuclear receptors RAR-alpha and their transcripts are expressed in oligodendrocyte precursor cells as seen by Western blot, Northern blot and in situ hybridization. The expression of RAR-alpha transcripts is stimulated transiently by RA alone or associated to bFGF. The expression of RAR-beta transcripts is not constitutive and is induced by RA alone or associated to bFGF and to a lesser extent by bFGF alone. These results suggest that retinoids participate in the control of the development of glial cells of the oligodendrocyte lineage.