Proc. Nadl. Acad. Sci. USAVol. 91, pp. 3067-3071, April 1994Developmental Biology

The retinoid X receptor ligand, 9-ci8-retinoic acid, is a potentialregulator of early Xenopus developmentJOAN CREECH KRAFT*, TIM SCHUHt, MONT JUCHAU*, AND DAVID KIMELMANtDepartments of *Pharmacology SJ-30 and tBiochemistry SJ-70, School of Medicine, University of Washington, Seattle, WA 98195

Communicated by Marc W. Kirschner, November 24, 1993

ABSTRACT Endogenous retinoids are potential regula-tors of vertebrate embryogenesis that have been implicated inearly anterior-posterior patterning and limb-bud develop-ment. We have characterized the temporal and spatial distri-bution of 9-cis-retinoic acid in the Xenopus embryo and com-pared it to two other retinoids, afl-tmns-retinoic acid andalI-frns-retinoyl-3glucuronide. 9-cis-Retinoic acid is first de-tected after the midblastula transition and by the end ofgastruation is localz primarily within the anterior andposterior dorsal regions ofthe embryo. Since 9-cis-retinoic acidis a 6-fold more potent dysmorphogen than trans-retinoic acid,we suggest that it is involved in the early specification of theXenopus anterior-posterior aids.

The organization of the vertebrate body plan is dependentupon communication between groups of cells in the earlyembryo. It has been proposed that all-trans-retinoic acid(trans-RA), a metabolite of retinol (vitamin A) and a memberof a class ofcompounds known as retinoids, functions as oneof the molecular signals in early vertebrate development(1-9). In support of this proposal, experiments have shownthat trans-RA affects cellular differentiation (10) and thatembryonic exposure to trans-RA causes severe malforma-tions in a number ofvertebrate species (11-13). Furthermore,retinoid deficiency during embryonic development also pro-duces malformations (14, 15). The cellular effects of RAisomers appear to be mediated by two distinct classes ofreceptors within the steroid-thyroid nuclear receptor super-family, the RA receptors (RARs) and the retinoid X receptors(RXRs), which presumably function by altering the transcrip-tion of specific target genes (16). In addition, cellular RA andretinol binding proteins (CRABPs and CRBPs) appear tomodulate the localization, availability, and metabolism ofthese retinoids within the organism (17).

Despite the ability of exogenous all-trans-RA to causemajor alterations to the anterior-posterior (A-P) axis of mice(2, 3) and frogs (1, 4-6), evidence that endogenous retinoidsare important modulators of early development has beendifficult to obtain. Indirect evidence that retinoids are presentin restricted regions ofthe embryo has been obtained from invivo studies with transgenic mice (18-21) and in vitro studieswith rat (22) and chicken (23) tissue explants. Chickenembryos also exhibit regional differences in the capacity toconvert retinol to trans-RA (24), with the maximum activityfound in Hensen's node. Since these studies have not directlyidentified the specific retinoids in the early embryo or mea-sured their spatial distribution during early embryonic stages,we have addressed these questions using the amphibianembryo. The results from these studies indicate that a num-ber of retinoids were present in the early amphibian embryoand that the levels of three of these retinoids vary bothspatially and temporally in early development. Notably,9-cis-RA was first detected after the midblastula transition

and at the end of gastrulation was asymmetrically distributedalong both the A-P and dorsal-ventral (D-V) axes. In addi-tion, 9-cis-RA was a 6-fold more potent dysmorphogen thanall-trans-RA. These results suggest that 9-cis-RA is a likelyendogenous regulator of early Xenopus development.

MATERIALS AND METHODSRetinoid Extraction. All procedures were carried out using

amber light. One hundred Xenopus embryos (=100-120 mgof wet weight) from the designated stages of developmentwere collected into a vial and rapidly frozen. To eliminatevariability between different frogs in our outbred colony, allembryos for a single experiment were collected from a singlemother. Immediately prior to HPLC analysis,- 100 ,P ofisopropyl alcohol was added to the embryos and they werethen sonicated on ice. The homogenate was dispersed in aVortex for 1 min and centrifuged at 40C for 20 min at 4000 xg. The supernatant was injected directly into the HPLCsystem.HPLC Analysis. A binary gradient system allowed separa-

tion of 10 retinoids within 20 min (25). A precolumn (20 x 4mm cartridge prepacked with Lichrosorb RB 18 10 pum) andan analytical column (125 x 4.6 mm slurry packed withSpherisorb ODS II 3 pkm) were used. The mobile phase wasa mixture of 40 mM ammonium acetate (pH 7.3) and meth-anol. The gradient was formed by mixing these solvents suchthat the initial methanol concentration was 55%, reaching100%o at the termination of the run. The flow rate was 1.6ml/min and the eluate was monitored at 354 nm. Overallrecovery was quantitative allowing for external standardiza-tion (25). Each HPLC analysis was run in duplicate. Run-to-run variation was =z2% (26).

Retinoid Treatment of Embryos. Embryos were treatedwith 9-cis-RA in 0.1 x MMR for 30 min at 210C in the dark (lxMMR = 0.1 M NaCl/2 mM KCl/1 mM MgSO4/2 mMCaCl2/5 mM Hepes, pH 7.8/0.1 mM EDTA). Embryos wererinsed with 0.1 x MMR (five times) and allowed to develop atroom temperature.RNase Protection Assays. Ten animal cap explants isolated

from stage 9 embryos (27) were cultured in 1x MMR plus theindicated concentrations of retinoids for 7 hr at 210C in thedark. The animal pole cell explants were harvested and RNAwas isolated and analyzed by RNase protection assay (28)using a 254-bp antisense transcript from the Xlim-1 gene (29)(Pst I-Ssp I fragment encoding amino acids 285-369) syn-thesized using T7 RNA polymerase in the presence of[32P]UTP. An EF-la probe prepared at reduced specificactivity was added to the RNase protection assays as acontrol for sample loading. RNase treatment of hybridizedtranscripts was for 1 hr at 300C with RNase A (10 pg/ml) andT1 (0.5 pg/ml), respectively. Protected RNA species were

Abbreviations: RA, retinoic acid; RAR, RA receptor; RXR, retinoidX receptor; CRABP, cellular RA binding protein; CRBP, cellularretinol binding protein; trans-RAG, all-trans-retinoyl-p-glucuronide;A-P, anterior-posterior; D-V, dorsal-ventral.

3067

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3068 Developmental Biology: Creech Kraft et al.

detected by autoradiography of dried gels exposed for 2-4days with an intensifying screen.

RESULTS AND DISCUSSIONPreliminary analyses utilizing a sensitive HPLC detectionsystem that measures the levels of 10 natural retinoids (Fig.1A) (25, 26), reconfirmed the presence of trans-RA in Xeno-pus embryos and indicated the presence of 9-cis-RA andall-trans-retinoyl-3,-glucuronide (trans-RAG) in these em-

A

C

It

0

Bab d hi

5 10 15 20 0 5Retention time, min

hi

def

c

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bryos, as shown by coelution with external (Fig. 1 A and B)and internal standards (not shown). Other retinoid metabo-lites were also observed (Fig. 1B). The presence of 9-cis-RAin the embryo was of particular interest, since it has beenrecently identified as the ligand ofthe RXR (30, 31) and sincetranscripts encoding RXRs have been found in the earlyXenopus embryo (32).To further identify 9-cis-RA, peak e (Fig. 1B) was collected

from several HPLC runs of retinoids extracted from stage 12Xenopus embryos and chemically derivatized to the methylester. Rechromatography showed that the derivatized peak e(Fig. ID, peak j) comigrated with authentic 9-cis-RA methylester (Fig. 1C, peak j). The concentration of the derivatized9-cis-RA from Xenopus embryos was 60-fold higher than thelimit of detection for this HPLC system (25). To furtheridentify trans-RAG, retinoids extracted from unfertilizedXenopus eggs were analyzed using HPLC, before and aftertreatment with the enzyme (-glucuronidase for 30 min at37TC. Peak c (Fig. 1B) was reduced significantly followingtreatment with (-glucuronidase, whereas all-trans-RA in-creased (not shown).Exogenous trans-RA causes dysmorphogenic effects in

Xenopus embryos in the period between the start of zygotic

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FIG. 1. Analysis of retinoids in early Xenopus embryos. (A)Chromatogram of reversed-phase HPLC separation of a mixture ofauthentic retinoid standards. The retinoid standards showed identi-cal retention times when added to Xenopus embryo extracts. Peaks:a, 4-oxo-all-trans-RA (740 ng/ml); b, 4-oxo-13-cis-RA (650 ng/ml); c,all-trans-RAG (180 ng/ml); d, 13-cis-RA (580 ng/ml); e, 9-cis-RA(410 ng/ml); f, all-trans-RA (390 ng/ml); g, didehydroretinol (25ng/ml); h, retinol (490 ng/ml); i, retinal (666 ng/ml). Sensitivity: 0.02absorbance units full scale (AUFS). The limit of detection in thissystem is 2 ng/ml or g ofwet weight with a sample size of 0.1 ml (25).(B) Sample chromatogram ofHPLC analysis of 100Xenopus stage 12embryos. Retinoids were extracted from rapidly frozen Xenopusembryos and analyzed by HPLC. Peak c coeluted with authenticall-trans-RAG (35 ng/g); peak d coeluted with authentic 13-cis-RA(138 ng/g); peak e coeluted with the authentic 9-cis-RA (220 ng/g);peak f coeluted with authentic all-trans-RA (136 ng/g); peak gcoeluted with authentic didehydroretinol (108 ng/g); peak h coelutedwith authentic retinol (520 ng/g); peak i coeluted with authenticretinal (720 ng/g). Sensitivity: 0.0025 AUFS. (C and D) HPLCanalysis of the chemically derivatized methyl ester of authentic9-cis-RA (C) and HPLC-purified peak e from several batches of stage12 Xenopus embryos (D). Note that the methyl ester of peak e (D,peak j, 127 ng/g, 0.005 AUFS) comigrated with authentic 9-cis-RAmethyl ester (C, peak j, 1530 ng/ml, 0.02 AUFS).

60o -

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8 10 13 16 18

8 10 13 16 18

13Stage

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FIG. 2. Developmental changes in the levels of all-trans RA (A),9-cis-RA (B), and all-trans RAG (C). Stage 8, midblastula; stage 10,early gastrula; stage 13, early neurula; stage 16, mid-neurula; stage18, late neurula. Each point represents the mean from three to sixindependent experiments; error bars indicate the SEM. Concentra-tions are expressed as ng/g of wet weight.

J

Proc. NatL Acad. Sci. USA 91 (1994)

Developmental Biology: Creech Kraft et al.

transcription (stage 8) and the end of neurulation (stage 18),and it has been proposed to play a role in pattern formationduring this period of development (1, 4-6). To determinewhether the levels of 9-cis-RA, trans-RA, and trans-RAGvaried during this period, the levels of these three retinoidswere measured at five stages between stage 8 and stage 18.During these early embryonic stages, trans-RA levels re-mained relatively constant, while trans-RAG levels contin-ually decreased. In contrast, 9-cis-RA levels increased afterthe start of zygotic transcription and peaked at the end ofgastrulation (stage 13; Fig. 2).

If retinoids act as endogenous morphogens, we expectedthat they would be present in restricted regions ofthe embryo

Stage 1 0trans-RA trans-RAG

DORSAL

VENTRAL

Stage 13

DORSAL

VENTRAL

Stage 18

DORSAL

Proc. Natl. Acad. Sci. USA 91 (1994) 3069

during the stages that exogenous trans-RA can promotedysmorphogenesis. To determine whether the retinoids wereasymmetrically distributed along the D-V axis, embryoswere dissected into dorsal and ventral halves and the levelsof endogenous retinoids were measured. At the start ofgastrulation (stage 10) there was 3.5-fold more 9-cis-RAdorsally than ventrally and by the end of neurulation it waspredominantly localized dorsally (Fig. 3). trans-RA andtrans-RAG were also asymmetrically distributed along theD-V axis during these stages, but by the end of neurulationthey were found mostly in the ventral region (Fig. 3).

Stage 13

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FIG. 3. Relative levels of retinoids in dorsal (Dors) and ventral(Vent) halves of gastrula and neurula stage embryos. The vitellinemembranes were manually removed from 200 embryos at the des-ignated stages (stage 10, early gastrula; stage 13, early neurula; stage18, late neurula) and embryos were dissected into dorsal and ventralhalves. Retinoids were extracted from the combined dorsal or ventralhalves and analyzed by HPLC. The results were corrected for totalprotein in the extracted sections to control for variations in tissuemass from different regions of the embryo. The data presented foreach experiment at each stage were obtained from embryos from asingle mother. The levels of each retinoid at each stage werenormalized to 100%16 to account for differences in retinoid levels inembryos from different mothers. Data presented are the means fromtwo experiments (stages 13 and 18) or three experiments (stage 10).Error bars represent SEM for stage 10 or the range ofvalues from themean for stages 13 and 18.

I

I.,NMD !

I

ANT!I MID!I POST

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FIG. 4. Relative levels of retinoids along the A-P axis of neurulastage embryos. The vitelline membranes were manually removedfrom 300 embryos at the designated stages (stage 13, early neurla;stage 18, late neurula) and embryos were dissected into approxi-mately equal anterior (Ant), middle (Mid), and posterior (Post)thirds. Retinoids were extracted from the combined anterior, middle,or posterior thirds and analyzed by HPLC. The results were cor-rected for total protein in the extracted sections to control forvariations in tissue mass from different regions of the embryo. Thedata presented for each experiment at each stage were obtained fromembryos from a single mother. The levels of each retinoid at eachstage were normalized to 100%o to account for differences in retinoidlevels in embryos from different mothers. Data presented are themean from at least three experiments. Error bars indicate the SEM.

3070 Developmental Biology: Creech Kraft et al.

A....

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FIG. 5. (A) Morphology of 9-cis-RA-treated Xenopus embryos.Twelve stage 9 embryos (late blastula) were treated with variousconcentrations of 9-cis-RA and allowed to develop to the tadpolestage (3 days), at which time a representative tadpole from eachtreatment group was photographed. Top to bottom: embryos treatedwith 0 (solvent only), 0.01, 0.03, 0.09, 0.3, 0.9, 3, and 9 pM (0, 3, 9,27, 40, 270, 900, and 2700 ng/ml) 9-cis-RA. (B) Comparison ofXlim-1mRNA expression induced by 9-cis-RA and all-trans-RA inXenopusanimal cap explants. Levels ofXlim-1 RNA were measured in animalcap explants treated with the indicated retinoids for 7 hr beginning atstage 9. An EF-la probe was included to control for variations insample preparation.

The A-P axis in Xenopus is clearly established by stage 13when the major movements of gastrulation have terminated.To assess the distribution of retinoids along the A-P axis,embryos were dissected into equal thirds beginning at stage13. At this time, 9-cis-RA was found exclusively in theanterior and posterior regions (Fig. 4). By stage 18, it wasmore evenly distributed along the A-P axis. trans-RA was

found in a shallow gradient at stages 13 and 18, with thehighest levels anteriorly (Fig. 4). trans-RAG, in contrast, wasevenly distributed along the A-P axis at stage 13, but by stage18 it was present predominantly in the middle third of theembryo (Fig. 4). We note, however, that the levels ofretinoids in different regions of the embryos were morevariable at stage 18 than stage 10 or 13 (Figs. 3 and 4).

Since the distribution of 9-cis-RA suggested that it mayplay a role in embryogenesis, we examined its effects whenadded exogenously to embryos. Addition of9-cis-RA at stage9 caused concentration-dependent alterations along the A-Paxis (Fig. 5A). Embryos treated with 9-cis-RA were mor-phologically indistinguishable from embryos treated with6-fold higher concentrations of trans-RA (not shown), al-though subtle differences cannot be ruled out. At the lowestconcentration of 9-cis-RA added to the embryos (3 ng/ml),only minor defects in the anterior region were observed. Thisconcentration of 9-cis-RA was equivalent to the endogenouslevel of 9-cis-RA at stage 9 (Fig. 2), the time at which theexogenous retinoids were added. With increasing levels ofexogenous 9-cis-RA, embryos failed to develop cementglands, eyes, and forebrains (Fig. SA). When levels ofexogenous 9-cis-RA were 100- to 1000-fold the endogenousstage 9 level, truncations of posterior structures were alsoobserved (Fig. SA). To directly assess the differences be-tween 9-cis-RA and trans-RA, expression of the homeo6oxgene Xlim-1 (29) was measured in animal cap explants. TheRA-mediated induction of Xlim-1 in animal caps may berelated to its normal induction in the organizer region duringgastrulation and in the anterior regions during neurulation(29). -Consistent with its dysmorphogenic properties,9-cis-RA induced the expression of Xlim-1 at 6-fold lowerconcentrations than trans-RA (Fig. SB). The rise in levels of9-cis-RA during the period in which the A-P axis specifica-tion has been proposed to occur (33), combined with itslocalized distribution at the end of gastrulation and its potent

dysmorphogenic effects, suggests that endogenous 9-cis-RAis an important regulator of early Xenopus development.The early Xenopus embryo contains transcripts encoding

RARs and RXRs (32, 34), consistent with the ability of bothtrans-RA and 9-cis-RA to alter the development of the A-Paxis. Although the locations of all the Xenopus RARs andRXRs have not yet been described, Xenopus RARy isrestricted to the anterior and posterior regions of gastrula andneurula stage embryos (34). We have now shown that theligands for these receptors are also asymmetrically distrib-uted and appear to be tightly controlled during early embryo-genesis. Since different RARs and RXRs respond preferen-tially to various ligands (30, 31, 35, 36) and exhibit differentialpromoter-specific trans-activating capabilities (16, 37), earlyaxial specification may occur through local interactions be-tween various retinoids and their receptors. This may occurthrough localized synthesis of various retinoids and/or lo-calized concentrations of specific cellular retinoid bindingproteins that are responsible for the restricted distribution of9-cis-RA and trans-RA during early development.

It was surprising to find that the highest endogenous levelsof 9-cis-RA were found in the regions of the embryo that arethe most sensitive to treatment with exogenous retinoids-namely, the future anterior and posterior ends. Three expla-nations might account for these results. (i) The retinoidreceptors might be predominantly localized in the anteriorand posterior ends of the embryo, making these regions themost sensitive to exogenous retinoids. Only the distributionof the Xenopus RARy receptor has been described, and thisis found at both ends of the embryo (34). (ii) The anterior andposterior regions of the embryo may be most affected by theaddition of retinoids, since the endogenous concentration of9-cis-RA is the highest at the ends of the embryo and perhapsclosest to a tightly regulated threshold level. It is interestingto note that the anterior end of the embryo has a highercombined level of 9-cis-RA and trans-RA than the posteriorend and is more sensitive to perturbation by exogenousretinoids than is the posterior region (5, 38). (iii) The terato-genicity of exogenous retinoids may be due in part to apremature activation of the retinoid receptors. Resistance tothe teratogenic effects of trans-RA is acquired graduallyduring the gastrula and neurula stages (5, 38). Similarly, thelevels of 9-cis-RA gradually increase during the gastrulastages. Treatment ofembryos with exogenous retinoids at thebeginning of gastrulation may activate the expression ofRA-dependent genes prior to their normal time of activation,resulting in developmental aberrations.

It is important to note that exogenous retinoids are terato-genic only when applied at higher concentrations than theendogenous levels observed here. The embryo appears tocarefully regulate the levels and distribution of each retinoidso that they can function without causing abnormal effects.Although the teratogenic effects ofexogenous retinoids haveoften been thought to be due to a mislocalization of theretinoid receptor signal, it may equally well be true that thedysmorphogenic effects are due to an excess of retinoids inthe region in which they are normally used and localized.These views will eventually be reconciled as more becomesknown about the distribution of the retinoids and theirreceptors during embryogenesis.

J.C.K. and T.M. contributed equally to this work. We thank JackSaari for his advice during this project, Hoffmann-La Roche forsupplying retinoid standards, and James A. Olson for providing transRAG. We gratefully acknowledge Steve Hauschka, Randall Moon,and Jack Saari for critically reviewing this manuscript. This workwas supported by grants from the National Institutes of Health (toM.J.) and the March of Dimes (to D.K.). D.K. is a Scholar of theSearle Community Trust.

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