2807Development 124, 2807-2817 (1997)Printed in Great Britain © The Company of Biologists Limited 1997DEV4822

The bicoid-related homeoprotein Ptx1 defines the most anterior domain of the

embryo and differentiates posterior from anterior lateral mesoderm

Christian Lanctôt1,2, Bruno Lamolet1 and Jacques Drouin1,2,*1Laboratoire de génétique moléculaire, Institut de recherches cliniques de Montréal, 110 avenue des Pins ouest, Montréal,Québec, Canada, H2W 1R72Département de Biochimie, Université de Montréal

*Author for correspondence (e-mail: drouinj@ircm.umontreal.ca)

Ptx1 is a member of the small bicoid family of homeobox-containing genes; it was isolated as a tissue-restricted tran-scription factor of the pro-opiomelanocortin gene. Itsexpression during mouse and chick embryogenesis wasdetermined by in situ hybridization in order to delineate itsputative role in development. In the head, Ptx1 expressionis first detected in the ectoderm-derived stomodeal epi-thelium at E8.0. Initially, expression is only present in thestomodeum and in a few cells of the rostroventral foregutendoderm. A day later, Ptx1 mRNA is detected in theepithelium and in a streak of mesenchyme of the firstbranchial arch, but not in other arches. Ptx1 expression ismaintained in all derivatives of these structures, includingthe epithelia of the tongue, palate, teeth and olfactorysystem, and in Rathke’s pouch. Expression of Ptx1 incraniofacial structures is strikingly complementary to thepattern of goosecoid expression. In addition, Ptx1 is

expressed early (E6.8) in posterior and extraembryonicmesoderm, and in structures that derive from these. Therestriction of expression to the posterior lateral plate islater evidenced by exclusive labelling of the hindlimb butnot forelimb mesenchyme. In the anterior domain ofexpression, the stomodeum was shown by fate mapping toderive from the anterior neural ridge (ANR) which repre-sents the most anterior domain of the embryo. The con-cordance between these fate maps and the stomodealpattern of Ptx1 expression supports the hypothesis thatPtx1 defines a stomodeal ectomere, which lies anteriorly tothe neuromeres that have been suggested to constitute unitsof a segmented plan directing head formation.

Key words: stomodeum, homeobox, bicoid, Ptx1, mouse, chick.craniofacial development, fate map

SUMMARY

INTRODUCTION

Homeobox-containing genes encode transcription factors thatare essential for proper development. The current models forthe role of these genes in development postulate that differentcombinations of homeoproteins define segments within thedeveloping organism. The genes of the Hox cluster areexpressed in overlapping domains and their anterior borders ofexpression differ according to their position in the cluster, thoselocated more 3′ being the more rostrally expressed (Krumlauf,1994). Numerous gain- and loss-of-function experimentsinvolving Hox genes have validated the concept of a segmentedpattern of development. Thus, inactivation or ectopicexpression of genes of the Hox cluster lead to homeotic trans-formations of the trunk where those genes are expressed(Chisaka and Capecchi, 1991; Le Mouellic et al., 1992). Inter-estingly, Hox genes are not expressed in structures anterior tothe hindbrain (rhombomere 1). It is not yet clear whether devel-opment of head structures results from a segmented plan,although this idea has been proposed, largely based on thepatterns of expression of non-cluster homeobox-containinggenes (Puelles and Rubenstein, 1993; Rubenstein et al., 1994).

These include, among others, members of the Dlx, Otx andEmx families (Puelles and Rubenstein, 1993; Simeone et al.,1992a,b). Thus, the developing neuroepithelium has been sub-divided into neuromeres, which are defined by the comple-mentary patterns of expression of these genes.

In the chick, the most anterior region of the early embryocomprises a ridge which appears as a thickening at the marginof the neural plate. The fate of this anterior neural ridge (ANR)was determined by Couly and LeDouarin using quail-chickchimeras and it was found that the olfactory and oral epithelia,as well as the pituitary gland, derive from the ANR (Couly andLe Douarin, 1985, 1987). Upon expansion and subsequentbending of the anterior neural tube, the ANR is displacedventrally and becomes the stomodeum. Thus, the stomodeumis the ectoderm-derived epithelium that forms the earliestmouth structures. Its point of contact with the foregutendoderm, the oral membrane, ruptures at E9 to allow com-munication between the forming mouth and digestive tube.Two placodes will form within the stomodeum and their invagi-nations will give rise to the nasal pit and to Rathke’s pouch,the anterior pituitary primordium. Both the olfactory and neu-roendocrine hypothalamo-pituitary systems are formed from

2808 C. Lanctôt, B. Lamolet and J. Drouin

the association of stomodeal and neural ectoderms, i.e. theolfactory epithelium and the olfactory bulb and the pituitaryand hypothalamus, respectively. It is interesting to note thateach of these components derive from territories that wereadjacent at the neural plate stage (Couly and Le Douarin,1985). This observation led Takor and Pearse to suggest thatthese composite organs may in fact arise from a single embry-ological unit (Takor and Pearse, 1975).

There is no molecular marker or gene that may be acandidate for specification of a ‘stomodeal segment’, should itexist, although fate map studies are consistent with this possi-bility (Couly and Le Douarin, 1985; Osumi-Yamashita et al.,1994). This contrasts with the growing number of homeopro-teins found to be expressed in Rathke’s pouch (Hermesz et al.,1996; Sheng et al., 1996). In particular, Rpx expression ishighly restricted to the developing Rathke’s pouch and clearlydifferentiates the pituitary from stomodeal fate (Hermesz et al.,1996). We have recently cloned a novel homeoprotein by itsproperty to bind a cis-acting element of a pituitary hormonegene promoter and we named it Ptx1 (pituitary homeobox 1)(Lamonerie et al., 1996). Ptx1 acts as a transcriptional activatorand binds sequences with a TAAT/GCC core motif. Thehomeodomain of Ptx1 contains a lysine at position 9 of therecognition helix (position 50 of the homeodomain). Thisresidue is strategically placed in the major groove of DNA andit is a major determinant of DNA-binding specificity recog-

Fig. 1. Ptx1 expression in the stomodeum. Planes of sections are illustratembryo. Ptx1 is strongly expressed in the stomodeum (st) and more weak(C,D) Transverse section of an E8.25 embryo through the forming mouth(st). (E,F) Sagittal section of an E9 embryo. Arrowheads in F demarcate mount in situ hybridization to an E9 embryo. Specific signal in the stomoanterior is at the top and ventral is on the left unless indicated otherwise.yolk sac.

nizing the CC doublet of the target site (Treisman et al., 1989).This lysine residue defines the bicoid subfamily of home-oboxes. This subfamily has relatively few members, and mostof them have been implicated in axis and pattern formation.Bicoid is essential for development of anterior structures inDrosophila (Driever and Nusslein-Volhard, 1988); Otx1 andOtx2 are predominantly expressed in forebrain and midbrain(Simeone et al., 1992a, 1993) and disruption of the Otx2 generesults in headless embryos (Acampora et al., 1995; Ang et al.,1996; Matsuo et al., 1995); goosecoid (Gsc) is an importantmolecular component of the gastrulation organizer (Blum etal., 1992). We report here that, both in mouse and chicken, Ptx1is expressed in the stomodeum as it forms and that itsexpression is maintained in all stomodeal derivatives.

MATERIALS AND METHODSTissue preparationCBA × C57Bl6 mice were mated and the morning when a vaginalplug was detected was considered E0.5. Pregnant mice were killed bycervical dislocation, embryos were dissected, fixed overnight at 4°Cin 4% paraformaldehyde and embedded in paraffin. Sections (5 µm)were mounted on aminopropylethoxysilane-treated slides.

In situ hybridizationPrehybridization treatment of the slides was adapted from (Wilkinson,1992) and performed as follows: rehydration, digestion with proteinase

ed on the diagrams on the left. (A,B) Sagittal section of an E8.25ly in a patch of rostroventral foregut endoderm (arrowhead).. Arrows in D indicate lateral limits of expression in the stomodeumthe domain of Ptx1 expression in the foregut endoderm. (G) Whole-deal epithelium is indicated by an arrow. In this and following figures,

fb, forebrain; fg, foregut; hb, hindbrain; he, heart; st, stomodeum; ys,

2809Ptx1 defines a stomodeal ectomere

lar mesenchyme. (A,B) Transverse section of an E9.5 embryo, as showned in the mandibular (md) and maxillary (mx) epithelia as well as in as in B). (C) Whole-mount in situ hybridization of Ptx1 mRNA in a 10.5on in central stripes of mandibular mesenchyme as well as in the oraln E10.5 embryo. (F,G) Sagittal section of an E10.5 embryo. Ptx1 is such as Rathke’s pouch (rp). Ptx1 expression in the epithelium of the

erm and endoderm (arrowheads, D-G). II, second branchial arch; fb,ponent of 1st branchial arch; mx, maxillary component of 1st branchialrp, Rathke’s pouch.

K (5 µg/ml, 15 minutes, 22°C), refixation in paraformaldehyde 4% andacetylation with 0.25% acetic anhydride in 0.1 M triethanolamine.Hybridization was carried out at 52°C with 50 ng/ml of 35S-labelledcRNA probe (sp. act. >109 cts/minute/µg) in 50% formamide, 4×SET,10 mM DTT, 0.1% SDS,1× Denhardt’s, 100 µg/ml yeast tRNA,100 µg/ml denatured calf thymus DNA. Slides were washed at highstringency with 50% formamide/1× SSC/10 mM DTT at 62°C, treatedwith RNAse A (20 µg/ml, 30 minutes, 37°C) and washed again at highstringency. Slides were dipped in K.5 autoradiographic emulsion(Ilford). Exposure time at 4°C was between 12 and 21 days. After rev-elation with Kodak D-19, slides were counterstained with haema-toxylin-eosin. Two cDNA fragments were transcribed into cRNAprobes: a 573 nt fragment (from nt 604-1177) encompassing thehomeodomain and a 310 nt fragment encoding the last 14 amino acidsand 268 bp of 3′ untranslated region. Most figures presented here wereobtained using the longest cRNA probe because it gave a strongersignal; however, labelling was identical with the 3′ probe. In addition,the specificity of our hybridization signal is demonstrated by the factthat we did not observe cross-reaction with a recently cloned memberof the Ptx family (unpublished observations). Other probes used in thisstudy include Gsc (909 bp containing 180 bp of exon 2, intron 2 andexon 3), Msx2 (660 bp, 3′ portion of cDNA), Dlx3 (1100 bp) and Myf5(1900 bp, full-length cDNA). These probes were alkaline hydrolyzedto an average length of 500 bp.

Cloning of chicken Ptx1A chicken pituitary cDNAlibrary (kindly provided byNicci Illing, University ofCapeTown) was screenedusing a 1510 nt fragment ofthe mouse Ptx1 cDNA con-taining the homeodomain.Filters were washed atmoderate stringency (1×SSC/0.1% SDS, 55°C).Plaques were purifiedaccording to standardmethods (22). A total of 5overlapping clones wereobtained and sequenced.

RESULTS

Ptx1 was isolated as aputative cell-specific tran-scription factor of thepituitary pro-opiomelano-cortin (POMC) gene(Lamonerie et al., 1996).Although Ptx1 contributesto corticotroph-specifictranscription of the POMCgene, it is clear that Ptx1 isnot the sole determinant ofthis specificity and that itsearly expression through-out Rathke’s pouch(Lamonerie et al., 1996;Szeto et al., 1996) suggestsa wider role during mouseembryogenesis, forinstance in pituitarylineage specification, in

Fig. 2. Expression of Ptx1 in the mandibuon the diagram on the left. Ptx1 is expressstreak of mandibular mesenchyme (arrowdpc embryo. Note the prominent expressiepithelium. (D,E) Parasagittal section of aexpressed in derivatives of the stomodeummouth ends at the junction between ectodforebrain; he, heart; md, mandibular comarch; nt, neural tube; oe, oral epithelium;

Rathke’s pouch and pituitary morphogenesis or in patterningof anterior structures. In order to better define these hypoth-eses, we determined the pattern of Ptx1 expression throughoutmouse embryogenesis by in situ hybridization. Expression ofPtx1 is restricted to two domains of the developing embryo. Atthe anterior end, Ptx1 is initially expressed throughout thestomodeum and, at a weaker level, in a patch of foregutendoderm that contacts the stomodeum. At the posterior end,lateral plate and extraembryonic mesoderm and their deriva-tives also express Ptx1. Each expression profile is describedbelow.

Early expression of Ptx1 in the stomodeum andforegut endodermPtx1 expression is detected at E8.0 in the stomodeal epithe-lium and in a rostroventral portion of the foregut endodermwhere the two tissue layers meet (Fig. 1B). Cross-sectionanalysis shows the lateral extent of the signal in thestomodeum (Fig. 1D). At this early stage, the stomodealepithelium is continuous with the surface ectoderm and Ptx1expression abruptly stops where the epithelium becomes moresquamous (Fig. 1D, arrows). As assessed by whole-mount in

2810 C. Lanctôt, B. Lamolet and J. Drouin

Fig. 3. Expression of Ptx1 in thehead at later stages of development.(A,B) Transverse section of E13.5embryo through the mouth, asshown on the diagram on the left.Note the unlabelled mesenchyme(arrow, B) surrounding the dentalepithelium (de) and the absence ofexpression in the respiratoryepithelium (arrowhead, B).(C,D) Sagittal section of an E15.5head. Expression of Ptx1 ismaintained in all derivatives of thestomodeum. Also note stripes ofexpression in the tongue (to). de,dental epithelium; en, enamel organ;me, Meckel’s cartilage; np,nasopharynx; oe, oesophagus; ol,olfactory epithelium; pa, palate; pit,pituitary; sg, submandibular glands;to, tongue.

Fig. 4. Comparison of Ptx1 expression with other homeobox markers of the first branchial arch. Consecutive transverse sections through themouth of an E11.5 embryo (A-E) or consecutive sagittal sections of E13.5 head (F-H) were hybridized with the indicated probes. Ventral is tothe right. Note the complementarity between the expression patterns of Ptx1 (G) and Gsc (H), particularly in the nasal capsule (na) andoesophagus (oe)/trachea (tr). Arrow in F points to the tongue epithelium which is labelled by the Ptx1 probe (G) but not by the Gsc probe (H).II, second branchial arch; ma, maxillary component of the first arch; md, mandibular component of the first arch; na, nasal capsule; np, nasalpit; oe, oesophagus; pi, pituitary; to, tongue; tr, trachea.

2811Ptx1 defines a stomodeal ectomere

arly mouse embryo. Planes of sections are illustrated. Anterior is to the through the extraembryonic region of an E6.8 embryo. Expression ofing posterior extraembryonic mesoderm (p). (B,C) Sagittal section ofge, Ptx1 is expressed in the allantois (al) but not at the anterior levelerse section at the level of the extraembryonic and embryonic junctione of the allantois is strongly labelled. (F,G) Transverse section of anore distal level than in D,E. Expression is only detected in theerm. a, anterior; al, allantois; am, amnion; cp, cardiogenic plate; hf,

mesoderm; p, posterior; ps, primitive streak.

situ hybridization (Fig. 1G) and tissue sections, Ptx1expression defines a V-shaped segment of facial ectodermextending from underneath the future lens placode to thedeveloping first branchial arch. Expression in the oral epi-thelium seems to be more intense at the oral (buccopharyn-geal) membrane, the site of contact between stomodeal andforegut epithelia. Weaker labelling of the foregut endoderm islimited to a few cuboidal cells of the ventral wall that arecentered around the buccopharyngeal membrane (Fig. 1F,arrowheads). Ptx1 expression was not detected in anteriorregions prior to neural tube bending.

Expression in the first branchial archWhile Ptx1 mRNA is only detected in epithelial layers on E8,expression appears in the first branchial arch mesenchymestarting at E9 in a proximal-to-distal gradient and it isrestricted to a central stripe in the mandibular component (Fig.2B). No labelling is seen in the mesenchyme of the maxillarycomponent or in other branchialarches (Fig. 2B,E). The epitheliumsurrounding the mandibular arch isonly labelled in its inner half. Theepithelium of the maxillarycomponent is also labelled but thesignal decreases towards themidline. Fig. 2C shows a whole-mount in situ hybridization andFigure 2E a parasagittal section of10.5 dpc embryos. The epitheliumof the mouth is strongly labelledand the signal is restricted to tissuesof stomodeal origin: it endsprecisely at the junction with theendoderm (Fig. 2E, arrowhead).Discontinuity in labelling of theepithelium of the mouth reflects therupture of the stomodeum at the oralmembrane at E9 to allow communi-cation between mouth and foregut.At E10.5, both invaginations withinthe stomodeal epithelium arelabelled: the nasal pit and Rathke’spouch, the pituitary primordium(Fig. 2C,G). Labelling in the mes-enchyme of the future lower jaw isin the rostral two-thirds of the arch(Fig. 2E).

Derivatives of stomodeumand first branchial archLater in development, all epitheliathat derive from the stomodeummaintain Ptx1 expression. Theseinclude the epithelia of thenasopharynx, palate and tongue aswell as the adenohypophysis, thedental epithelium, the olfactoryepithelium and the epithelium of thesubmandibular glands (Fig. 3B,D).It is noteworthy that the tooth budsare not labelled and that Ptx1 is

Fig. 5. Ptx1 expression in eleft. (A) Transverse sectionPtx1 is detected in the forman E7.5 embryo. At this sta(headfold, hf). (D,E) Transvof an E7.5 embryo. The basE7.5 embryo at a slightly mposterior lateral plate mesodheadfold; lpm, lateral plate

expressed only in the dental epithelium (Fig. 3D). In the nasalsystem, Ptx1 is expressed in the olfactory but not in the respi-ratory epithelium (Fig. 3B). The Ptx1-positive rostral foregut(Fig. 1D) develops into the oesophagal epithelium, whichmaintains expression of Ptx1 at E15.5 (Fig. 3D). Derivativesof the first arch mesenchyme that express Ptx1 at E13.5-E15.5include portions of Meckel’s cartilage and surroundingmuscles (Fig. 3B), extrinsic muscles of the tongue and thedistal portion of the tongue proper (Fig. 3B,D). It should benoted that the apparently weaker labelling in cells of Meckel’scartilage is due to much lower cell density in this structure.

Complementarity between Ptx1 and goosecoidexpression domainsThe expression of Ptx1 in the first branchial arch prompted usto compare it with the expression of other homeobox-contain-ing genes known to be expressed in this structure. Consecutivetransverse sections through the mouth of a 11.5 dpc embryo

2812 C. Lanctôt, B. Lamolet and J. Drouin

Fig. 6. Expression of Ptx1 in the posteriorregion and limbs. Transverse sections in theposterior region of the embryo taken at levelsshown on the left. In these sections, dorsal isto the top. (A-D) At E8.5 and E9.5, Ptx1 isexpressed in the splanchnic (sp) and somatic(sm) layers of the lateral plate mesoderm. Nolabelling is seen in hindgut endoderm (hg).(E,F) Expression is not detected in a morerostral section of an E9.5 embryo. Inparticular, the newly formed forelimb buds (fl)are not labelled. Other sections through theforelimbs never showed labelling. (G,H) AtE11.5, expression of Ptx1 in the hindlimbs (hl)is mainly confined to the distalundifferentiated mesenchyme of the bud. Theadjacent body wall (bw) is labelled. am,amnion; bw, bodywall; fl, forelimb bud; hg,hindgut; hl, hindlimb bud; mg, midgut; nt,neural tube/plate; sm, somatic layer of lateralplate mesoderm; sp, splanchnic layer of lateralplate mesoderm; va, vitelline artery; uv,umbilical vessels.

were hybridized with probes for Ptx1, Gsc, Msx2 and Dlx3(Fig. 4). As previously reported by others (Hill et al., 1989;Robinson and Mahon, 1994), we observe expression of Msx2throughout the mesenchyme of the frontonasal mass andexpression of Dlx3 in the epithelium of the first arch and in thelateral region of the second arch (Fig. 4D,E). No obvious cor-relation can be made with the pattern of Ptx1. However, thepatterns of Gsc and Ptx1 expression appear to be complemen-tary. Indeed, Gsc labelling in the mandibular component isconfined to a central stripe of mesenchyme (Fig. 4C) whereasPtx1 labelling is observed more laterally (Fig. 4B). Similarly,the epithelium of the first arch, a site of strong Ptx1 expression,is not labelled by the Gsc probe (Gaunt et al., 1993). Thesignals may overlap at the margin of each gene’s expressiondomain, although it cannot be excluded that the two genes areexpressed by distinct adjacent cells. Complementarity betweenGsc and Ptx1 expression domains is also clearly seen onsagittal sections of E13.5 embryos (Fig. 4F-H). The exclusiveexpression of Gsc and Ptx1 is particularly striking in the nasalcapsule and olfactory epithelium, respectively, and in thedescending trachea (Gsc) and oesophagus (Ptx1). In thetongue, the Ptx1 probe labels the ventral/distal region whereasthe Gsc probe labels the dorsal aspect and the distal tip. Thesecomplementary patterns of expression suggest that the genesmight fulfill complementary roles during craniofacial develop-ment.

Expression in the posterior lateral plate andextraembryonic mesodermPtx1 expression is first detected in the gastrulating embryo atE6.8 in the newly formed mesoderm located at the posteriormargin of the embryonic ectoderm where the primitive streakinitially appears (Fig. 5A). Later (E7.5), the allantois andposterior lateral mesoderm will derive from this tissue (Fig.5B-G). Ptx1 labelling is restricted to the lateral platemesoderm (Fig. 5G) and is absent from the primitive streakand the paraxial mesoderm. More posteriorly, the labelledmesoderm joins in the midline (Fig. 5E) and forms theextraembryonic allantois (Fig. 5C). 12 hours later (E8.0), thecaudal region has elongated and the hindgut has formed. Fig.6B shows that Ptx1 is expressed in the somatic and splanch-nic mesoderm; this expression extends up to the opening ofthe hindgut, the posterior intestinal portal (data not shown).By E9.0-9.5, expression of Ptx1 is evident in the thickenedsomatic mesoderm (body wall), in the splanchnic mesodermand in the umbilical vessels (Fig. 6D). At this stage,expression of Ptx1 in the posterior region of the embryo isspecific to the lateral plate mesoderm: no labelling is seen inthe ectoderm or the endoderm, or in the notochord, the somitesor the nephrogenic duct. Later in development (E11.5, Fig.6G,H), the hindlimb mesenchyme expresses Ptx1. At a similarstage of forelimb development, Ptx1 is not expressed in theforelimb buds (Fig. 6E,F), nor is it ever expressed in the

2813Ptx1 defines a stomodeal ectomere

Fig. 7. Expression of Ptx1 in derivatives of the posterior lateral plate mesoderm.(A,B) Frontal section of E11.5 mouse embryo. The hindlimb buds (hl) and the posteriorbodywall (bw) are strongly labelled. Note that labelling in the nasal pit (np) is restrictedto the olfactory epithelium (ol). (C,D) Frontal section of the same E11.5 embryo takenmore dorsally. Note that expression is not detected in the mesenchyme of the maxillaryarch (mx) nor in the forelimb buds (fl). (E,F) Transverse section at the level of thehindlimbs of E15.5 embryo. Expression is maintained in hindlimbs (hl) and in musclelayers of the bodywall (bw), hindgut (hg), stomach (st), bladder (bl) and penis (pe).Expression is also detected in the epithelial lining of the intestine (in). Ptx1 is notexpressed in the adrenals (ad), kidney (ki), liver (li) or pancreas (pc). ad, adrenals; bl,bladder; bw, bodywall; fl, forelimb; he, heart; hg, hindgut; hl, hindlimb; in, intestine; ki,kidney; li, liver; md, mandibular component of the first branchial arch, mx, maxillarycomponent of the first branchial arch; np, nasal pit; ol, olfactory epithelium; pc,pancreas; pe, penis; rp, Rathke’s pouch; st, stomach; ta, tail.

forelimbs (e.g. E11.5, Fig. 7C,D). It appearsas though the domain of Ptx1 expression inthe lateral plate mesoderm extends from theumbilical stalk up to, but not inclusive of, theforelimb buds (Fig. 7D). At E15.5, Ptx1 isexpressed in derivatives of posterior lateralplate mesoderm like the muscle layers of thebody wall, bladder, stomach, hindgut andgenital tubercle (Fig. 7F). Ptx1 is notexpressed in liver, kidney, adrenals, spleen orpancreas. Ptx1 is expressed by the epithelia ofthe stomach and the intestine rostrally frommidgut level. Interestingly, expression of Ptx1in the digestive system occurs in differenttissue layers depending on their rostrocaudalposition.

Ptx1 and its expression pattern arehighly conservedIn order to assess conservation and to studyPtx1 function in chick embryo, we clonedPtx1 from a chicken anterior pituitary library.The chicken protein (cPtx1) is 84% identicaland 92% similar to its mouse homolog (Fig.8A). The homeodomain is 100% conservedand homology is 68% N-terminal to thehomeodomain and 85% C-terminal. As inmice, Ptx1 expression in the head of 10-somite chick embryos was only detected in thestomodeum and rostral foregut (data notshown). Later at day 3 of development, cPtx1expression mirrors the mouse pattern: it isexpressed in Rathke’s pouch, in the mandibu-lar and oral epithelia, in the foregut endodermand in the posterior lateral plate mesoderm(Fig. 8C) up to the opening of the gut in theyolk sac. Ptx1 expression is maintained inderivatives of these structures at later stages(not shown). In particular, limb expression isrestricted to the undifferentiated hindlimbmesenchyme (Fig. 9). In a transverse sectionof a day 5 embryo (Fig. 9A,B), expression isobserved in the distal portion of the hindlimbexcept in condensing chondrogenic centersand possibly in migrating myoblasts (Nelsonet al., 1996). This is also seen in a frontalsection of a similar age embryo (Fig. 9C,D).As in mice, Ptx1 is never expressed inforelimbs (e.g. at E5, Fig. 9E,F). Thus, thepattern of Ptx1 expression is highly conservedin mice and chicken in agreement with itssequence conservation.

DISCUSSION

Ptx1 defines a stomodeal ectomereduring mouse embryogenesisExpression of genes from the Hox clusterdefines overlapping segments along the trunkthat are delimited anteriorly by anatomical

2814 C. Lanctôt, B. Lamolet and J. Drouin

Fig. 8. Conservation of chicken and mouse Ptx1 sequence andexpression. (A) Alignment of amino acid sequences of mouse andchicken Ptx1 derived from cDNA sequences. A dash (−) indicates aconserved residue, a dot (.) indicates a gap. The homeodomain isboxed and serine-rich regions are underlined. (B,C) In situhybridization analysis on chick embryo at day 3 of incubation.Expression of cPtx1 is detected in the lateral plate mesoderm (lpm),the foregut endoderm (fg) and in derivatives of the stomodeum[Rathke’s pouch (rp), oral epithelium (oe), mandibular epithelium(md)]. fb, forebrain; fg, foregut; he, heart; lpm, lateral platemesoderm; md, mandibular/oral epithelium; rp, Rathke’s pouch.

boundaries, the developing vertebrae. The cellular basis for thisproperty is unknown although it has recently been suggestedthat homeoproteins act by differentially controlling the rate ofproliferation in specific regions of the embryo (Duboule, 1995)or by affecting the morphogenetic behavior of cells (Niehrs etal., 1993). Non-clustered homeobox genes also frequentlydefine morphological entities in the developing mouse. Someof these genes are associated with organogenesis of specifictissues [for example, Pdx1 in the pancreas (Jonsson et al.,1994) and Hox11 in the spleen (Roberts et al., 1994)], whereasothers may determine identity of one or more developmentalunit(s) [for example, Dlx2 in mandible and forebrain (Qiu etal., 1995), and lim-1 in head and kidney (Shawlot andBehringer, 1995)]. The present work has shown that Ptx1expression is associated with two domains of the developingmouse, the stomodeum and the posterior lateral plate andextraembryonic mesoderm.

The stomodeum is a distinct structure that is first apparentat E8.0 after bending of the neural tube at the cephalic level.As soon as it can be recognized, it expresses Ptx1. Expressionof Ptx1 is maintained throughout development in thestomodeum and its derivatives, including two placodal invagi-nations that lead to formation of the olfactory organ and thepituitary gland. Expression could not be detected earlier inmouse embryos where a structure equivalent to the chick ANRis not readily identifiable; indeed, the rapid bending of thecephalic neural tube may prevent the formation of a distinctstructure. Further, fate map studies in Bufo japonicus haveshown that the endoderm subjacent to the ANR gives rise tothe rostral foregut (Kawamura and Kikuyama, 1992).Expression of Ptx1 in this rostral foregut (see Fig. 1F) providesfurther support that expression of Ptx1 truly defines derivativesof the anteriormost segment of the embryo. Within the contextof a neuromeric model of brain development (Puelles andRubenstein, 1993; Rubenstein et al., 1994), the stomodeumconstitutes this anteriormost domain and it is a distinct devel-opmental unit that is composed of ectoderm rather than neu-roectoderm: for this reason, we have called this unit thestomodeal ectomere.

Expression of Ptx1 in mesenchymal cells of the first archraises the question of their origin. Indeed, the mesenchyme ofthe branchial arches contains cells of both mesoderm andneural crest origin. Vital dye labelling studies have shown thatthe population of neural crest that colonizes the first branchialarch originates from the posterior midbrain (Osumi-Yamashitaet al., 1994; Trainor and Tam, 1995). Initially, Ptx1 labellingis confined to a central stripe of mandibular mesenchyme (E9,Fig. 2) but it progressively extends laterally (E11.5, Fig.4).Although mesoderm and crest cells become intermingled in thearch mesenchyme, a cluster of mesoderm-derived cells in thecenter of the arch was found to transiently express Myf5 (Ottet al., 1991). However, the absence of co-localization of Myf5and Ptx1 (data not shown) did not help to resolve the origin ofPtx1-positive mesenchymal cells. Nonetheless, it should benoted that Ptx1 is expressed in the extrinsic muscle of thetongue (Fig. 3B), which is thought to derive from mesoderm(Trainor et al., 1994). In contrast, Ptx1 is also expressed in cellsof Meckel’s cartilage that were shown to derive from neuralcrest (Le Lièvre, 1978). It may thus be that both mesenchymalcell types eventually express Ptx1 not because of cell originbut rather because of their position in the mandibular arch.Whatever the mechanism of Ptx1 expression in the mes-enchyme, it is clear that Ptx1 marks mandibular arch mes-enchyme at the exclusion of other mesenchymes.

Complementarity between expression patterns ofPtx1 and GscWe have observed complementary patterns of Ptx1 and Gscexpression in the craniofacial region (Gaunt et al., 1993). SincePtx1 and Gsc are expressed in different components of thesame organ (i.e. mesenchyme and epithelium of olfactorysystem, or different parts of tongue muscle), it may be thatexpression of one gene is incompatible with expression of theother. The importance of Gsc expression for craniofacial devel-opment was revealed by the severe abnormalities observed inGsc null mice (Rivera-Perez et al., 1995; Yamada et al., 1995).In particular, defects in the mandible, tongue and nasal cavitiescorrespond to structures where Ptx1 and Gsc expression exhibit

2815Ptx1 defines a stomodeal ectomere

Fig. 9. Expression of Ptx1 in chick limbs. (A,B) Transversesection through the hindlimb region of an E3 chick embryo.Anterior is to the left and distal is to the top. Expression isexcluded from the cartilaginous mesenchyme (cm) and ismainly confined to the surrounding undifferentiatedmesenchyme. (C,D) Frontal section of an E3 chick embryo.The amnion (am) and hindlimb (hl) mesenchyme are labelled.(E,F) Transverse section at the forelimb level of a similarstage embryo shows that Ptx1 is not expressed in forelimbs(fl). Similarly, expression was not detected in any othersection through the forelimbs. am, amnion; cm, cartilaginousmesenchyme; fl, forelimb; hl, hindlimb; nt, neural tube.

complementary patterns. Although we cannot yet appreciatethe significance of this complementarity, it is sufficientlystriking to warrant further analysis, particularly as both belongto the same class of homeoproteins and as they are expressedin different areas of mesoderm in gastrulating embryos.

Expression in posterior primitive streak and lateralplate mesodermThroughout embryogenesis, Ptx1 marks posterior lateral andextraembryonic mesoderm. Ptx1 is expressed in the earliestmesoderm formed during gastrulation (Tam and Trainor, 1994)in the posterior primitive streak. Expression is maintained inthis tissue as it gives rise to the allantois and the posteriorlateral plate mesoderm. At E6.8, expression of Gsc is restrictedto the node area of the primitive streak (Blum et al., 1992) andthere is no overlap in the patterns of Gsc and Ptx1 expression(not shown). Thus, it may be that Gsc and Ptx1 determinedifferent mesodermal cell populations in the primitive streak,one destined to migrate anteriorly and the other destined toform posterior and extraembryonic structures. The domain ofPtx1 expression does however seem to be very similar, if notthe same, as that of BMP-4 in the gastrulating embryo (Winnieret al., 1995) and thus the apparent functional complementaritybetween BMP-4 and Gsc in patterning the early embryo (Stein-beisser et al., 1995) may actually reflect a link between BMP-4 and Ptx1 expression. This correlation suggests a role for Ptx1in early gastrulation, in addition to its role in patterning anteriorstructures.

Expression in hindlimbsThe most striking consequence of the restricted expression ofPtx1 in the posterior half of the lateral mesoderm is thathindlimb mesenchyme expresses Ptx1 whereas there is never

any expression in the forelimbs (Figs 6E,F, 9). The pattern ofPtx1 expression throughout the hindlimb is reminiscent of theexpression of Hoxc-10 (Nelson et al., 1996). Together withHoxc-9 and Hoxc-11, these Hox genes also exhibit expressiononly in hindlimbs, not in forelimbs. A pair of T-box genes,Tbx4 and Tbx5, also exhibit restricted expression to hindlimbsand forelimbs, respectively (Gibson-Brown et al., 1996).Within the posterior half of the embryo, the domain of Ptx1expression overlaps and includes many Hox-defined segmentsand, as such, it may provide a determinant for identity ofposterior structures like the hindlimbs. As discussed below, itmay be that Ptx1 expression in the posterior mesoderm is evo-lutionary older and that Ptx1 was recruited at the anterior levelto define a stomodeum segment during cephalization of thechordates.

Evolutionary considerationsThe nineteenth-century idea that vertebrates are ‘arthropodswalking on their back’ has recently been revived in molecularterms. Indeed, it was found that vertebrate homologs ofDrosophila dorsal markers are expressed ventrally and con-versely homologs of Drosophila ventral markers are expresseddorsally in mouse and Xenopus (Holley et al., 1995). Theformer include the pair dpp/BMP-4 while the best example ofthe latter is given by sog (ventral in Drosophila) and itshomolog, chordin (dorsal in Xenopus). Geoffroy Saint-Hilaire,the French naturalist who proposed this idea in 1820, alreadyrealized that the mouth poses a special problem in the contextof a complete inversion of the body plan since it lies ventrallyin both phylae. Two hypotheses were put forward: either theposition of the mouth was reversed during evolution and even-tually shifted ventrally or the vertebrate mouth is an altogethernew structure that has no homolog in invertebrates. Expression

2816 C. Lanctôt, B. Lamolet and J. Drouin

of homeobox genes, and particularly of Ptx1 and Gsc, mightoffer some clues to answer this unresolved question.

A Drosophila Gsc has recently been cloned and shown to beexpressed in the stomodeal invagination (Goriely et al., 1996;Hahn and Jäckle, 1996). Gsc is not expressed in the vertebratestomodeum, nor are homologs of other genes expressed in theDrosophila stomodeum. Based on these gene expressionpatterns, we favor the hypothesis that the vertebratestomodeum is an evolutionary innovation. Thus, it is plausiblethat the ANR/stomodeum represents a new segment thatevolved at the anterior end of the neural plate and that isdestined to form the mouth invagination after bending of theneural tube. Ptx1, the earliest known homeobox marker of thestomodeum, may therefore play an important transcriptionalrole in the cascade of events leading to mouth formation. Inac-tivation of the gene will be used to test this hypothesis. Fur-thermore, because Ptx1 presumably marks vertebrate featuresat the anterior level, we speculate that a Ptx1 homolog inDrosophila, should it exist, will only recapitulate the posteriorpattern of expression seen in the mouse embryo.

We are indebted to Dr Kathleen Mahon (National Institute of ChildHealth and Human Development) and to members of our laboratoryfor helpful discussions. We appreciate the useful comments of KathiMahon and Catherine Dulac on the manuscript. We thank K. Mahon,R. Moss, E. de Robertis, and M.A. Rudnicki for generous gifts ofprobes and Nicola Illing for kindly supplying the chicken anteriorpituitary cDNA library. We also thank Ms B. Banaszak for help inpreparing tissue sections. C. L. is a Research Student of the NationalCancer Institute of Canada. B. L. was supported by a studentship fromNSERC. This work was funded by the National Cancer Institute ofCanada supported with funds provided by the Canadian CancerSociety.

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(Accepted 1 May 1997)