3235Development 125, 3235-3246 (1998)Printed in Great Britain © The Company of Biologists Limited 1998DEV5215

A conserved retinoic acid responsive element in the murine Hoxb-1 gene is

required for expression in the developing gut

Danyang Huang, Siming W. Chen, Alexander W. Langston and Lorraine J. Gudas*

Department of Pharmacology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, USA*Author for correspondence (e-mail: ljgudas@mail.med.cornell.edu)

Accepted 9 June; published on WWW 21 July 1998

The murine Hoxb-1 gene contains a homeobox sequenceand is expressed in a spatiotemporal specific pattern inneuroectoderm, mesoderm and gut endoderm duringdevelopment. We previously identified a conserved retinoicacid (RA)-inducible enhancer, named the RAIDR5, whichcontains a DR5 RARE; this RAIDR 5 enhancer is located 3′of the Hoxb-1-coding region in both the mouse and chick.In the F9 murine teratocarcinoma cell line, this DR5 RAREis required for the RA response of the Hoxb-1 gene,suggesting a functional role of the DR5 RARE in Hoxb-1gene expression during embryogenesis. From the analysisof Hoxb-1/lacZ reporter genes in transgenic mice, we haveshown that a wild-type (WT) transgene with 15 kb of Hoxb-1 genomic DNA, including this Hoxb-1 3′ RAIDR 5, isexpressed in the same tissues and at the same times as theendogenous Hoxb-1 gene. However, a transgene constructwith point mutations in the DR5 RARE (DR5mu) was notexpressed in the developing foregut, which gives rise toorgans such as the esophagus, lung, stomach, liver andpancreas. Like the wild-type transgene, this DR5 RAREmutated transgene wasexpressed in rhombomere 4 in 9.5

day postcoitum (d.p.c.) embryos. Similarly, transgenestaining in the foregut of animals carrying a deletion of theentire Hox-b1 RAIDR 5 enhancer (3′-del) was greatlyreduced relative to that seen with the WT transgene. Wealso demonstrated that expression of the WT transgene inthe gut increases in response to exogenous RA, resulting inanterior expansion of the expression in the gut. Theseobservations that the Hoxb-1gene is expressed in thedeveloping gut and that this expression is regulatedthrough a DR5 RARE strongly suggest a role for Hoxb-1 inthe anteroposterior axis patterning of the gut and a criticalrole for endogenous retinoids in early gut development.

Abbreviations: RA, retinoic acid; Hox, homeobox gene; RAIDR5,retinoic acid RARE direct repeat5 enhancer; RAR, retinoic acidreceptor; RXR, retinoid X receptor.

Key words: Homeobox, Homeodomain, Hox, Retinoid, Rhombomere4, Digestive tract, Foregut, Lung, Stomach, Liver, Pancreas,Intestine, Mouse, Gut

SUMMARY

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INTRODUCTION

Retinoic acid (RA) is required during normal embryonidevelopment in vertebrates. Both a deficiency and an excesRA lead to a variety of developmental defects in orgaincluding the brain, heart, pituitary, thyroid and limbs (Guda1994; Hofmann and Eichele, 1994). The potent teratogeeffects of exogenous RA during development have implicatendogenous retinoids in the patterning of the anteroposteaxis of the embryo (Kessel and Gruss, 1991; Krumlauf, 199

In vertebrates, 39 genes related to those of the Drosophilahomeotic gene complex (HOM-C) have been identified. mammals, many of the Hoxgenes are organized in fourclusters, HoxA, HoxB, HoxC and HoxD. The conservation ofgene organization, expression and function of the Hoxgenes iswell documented in all vertebrate species studied (reviewedKrumlauf, 1994; McGinnis and Krumlauf, 1992). Hox genesencode transcription factors containing the homeobox domaExperiments involving the targeted disruption o

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misexpression of Hox genes suggest that Hoxgenes specifyregional identity along the anteroposterior body axis(Krumlauf, 1994; McGinnis and Krumlauf, 1992). Hox genesare turned on sequentially from the hindbrain to the posterioend of the embryo in a nested fashion, colinear with thepositions in the clusters. Hox genes at the 3′ends of thechromosome clusters are expressed earlier and in more anteregions of the embryo, while Hox genes at the more 5′ends ofthe clusters are expressed at later times in embryogenesis in more posterior regions of the embryo (Duboule and Morat1994; Krumlauf, 1994; Lewis, 1978). Alterations in normalexpression patterns lead to homeotic transformations amalformations (McGinnis and Krumlauf, 1992).

The murine Hoxb-1gene is located at the 3′ end of the Hoxbgene cluster on mouse chromosome 11 (Frohman et al., 19Wilkinson et al., 1989). The Hoxb-1gene shows two phases ofexpression in the developing embryo. Hoxb-1 mRNA is firstdetectable at 7.5 d.p.c. in the posterior half of the embryo, the neuroectoderm, in the primitive streak and in newly forme

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mesoderm rostral to the node. By the early somite stage ad.p.c., Hoxb-1expression becomes divided into two domainthe prospective rhombomere 4 in the hindbrain and posterior half of the embryo, which includes the posterneural tube, paraxial (somite) mesoderm and gut (Frohmaal., 1990; Murphy and Hill, 1991; Wilkinson et al., 1989). Thanterior expression of Hoxb-1 induced by exogenous RAtransforms rhombomere 2 to a rhombomere 4 ident(Marshall et al., 1992). Therefore, in the developing hindbrathe Hoxb-1 gene, like its Drosophilacounterpart, labial, isinvolved in specifying the identities of segments. Consistewith this, a Hoxb-1null mutation results in neonatal lethalitywith alterations in rhombomeric identity and failure to formfunctional facial (VIIth) nerve (Goddard et al., 1996; Studeral., 1996).

Several lines of evidence implicate RA as one of the ksignaling molecules in determining the anteroposterpatterning through the regulation of Hoxgene expression. First,during the RA-induced differentiation of teratocarcinoma celmost Hoxgenes are activated by RA sequentially, colinear wtheir positions in the clusters (Simeone et al., 1990, 199Second, in early development 3′ Hoxgenes such as Hoxa-1andHoxb-1are expressed in the neural tube with distinct anterboundaries in the hindbrain. In embryos treated wexogenous RA, an anterior expansion of the expression of thgenes is observed and this expansion is associated withhomeotic transformation of segments in the hindbrain (Cho aDe Robertis, 1990; Conlon and Rossant, 1992; Holder and H1991; Kessel, 1992; Kessel and Gruss, 1991; Marshall et1992; Morriss-Kay et al., 1991; Papalopulu et al., 1991; Sand Cheng, 1991). Third, RA has been detected in the Xenopusdorsal lip, in Hensen’s node of the chick and in the primitistreak in the mouse, an area responsible for organizing vertebrate embryo and likely the site of Hoxinduction (Chenet al., 1992; Thaller and Eichele, 1987; Wagner et al., 199Thus, RA and potentially other bioactive retinoids are likely be the natural morphogens. Fourth, the discovery of retinacid-response elements (RAREs) in the Hoxa-1 (Dupé et al.,1997; Frasch et al., 1995; Langston and Gudas, 1992; Langet al., 1997), Hoxb-1 enhancers (Langston et al., 1997Marshall et al., 1994; Studer et al., 1994), Hoxa-4 promoter(Doerksen et al., 1996) and Hoxd-4promoter (Popperl andFeatherstone, 1993) suggests that transcriptional activatioat least some Hox genes by RA is via a direct mechanisminvolving the RARs (Conlon, 1995; Gudas, 1994).

Most of the retinoic acid responses in the embryo amediated through retinoic acid receptor proteins (RARs aRXRs) that are members of the steroid/thyroid nuclear recepsuperfamily (reviewed in Leid et al., 1992; Mangelsdorf et a1994). RARs form heterodimers with RXRs and bincooperatively to cis-acting RAREs on many of the responsgenes to activate transcription. Several different RARcontaining enhancers that contribute to the expression ofhuman and murine Hoxb-1genes have been identified(Langston et al., 1997; Marshall et al., 1994; Ogura and Eva1995a,b; Studer et al., 1994). Marshall et al. (1994) descria functional DR2-type RARE, conserved in the mouse, chicand pufferfish, which was able to mediate the Rresponsiveness of the early expression of the Hoxb-1gene intransgenic animals. We identified an RA-inducible enhan(RAIDR5) containing a DR5-type RARE as part of a DNAse I

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hypersensitive site located approximately 6.5 kb 3′ of themurine Hoxb-1-coding region. This DR5 RARE is required forRA-induction of Hoxb-1in teratocarcinoma cells (Langston etal., 1997). In addition to this DR5 RARE, the RAIDR5enhancer contains two other blocks of highly conservesequences called CE1 and CE2. We have shown that the Celement in the RAIDR5 enhancer of the Hoxa-1gene, a paralogof Hoxb-1, contributes to Hoxa-1 expression in somites andadjacent mesenchymal tissue during development (Thompset al., 1998).

There is accumulating evidence that suggests that Hoxgenesmay be involved in the regional specification of the digestivtract (gut) along the anteroposterior axis (Roberts et al., 199Yokouchi et al., 1995). The digestive tract is composed various organs including the esophagus, stomach, livepancreas, intestine and colon. These organs originate fromsimple tube of endoderm, surrounded by visceral mesoderm, sequential induction between these two germ layers (Haffenal., 1987). The region-specific differentiation of gut epitheliumis dependent on signals from the surrounding visceral mesode(Haffen et al., 1983). Conversely, primitive endoderm signals thadjacent mesenchymal tissues to undergo gut-specimesodermal differentiation (Haffen et al., 1983; Kedinger et a1986). For gut morphogenesis, one endoderm invagination frothe anterior end, the anterior intestinal portal (AIP), extendposteriorly to form the foregut at ~7.5 d.p.c., and anotheendoderm invagination from the posterior end, the caudintestinal portal (CIP), extends anteriorly to form the hindgut aaround 8.5 d.p.c. The midgut is then derived by fusion of thforegut and hindgut at the midline around 9 d.p.c. (Rugh, 1996Simple ducts subsequently begin to protrude from the primagut tube to form structures such as the lung buds, hepadiverticulum and pancreas evaginations. At later stages, tmesodermal layer of the tube differentiates into muscle aconnective tissue, and the endoderm layer differentiates inepithelium with specific histological and biochemical featuresFor example, the foregut gives rise to organs such as tesophagus, lung, stomach, liver and pancreas. The molecumechanisms underlying the generation of positional cues specify duct budding and regional specification of the digestivtract in vertebrates are still unknown. Transcripts of many Hoxgenes are expressed in a restricted region in the developing (reviewed in Roberts et al., 1995; Yokouchi et al., 1995Moreover, targeted disruptions of some 5′ Hox genes result inmalformations of the posterior structures in the digestive trasuch as the anus sphincter (Kondo et al., 1996). These findisuggest that Hoxgenes play significant roles in theanteroposterior patterning of gut.

In the studies presented here, we examined the role of DR5 RARE in the Hoxb-13′ RAIDR5 enhancer in vivo throughanalysis of transgenic mice. We show that a 15 kb fragmentmurine Hoxb-1 genomic DNA, which includes this DR5RARE, directs the expression of a lacZ reporter gene in atemporal and spatial pattern that reflects that of the endogenHoxb-1gene. We also demonstrate that the 3′ DR5 RARE isrequired for the regulation of Hoxb-1 region-specificexpression in the gut and for the RA-induced anteriorization Hoxb-1 expression in the gut. This suggests that the Rsignaling pathway is involved in the specification of the regionof Hoxgene expression and consequently in the anteroposterpatterning of gut.

3237Hoxb-1 expression in development

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MATERIALS AND METHODS

Transgene constructionThe genomic DNA fragments containing the Hoxb-1 locus wereisolated from a lambda FIXII mouse genomic library. A 15 kb NotIfragment containing the 3′ DR5 RARE was subcloned into pBluescripKS, generating pHoxb-1. lacZwas inserted into the Hoxb-1-codingsequence as follows. The E. coli lacZgene from pMC1871(Pharmacia) was excised by digestion with BamHI and ligated to aBamHI-EagI adaptor; it was purified and ligated to the partialdigested pHoxb-1 in the EagI site in exon I, creating an in-framfusion at the 34th amino acid of Hoxb-1(pHoxb-1RAIDR5WT/lacZ).A stop codon from pMC1871, located downstream of the lacZ openreading frame, is included.

The DR5 RARE mutation was made by PCR-mediated mutageneas described (Prelich, 1993). The RARE was mutated by Pamplification of the subcloned enhancer fragment with the followioligonucleotides, which were paired with primers to outer flankiplasmid sequences (mutated nucleotides are in lower case letunderlined sequence is the DR5 RARE):

RARE top strand: 5′-tcT agc TAG AGA aTT acG CTC TGA AATGCT TGC AGC-3′

RARE bottom strand: 5′-AGC ATT TCA GAG Cgc AAt TCT CTAgct Aga AGG AAG GAA AGG G-3′.

Sequences of the 5′ and 3′outer flanking primers are: 5′-TGG TGGCTC ACA ATC TC-3′ and 5′-CGC TCT AGA ACT AGT GGA TC-3′ (SK primer of pBluescript vector), respectively.

Following amplification of both top and bottom strands, the PCproducts were mixed and another round of amplification was carrout using only the outer flanking primers. The resulting 1 kb PCproduct containing the RARE mutation was cut with SphI and BglII,purified, and ligated to the compatible vector, which was prepareddigestion of a 2 kb SphI-KpnI subclone of Hoxb-13′ flanking sequencein pBluescript with SphI and BglII; this created pKS-DR5m. Then, anapproximately 2 kb Hoxb-1 3′ sequence containing the DR5 RAREmutations was cut from the pKS-DR5m with SphI and KpnI and ligatedto the 3′ portion and the 5′ portion of the Hoxb-1 chimeric gene asfollows. The 3′ portion was derived from pHoxb-1RAIDR5WT/lacZby digestion with ClaI and KpnI. The 5′ portion of the chimeric genewas prepared by digestion of pHoxb-1RAIDR5WT/lacZ by ClaI andSphI. This three fragment ligation generated pHoxb-1RAIDR5mu/lacZ, which is identical to pHoxb-1RAIDR5WT/lacZexcept for the DR5 RARE mutation. DNA sequencing analysisconfirmed that pHoxb-1RAIDR5mu/lacZ contained the desired RAREmutations but no other sequence changes.

The pHoxb1RAIDR5 3-del/lacZconstruct was prepared as followsThe 5′portion ClaI-SphI fragment containing the Hoxb-13′ proximalDR2 RARE, but not the RAIDR5 RARE, was ligated to a SphI-KpnIadaptor, and was then ligated to the Hoxb-15′ portion, the ClaI-KpnIfragment derived from the pHoxb-1RAIDR5WT/lacZ. This 3′ deletionconstruct is identical to pHoxb-1RAIDR5WT/lacZ except for a ~2 kbdeletion of Hoxb-13′-flanking sequence.

Cell culture and transient transfectionsF9 teratocarcinoma cells were cultured in DMEM with 10% caserum as previously described (Langston et al., 1997). F9 cells wtransfected with Hoxb-1/lacZplasmids by calcium phosphatecoprecipitation. β-galactosidase activities were normalized to reportactivity of a plasmid containing the β-actin/CAT. Transienttransfections, CAT assays and β-galactosidase assays were performeas described previously (Langston et al., 1997). Quantitation of Cassays was carried out using a PhosphorImager (MolecDynamics).

Generation of transgenic miceDNA was prepared as a linearized NotI fragment with vector

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sequence removed and microinjected into 1-cell mouse embry(C57B/6×CBA/J F1) at the Rockefeller University TransgenicMouse Facility. To identify the transgenic offspring, tail DNA wasdigested with EcoRI, Southern blotted to Hybond N nylonmembrane (Amersham) and probed with a random-prime[32P]dCTP-labeled lacZ fragment as described (Sambrook et al.1989). Production of transgenic embryos was performed by matitransgenic males with non-transgenic females (CBA57/J6) in thevening and monitoring the plugs the following morning; a plufound the following morning is considered 0.5 d.p.c. Embryos weremoved at the appropriate gestational ages and stained forβ-galactosidase activity. The expression patterns of the Hoxb-1RAIDR5WT/lacZ, Hoxb-1RAIDR5mu/lacZ, and Hoxb-1RAIDR53′del/lacZ transgenes were confirmed at all stages of developmein at least two independent founder lines of transgenic mice. Ttwo transgenic lines of Hoxb-1RAIDR5WT/lacZare Tg11 and Tg21;the lines of Hoxb-1RAIDR5mu/lacZ are Tg49, Tg50, Tg63 andTg75, and the lines of the Hoxb-1 RAIDR5 3′-del/lacZ are Tg103and Tg109.

β-galactosidase staining and embryo sectioningEmbryos were fixed as previously described (Conlon and Rossa1992; Means and Gudas, 1997) in 1% formaldehyde, 0.2glutaraldehyde, 2 mM MgCl2, 5 mM EDTA, and 0.02% NP-40 inPBS, for 30-90 minutes at 4°C. The embryos were then washed thtimes in PBS plus 0.02% NP-40, and stained in 1 mg/ml X-gal, 5 mK3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 0.01% sodiumdeoxycholate and 0.02% NP-40 in PBS, at room temperature andthe dark. Stained embryos were washed with several changes of P+ 0.02% NP-40. Photographs were taken on a Leica dissectmicroscope.

For sectioning, the embryos were dehydrated and paraffiembedded as previously described (Means and Gudas, 1997). Sectwere cut (7 µm) and mounted on glass slides. Slides were deparaffinand counterstained with eosin as described previously (Means aGudas, 1997).

Whole-mount in situ hybridizationHoxb-1plasmid was kindly provided by Dr Joe Grippo. The plasmiDNA contains a 435 bp EcoRI-HindIII fragment of Hoxb-1cDNA.Digoxigenin-labeled antisense probes were synthesized frolinearized plasmid with HindIII digestion and transcribed in vitrousing T7 RNA polymerase as described by the manufactur(Boehringer Mannheim Biochemicals, St Louis, MO). The Hoxb-1plasmid was linearized with EcoRI and transcribed with SP6 RNApolymerase in order to obtain the sense probe.

Whole-mount in situ hybridization was performed as previousldescribed (Conlon and Rossant, 1992) with the following changeEmbryos were treated with 10 µg/ml proteinase K for 8 minutes atroom temperature and prehybridized for at least 1 hour at 68°Chybridization buffer (50% formamide, 0.75 M NaCl, 1 mM EDTA,50 µg/ml tRNA, 0.05% heparin, 1% sodium dodecyl sulfate). Thhybridization buffer was replaced and single-stranded RNA problabeled with digoxigenin were added to 1 µg/ml; embryos werehybridized overnight at 68°C.

Retinoic acid treatmentPregnant females were treated with RA at various times befoembryo dissection. RA was administrated at a dose of 20 mg perof maternal body weight by oral gavage (Conlon and Rossant, 199A stock solution of 25 mg/ml of all-trans-RA (Sigma) indimethylsulfoxide was dissolved just before use in corn oil so that0.2 ml dose of oil contained the requisite amount of RA. Control micreceived corn oil alone. Embryos were isolated 12-16 hours later astained for lacZ expression for 12-18 hours unless mentionespecifically in the text.

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Fig. 1.Structure and map of theHoxb-1/lacZtransgene reporterconstructs. The wild-type (WT)construct contains a total of 15 kbof Hoxb-1genomic DNA with ~6kb of 5′- and ~7.5 kb of 3′-flankingsequence; all exons and intronsequences are also included. Exonsand the region containing thehomeobox are depicted as openboxes and a dotted box,respectively. lacZ(diagonal stripedbox) is fused in frame to exon 1 atthe 34th amino acid of Hoxb-1. Theconstruction of the Hoxb-1RAIDR5WT/lacZ, Hoxb-1RAIDR5mu/lacZand Hoxb-1RAIDR5 3′del/lacZ transgenes is described in the Materialsand Methods. Restriction enzyme sites are: E, EcoRI; C, ClaI; S, SacI; Sp,SphI, N, NotI. The locations of the 3′ RAREs are indicated as DR2and DR5, respectively.

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Fig. 2.RA responses of the Hoxb-1/lacZreporter constructs in F9cells. F9 cells were transiently transfected with wild-type Hoxb-1/lacZ(WT), Hoxb-1RAIDR5mu (DR5mu) and Hoxb-1RAIDR5 3′-deletion (3′-del) reporter constructs for 48 hours in the presence(open bar) or absence (shaded bar) of 1 µM RA (see Materials andMethods). The results are expressed in arbitrary units of β-galatosidase enzyme activity normalized for transfection efficiency,with CAT reporter activity as an internal control. Bars representstandard error.

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RESULTS

The responses of the Hoxb-1 /lacZ constructs to RAin cultured F9 cellsPreviously we identified a single RA-inducible DNAse hypersensitive site located 6.5 kb 3′ of the murine Hoxb-1-coding sequence (Langston et al., 1997). Further analydemonstrated that an enhancer, called RAIDR5, at this siteregulates the RA responsiveness of the Hoxb-1 gene in F9teratocarcinoma and embryonic stem cells. This newidentified DR5 RARE is different in location and sequencefrom the RA-regulated 3′Hoxb-1 enhancers previouslydescribed (Marshall et al., 1994; Ogura and Evans, 1995Sequence comparison also shows a conserved DR5 RARE inthe chick Hoxb-13′ enhancer, and these Hoxb-1DR5 RAREsare identical to the DR5 RAREs in the human and murineHoxa-1 3′ RAIDR5 enhancers (Langston and Gudas, 199Langston et al., 1997).

To elucidate the role of the Hoxb-13′ DR5 RARE in animals,we constructed a wild-type Hoxb-1/lacZtransgene thatincludes 6 kb of 5′ and 7.5 kb of 3′ flanking sequences in themurine Hoxb-1gene; this transgene includes the distal 3′ DR5-type RARE (Fig. 1, RAIDR5 WT). The lacZgene was insertedinto the Hoxb-1-coding sequence so that an in-frame fusiowas generated that contained the first 34 amino acids of Ho1 fused to the β-galactosidase-coding sequence. We madesecond construct with point mutations in the DR5 RARE (Fig.1, RAIDR5mu), and a third construct with the 3′-most 2 kbfragment containing the RAIDR5 enhancer deleted (Fig. 1, 3′-deletion). The same DR5 RARE point mutations in theconserved Hoxa-1gene result in a loss of RAR binding in geshift assays (Langston and Gudas, 1992).

To test the RA inducibility of the reporter constructs, thesHoxb-1/lacZ constructs were transfected into F9 cellsTransiently transfected F9 cells were treated with RA or leuntreated. The pHoxb-1RAIDR5WT/lacZ transgene was ableto confer RA-responsiveness (~5-fold) as measured by β-galactosidase activity (Fig. 2). In contrast, both the RAIDR5muand the RAIDR5 3′ deletion constructs showed only minimaRA induction. These results are consistent with our previodata that a ~300 bp Hoxb-1DNA fragment containing the DR5RARE is sufficient for RA inducibility in F9 cells (Langstonet al., 1997) and they show that the 3′ DR5 RARE is required

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Point mutations within the DR 5 RARE of the Hoxb-1RAIDR5 enhancer abolish expression in the foregutof the mouse embryoTo determine the role of the Hoxb-1 3′ DR5 RARE in vivo,transgenic mice carrying the various Hoxb-1/lacZtransgeneswere generated. Transgene expression patterns were analat all stages of development during which the endogenoHoxb-1gene is expressed in the embryo.

Hoxb-1 is one of the earliest expressed Hox genes. Thenormal pattern of expression of Hoxb-1 mRNA in the

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Fig. 3.Transgenic embryos expressing the Hoxb-1RAIDR5WT/lacZor Hoxb-1RAIDR5mu/lacZconstructs at 8-8.5 d.p.c.. Whole-mountX-gal staining of 8 d.p.c. embryos transgenic for WT construct (A)and the DR5mu construct (B). Whole-mount X-gal staining of ~8.5d.p.c. embryos transgenic for the WT construct (C) and the DR5muconstruct (D). Arrowheads, pharyngeal foregut. Scale bars: (A-C)200 µm; (D) 150 µm.

ic embryos expressing the Hoxb-1/lacZconstructs at 9.5 d.p.c. Side viewiew (D) of 9.5 d.p.c. embryos transgenic for the WT construct. Side viewiew (E) of 9.5 d.p.c. embryos transgenic for the DR5mu construct. Sidesal view (F) of 9.5 d.p.c. embryos transgenic for the 3′-deletion (3′del)head, foregut; arrow, rhombomere 4. Scale bars: (A,B,D,E) 500 µm, (C)µm. Abbreviations: b1, branchial arch 1; b2, branchial arch 2; fg,; ms, mesencephalon; ov, otic vesicle; s, somitic mesenchyme; r4,Representative illustrations are shown. At least two different transgenicnt founders were examined for each of the transgene constructs. Severalt developmental stages were examined for each transgenic line; no observed when experiments were repeated on multiple litters.

gastrulation stage of development has been well defin(Frohman et al., 1990; Murphy and Hill, 1991; Wilkinson eal., 1989). Hoxb-1mRNA is expressed in embryonic ectodermand mesoderm in the posterior half of the embryo by laprimitive streak stages. By the headfold stage, Hoxb-1mRNAexpression extends slightly anteriorly to the primitive streakthe neuroepithelium and the underlying mesoderm. At abthe time of formation of the first somite (8.5 d.p.c.), the moanterior neural staining at the preotic sulcus becomes separfrom the staining at the posterior of the embryo and tmesoderm expression retreats posteriorly toabout the level of the primitive streak. Thefocus of this study is on Hoxb-1 transgeneexpression between 8 and 12 d.p.c.

The expression patterns of the Hoxb-1RAIDR5WT/lacZ transgene (designated asWT) and the Hoxb-1RAIDR5mu/lacZtransgene (designated as DR5mu) at 8-8.5d.p.c. are shown in Fig. 3. At 8.0 d.p.c., theexpression of the WT transgene extendsfrom the posterior of the embryo up to awell-defined anterior boundary in theheadfolds. Expression of the WT transgeneis restricted to the neural tube, the lateralmesoderm and the primitive streak (Fig.3A). A similar pattern is seen with theDR5mu transgene. The anterior boundary ofthe DR5mu transgene expression in theneuroepithelium and mesoderm is similar tothat observed for the WT transgene, i.e. justup to the posterior edge of the preotic sulcus(Fig. 3B).

At 8.5 d.p.c. the most anterior neuralstaining of the Hoxb-1RAIDR5WT/lacZtransgene at the preotic sulcus is separatedfrom the staining in the posterior of theembryo. Meanwhile, the mesodermal

Fig. 4.Transgen(A) and dorsal v(B) and dorsal vview (C) and dorconstruct. Arrow400 µm, (F) 450 foregut; h, heartrhombomere 4. lines from differelitters at differendifferences were

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expression retreats toward the posterior (Fig. 3C). In additioexpression of the WT transgene is observed in the foregpocket (Fig. 3C).

A different, distinct pattern of expression is seen with thRAIDR5mu transgene; β-gal staining is observed in the neuraepithelium and the lateral mesoderm but not in the developigut epithelium (Fig. 3D). These data suggest that the D5RARE is required for initiating Hoxb-1expression in the gutepithelium but is not required for setting the correct anteriboundary of Hoxb-1expression in the neural tube andmesoderm.

By 9.5 d.p.c., the pattern of expression of the Hoxb1RAIDR5WT/lacZ transgene is similar to that at 8.5 d.p.c., buthe intensity of the β-gal staining is higher. At this stage,expression in the neural epithelium is in a discrete band rhombomere 4, which is one segment anterior to the ovesicle and a domain in the posterior neural tube (Fig. 4A,DWT transgene expression is also seen in the notochord paraxial (somitic) mesoderm. It is worth noting that, inrhombomere 4, there is staining of the neural crest cells thave migrated from rhombomere 4 (Fig. 4D). The Wtransgene expression is also detected in the gut region at below the level of the heart and in the paraxial mesodermthe posterior region of the embryo.

In embryo sections, expression of the WT transgene in tforegut epithelium can be seen, and expression is also obsein the gut-associated mesoderm and some ectoderm in clproximity (Fig. 5A-C). At ~9.5 d.p.c., the gut is a simple tubewider at both ends than in the middle. The foregut includprimarily the pharynx, pharyngeal pouches and esophagregion at this stage. Expression in the gut epithelium, guassociated mesoderm and the adjacent ectoderm hasapparent anteroposterior restriction in the foregut from ththird pharyngeal arch throughout the pharynx to the esophag

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Table 1. Summary of expression patterns of theHoxb1/lacZ transgenes in 9.5 dpc embryos (-RA)

Transgene construct

Transgene expression domain WT DR5mu 3′-del

rhombomere 4neural epithelium + + +neural crest + + +

gutgut epithelium + −* +/−gut associated mesoderm + − −gut adjacent ectoderm + − −

posterior neural tube + + +notochord + + +somites + + +

*Only a very low level of lacZ expression was detected in embryos withthe Hoxb-1RAIDR5mu/lacZ transgene.

+, expression; −, no expression; +/−, low expression of the transgene ascompared to the Hoxb-1RAIDR5 WT transgenic embryos. Embryos werefixed first and then stained with X-gal (see Materials and Methods). For eachtransgene construct, 5-10 β-gal-stained embryos were examined andphotographed. Abbreviations are: WT, Hoxb-1RAIDR5WT/lacZ; DR5mu,Hoxb-1RAIDR5mu/lacZ; 3′-del, Hoxb-1RAIDR5 3′-deletion/lacZ.

region, with the most intense staining found in the epithecells in the lateral walls of the most posterior part of the fore(Figs 4A, 5B). There appears to be a coordinated expresof the Hoxb-1 RAIDR5WT/lacZ transgene in the endodermmesoderm and ectodermal tissue layers in the gut at this t

At 9.5 d.p.c., expression of the Hoxb-1 RAIDR5mu/lacZtransgene is observed in the neural tube, notochord and sommesoderm at a level similar to that observed in animcarrying the Hoxb-1RAIDR5WT/lacZ transgene (Fig. 4B,E).The most striking change, as compared to the embryos wthe WT transgene, is the lack of DR5mu transgene expressionin the gut region. Only very weak β-gal staining is seen in theforegut epithelium and in the most posterior region of tforegut of the DR5mu transgenic animals (Fig. 4B). Nostaining is observed in the gut-associated mesoderm andadjacent ectoderm epithelium (Fig. 5E-H). In the foindependent lines of DR5mu transgenic animals analyzedthree lines exhibited essentially no β-gal staining in the foregutepithelium (Fig. 4B). One DR5mu transgenic line showed a lowlevel of transgene expression in the gut epithelium, a level twas significantly lower than that observed in the embryos wthe Hoxb-1RAIDR5WT/lacZ transgene (data not shown).

The expression pattern of the Hoxb-1RAIDR5 3′-del/lacZtransgene (designated as 3′-del) is similar to the pattern of theHoxb-1RAIDR5mu/lacZ transgene (Fig. 4C,F). Expression othe 3′-del transgene was found in the neural tube, notochand somitic mesoderm at a level similar to that observedanimals carrying the Hoxb-1RAIDR5WT/lacZ transgene.However, staining in the foregut of the animals carrying the ′-del construct is significantly reduced as compared to the Hoxb-1RAIDR5WT/lacZ transgene (Fig. 4C versus A). We concludthat the DR5 RARE is required for region-specific expressioin the gut, including the gut epithelium, and in cells derivfrom all three germ layers. This is consistent with the notithat Hoxb-1 expression in different germ layers in the gut coordinately regulated by bioactive retinoids such as RA.

In 10.5-11.5 d.p.c. embryos, expression of the Hox1RAIDR5WT/lacZ transgene is reduced relative to earliestages, but expression is still observed in all of the same regseen in earlier embryos, i.e. in rhombomere 4, the notochthe most posterior somitic mesoderm, and in a very restricregion of the foregut epithelium and gut-associated mesod(data not shown). After 11.5 d.p.c., lacZactivity is significantlyreduced, but is still detectable in small, diffuse groups of cein the hindbrain, paraxial mesoderm, posterior neural tunotochord and root of the forelimb of the embryos carryieither the Hoxb-1RAIDR5WT/lacZ or the Hoxb-1RAIDR5mu/lacZtransgenes (data not shown).

Taken together, our data show that a 15 kb DNA fragmof the Hoxb-1gene is sufficient to recapitulate the expressiof the endogenous Hoxb-1gene both in the early and the latstages of development. We demonstrate that the conserveDR5 RARE in the murine Hoxb-1 RAIDR5 is essential forHoxb-1region-specific expression in the developing gut (Tab1).

Alterations in the expression of the endogenousHoxb-1 gene are associated with exogenous RAadditionThe RA-induced expression pattern of the endogenous Hoxb-1 mRNA has been reported previously (Conlon and Rossa

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1992; Marshall et al., 1992; Morriss-Kay et al., 1991; Wood eal., 1994). RA treatment resulted in extension of thendogenous Hoxb-1mRNA expression in the anterior directionrelative to the expression seen in untreated embryos both prto and after the formation of the headfolds. Ectopic anteriexpression of Hoxb-1was induced in both neuroectodermaand mesodermal cell layers of headfold embryos. The timinof RA treatment was critical for its effects on Hoxb-1 geneexpression and for its teratogenic effects (Conlon and Rossa1992; Morriss-Kay et al., 1991; Popperl et al., 1995)Expression of Hoxb-1in rhombomere 4 became largelyrefractory to the RA treatment if the embryos were treated wiRA after 8 d.p.c. (Conlon, 1995; Wood et al., 1994).

We performed whole-mount in situ hybridization to showthe expression pattern of the endogenous Hoxb-1mRNA afterRA treatment (Fig. 6). In 8.5 d.p.c. embryos, the endogenoHoxb-1 mRNA was expressed in a pattern identical to thadescribed above by other laboratories. For example, justhours of RA treatment at 8.5 d.p.c. resulted in a dramaanterior shift ofHoxb-1mRNA expression in the foregut (Fig.6A,B), as shown previously (Conlon and Rossant, 1992indicating that the RA effect on Hoxb-1 gut expression isrelatively rapid. By ~9.5 d.p.c., exogenous RA can still causanteriorization of endogenous Hoxb-1mRNA expression in thegut (Fig. 6D,E).

The DR5 RARE is required for Hoxb-1 transgeneresponse to exogenous RA in the gutTo determine if the RAIDR5 RARE was responsive to RA invivo, we treated pregnant mothers carrying the Hoxb1RAIDR5WT/lacZ or Hoxb-1RAIDR5mu/lacZtransgene withRA for 12-16 hours. The expression patterns of the WT anDR5mu transgenes at 8 d.p.c. in untreated embryos andembryos exposed to RA at ~7.5 d.p.c. for 12 hours anharvested at 8 d.p.c. are shown in Fig. 7. RA treatment caua dramatic anterior expansion of the WT transgene in the neutube, somites and lateral plate mesoderm, such that even most anterior structures such as the midbrain and forebra

3241Hoxb-1 expression in development

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Fig. 5.Sections of transgenicembryos at 9.5 d.p.c. Midsagittalsections of Hoxb-1RAIDR5 WTconstruct at low (A) and highermagnification (B). Transversesections of WT construct in anterior(C) and posterior (D) portions of theembryo. Mid-sagittal sections of theDR5mu construct at low (E) andhigher (F) magnification. Transversesections of the DR5mu construct inanterior (G) and anterior,rhombomere 4 containing (on theright) and posterior (on the left) (H)portions of the embryos. Transversesection of the 3′-deletion construct inthe anterior portion (I) of theembryo. Scale bars: (A) 500 µm,(B,F) 200 µm, (C,D,G-I) 250 µm,(E) 700 µm. Abbreviations: ec, gutadjacent ectoderm; fg, foregut; h,heart; ge, gut epithelium; gm, gut-associated mesoderm; nc, notochord;nt, posterior neural tube; s,somiticmesenchyme; r4, rhombomere 4.

of endogenous Hoxb-1mRNA in 8.5-9.5 d.p.c. RA-treated embryosle-mount in situ hybridization using an antisense strand Hoxb-1probe.ion in an 8.5 d.p.c. embryo. (B) Hoxb-1expression in an 8.5 d.p.c. RA for 4 hours in utero. (C) Control embryo at 8.5 d.p.c. hybridizedxb-1probe. (D) Hoxb-1expression pattern in a 9.5 d.p.c. embryo.ion pattern in a 9.5 d.p.c. embryo after 12 hours of RA treatment. at 9.5 d.p.c. hybridized to the sense strand Hoxb-1probe. ) 200 µm. Abbreviations: b2, branchial arch 2; fg, foregut; h, heart; r4,rowheads mark the anterior boundary of lacZstaining in the foregut.

express the WT transgene (Fig. 7B). There is no significdifference in expression between the WT and the DR5mutransgenes in response to RA at this stage except thaactivity of lacZ in the foregut pocket was observed in thDR5mu transgenic embryos (Fig. 7B,C).

Treatment with RA at the early somite stage (8.5-8.d.p.c.) for 12-16 hours prior to embryo harvesting at ~9d.p.c. did not result in any apparent teratogenic effects oncentral nervous system or the overall body structure. Howethe expression pattern of the Hoxb-1RAIDR5WT/lacZtransgene was altered only in the gut of the embryos followingRA treatment (Fig. 7D versus E). Exogenous RA treatmedramatically anteriorized the expressionof the WT transgene in the foregut, sothat WT transgene expression wasdetected extensively in the branchialarches, along the anteroposterior axis tothe first branchial arch (Fig. 7E,I; notedifference in locations of transgeneexpression relative to rhombomere 4 inFig. 7D versus E). The expression oflacZ was most abundant in thepharyngeal pouch, which is an extensionof the pharyngeal foregut endoderm.Upon careful examination of thetransgene staining in the sections fromthe RA-treated WT transgenic embryos,lacZ expression was detected in all threegerm layers in the foregut region (Fig.8A,B).

In contrast, when mice carrying theHoxb-1RAIDR5mu/lacZ transgene weretreated with exogenous RA at the earlysomite stage (~8.75 d.p.c.) for 12-16hours, there were no detectable changesin the transgene expression in the foregut,

Fig. 6. Expression determined by who(A) Hoxb-1expressembryo exposed towith sense strand Ho(E) Hoxb-1express(F) Control embryo(A,B) 100 µm; (D,Erhombomere 4. Ar

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somitic mesoderm and other expression domains in tembryos by 9.5 d.p.c.; the only exception was the minanterior extension in r4 (Fig. 7F,J). No ectopic transgeexpression was observed in the foregut (Fig. 8C,D). No β-galstaining was seen in the gut-associated mesoderm andadjacent ectoderm (Figs 7F,J, 8C,D); only the notochoexpressed the transgene (Fig. 8D). No anterior extension oftransgene expression in the foregut was detected after 12hours RA treatment (Fig. 7F,J). Therefore, in the early somstage, the DR5mu transgene does not respond to RA. Similarlembryos carrying the RAIDR53′-del/lacZ transgene did notrespond to exogenous RA (Fig. 7G,K). We conclude that t

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RAIDR5 RARE is essential for anterior extension of Hoxbexpression in the foregut in response to exogenous RA.

In 10-11.5 d.p.c. embryos, the lacZexpression patterns andthe RA responses of both the Hoxb-1RAIDR5WT/lacZ andHoxb-1RAIDR5mu/lacZ transgenic embryos are similar tothose in earlier stage embryos. Embryos carrying the Wtransgene still show restricted transgene expression in theand RA causes some detectable anterior expansion of theexpression, whereas in the DR5mu transgenic embryos noexpression of lacZis detectable in the gut. There is no ectoptransgene expression in the DR5mu transgenic embryos afteRA treatment (data not shown).

Collectively, our results show that the 3′ DR5 RARE directlymediates part of the Hoxb-1 RA response and that it is anessential regulatory element for the correct Hoxb-1expressionpattern in the gut epithelium and the associated mesodermectoderm during embryogenesis. Our data also stronsuggest that endogenous retinoids play a significant role inregulation of Hoxb-1expression in the foregut. Moreover, thobservations that the Hoxb-1 gene is expressed in thedeveloping gut and that this expression is regulated by through a DR5 RARE suggest a role for Hoxb-1 in theanteroposterior patterning of the gut.

DISCUSSION

The DR5 RARE is essential for Hoxb-1 regionalexpression in the gut and for the RA-associatedanteriorization of Hoxb-1 expression in the gutIn the present studies, we have shown that the Hoxb-13′ DR5RARE is required for the expression of the Hoxb-1/lacZtransgene in the gut. Hoxb-1expression in gut endoderm, gutassociated mesoderm and the adjacent ectoderm appearsregulated in a coordinated manner corresponding to anteroposterior axis at this stage (8.5-10.5 d.p.c.).

We demonstrate that Hoxb-1expression in the gut respondto exogenous RA, resulting in anterior expansion of Hoxbexpression in the gut similar to the well-documented Rresponse in the neural tube at earlier stages. We have shthat this RA response of the lacZreporter gene in the gut regionis dependent on the DR5 RARE in the Hoxb-13′ RAIDR5enhancer (Fig. 7). Therefore, the DR5 RARE 3′ of the Hoxb-1gene is the major determinant of the anteroposterior limitHoxb-1expression in the three germ layers in the gut. Howevthe RAIDR5 RARE is dispensable for Hoxb-1 neural tubeexpression and for setting the anterior expression boundarthe neuroepithelium.

These data are consistent with a model that Hoxexpression in the gut is defined by a developmental field tis independent of the early neural tube expression. By analto the previously defined role of RA in the hindbrain, it conceivable that a retinoid gradient in the early somite stais required for the coordinated expression of the Hoxb-1geneand subsequent pattern formation in the gut. The requiremfor a DR5 RARE for Hoxb-1expression in the gut providesanother illustration that retinoids such as RA are able regulate the expression of Hox genes at the 3′ends of thechromosomal clusters directly, thus suggesting thendogenous retinoids may be required for the approprspatial and temporal regulation of Hoxb-1 expression in thegut. We think that this regulation is an important compone

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The role of Hoxb- 1 in gut anteroposterior patterningDuring mouse embryo gastrulation, the foregut forms as earas 7.5 d.p.c. when differential growth produces a ventrinvagination of the anterior definitive endoderm (Rugh, 1996At 8.5 d.p.c., a second endodermal invagination starts from tposterior end to form the hindgut. The midgut is derived fromboth foregut and hindgut primordia and forms from the fusioof the primitive gut tubes. The foregut gives rise to organs suas the esophagus, lung, stomach, liver and pancreas. Tmidgut develops into the small intestine. The hindgut developinto the large intestine and forms part of the cloaca, thcommon gut-urogenital opening. The molecular mechanismunderlying the positional cues to specify regionadifferentiation of the digestive tract are poorly understoodOrgan-specific differentiation of the endoderm-derivedepithelium is induced by signals from the surrounding viscermesoderm and partially depends on the reactive potential of tendoderm to the inductive signal (Haffen et al., 1983; Kedingeet al., 1986).

Transcripts of some Hox genes have been found in arestricted region in the developing digestive tract (Conlon anRossant, 1992; Dollé et al., 1991; Gaunt et al., 1989, 199Holland and Hogan, 1988; Toth et al., 1987). Recently, Hogenes from paralog groups 9-13, located at the 5′ end of the Hoxclusters, have been shown to be expressed in progressivposterior domains in the visceral mesoderm in the chichindgut, colinear with their order on the chromosome (Roberet al., 1995; Yokouchi et al., 1995). Moreover, Hoxd-12 andHoxd-13 targeted disruptions result in malformations of thanus sphincter (Kondo et al., 1996). In addition, ectopiexpression of Hoxa-4 and Hoxc-8 in transgenic mice causabnormal development in the digestive system (Pollock et a1992; Wolgemuth et al., 1989). Our data that the DR5 RARE isrequired for Hoxb-1regional expression in the foregut and itsanteriorization in the gut epithelium in response to RA arconsistent with the concept of the classical Hoxcode applied tothe developing gut. In such a model, Hoxgenes are coordinatelyexpressed in the gut from the anterior to the posterior end incolinear fashion, as they are expressed in the neural tube (Konet al., 1996; Roberts et al., 1995; Yokouchi et al., 1995).

In Drosophila, homeotic genes are expressed in restricteparts of the visceral mesoderm along the anteroposterior aduring gut morphogenesis. The restricted homeotic genexpression is known to determine the morphological bordeof the gut (reviewed in Bienz, 1994). Labial, the Drosophilahomolog of Hoxb-1, is expressed in a restricted domain in theepithelium in the midgut that is required for constriction of theendoderm (Panganiban et al., 1990). Decapentaplegicprotein(DPP), produced and secreted from the visceral mesodecells, locally induces the region-restricted expression of labial.There are intriguing parallels between the expression patterof Hoxb-1, sonic hedgehogand bone morphogenic protein(BMP) genes in the vertebrate gut and those of their Drosophihomologs, labial, hedgehog, and dpp during Drosophilamorphogenesis (Roberts et al., 1995). Given the remarkabcorrelation of Hox gene expression with morphologicalborders, it is likely that Hox gene expression also regulatesaspects of vertebrate gut morphology.

3243Hoxb-1 expression in development

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Fig. 7.Expression patterns of untreated and RA-treated transgenic embryos at 8-9.5 d.p.c.. Whole-mount X-gal staining of 8 d.p.c. embryostransgenic for (A) the Hoxb-1RAIDR5WT/lacZ construct, untreated; (B) the WT construct in an 8 d.p.c. embryo which was treated at 7.5 d.p.c.with RA; (C) expression of the Hoxb-1RAIDR5mu/lacZ(DR5mu) construct in an embryo treated with RA as in B. Whole-mount X-gal stainingof 9.5 d.p.c. embryos transgenic for (D) the WT construct, untreated embryo; (E) the WT construct in an embryo that was treated at ~8.75 d.p.c.with RA in utero; (F) the expression of the DR5mu construct in an embryo treated with RA as in E; (G) the DR5 3′-deletion construct in anembryo treated with RA as in E; (H) dorsal view of an untreated embryo carrying the WT transgene, 9.5 d.p.c.; (I) dorsal view of a 9.5 d.p.c.embryo, carrying the WT transgene, treated with RA at ~8.75 d.p.c. in utero; (J) dorsal view of a 9.5 d.p.c. embryo carrying the DR5mutransgene, treated with RA as in I; (K) dorsal view of a ~9.5 d.p.c. embryo carrying the 3′-del transgene, treated with RA as in I. Abbreviations:r4, rhombomere 4; h, heart; ms, mesencephalon; arrows mark rhombomere 4; arrowheads mark the anterior boundary of lacZ transgeneexpression in A-C or the position of the foregut in D-K. Scale bars: B,C, 200 µm; D,H, 400 µm; E,F,G,I,J,K, same magnification as D,H.A, same magnification as B.

Our results also suggest that the retinoic acid receptors, Rand RXR, may function in the anteroposterior patterniduring gut development. RAR and RXR mRNA expressionpatterns during early embryogenesis have been w

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documented (reviewed in Mangelsdorf et al., 1994). Fexample, RARα is ubiquitously expressed while transcripts oRARβ and RARγ are temporally and spatially restricted(Ruberte et al., 1990). The expression patterns of RARand RXR

Fig. 8.Sections of RA-treated embryos transgenic for theHoxb-1RAIDR5WT/lacZand Hoxb-1RAIDR5mu/lacZconstructs at 9.5 d.p.c.. Embryos were treated at ~8.75 d.p.c.in utero with RA and harvested 12-16 hours later. Tranversesections of an RA-treated embryo carrying a WT transgene(A,B). Transverse sections of an RA-treated embryo carryingthe DR5mu transgene (C,D). Section (C) is throughrhombomere 4 on the right; section (A) is slightly posterior tor4. The anterior portion (marked A) and posterior portion(marked P) of the same embryo in B and D, but B and D areboth more posterior cuts than A and C. Scale bars: (A-D) 500µm. Abbreviations: fg, foregut; nc, notochord; nt, posteriorneural tube; s, somitic mesenchyme; r4, rhombomere; ge, gutepithelium. Arrowhead in A points to foregut and arrowheadin C points to neural crest cells.

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transcripts show some overlap with Hoxb-1, including theexpression in the embryonic gut and many endoderm-deritissues. More specifically, at ~8.5 d.p.c., embryos exprRARβ mRNA in the foregut endoderm and CRBPI transcriptswere detected in the gut endoderm (Ruberte et al., 199CRABP II transcripts were observed in the foregut endodeat ~8.5 d.p.c. embryos, whereas CRABP ImRNA was not seen(Ruberte et al., 1992). Moreover, mice with RAR/RARorRAR/RXRdouble mutations show abnormalities in severorgans derived from the embryonic foregut durindevelopment (Kastner et al., 1997; Mendelsohn et al., 199Thus, RARα and/or RARβ are implicated in Hoxb1regulationby RA in the developing foregut, whereas RARγ appears not tobe involved. Consistent with these genetic and expression dteratogenic doses of RA have been shown to cauabnormalities in the digestive tract in Syrian hamste(Shenefelt, 1972). Therefore, it is likely that the endogenoretinoid signaling pathway plays an important role in gdevelopment through the regulation of Hox gene expression.We are currently investigating whether there are aabnormalities in gut morphology, especially in the foregregion, after exogenous RA treatment of 8.5-9.5 d.p.c. moembryos.

It is worth noting that Hoxgene knockout and misexpressioexperiments have primarily reported phenotypic abnormalitin the craniofacial region, hindbrain, axial skeleton and lim(reviewed in Krumlauf, 1994). There have been few reportsabnormalities in other regions of the embryo, such as the gassociated with alterations in Hox gene expression. This maybe because in certain tissues, like the digestive tract, thersignificant functional redundancy among many different Hoxgenes, especially paralogous genes, so that an alteration inexpression of any one gene would not significantly affect tdevelopment of the gut. Alternatively, there may be subfunctional abnormalities in many of these organs that hagone unrecognized because the evaluation of the Hox mutantsthus far has been focused on more obvious abnormalities.

The expression of the murine Hoxb-1 gene in bothearly and late stages can be recapitulated with aHoxb-1 /lacZ transgene that contains 6.5 kb 5 ′ and7.5 kb of DNA 3′ of the Hoxb-1 -coding regionPreviously several regulatory elements have been identifiedboth the 5′and 3′flanking regions of murine and human Hoxb1 genes (Marshall et al., 1994; Ogura and Evans, 1995aStuder et al., 1994). Marshall et al. (1994) found a DR2-typeRARE ~2 kb 3′ of the murine Hoxb-1-coding sequence that conserved in chick and pufferfish. Transgenes with this D2RARE can partially recapitulate the earlyexpression pattern ofthe endogenous Hoxb-1gene and can mediate the responseexogenous RA in the neural tube at early times (~7.5-8d.p.c.). The Hoxb-13′ RAIDR5 RARE was not included in thetransgene constructs used in these prior studies. The restrrhombomere 4 expression of the endogenous Hoxb-1gene hasbeen shown to be dependent on two enhancers in the Ho5′ flanking region, an r4 enhancer with a Hoxb-1autoregulatoryloop and a 5′ DR2 RARE repressor element (Popperl et a1995; Studer et al., 1994).

Our studies show that the RAIDR5 RARE, located further3′ of the previously studied DR2 RARE, is also required forthe lacZ transgene to recapitulate the correct region

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expression of the endogenous Hoxb-1gene in the gut in bothearly and late stages (Figs 3, 4). Therefore, the regulationHoxb-1 expression and its response to RA involves multipRAREs and other regulatory elements.

Hoxb-1 expression is determined by sequentialactivation of different RAREs in different germlayersFrom the studies described here and elsewhere (Marshall et1994), it is clear that expression of Hoxb-1 in bothneuroectoderm and endoderm is directly regulated by Rsignaling. There are two phases of Hoxb-1sensitivity toexogenous RA. The Hoxb-1expression boundary in r4 exhibitsa period of sensitivity to exogenous RA when embryos atreated before 8.5 d.p.c.. This initial RA-response is dependon the Hoxb-13′ DR2 RARE. During the second phase at ~9.5d.p.c., Hoxb-1 expression in r4 becomes insensitive toexogenous RA, but Hoxb-1 expression in the foregut is stillsensitive to alteration by RA. This later RA response dependent on the 3′DR5 RARE. Thus, Hoxb-1expression isestablished by the sequential activation of different RAREs different germ layers. The multiple RAREs found in the Hoxb-1 enhancers suggest that endogenous retinoids play an esserole in the regulation of Hoxb-1and hence in theanteroposterior patterning of the embryo.

The Hoxb-1 DR5 RARE is conserved in other geneswith gut expressionHoxb-1, Hoxa-1 and Hoxd-1 constitute the paralog group 1,evolutionarily related to the Drosophila labialhomeotic gene.Hoxb-1shares several features of its early expression pattewith Hoxa-1, both spatially and temporally (Frohman et al.1990; Murphy and Hill, 1991). In contrast to the compleregulatory elements found in Hoxb-1, only one functional DR5RARE has been identified in the 3′ flanking sequence of Hoxa-1 to date (Frasch et al., 1995; Langston and Gudas, 19Langston et al., 1997). We have previously shown that tHoxb-1 3′ RAIDR5 enhancer is conserved in the chick anmurine Hoxb-1 genes, as well as in the human and murinHoxa-1genes. There are three blocks of conserved sequenin the enhancer: a DR5 RARE, a conserved element 1 (CE1)and a conserved element 2 (CE2). The Hoxb-1DR5 RAREsequence is 5′-GGTTCA (N)5 AGTTCA-3′ and is identical tothe DR5 RARE found in the Hoxa-1 3′ enhancers. The highdegree of homology among the three conserved sequensuggests that the RAIDR5 enhancers in the Hoxb-1and Hoxa-1 genes have been conserved during the duplication adivergence of the Hoxgene complexes. Frasch et al. (1995demonstrated that the Hoxa-1 3′ enhancer directs lacZtransgene expression in the floor plate, notochord, gepithelium and the posterior neural tube in transgenic micMore recently, Dupé et al. (1997) used a cre-lox targetinmutation of the Hoxa-13′ DR5 RARE to demonstrate that thisenhancer plays an important role in the early establishmentthe Hoxa-1 anterior expression boundary in the neural platThese data show that the function of the Hoxa-1DR5 RAREappears to be different from that of the Hoxb-1DR5 RARE, aswe have shown in this study. It is possible that the Hoxb-13′DR2 and DR5 RAREs have some redundant functions indetermining Hoxb-1 expression in the neural tube andnotochord, but not in the gut. Alternatively, during the Hox

3245Hoxb-1 expression in development

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d

gene divergence these two enhancers may have acquired different functions in the control of the region-specifiexpression of these genes.

RARβis known to be directly transcriptionally activated bRA through a DR5 RARE identical to the Hoxb-1DR5 RARE(de Thé et al., 1990; Sucov et al., 1990). By 8 d.p.c., RARβexpression is seen in many regions of the embryo that ovewith regions expressing Hoxb-1, including the neural tubelateral mesoderm and the foregut (Ruberte et al., 1990). overlapping expression domains in the gut among RARβ,Hoxa-1and Hoxb-1may result from the presence of a commoDR5 RARE in their regulatory regions. Alternatively, the RARβreceptor itself may play a role in the responses of the Hoxb-1and Hoxa-1 genes to bioactive retinoids in the gut. Thpresence of a DR5 RARE in several genes expressed in tembryonic gut suggests that endogenous retinoids such asplay a regulatory role in the developing gut.

We thank all the members of the Gudas laboratory for helpdiscussions and Taryn Resnick for editorial assistance. This resewas supported by NIH grant R01CA39036 to L. J. G., and in parta Leukemia Research Foundation fellowship to D. H. and an Nfellowship (1F32CA71153) to S. W. C.

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