Cloning of a novel retinoic-acid metabolizing cytochrome P450, Cyp26B1, and comparative expression analysis with Cyp26A1 during early murine development.

Tight regulation of retinoic acid (RA) distribution in the embryo is critical for normal morphogenesis. The RA-metabolizing enzymes Cyp26A1 and Cyp26B1 are believed to play important roles in protecting certain embryonic tissues from inappropriate RA signaling. We have cloned the murine Cyp26B1 cDNA and compared its expression pattern to that of Cyp26A1 from embryonic day (E) E7-E11.5 using in situ hybridization. Northern blot analysis shows the presence of two Cyp26B1 transcripts of approximately 2.3 and 3.5 kb in embryonic limb bud. Whereas Cyp26A1 is expressed in gastrulating embryos by E7, Cyp26B1 is first expressed at E8.0 in prospective rhombomeres 3 and 5. Cyp26B1 expression expands to specific dorso-ventral locations in rhombomeres 2-6 between E8.5 and E9.5, whereas Cyp26A1 hindbrain expression is limited to rhombomere 2 at E8.5. No (or very weak) Cyp26B1 expression is observed in the tail bud, a major site of Cyp26A1 expression. Differential expression is seen in branchial arches, with Cyp26A1 being mainly expressed in neural crest-derived mesenchyme, and Cyp26B1 in specific ectodermal and endodermal areas. Cyp26B1 is markedly expressed in the ectoderm and distal mesoderm of the limb buds from the beginning of their outgrowth. Cyp26A1 transcripts are seen later and at lower levels in limb ectoderm, and both transcripts are excluded from the apical ectodermal ridge.

The retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain patterning, vertebral identity, and development of posterior structures.

The active derivative of vitamin A, retinoic acid (RA), is essential for normal embryonic development. The spatio-temporal distribution of embryonic RA results from regulated expression of RA-synthesizing retinaldehyde dehydrogenases and RA-metabolizing cytochrome P450s (CYP26). Excess RA administration or RA deficiency results in a complex spectrum of embryonic abnormalities. As a first step in understanding the developmental function of RA-metabolizing enzymes, we have disrupted the murine Cyp26A1 gene. We report that Cyp26A1-null mutants die during mid-late gestation and show a number of major morphogenetic defects. Spina bifida and truncation of the tail and lumbosacral region (including abnormalities of the kidneys, urogenital tract, and hindgut) are the most conspicuous defects, leading in extreme cases to a sirenomelia ("mermaid tail") phenotype. Cyp26A1 mutants also show posterior transformations of cervical vertebrae and abnormal patterning of the rostral hindbrain, which appears to be partially posteriorly transformed. These defects correlate with two major sites of Cyp26A1 expression in the rostral neural plate and embryonic tail bud. Because all of the Cyp26A1(-/-) abnormalities closely resemble RA teratogenic effects, we postulate that the key function of CYP26A1 is to maintain specific embryonic areas in a RA-depleted state, to protect them against the deleterious effect of ectopic RA signaling.

Cytochrome P450RAI(CYP26) promoter: a distinct composite retinoic acid response element underlies the complex regulation of retinoic acid metabolism.

The catabolism of retinoic acid (RA) is an essential mechanism for restricting the exposure of specific tissues and cells to RA. We recently reported the identification of a RA-inducible cytochrome P450 [P450RAI(CYP26)], in zebrafish, mouse, and human, which was shown to be responsible for RA catabolism. P450RAI exhibits a complex spatiotemporal pattern of expression during development and is highly inducible by exogenous RA treatment in certain tissues and cell lines. Sequence analysis of the proximal upstream region of the P450RAI promoter revealed a high degree of conservation between zebrafish, mouse, and human. This region of the promoter contains a canonical retinoic acid response element (5'-AGT-TCA-(n)5-AGTTCA-3'), embedded within a 32-bp region (designated R1), which is conserved among all three species. Electrophoretic mobility shift assays using this element demonstrated the specific binding of murine retinoic acid receptor-gamma (RARgamma) and retinoid X receptor-alpha (RXRalpha) proteins. Transient transfection experiments with the mouse P450RAI promoter fused to a luciferase reporter gene showed transcriptional activation in the presence of RA in HeLa, Cos-1, and F9 wild-type cells. This activation, as well as basal promoter activity, was abolished upon mutation of the RARE. Deletion and mutational analyses of the P450RAI promoter, as well as DNase I footprinting studies, revealed potential binding sites for several other proteins in conserved regions of the promoter. Also, two conserved 5'-TAAT-3' sequences flanking the RARE were investigated for their potential importance in P450RAI promoter activity. Moreover, these studies revealed an essential requirement for a G-rich element (designated GGRE), located just upstream of the RARE, for RA inducibility. This element was demonstrated to form complexes with Sp1 and Sp3 using nuclear extracts from either murine F9 or P19 cells. Together, these results indicate that the P450RAI-RARE is atypical in that conserved flanking sequences may play a very important role in regulating RA inducibility and expression of P450RAI(CYP26).

Identification of the human cytochrome P450, P450RAI-2, which is predominantly expressed in the adult cerebellum and is responsible for all-trans-retinoic acid metabolism.

Retinoids, particularly all-trans-retinoic acid (RA), are potent regulators of cell differentiation, cell proliferation, and apoptosis. The role of all-trans-RA during development and in the maintenance of adult tissues has been well established. The control of all-trans-RA levels in cells and tissues is regulated by the balance between its biosynthesis and its catabolism to inactive metabolites. The cytochrome P450 enzyme P450RAI (herein renamed P450RAI-1) is partially responsible for this inactivation of all-trans-RA. In this report, we describe the identification, molecular cloning, and characterization of a second related enzyme, P450RAI-2, which is also involved in the specific inactivation of all-trans-RA. Transiently transfected P450RAI-2 can convert all-trans-RA to more polar metabolites including 4-oxo-, 4-OH-, and 18-OH-all-trans-RA. Competition experiments with other retinoids suggest that all-trans-RA is the preferred substrate. The high level of expression of P450RAI-2, particularly in the cerebellum and pons of human adult brain, suggests a unique role for this enzyme in the protection of specific tissues from exposure to retinoids.

Complementary domains of retinoic acid production and degradation in the early chick embryo.

Excess retinoids as well as retinoid deprivation cause abnormal development, suggesting that retinoid homeostasis is critical for proper morphogenesis. RALDH-2 and CYP26, two key enzymes that carry out retinoic acid (RA) synthesis and degradation, respectively, were cloned from the chick and show significant homology with their orthologs in other vertebrates. Expression patterns of RALDH-2 and CYP26 genes were determined in the early chick embryo by in situ hybridization. During gastrulation and neurulation RALDH-2 and CYP26 were expressed in nonoverlapping regions, with RALDH-2 transcripts localized to the presumptive presomitic and lateral plate mesoderm and CYP26 mRNA to the presumptive mid- and forebrain. The two domains of expression were separated by an approximately 300-micrometer-wide gap, encompassing the presumptive hindbrain. In the limb region, a similar spatial segregation of RALDH-2 and CYP26 expression was found at stages 14 and 15. Limb region mesoderm expressed RALDH-2, whereas the overlying limb ectoderm expressed CYP26. RA-synthesizing and -degrading enzymatic activities were measured biochemically in regions expressing RALDH-2 or CYP26. Regions expressing RALDH-2 generated RA efficiently from precursor retinal but degraded RA only inefficiently. Conversely, tissue expressing CYP26 efficiently degraded but did not synthesize RA. Localized regions of RA synthesis and degradation mediated by these two enzymes may therefore provide a mechanism to regulate RA homeostasis spatially in vertebrate embryos.

Dorsal and ventral rentinoic territories defined by retinoic acid synthesis, break-down and nuclear receptor expression.

Determination of the dorso-ventral dimension of the vertebrate retina is known to involve retinoic acid (RA), in that high RA activates expression of a ventral retinaldehyde dehydrogenase and low RA of a dorsal dehydrogenase. Here we show that 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 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 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.

Dorsal and ventral retinal territories defined by retinoic acid synthesis, break-down and nuclear receptor expression.

Determination of the dorso-ventral dimension of the vertebrate retina is known to involve retinoic acid (RA), in that high RA activates expression of a ventral retinaldehyde dehydrogenase and low RA of a dorsal dehydrogenase. Here we show that 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.

Expression and activity of vitamin D-metabolizing cytochrome P450s (CYP1alpha and CYP24) in human nonsmall cell lung carcinomas.

Extrarenal 25-hydroxyvitamin D3-1alpha-hydroxylase is believed to play a major role in the pathogenesis of hypercalcemia associated with various types of granulomatous and lymphoproliferative diseases and certain solid tumors. In this paper, we describe the cloning of the cytochrome P450 component of the extrarenal enzyme from a human nonsmall cell lung carcinoma, SW 900. The cytochrome P450 for the extrarenal 1alpha-hydroxylase has an amino acid sequence identical to that of the cytochrome P450 component of the CYP1alpha, the renal form of the enzyme, and appears to be a product of the same gene. CYP1alpha messenger RNA (mRNA) and 1alpha-hydroxylase enzyme activity were detected in two (SW 900, SK-Luci-6) of a series of five nonsmall cell lung carcinoma cell lines. All five lung cell lines were cultured with the same medium under the same conditions, but only two of the five expressed 1alpha-hydroxylase enzyme; two others (WT-E, Calu-1) expressed high levels of the reciprocally regulated enzyme, 25-hydroxyvitamin D3-24-hydroxylase, with its specific cytochrome P450 component, CYP24. Although under basal conditions the lung cell line SW 900 expressed only CYP1alpha and showed 1alpha-hydroxylase enzyme activity, when treated with small concentrations of 1alpha,25-dihydroxyvitamin D3 or high concentrations of 25-hydroxyvitamin D3, it began to express CYP24 and exhibit 24-hydroxylase enzyme activity. Somewhat surprisingly, SW 900 cells still had detectable CYP1alpha mRNA some 24 h after vitamin D treatment despite the fact that 1alpha-hydroxylase enzyme activity was unmeasurable. These data are consistent with the emerging hypothesis that vitamin D through its active form does not directly turn off CYP1alpha mRNA production but, rather, strongly stimulates CYP24, thereby masking CYP1alpha activity. The factor(s) responsible for the basal expression of CYP1alpha in SW 900 and SK-Luci-6 is currently unknown.

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 receptors regulate expression of retinoic acid 4-hydroxylase that specifically inactivates all-trans retinoic acid in human keratinocyte HaCaT cells.

Tissue levels of all-trans retinoic acid (RA) are maintained through coordinated regulation of biosynthesis and breakdown. The major pathway for all-trans RA inactivation is initiated by 4-hydroxylation. A novel cytochrome P-450 (CYP26) that catalyzes 4-hydroxylation of all-trans RA has recently been cloned. We have investigated regulation and properties of RA 4-hydroxylase in immortalized human keratinocyte HaCaT cells. In the absence of added retinoid, RA 4-hydroxylase (CYP26) mRNA and protein were minimally detected. Addition of all-trans RA rapidly induced RA 4-hydroxylase mRNA (within 2 h) and activity (within 6 h). Induction of both mRNA and activity was transient, returning to baseline within 48 h, and completely dependent on mRNA synthesis (i.e., blocked by actinomycin D). The synthetic retinoid CD367, which specifically activates nuclear RA receptors, also rapidly induced RA 4-hydroxylase activity. This induction, however, unlike that of all-trans RA, was long-lived (>48 h). This difference was attributable to lack of metabolic inactivation of CD367 in HaCaT cells. CD2665, which inhibits RA receptor-dependent gene transcription, blocked retinoid induction of RA 4-hydroxylase, indicating that it is mediated by RA receptors. Addition of excess unlabeled substrates specific for 10 distinct mammalian P-450 subfamilies did not compete with all-trans RA for RA 4-hydroxylase activity. RA 4-hydroxylase did not hydroxylate 9-cis RA or 13-cis RA. Inhibition of RA 4-hydroxylase activity by ketoconazole potentiated activation of RA receptors by all-trans RA. In summary, RA 4-hydroxylase is a unique, highly specific cytochrome P-450 isoenzyme, whose expression is regulated by its natural substrate, all-trans RA, through activation of RA receptors. RA 4-hydroxylase functions to limit the levels, and thereby the biologic activity of all-trans RA in HaCaT cells.

Human retinoic acid (RA) 4-hydroxylase (CYP26) is highly specific for all-trans-RA and can be induced through RA receptors in human breast and colon carcinoma cells.

We report on the isolation of a cytochrome P450 (CYP)-like retinoic acid (RA) 4-hydroxylase cDNA from T-47D human breast cancer cells that is identical to the recently cloned hCYP26, which is involved in the metabolic breakdown of RA. Northern analysis showed that this novel human CYP26 is induced within 1 h upon RA treatment in RA-sensitive T-47D breast carcinoma cells but not in RA-resistant MDA-MB-231 breast cancer cells and HCT 116 colon cancer cells. Stable introduction of different RA receptor (RAR) subtypes in HCT 116 cells showed that CYP26 expression is dependent on RARalpha and RARgamma and, to a lesser extent, on RARbeta and closely paralleled RA metabolism, suggesting that it represents the major RA 4-hydroxylase in these human cells. Furthermore, stable introduction of all three RAR subtypes in HCT 116 cells resulted in restored RA sensitivity as assayed by growth inhibition. Interestingly, CYP26 activity was efficiently inhibited by liarozole, an inhibitor of RA metabolism, leading to enhanced growth inhibition by RA. The RA-induced CYP26 was shown to be highly specific for the hydroxylation of all-trans-RA and did not recognize the 13-cis and 9-cis isomers. This substrate specificity is promising for finding retinoids that are not recognized by this enzyme and, therefore, could be more effective in growth inhibition of susceptible cancer cells.

Mouse P450RAI (CYP26) expression and retinoic acid-inducible retinoic acid metabolism in F9 cells are regulated by retinoic acid receptor gamma and retinoid X receptor alpha.

We have cloned a mouse cDNA homolog of P450RAI, a cytochrome P450 belonging to a new family (CYP26), which has previously been isolated from zebrafish and human cDNAs and found to encode a retinoic acid-inducible retinoic acid hydroxylase activity. The cross-species conservation of the amino acid sequence is high, particularly between the mouse and the human enzymes, in which it is over 90%. Like its human and zibrafish counterparts, the mouse P450RAI cDNA catalyzes metabolism of retinoic acid into 4-OH-retinoic acid, 4-oxo-retinoic acid, 18-OH-retinoic acid, and unidentified water-soluble metabolites when transfected into COS-1 cells. Retinoic acid-inducible retinoic acid metabolism has previously been observed in F9 murine embryonal carcinoma cells and some derivatives lacking retinoid receptors. We were interested in determining whether P450RAI could be responsible for retinoic acid metabolism in F9 cells and in studying the effect of retinoid receptor ablation on P450RAI expression. In wild-type F9 cells and derivatives lacking RAR gamma, RAR alpha, and/or RXR alpha, we observed a direct relationship between the level of retinoic acid metabolic activity and retinoic acid-induced P450RAI mRNA. These experiments, as well as others using synthetic receptor subtype-specific retinoids, suggest that the RAR gamma and RXR alpha receptors mediate the effects of retinoic acid on the expression of the P450RAI gene.

cDNA cloning of human retinoic acid-metabolizing enzyme (hP450RAI) identifies a novel family of cytochromes P450.

Retinoids, including all-trans-retinoic acid (RA) and its stereoisomer 9-cis-RA play important roles in regulating gene expression, through interactions with nuclear receptors, during embryonic development and in the maintenance of adult epithelial tissues (Chambon, P. (1995) Rec. Prog. Horm. Res. 50, 317-32; Mangelsdorf, D. J., and Evans, R. M. (1995) Cell 83, 841-850; Petkovich, M. (1992) Annu. Rev. Nutr. 12, 443-471). Evidence suggests that 4-hydroxylation of RA inside the target cell limits its biological activity and initiates a degradative process of RA leading to its eventual elimination. However, 18-hydroxylation and glucuronidation may also be important steps in this process. In this paper, we describe the cloning and characterization of the first mammalian retinoic acid-inducible retinoic acid-metabolizing cytochrome P450 (hP450RAI), which belongs to a novel class of cytochromes (CYP26). We demonstrate that hP450RAI is responsible for generation of several hydroxylated forms of RA, including 4-OH-RA, 4-oxo-RA, and 18-OH-RA. We also show that hP450RAI mRNA expression is highly induced by RA in certain human tumor cell lines and further show that RA-inducible RA metabolism may correlate with P450RAI expression. We conclude that this enzyme plays a key role in RA metabolism, functioning in a feedback loop where RA levels are controlled in an autoregulatory manner.

Aryl hydrocarbon receptor knockout mice (AHR-/-) exhibit liver retinoid accumulation and reduced retinoic acid metabolism.

Livers from aryl hydrocarbon receptor-null mice showed a 3-fold increase in retinoids and a 65% decrease in retinoic acid metabolism. Levels of expression of the retinoic acid 4-hydroxylase, P450RAI, did not change, whereas cytochrome P4501A2 levels were lower in the null mouse, as shown earlier; however, this enzyme was found not to be active toward retinoic acid. These data suggest that aryl hydrocarbon receptor controls retinoic acid catabolism, through modulation of an unidentified target gene. Aldehyde dehydrogenases 1 and 2 were down-regulated markedly in the aryl hydrocarbon receptor-deficient mouse liver. 2,3,7,8-Tetrachlorodibenzo-p-dioxin induced cytochrome P4501A2 but not the aldehyde dehydrogenases in wild-type mice, suggesting that aryl hydrocarbon receptor is not involved directly in the down-regulation of this gene. Transglutaminase II, a retinoic acid-responsive gene product, was increased 2-fold, consistent with the liver fibrosis phenotype observed in the null mice. These findings suggest a molecular connection between xenobiotic-activated receptor signaling and retinoid homeostasis.

Identification of the retinoic acid-inducible all-trans-retinoic acid 4-hydroxylase.

Retinoic acid (RA) metabolites of vitamin A are key regulators of gene expression involved in embryonic development and maintenance of epithelial tissues. The cellular effects of RA are dependent upon the complement of nuclear receptors expressed (RARs and RXRs), which transduce retinoid signals into transcriptional regulation, the presence of cellular retinoid-binding proteins (CRABP and CRBP), which may be involved in RA metabolism, and the activity of RA metabolizing enzymes. We have been using the zebrafish as a model to study these processes. To identify genes regulated by RA during exogenous RA exposure, we utilized mRNA differential display. We describe the isolation and characterization of a cDNA, P450RAI, encoding a novel member of the cytochrome P450 family. mRNA transcripts for P450RAI are expressed normally during gastrulation, and in a defined pattern in epithelial cells of the regenerating caudal fin in response to exogenous RA. In COS-1 cells transfected with the P450RAI cDNA, all-trans-RA is rapidly metabolized to more polar metabolites. We have identified 4-oxo-RA and 4-OH-RA as major metabolic products of this enzyme. P450RAI represents the first enzymatic component of RA metabolism to be isolated and characterized at the molecular level and provides key insight into regulation of retinoid homeostasis.

New retinoid X receptor subtypes in zebra fish (Danio rerio) differentially modulate transcription and do not bind 9-cis retinoic acid.

Retinoid X receptors (RXRs), along with retinoic acid (RA) receptors (RARs), mediate the effects of RA on gene expression. Three subtypes of RXRs (alpha, beta, and gamma) which bind to and are activated by the 9-cis stereoisomer of RA have been characterized. They activate gene transcription by binding to specific sites on DNA as homodimers or as heterodimers with RARs and other related nuclear receptors, including the vitamin D receptor, thyroid hormone receptors (TRs), and peroxisome proliferator-activated receptors. Two additional RXR subtypes (delta and epsilon) isolated from zebra fish cDNA libraries are described here; although both subtypes form DNA-binding heterodimers with RARs and TR, neither binds 9-cis RA, and both are transcriptionally inactive on RXR response elements. In cotransfection studies with TR, the delta subtype was found to function in a dominant negative manner, while the epsilon subtype had a slight stimulatory effect on thyroid hormone (T3)-dependent transcriptional activity. The discovery of these two novel receptors in zebra fish expands the functional repertoire of RXRs to include ligand-independent and dominant negative modulation of type II receptor function.

A zebrafish retinoic acid receptor expressed in the regenerating caudal fin.

Retinoic acid (RA) is an important signalling molecule in vertebrate pattern formation both in developing and regenerating tissues. The effects of RA are due largely to regulation of gene transcription, mediated by retinoic acid receptors (RAR-alpha, RAR-beta, RAR-gamma) and retinoid X receptors (RXR-alpha, RXR-beta, RXR-gamma). We have been using zebrafish as a model of regeneration to study the role of retinoic acid and its receptors in vertebrate pattern formation. In this report, we describe the molecular cloning and characterization of one of the zebrafish RARs that is the predominant receptor in the regenerating caudal fin and corresponds most closely to the RAR-gamma subtype isolated from mouse and human and to RAR-delta from newt. Zebrafish RAR-gamma (zfRAR-gamma) exhibits both structural and functional conservation with its mammalian counterparts. Studies utilizing both normal and regenerating caudal fins of the zebrafish have indicated that it is the RAR-gamma subtype, compared to RAR-alpha or RAR-beta, which is expressed at the highest levels in the tail fin. To localize the expression pattern of RAR-gamma during fin regeneration, we have carried out whole-mount in situ hybridization. ZfRAR-gamma transcripts, during fin regeneration, are localized in the blastemal tissue formed at the distal ends of the bony rays following amputation. Treatment of fish with RA during fin regeneration induces a number of striking morphological effects on the regenerate. When amputations are performed distal to the branch points or dichotomies, where a single ray bifurcates to extend two individual 'daughter' rays, RA treatment causes a dichotomy reduction where the two 'daughter' rays fuse to once again form a single ray. The single ray subsequently bifurcates in a comparatively normal manner. Our data suggest that exogenous RA can respecify pattern in the regenerating caudal fin and identifies the blastemae as possible RA target tissues.