PATTERNS & PHENOTYPES

Contribution of Cellular Retinol-BindingProtein Type 1 to Retinol Metabolism DuringMouse DevelopmentNicolas Matt,1 Carsten K. Schmidt,2 Valerie Dupe,1 Christine Dennefeld,1 Heinz Nau,2

Pierre Chambon,1 Manuel Mark,1 and Norbert B. Ghyselinck1*

Within cells, retinol (ROL) is bound to cytoplasmic proteins (cellular retinol-binding proteins [CRBPs]),whose proposed function is to protect it from unspecific enzymes through channeling to retinoid-metabolizing pathways. We show that, during development, ROL and retinyl ester levels are decreased inCRBP type 1 (CRBP1) -deficient embryos and fetuses by 50% and 80%, respectively. The steady state level ofretinoic acid (RA) is also decreased but to a lesser extent. However, CRBP1-null fetuses do not exhibit theabnormalities characteristic of a vitamin A-deficiency syndrome. Neither CRBP1 deficiency alters theexpression patterns of RA-responding genes during development, nor does CRBP1 availability modify theexpression of an RA-dependent gene in primary embryonic fibroblasts treated with ROL. Therefore, CRBP1is required in prenatal life to maintain normal amounts of ROL and to ensure its efficient storage but seemsof secondary importance for RA synthesis, at least under conditions of maternal vitamin A sufficiency.Developmental Dynamics 233:167–176, 2005. © 2005 Wiley-Liss, Inc.

Key words: branchial arch; Cyp26; embryo; expression pattern; eye; HPLC; knockout; RARbeta; retinoic acid; retinylester; spinal cord; vitamin A

Received 19 July 2004; Revised 18 October 2004; Accepted 19 October 2004

INTRODUCTION

Retinol (ROL) is an essential dietarycomponent necessary for vision,growth, development, reproduction,and tissue differentiation (Blomhoff etal., 1991). Within cells, ROL can beeither esterified with long chain fattyacids for storage, or oxidized to reti-naldehyde (RAL) and subsequently toretinoic acid (RA). RAL is required for

the visual process, whereas RA exertsits biological effects through bindingto nuclear retinoid receptors (RARsand RXRs). The latter are ligand-de-pendent transcriptional factors thatbelong to the superfamily of nuclearhormone receptors and control targetgene expression (Mangelsdorf andEvans, 1995; Chambon, 1996). In thecytoplasm, ROL and RAL are associ-

ated with one of the several isotypes ofcellular retinol-binding proteins(CRBPs). Each of them has a distinctexpression pattern and may play aspecific role in ROL metabolism.CRBP type 1 (CRBP1, encoded byRbp1 gene) is the most widespread,but it nevertheless displays some tis-sue specificity (Dolle et al., 1990; Ma-den et al., 1990; Ruberte et al., 1991;

1Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), Institut Clinique de la Souris (ICS), CNRS/INSERM/ULP, Collegede France, BP10142, Strasbourg, France2Department of Food Toxicology, School of Veterinary Medicine Hannover, Hannover, GermanyGrant sponsor: Centre National de la Recherche Scientifique (CNRS); Grant sponsor: Institut National de la Sante et de la RechercheMedicale (INSERM); Grant sponsor: Hopital Universitaire de Strasbourg; Grant sponsor: College de France; Grant sponsor: InstitutUniversitaire de France; Grant sponsor: European Economic Community; Grant number: EU-QLK4-CT-02-02528; Grant sponsor: Ministerede l’Education Nationale et de la Recherche and the Association de la Recherche contre le Cancer; Grant sponsor: European Commission;Grant number: Research Training Network RTN2-2001-00370/HPRN-CT-2002-00268.Dr. Schmidt’s current address is Water Technology Center (TZW), Karlsruher Strasse 84, D-76139 Karlsruhe, Germany.*Correspondence to: Norbert B. Ghyselinck, Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), InstitutClinique de la Souris (ICS), CNRS/INSERM/ULP, College de France, BP10142, 67404 Illkirch Cedex, CU de Strasbourg, France.E-mail: norbert@igbmc.u-strasbg.fr

DOI 10.1002/dvdy.20313Published online 11 March 2005 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 233:167–176, 2005

© 2005 Wiley-Liss, Inc.

Gustafson et al., 1993). The otherCRBPs have more restricted expres-sion domains: CRBP2 is expressed inthe small intestine (Levin et al., 1987;Schaefer et al., 1989), whereas mouseCRBP3 (called CRBP4 in human) isexpressed in heart, skeletal muscle,adipose tissue, and mammary gland(Vogel et al., 2001; Folli et al., 2002).

In vitro, the cellular uptake of ROLinvolves CRBP1 (Ottonello et al.,1987; Noy and Blaner, 1991;Sundaram et al., 1998) and CRBP1interacts with enzymes involved in es-terification of ROL with long chainfatty acids (Yost et al., 1988; Herr andOng, 1992) and in hydrolysis of retinylesters (RE) into ROL (Boerman andNapoli, 1991). Thus, CRBP1 is viewedas a chaperone for ROL, facilitatingits uptake, storage, and mobilization(Napoli, 2000). In vitro, CRBP1 canalso channel (1) ROL toward microso-mal dehydrogenases (e.g., RDH1,RDH2, and CRAD1) that catalyze theoxidation of ROL into RAL (Ottonelloet al., 1993; Boerman and Napoli,1996; Napoli, 2000; Lapshina et al.,2003); and (2) RAL toward cytosolicdehydrogenases (RALDHs) that oxi-dize RAL into RA (Posch et al., 1992;Napoli, 1999). The observation thatCRBP1 and RDH1 or RDH2 are coex-pressed in several adult or embryonictissues has provided support to thehypothesis that CRBP1-bound ROLmight serve as substrate for and limitRAL synthesis in vivo (Zhai et al.,1997; Båvik et al., 1997). Finally, asthe concentration of CRBP1 alwaysexceeds that of ROL in vivo (reviewed

in Napoli, 1999), free ROL shouldnever accumulate within cells butshould be protected from cytosolic al-cohol dehydrogenases (ADHs) thatuse free ROL as substrate (reviewedin Duester et al., 2003).

In the mouse, CRBP1 deficiency de-creases the capacity of hepatic stellatecells to take up incoming ROL and tomaintain RE stores, notably becauseof an accelerated rate of RE turnover,indicating that CRBP1 is essential forefficient ROL storage during postna-tal life. However, CRBP1-deficientmice did not show any morphologicaldefects related to alteration of RA ho-meostasis, at least under conditions ofdietary vitamin A sufficiency (Ghy-selinck et al., 1999). To account forthis observation, it was hypothesizedthat the lack of CRBP1 might increaseRA synthesis, through increasing theamount of free ROL available for non-specific ADHs, without inducingsymptoms of retinoid toxicity (Napoli,2000). To gain further insight into thefunctions of CRBP1 during develop-ment, we have analyzed the retinoidcontent in wild-type (WT) and CRBP1-null (Rbp1�/�) embryos and fetuses.We thought that, if CRBP1 signifi-cantly contributes to RA synthesis invivo, then expression of RA-targetgenes should be altered in Rbp1�/�

embryos and fetuses, in areas corre-sponding to tissues normally express-ing CRBP1. Therefore, we have exam-ined the ability of Rbp1�/� embryoniccells and tissues to express selectedRA-target genes. Altogether, our re-sults indicate that CRBP1 deficiency

dramatically reduces ROL storageand slightly decreases the RA steadystate level, but without altering orga-nogenesis and RA-dependent gene ex-pression.

RESULTS

Altered Retinol Storage inRbp1-Null Mutants

We have reported previously thatROL and RE levels are lower in theliver of Rbp1�/� fetuses than in WTat embryonic day (E) 16.5 (Ghyselincket al., 1999). To investigate the effectof CRBP1 deficiency on ROL metabo-lism at stages earlier than E16.5, wehave quantified by high performanceliquid chromatography (HPLC) theretinoid content in embryos and fe-tuses between E10.5 and E14.5. InWT, the level of ROL remained con-stant, at approximately 300 pmol/g oftissue, throughout development. Incontrast, RE accumulated from 25 to1,400 pmol/g of tissue between E10.5and E14.5, whereas RA levels progres-sively decreased from 40 to 20 pmol/gof tissue during the same period (Ta-ble 1). It is noteworthy that 9cis-RA,the putative RXR ligand (reviewed inMangelsdorf and Evans, 1995), wasnever detected, although the HPLCprocedure was sensitive enough to de-tect 1.5 pmol/g of tissue (Schmidt etal., 2003). In E10.5 Rbp1�/� embryos,ROL and RE levels were reduced byapproximately 25% and 40%, respec-tively. At later stages (E12.5 toE14.5), Rbp1�/� fetuses took up and

TABLE 1. Retinoid Amounts in Embryos and Fetuses With the Indicated Genotypesa

Stage(embryonic day)

Rbp1genotype

Retinoids (pmol/g of tissue)

ROL RE all trans-RA

10.5�/� (n�1) 334 25.5 43.9�/� (n�1) 243 15.2 41.3

12.5�/� (n�4) 301 � 8 205 � 16 30.3 � 1.2�/� (n�5) 151 � 8* 59 � 5* 24.6 � 0.2*

13.5�/� (n�6) 302 � 12 781 � 51 22.0 � 0.6�/� (n�5) 153 � 7* 125 � 10* 20.4 � 0.2NS

14.5�/� (n�6) 347 � 15 1407 � 80 19.0 � 0.7�/� (n�6) 177 � 12* 201 � 21* 17.8 � 0.9NS

aThe number of pools that were analyzed by HPLC are indicated between parentheses. These pools were composed of eightindividuals at E10.5, four to five individuals at E12.5, three to four individuals at E13.5, and two individuals at E14.5. ROL, retinol;RE, retinyl esters (sum of retinyl linoleate, oleate, palmitate, and stearate); RA, retinoic acid; NS, not statistically significant. Anasterisk indicates a significant difference with wild-type values (P � 0.001).

168 MATT ET AL.

stored ROL, but with an efficacy six-to seven-times lower than WT (ROLand RE amounts were decreased by50% and 80%, respectively; Table 1).Therefore, CRBP1 deficiency signifi-cantly alters ROL homeostasisthroughout the period of organogene-sis. In contrast, the amount of RA wasonly slightly reduced (approximately7%) in Rbp1�/� mutants at E10.5,E13.5, and E14.5, and decreased byapproximately 20% at E12.5 (Table 1).Thus, mice can synthesize near nor-mal amounts of RA from ROL, eventhough ROL is present at only half anormal level and not bound to CRBP1.

Unaltered Expression of RA-Target Genes in Rbp1-NullMutants

To test the possibility that CRBP1 defi-ciency altered RA synthesis at discreteplaces in the embryo, without dramati-cally changing the total amount of RAmeasured by HPLC, we analyzedRbp1�/� embryos and fetuses betweenE9.5 and E14.5. They were recovered atthe expected Mendelian ratio (Table 2)and were normal with respect toweight, size, morphology, and histology(data not shown).

To detect potential embryonic tis-sues lacking RA synthesis in Rbp1-null mutants, we analyzed the expres-sion pattern of the RA-regulated Rarbgene (Sucov et al., 1990), and of twodistinct RA-regulated reporter trans-genes: Rarb2-lacZ, indicative of theRAR�2 isoform expression profile(Mendelsohn et al., 1991), and Rare-hsp68-lacZ (hereafter designated asRare-lacZ), which is commonly used to

identify RA-synthesizing tissues, be-cause its expression closely matchesthe distribution of endogenous RA(Rossant et al., 1991) and its activityis abolished in the presence of a pan-RAR antagonist (Wendling et al.,2000). In E9.5 WT embryos, the ex-pression of Rbp1 and Rarb genes over-lapped in the developing fourthbranchial arch and in abdominal vis-cera (Fig. 1A,B, B4 and V). The �-ga-lactosidase activity driven by Rarb2-lacZ also overlapped with the Rbp1expression domain in these structures(Fig. 1C). Both Rarb mRNA distribu-tion (Fig. 1H) and Rarb2-lacZ activity(Fig. 1I) were unaffected in E9.5Rbp1�/� embryos. Similarly at E10.5,the Rbp1 expression domain (Fig. 1D)overlapped with the Rare-lacZ activ-ity (Fig. 1E) in abdominal viscera, butagain the reporter expression wassimilar in WT and Rbp1�/� mutants(compare Fig. 1E with 1K, and datanot shown).

At E12.5, a stage at which RA syn-thesis is reduced, expression of Rare-lacZ was not altered in Rbp1�/� mu-tants (compare Fig. 1F with 1L),notably in tissues that normally ex-press both the transgene and Rbp1.For example in the heart, Rbp1mRNA (Fig. 2A) and Rare-lacZ activ-ity (Fig. 2B) were overlapping in theventricular epicardium, but the re-porter activity was not changed uponCRBP1 inactivation (Fig. 2H). Simi-larly at E13.5, the lack of CRBP1 didnot alter activity of Rare-lacZ (Fig.2D,J) or expression of Rarb gene (Fig.2F,L) in organs where these genes arenormally coexpressed with Rbp1 (Fig.2C,E), such as eye and spinal cord. In

the latter case, Rbp1 mRNA (see Fig.6A) and Rare-lacZ expression wereoverlapping in the roof plate and inneural progenitors, but �-galactosi-dase activity was not changed inRbp1�/� fetuses (data not shown).Similar observations were made in allother CRBP1-expressing tissues ex-amined between E9.5 and E14.5 (datanot shown).

To provide more quantitative data,we analyzed the level of mRNA ex-pressed from direct RA-target genes,namely Rarb (Sucov et al., 1990),Crabp1 (Kleinjan et al., 1997), andHoxa1 and Hoxb1 (Langston et al.,1997), by real-time reverse transcrip-tase-polymerase chain reaction (RT-PCR) using total RNA extracted fromE12.5 fetuses, when the RA amountwas decreased by approximately 20%in Rbp1-null mutants (see Table 1).Expression of Rarb was slightly butnot significantly reduced in Rbp1�/�

fetuses, whereas expression of allthe other tested RA-target genes wasnot changed by CRBP1 inactivation(Fig. 3).

In a given tissue, the level of RA isdetermined by the balance between its

TABLE 2. Viability of Rbp1-null Mutantsa

Stage(embryonic day)

Rbp1 genotype Number oflitters�/� �/� �/�

9.5 8 (1.0) 17 (2.1) 9 (1.1) 410.5 7 (1.0) 14 (2.0) 9 (1.3) 411.5 17 (1.0) 30 (1.8) 20 (1.2) 612.5 16 (1.0) 27 (1.7) 14 (0.9) 813.5 34 (1.0) 71 (2.1) 30 (0.9) 1614.5 16 (1.0) 29 (1.8) 14 (0.9) 7Total 98 (1.0) 188 (1.9) 96 (1.0) 45

aThe number wild-type embryos or fetuses was arbitrarily assigned a value of 1.0, andthe relative ratio of heterozygotes and homozygotes were calculated accordingly(number between parentheses).

Fig. 3. Expression of endogenous retinoicacid (RA) -target genes is not altered in cellularretinol-binding protein type 1 (CRBP1) -null fe-tuses. Total RNA from four individual embryonicday 12.5 wild-type (WT) and Rbp1�/� fetuseswas subjected to real-time quantitative reversetranscriptase-polymerase chain reaction analy-sis for Rarb, Crabp1, Hoxa1, and Hoxb1 genes.The values (arbitrary units) correspond to themean amount � SD of RNA transcripts de-tected in the fetuses of each genotype (n � 4),relative to the amount of �-actin transcripts,whose expression is not changed by retinoids.Note that Hoxb1 mRNA level was 10-fold lowerthan that of Rarb and Hoxa1, whereas Crabp1level was 10-fold higher. NS, statistically notsignificant.

CRBP TYPE 1 AND RETINOL METABOLISM IN MOUSE 169

Fig. 1. Expression of Rarb and retinoic acid (RA) -responsive transgenes are not altered in cellular retinol-binding protein type 1 (CRBP1) -nullembryos. A,B,D,G,H,J: Whole-mount in situ hybridizations showing expression of Rbp1 (A,D,G,J) and Rarb (B,H) mRNA in WT (A,B,D) and Rbp1�/�

mutants (G,H,J). C,I,E,F,K,L: �-Galactosidase activity driven by Rarb2-lacZ (C,I) and Rare-lacZ, (E,F,K,L) transgenes in wild-type (C,E,F) and Rbp1�/�

mutants (I,K,L). Rarb expression and transgene promoter activities are not changed in Rbp1�/� embryos. B1m, mandibular process of the firstbranchial arch; B1x, maxillary process of the first branchial arch; B2, second branchial arch; B4, fourth branchial arch; E, embryonic day; F, frontonasalmass; SC, spinal cord; V, abdominal viscera; WT, wild-type.

Fig. 2. Expression of Rarb and Rare-lacZ transgene are not altered in cellular retinol-binding protein type 1 (CRBP1) -null fetuses. A,C,E,F,G,I,K,L:In situ hybridizations on histological sections from WT (A,C,E,F) and Rbp1�/� (G,I,K,L) fetuses, showing expression of Rbp1 mRNA (A,C,E,G,I,K) andRarb mRNA (F,L) in the heart (A,G), the eye (C,I), and the spinal cord (E,F,K,L). B,D,H,J: �-Galactosidase activity in WT (B,D) and Rbp1�/� (H,J) fetuses,showing transgene expression in the heart (B,H) and the eye (D,J). The �-galactosidase activity overlaps with the expression domain of Rbp1 mRNAin the epicardium, the dorsal and ventral fields of the retina, the optic nerve, the retinal pigment epithelium, and the periocular mesenchyme, but X-Galstaining is unchanged in Rbp1�/� mutants. In the spinal cord, Rbp1 and Rarb are coexpressed in the median motor column neurons (arrowheads) andin neural progenitors, but expression of Rarb is not changed in Rbp1�/� mutants. B,H: Sections are counterstained with safranin O. Arrowheads, motorcolumn neurons; C, cornea; D, dorsal retina; E, epicardium; L, lens; M, periocular mesenchyme; MY, myocardium; NP, neural progenitors; O, opticnerve; R, retinal pigment epithelium; SC, spinal cord; V, ventral retina. Scale bar in L � 80 �m A,B,G,H, 150 �m C–F,I–L.

170 MATT ET AL.

synthesis and its degradation. Thelatter is mediated by selective cyto-chrome P450 hydroxylases, belongingto the CYP26 family and convertingRA to polar metabolites (Fujii et al.,1997; White et al., 1997; MacLean etal., 2001; Tahayato et al., 2003). Toprotect the cells from a deleteriousovershoot of RA derived from anysource, expression of the Cyp26 genesis strongly and rapidly induced by RAitself (Loudig et al., 2000; Wang et al.,2002). In this context, CRBP1 inacti-vation could have resulted in the syn-thesis, from free ROL, of largeramounts of RA that could have in-duced, in turn, enhanced expression ofCYP26 enzymes to accelerate theclearance of RA, thereby masking theeffects on the Rare-lacZ transgene. Totest for this hypothesis, we checkedwhether the expression pattern ofCyp26 was altered in E10.5 Rbp1�/�

embryos. The distribution of Cyp26a1(Fig. 4A,D), Cyp26b1 (Fig. 4B,E), andCyp26c1 (Fig. 4C,F) mRNAs was iden-tical in WT and Rbp1�/� embryos, no-tably in the branchial arch region,where they colocalize with CRBP1(B1x and B1m, compare Fig. 1D withFig. 4A–C). It is unlikely, therefore,that the activity of ectopically synthe-sized RA, due to CRBP1 ablation, wasmasked because of a feed-back overex-pression of CYP26 enzymes.

CRBP1 Does Not AffectROL-Dependent ReporterActivation in PrimaryEmbryonic Fibroblasts

To check, at the cellular level, the in-fluence of CRBP1 on ROL oxidationinto RA, we generated primary embry-onic fibroblasts (PEFs) from E10.5Rbp1�/�embryos, forced CRBP1 ex-pression, and analyzed the transcrip-tional activation of a transfected re-porter (DR5-tk-CAT), containing acanonical RA-response element (DR5)cloned upstream of the chloramphen-icol acetyl transferase (CAT) reportergene. The level of Rbp1 mRNA wasassessed by Northern blot analysis oftotal RNA from cells transfected withempty pSG5 (Green et al., 1988) orwith pRbp1 (Fig. 5A). Using RT-PCR,we also checked that both pSG5- andpRbp1-transfected PEFs are able toproduce RA from ROL as they ex-pressed (1) RDH1 and/or CRAD1 to

oxidize CRBP1-bound ROL into RAL,(2) ADH4 to oxidize free ROL, and (3)RALDH2 to oxidize RAL into RA (Fig.5B). The CAT activity was consis-tently higher in pRbp1- than in pSG5-transfected cells, but the differenceswere not significant (Fig. 5C). Addi-tion of 10�5 M ROL was required toincrease basal CAT level (comparewhite to gray bars; P � 0.01). How-ever, CAT was similarly induced by10�5 M ROL without (left panel, 1.7-fold increase) or with CRBP1 (rightpanel, 1.5-fold increase). It is unlikelythat this lack of difference reflects alimitation in other cellular compo-nents required for RA synthesis, asincreasing ROL concentration from10�7 to 10�5 M increased induction ofthe reporter gene from 1.2- to 1.7-fold(Fig. 5C, and data not shown). Thus,PEFs actually displayed a functional,nonsaturated, enzymatic machineryto oxidize ROL to RA, and the pres-ence of CRBP1 did not significantlyalter its capability.

CRBP1 Expression DomainDoes Not Always CorrelateWith RA-Responsive Tissues

That CRBP1 and several RDHs dis-play overlapping expression patternsin some adult (Zhai et al., 1997) orembryonic tissues (Båvik et al., 1997)is consistent with the hypothesis thatholo-CRBP1 may promote RA synthe-sis (Napoli, 1999). However, in thepresent study, we often observedCRBP1 expression in structures inwhich RA synthesis was undetectable.For example, Rbp1 mRNA was readilydetected at E10.5 in the first and sec-ond branchial arches (B1x and B1m,Fig. 1D), in which the Rare-lacZtransgene was not expressed underphysiological conditions (Fig. 1E) butcan be induced by excess RA (Rossantet al., 1991; Matt et al., 2003). Con-versely, Rbp1 mRNA was not detectedin the forebrain region (F, Fig. 1D),which nevertheless synthesized RA asindicated by expression of the Rare-lacZ reporter (Fig. 1E; see also Matt etal., 2003, and references therein).Along the same lines, Rbp1 mRNAwas expressed in the ventral spinalcord (Fig. 6A, false red color) and inkidney glomeruli (Fig. 6B, false redcolor) at E13.5, while the Rare-lacZactivity was observed in the dorsal

zone of the spinal chord (Fig. 6A) andin the kidney tubules (Fig. 6B). Thus,CRBP1 was not systematically ex-pressed in RA-synthesizing tissues.

DISCUSSION

CRBP1 Is Important forRetinol Storage DuringPrenatal Development

Its restricted expression pattern inembryonic tissues and its conservedstructure in vertebrates have sug-gested that CRBP1 could play impor-tant roles in development (Ong, 1994).No developmental defect are detectedin Rbp1�/� mutants, indicating thatabsence of CRBP1 is not life-threaten-ing, at least under conditions of ma-ternal vitamin A dietary sufficiency.However, ROL and RE contents arelower in Rbp1�/� embryos and fe-tuses than in WT (from E10.5 toE14.5). As CRBP1 enhances ROL up-take by placental membranes in vitro(Sundaram et al., 1998), this decreasemay result from a reduced transfer ofROL from the maternal circulation tothe fetus, through the placenta, whereCRBP1 is normally expressed (Johan-sson et al., 1997; Sapin et al., 1997).Alternatively, as several of the mech-anisms proposed to account for ROLuptake by cells involve CRBP1 (Ot-tonello et al., 1987; Noy and Blaner,1991; Nilsson et al., 1997), the de-creased ROL and RE levels may resultfrom an altered uptake of ROL by thefetal cells themselves. In keeping withthis possibility, it was shown that tri-tiated ROL, administered to pregnantmice, specifically accumulates in re-gions of the embryo expressingCRBP1 (Gustafson et al., 1993). Fi-nally, the decreased capacity ofCRBP1-null fetuses to store ROL inthe form of RE may reflect an alteredmetabolic phenotype related either to(1) a reduced RE half-life, as in adultliver (Ghyselinck et al., 1999); (2) animpaired delivery of ROL to specificenzymes for esterification (Yost et al.,1988; Herr and Ong, 1992; Napoli,1999); or (3) an increase of free ROLdegradation through enzymes thatnormally would not have access toretinoids, such as reported for ADH4in adult liver (Molotkov et al., 2004).

In any event, our data indicate thatCRBP1 is required for efficient stor-

CRBP TYPE 1 AND RETINOL METABOLISM IN MOUSE 171

Fig. 4. Expression of Cyp26 is not altered in cellular retinol-binding protein type 1 (CRBP1) -nullembryos. A–F: Whole-mount in situ hybridizations showing expression of Cyp26a1 (A,D), Cyp26b1(B,E), and Cyp26c1 (C,F) mRNA in wild-type (WT, A–C) and Rbp1�/� mutants (D–F). Cyp26a1 isexpressed in the tail bud, the cervical mesenchyme, and the ectoderm of the maxillomandibularcleft. Cyp26b1 is strongly expressed in the limbs, the hindbrain region (R3/6), and the mandibularprocess of the first branchial arch. Cyp26c1 is detected in the cervical mesenchyme (M), theepibranchial placode, the hindbrain region (R2), and the mandibular process of the first branchialarch. In Rbp1�/� embryos, expression of Cyp26a1, Cyp26b1, and Cyp26c1 is not changed. B1m,mandibular process of the first branchial arch; B1x, maxillary process of the first branchial arch; EP,epibranchial placode; FL, forelimb; HL, hindlimb; M, branchial arch and cervical mesenchyme; R2,second rhombomere; R3/6, third to sixth rhombomere; T, tail bud.

Fig. 5.

Fig. 6. Sites of cellular retinol-binding proteintype 1 expression do not coincide with RA-synthesizing structures in embryonic day 13.5fetuses. A,B: In situ hybridization showing ex-pression of Rbp1 mRNA (red false color) andRare-lacZ activity (blue color) on superimposedadjacent sections through the spinal cord (A)and the kidney (B). Rbp1 mRNA is expressed inthe ventral motor column neurons, the menin-ges, and in the glomeruli, whereas the Rare-lacZ transgene is strongly expressed in the dor-sal part of the spinal cord and in the tubules.Note that �-galactosidase activity and Rbp1mRNA expression overlap (yellow false color) inneural progenitors and in the roof plate. G, kid-ney glomeruli; M, meninges; MC, motor col-umn; NP, neural progenitors; R, roof plate; SC,spinal cord; T, kidney tubules. Scale bar � 150�m in A, 80 �m in B.

172 MATT ET AL.

age of ROL during prenatal life. Byproviding a selective advantage toface a nutritional vitamin A defi-ciency, this function on its own is suf-ficient to account for the conservationof CRBP1 in vertebrates (Ong, 1994).

CRBP1 and Retinoic AcidHomeostasis

The present data show that CRBP1-null mutant embryos and fetuses dis-play reduced amounts of RA (by 7 to20%, depending on the developmentalstage), but do not exhibit any abnor-malities characteristic of RA defi-ciency (reviewed in Kastner et al.,1995). Thus, even though involved inRA homeostasis, CRBP1 is not criti-cally required for development, atleast under conditions of maternal vi-tamin A sufficiency. However, in vitrostudies (Ottonello et al., 1993; Napoli,1999; Lapshina et al., 2003) and ex-pression patterns (Båvik et al., 1997;Zhai et al., 1997) have suggested thatCRBP1 could be essential for RA syn-thesis through presenting ROL toRDHs. We reasoned that, if this wasthe case, RA synthesis should be de-creased in areas of Rbp1�/� normallyexpressing CRBP1. As an indicator ofRA availability, we have studied the ex-pression of selected RA-target genes.Our results show that (1) CRBP1 abla-tion does not change the expression ofthese genes during prenatal develop-ment and that (2) CRBP1 availabilityhas no effect on ROL-induced expres-sion of an RA-target gene in PEFs.These observations suggest thatCRBP1 is dispensable for RA synthesis.Therefore, the reduced levels of RA inRbp1�/� fetuses may simply resultfrom the lowered amount of ROL avail-able for oxidation.

Several studies have correlated theexpression pattern of retinoid bindingproteins (Dolle et al., 1990; Maden et

al., 1990; Ruberte et al., 1991;Gustafson et al., 1993; Horton andMaden, 1995; Båvik et al., 1997; Zhaiet al., 1997) and/or of enzymes thatmetabolize ROL (Vonesch et al., 1994;Ang et al., 1996; Båvik et al., 1997;Zhai et al., 1997; Haselbeck andDuester, 1998) with embryonic areaswhere RA should act as a signalingmolecule. In the present study, wecompared the expression profile ofCRBP1 with that of the functionaltransgene whose pattern indicates RAactivity (Rossant et al., 1991). Wehave observed both CRBP1 expressionin structures that do not synthesizeRA (e.g., first and second branchialarches), and CRBP1 absence from tis-sues that express the RA-responsivetransgene (e.g., the forebrain region).In the first branchial arch, it can behypothesized that CRBP1 expressionis required to protect ROL from oxida-tion by cytosolic ADH4, which is ex-pressed in the first and secondbranchial arches (Ang et al., 1996; Ha-selbeck and Duester, 1998). However,our results suggest that no RA is syn-thesized ectopically in these embry-onic structures upon CRBP1 ablation.In the forebrain region, diffusion ofRA from the maxillary process of thefirst branchial arch, i.e. the neareststructure expressing CRBP1, is un-likely. There, yet unidentified en-zymes whose activity do not requireCRBP1 and locally are involved in RAsynthesis should exist (Mic et al.,2002; Niederreither et al., 2002). Inconclusion, our comparative studydoes not support the proposal thatCRBP1 is needed at specific places forRA synthesis during prenatal life.

Each isotype of CRBP has a distinctexpression pattern, CRBP2 being re-stricted to the small intestine (Levinet al., 1987; Schaefer et al., 1989) andCRBP3 to heart, skeletal muscle, adi-pose tissue, and mammary gland (Vo-

gel et al., 2001; Folli et al., 2002).Therefore, it is unlikely that func-tional redundancies between CRBP1and other CRBPs could account forthe lack of obvious effect on RA syn-thesis in Rbp1�/� mutants. It israther tempting to speculate that eachCRBP may fulfill distinct functions invivo, although sharing common bio-chemical properties in vitro, such assubstrate affinity and presentation ofROL to selective enzymes. CRBP1might be required for ROL storage(present study, Ghyselinck et al.,1999), CRBP2 for ROL uptake in in-testine (E et al., 2002), and CRBP3and/or other yet unknown CRBP(s) inRA synthesis.

EXPERIMENTALPROCEDURES

Mice

Mice were on a mixed C57BL/6-129/Sv(50%–50%) genetic background. Theywere housed in an animal facility li-censed by the French Ministry of Ag-riculture (agreement no. B67-218-5).Animal experiments were supervisedby N.B.G. (agreement no. 67-205),who is qualified for experimentingwith mice, in compliance with the Eu-ropean legislation on care and use oflaboratory animals. The lines carryingthe Rbp1-null mutation (Ghyselincket al., 1999) or the Rarb2-lacZ (Men-delsohn et al., 1991) or Rare-hsp68-lacZ (Rossant et al., 1991) reportertransgenes have been described. Thebreeding diet contained 25,000 UI ofretinyl palmitate/kg (UAR; France)and was provided to the pregnantmice ad libitum. Noon of the day of avaginal plug was taken as E0.5. Em-bryos and fetuses were collected bycesarian section, and the yolk sacswere taken for DNA extraction.

Fig. 5. Induction of a retinoic acid (RA) -dependent reporter in Rbp1�/� primary embryonic fibroblasts (PEFs) without or with forced cellularretinol-binding protein type 1 (CRBP1) expression. A: Northern blot analysis of total RNA from Rbp1�/� PEFs, showing expression of Rbp1 mRNA onlyin pRbp1-transfected fibroblasts (upper panel, lane 2). The blot was subsequently probed with a �-actin cDNA (Actb, lower panel). B: Reversetranscriptase-polymerase chain reaction amplification obtained with RNA extracted from liver or from pSG5- and pRbp1-transfected PEFs with primersspecific for Rdh1/Rdh6 (RDH1/CRAD1), Adh1 (ADH1), Adh7 (ADH4), Aldh1a1 (RALDH1), Aldh1a2 (RALDH2), and the ubiquitous Arbp gene (36B4). Theidentities of the fragments are indicated. tRNA was used as negative control. C: Transfections of Rbp1�/� PEFs with pSG5 (left panel) or pRbp1 (rightpanel) vectors, together with the DR5-tk-CAT reporter. Retinol (ROL), RA, or vehicle (EtOH) were added as indicated. Bars show mean relativechloramphenicol acetyl transferase (CAT) expression � SD of 9 to 12 transfected samples (four independent experiments). The asterisks and bracketednumbers indicate difference (P � 0.01) and fold-induction compared with EtOH-treated cells (white bars), respectively. Treatment with 10�6 M RAinduces a threefold activation of CAT, showing that PEFs respond to RA.

CRBP TYPE 1 AND RETINOL METABOLISM IN MOUSE 173

Genotyping

Heterozygotes were crossed to generateRbp1�/� and Rbp1�/�, Rarb2-lacZtg/0/Rbp1�/� or Rare-lacZtg/0/Rbp1�/�,and Rarb2-lacZtg/0/Rbp1�/�, or Rare-lacZtg/0/Rbp1�/� embryos and fetuses.Genotyping was performed by PCR.For Rbp1, primer #1 (5�-GCACTTGCGGTCGTCTATGC-3�) and #2 (5�-AA-AAATGGAAAGGCAAGGCACAGAC-3�)were used to amplify a 338-bp productspecific for the WT allele. Primer #2was used with primer #3 (5�-GCCT-TCTATCGCCTTCTTGACGAGTTCT-TC-3�) to amplify a 593-bp productspecific for the Rbp1-null allele. ThePCR conditions used were 94°C for 15sec, 61°C for 30 sec, and 72°C for 30sec for 30 cycles. Amplification prod-ucts were resolved on agarose gels,transferred onto Hybond-N� (Amer-sham Biosciences), and hybridizedwith the 32P-labeled oligonucleotides#4 (5�-TGCAGGATGGCGACCACATG-3�) and #5 (5�-CTATTGCTTTAT-GATAATGTTTCATAGTTGGA-3�) tovisualize WT and Rbp1-null alleles,

respectively. For LacZ genotyping,primers 5�-CGCCGACGGCACGCTG-ATTG-3� and 5�-GTTTCAATATTG-GCTTCATC-3� were used to amplify a300-bp product. The PCR conditionsused were 92°C for 15 sec, 56°C for 30sec, and 72°C for 30 sec for 30 cycles.

Retinoid Analysis

Experiments involving retinoids wereperformed in a dark room under dimyellow light to prevent photoisomer-ization and photodegradation. Mouseembryos were frozen immediately ondried ice and kept at �80°C undernitrogen. Retinoids were extracted bya single liquid extraction, then sepa-rated from each other by means of sol-id-phase extraction using amino-pro-pyl phase, and injected onto twodifferent HPLC systems that werecoupled with ultraviolet detection(Schmidt et al., 2003). Standard reti-noids were from Sigma. Using thisprocedure, the detection limits (inpmol/g of tissue) were 1.5 for 9cis-RA,

1.0 for all trans-RA, 8.5 for ROL, and8.0 for RE.

Culture and Transfection ofPrimary Fibroblasts

PEFs were derived as described (Ab-bondanzo et al., 1993). The expandedPEFs were split into six-well tissueculture plates (3.105 cells/well) andtransfected using lipofectamine 2000(Invitrogen Life Technologies). After 5hr incubation with the transfectioncomplexes (containing 500 ng of DR5-tk-CAT reporter plasmid [Chen et al.,1995], 200 ng of pSV-�Gal [used as aninternal control to normalize for vari-ations in the transfection efficiency],and, when required, 300 ng of pRbp1[Dolle et al., 1990]), the culture me-dium was replaced by a fresh one con-taining 10% charcoal-stripped fetalcalf serum and ROL or RA dissolved inethanol. After 24 hr, CAT expressionwas quantified by ELISA (Roche) andnormalized to �-galactosidase activ-

TABLE 3. Primers for RT-PCR Amplificationsa

Gene ProteinAccessionno. Primers Position Size (nt)

Actb �-actin NM_0073935�-GACGGCCAGGTCATCACTAT-3� nt 810–829

2995�-CCACCGATCCACACAGAGTA-3� nt 1089–1108

Adh1 ADH1 NM_0074095�-ATGACCGACGGAGGGGTGGA-3� nt 846–865

3525�-AAGTCAGGACGGTACGGATG-3� nt 1178–1197

Adh7 ADH4 NM_0096265�-ATGGTTGATGCCCTCTCATC-3� nt 954–973

3245�-GAACACCCAGGTCTCTGGAA-3� nt 1258–1277

Aldh1a1 RALDH1 NM_0134675�-CTCCTCTCACGGCTCTTCAC-3� nt 634–653

2895�-CCATGGTGTGCAAACTCAAC-3� nt 903–922

Aldh1a2 RALDH2 NM_0090225�-ATGGGTGAGTTTGGCTTACG-3� nt 1482–1501

2745�-GGTTCATTGGAAGGCAGAAA-3� nt 1736–1755

Arbp 36B4 NM_0074755�-GTGTGAGGTCACTGTGCCAG-3� nt 428–447

4205�-GTGTACTCAGTCTCCACAGA-3� nt 828–847

Crabp1 CRABP1 NM_0134965�-CTTCGAGGAGGAGACAGTGG-3� nt 307–326

3335�-GGAAAATGGGGTTGCCTAAT-3� nt 620–639

Hoxa1 Hox-A1 AK0835755�-CGCACAATGTTCTGATGTCC-3� nt 2071–2090

3145�-TGCAAGCTTCATGACAGAGG-3� nt 2365–2384

Hoxb1 Hox-B1 NM_0082665�-TGACCAGTTCTCTCGAAGAC-3� nt 999–1018

3045�-CTCTCTAAGCTCAAAGGCAC-3� nt 1283–1302

Rarb RAR� NM_0112435�-AATGCCACCTCTCATTCAGG-3� nt-1390–1409

3695�-GGCATGAAATGGCTGATTTT-3� nt 1739–1758

Rdh1 RDH1 NM_0804365�-GAATTCTGCCAAGGGATTCA-3� nt 1268–1287

3115�-CATCACCGGGAACTGAACCT-3� nt 1559–1578

Rdh6 CRAD1 NM_0090405�-GAATTCTGCCAAGGGATTCA-3� nt 1182–1201

3115�-CATCACCGGGAACTGAACCT-3� nt 1473–1492

aThe gene names, the corresponding proteins, the accession numbers, the forward (upper line) and reverse (lower line) primers, theirpositions in the sequences, and the sizes of the amplified fragments are indicated. nt, nucleotide; RT-PCR, reverse transcriptase-polymerase chain reaction.

174 MATT ET AL.

ity. Significance of differences was cal-culated by unpaired Student’s t tests.

RNA Analysis

Total RNA was prepared from trans-fected fibroblasts (106 cells) or frozenembryos using Trizol reagent (Invitro-gen Life Technologies). Fifteen micro-grams of total RNA were separated on1% (w/v) agarose gels containing form-aldehyde, transferred onto Hybond-N(Amersham Biosciences), and hybrid-ized with Rbp1 and Actb probes, ac-cording to standard procedures. RT-PCR was performed according toBouillet et al. (1995). Liver RNA andtRNA were used as positive and neg-ative controls for amplification, re-spectively. Conditions were 30 cycleswith denaturation for 30 sec at 92°C,annealing for 30 sec at 55°C, and elon-gation for 30 sec at 72°C. Aliquotswere analyzed on 1.5% agarose gels.Quantitative analysis of RNA was car-ried out by one-step real-time RT-PCRusing a Light-Cycler (Roche MolecularBiochemicals) and the DNA double-strand-specific SYBR Green I dye fordetection. Reverse transcription of to-tal RNA from E12.5 fetuses (100 ng)was performed at 55°C using 1.5 UAMV reverse transcriptase (RocheMolecular Biochemicals), then ampli-fication was performed using theqPCR-&Go Mastermix (QBiogen,Illkirch France). Conditions were 40cycles with denaturation for 5 sec at95°C, annealing for 5 sec at 60°C, andelongation for 15 sec at 72°C. Thetranscripts levels were normalized ac-cording to Actb transcripts, which areunresponsive to retinoids treatment.The primers are indicated in Table 3.

Whole-Mount and In SituHybridizations

Samples were fixed in a phosphate-buffered saline (PBS) solution con-taining 4% (w/v) paraformaldehyde(PFA) at 4°C (2 hr for E9.5–E10.5 em-bryos and 16 hr for E13.5–E14.5 fe-tuses). In situ RNA hybridizationswere carried out as described (Matt etal., 2003). The Rarb2, Rbp1, Cyp26a1,Cyp26b1, and Cyp26c1 digoxigenin-la-beled riboprobes are as described(Dolle et al., 1990; MacLean et al.,2001; Tahayato et al., 2003). The �-ga-lactosidase activity was revealed as

described (Mendelsohn et al., 1991),and embryos or fetuses were post-fixed in 4% buffered paraformalde-hyde for 16 hr at 4°C and processed forhistology. Between three and eightembryos or fetuses of each genotypewere analyzed, at each developmentalstage.

ACKNOWLEDGMENTSWe thank B. Feret, G. Kimmich, B.Weber, and the staff of IGBMC andICS common services or facilities fortheir technical assistance. We alsothank Drs. A. Dierich, B. Chapellier,V. Vivat, and J.L. Plassat for advices.N.M. was supported by fellowshipsfrom the Ministere de l’Education Na-tionale et de la Recherche and the As-sociation de la Recherche contre leCancer. C.K.S. and H.N. were sup-ported by the European Commission.

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