forebrain and midbrain regions are deleted in otx2 …...3280 furthermore, in avians,...

3279Development 121, 3279-3290 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

Forebrain and midbrain regions are deleted in Otx2−/− mutants due to a

defective anterior neuroectoderm specification during gastrulation

Dario Acampora1,†, Sylvie Mazan2,†, Yvan Lallemand2, Virginia Avantaggiato1, Martine Maury2, Antonio Simeone1 and Philippe Brûlet2,*1International Institute of Genetics and Biophysics CNR, Via G. Marconi 10, 80125 - Naples, Italy2Unité d’Embryologie Moléculaire, URA 1947 du CNRS, Institut Pasteur, 25 rue du Dr Roux, 75724 - Paris Cedex 15, France

*Author for correspondence†Both first authors contributed equally to the work

We have replaced part of the mouse homeogene Otx2coding region with the E. coli lacZ coding sequence, thuscreating a null allele of Otx2. By 9.5 dpc, homozygousmutant embryos are characterized by the absence offorebrain and midbrain regions. From the early to mid-streak stages, endomesodermal cells expressing lacZ fail tobe properly localized anteriorly. In the ectodermal layer,lacZ transcription is progressively extinguished, beingbarely detectable by the late streak stage. These datasuggest that Otx2 expression in endomesoderm and

ectoderm is required for anterior neuroectoderm specifi-cation. In gastrulating heterozygous embryos, a post-tran-scriptional repression acts on lacZ transcripts in theectoderm, but not in the external layer, suggesting thatdifferent post-transcriptional mechanisms control Otx2expression in both layers.

Key words: neural induction, Otx2, mouse gastrulation, node,anterior neuroectoderm

SUMMARY

INTRODUCTION

The identification of the murine Hox genes, and the demon-stration that inactivation of some of them leads to homeotictransformations, have provided major clues to understand theestablishment of the body pattern in the vertebrate trunk andhindbrain (Krumlauf, 1993, 1994). By contrast, the geneticmechanisms underlying the development of more anteriorregions, fated to the adult forebrain and midbrain, are stillobscure. However, a number of regulatory genes, specificallyexpressed in morphogenetically defined regions of the devel-oping forebrain and midbrain, have been identified (Rubensteinet al., 1994). As in more posterior regions, most of them wereisolated as vertebrate homologs of Drosophila genes involvedin insect head formation.

In Drosophila, the head region consists of seven segments,identified by both the repetitive pattern of expression of twosegment polarity genes, engrailed and wingless, and morpho-logical considerations (Diederich et al., 1991; Schmidt-Ott andTechnau, 1992). Three genes, orthodenticle (Otd), emptyspiracles and button head, whose mutations specifically affectsome of these segments, were isolated by large-scale geneticscreens (Finkelstein and Perrimon, 1990, 1991; Finkelstein etal., 1990; Cohen and Jürgens, 1990). The mouse gene Otx2 wasidentified as one of the two murine homologs of Otd. BothOtx2 and Otd contain highly related homeodomains belongingto the bicoid class and, like its Drosophila cognate, Otx2 showsan expression pattern mainly restricted to specific head regions

during embryogenesis (Simeone et al., 1992a). Thus, by 10.5dpc, the Otx2 transcription domain covers most of the forebrainand midbrain neuroepithelium, with a sharp boundary at themidbrain-hindbrain junction. Moreover, Otx2 is transcribed inthe epiblast as early as 5.75 dpc and its expression domain pro-gressively regresses to anterior regions during gastrulation(Simeone et al., 1993). This pattern of expression is largelyconsistent with the results of whole-mount hybridizationexperiments recently reported in chick and Xenopus (Bally-Cuif et al., 1995; Pannese et al., 1995). Additionally, in thesetwo latter species, two distinct phases of Otx2 transcription canbe distinguished during gastrulation. In chick, during primitivestreak elongation, the transcripts become progressivelyrestricted to anterior regions but appear mainly associated withHensen’s node. At the streak maximal extension (stage HH4),Otx2 expression is restricted to the node. In a second phase,transcripts become highly abundant in anterior mesendodermand, at a slightly later stage, in anterior neuroectoderm (Bally-Cuif et al., 1995). Likewise, in Xenopus gastrulae, Xotx2 isexpressed at stage 10.25 in dorsal bottle cells and in cells fatedto prechordal mesendoderm at the dorsal lip of the blastopore.The transcripts can be detected in the presumptive anterior neu-roectoderm only later, at stage 10.5 (Pannese et al., 1995).

Both the chick Hensen’s node and the Xenopus dorsalblastopore lip, or Spemann’s organizer, show a remarkableproperty : when transplanted to other locations of the embryo,they are able to induce the formation of a complete secondneural axis (Spemann and Mangold, 1924; Waddington, 1932).

3280 D. Acampora and others

Furthermore, in avians, transplantation experiments haveestablished that the inductive properties of the node changethroughout gastrulation. Both anterior and posterior nervoussystem can be induced by young nodes (from stages HH2 toHH4), whereas older nodes have a reduced inductive capabil-ity and generate only posterior neural structures (Storey et al.,1992). The competence of the epiblast to respond to neuralinduction also declines after stage HH4 (Storey et al., 1992).The stages at which Hensen’s node transplants are able toinduce a neural axis exhibiting anterior characteristicstherefore correspond precisely to the time period when Otx2 isexpressed in the node (up to stage HH4). These Otx2expression patterns in chick and Xenopus gastrulae suggest aconserved role for this gene in the specification of anterior neu-roectoderm in vertebrates, in line with its highly conservednucleotide sequence during evolution.

To address directly the role of Otx2 during embryogenesis,we have inactivated Otx2 in mice, replacing part of its codingregion by a lacZ reporter gene. At 9.5 dpc, the Otx2−/− mutationresults in the absence of anteriormost regions of the neuraltube, corresponding to the midbrain and forebrain regions. Fur-thermore, homozygous Otx2−/− embryos exhibit marked abnor-malities at the early to mid-streak stages of gastrulation, sug-gesting that early steps in anterior neuroectoderm specificationare affected.

MATERIALS AND METHODS

Construction of an Otx2 targeting vectorA λ recombinant containing the mouse Otx2 gene was isolated froma 129/Sv genomic library using as a probe an Otx2 cDNA fragmentpreviously characterized (Simeone et al., 1992a). A 6.5 kb SmaIfragment, located 220 pb upstream from the translation initiation site,and a 1.2 kb NsiI fragment, located 240 pb upstream from the stopcodon, were excised from this recombinant phage and cloned respec-tively in the XmnI and ApaI sites of the mutagenesis pGN vector (LeMouellic et al., 1990). Both fragments were inserted in the same ori-entation as the neomycin resistance gene and the lacZ reporter gene.

Transfection of ES cells and selection of targeted clonesHM-1 embryonic stem (ES) cells (Magin et al., 1992) were culturedon neomycin-resistant mouse embryonic fibroblasts, according toRobertson, 1987. 10 µg of the pGN31 targeting vector were linearizedby digestion of the unique KpnI restriction site, and electroporatedinto 2×107 ES cells resuspended in 750 µl HeBS medium (20 mMHepes pH 7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mMglucose), at 200 V, 960 µF. Positive selection was carried out for 11days at 350 µg/ml G418. Resistant colonies were picked and DNAwas extracted from a fraction (1/5) of the cells for a PCR test ofhomologous recombination event. The primers, specific respectivelyfor the targeting vector (sense primer : 5′-TGCTGTGTTCCA-GAAGTGTT-3′, located immediately downstream from theneomycin resistance gene) and for the genomic Otx2 locus (antisenseprimer : 5′-CTGATTGAGATGGCTGGTAACAGC-3′, located 50 pbdownstream from the 3′ NsiI fragment) were used. As shown in Fig.1a, only homologous recombination events result in the amplificationof a 1.6 kb band, whose identity was confirmed by hybridization withthe NsiI 3′ fragment. 40 cycles were performed (denaturation : 1minute, 95°C, hybridization : 2 minutes, 65°C, elongation : 6 minutes,74°C) in a 50 µl volume containing 50 mmol KCl, 2 mM MgCl2, 1mmol DTT, 15 mM Taps-HCl, 0.2 mM dATP, dCTP, dGTP, dTTPeach pH 9.3, 2.5 u Taq DNA polymerase. Positive clones wereexpanded before freezing or DNA extraction for Southern blot

analysis. The probes used included fragments internal (1.3 kb BamHI-HindIII fragment containing the pGN31 neo gene) or external (120pb NsiI-HindIII located immediately downstream from the 3′ NsiI 1.6kb fragment) to the targeting vector.

Generation and genotyping of chimeric and mutant mice10-15 ES cells were microinjected into C57Bl/6 blastocysts. Injectedblastocysts were reimplanted in the uterine horn of pseudopregnantrecipient females. Chimeric animals were back-crossed to B6/D2mice and germ-line transmission was scored by the presence of agouticoat pigmentation. Heterozygous offspring were identified by twoPCR reactions, one to detect the presence of the lacZ gene, and oneto detect the presence of Otx2 sequences lost during homologousrecombination. Tail tips were incubated in lysis buffer (50 mM TrispH 8.0, 100 mM EDTA, 100 mM NaCl, 1% SDS, 0.6 mg/ml pro-teinase K) overnight at 55°C, phenol-chloroform extracted, ethanolprecipitated and redissolved in 10 mM Tris-HCl, 1 mM EDTA pH 8.0at a final concentration of 0.2-1.0 µg/µl. The presence of a mutatedallele was detected using two primers specific for the lacZ sequence(sense primer : 5′-GCGTTGATTTAACCGCC-3′, antisense primer :5′-CAGTTTACCCGCTCGCTAC-3′) and the presence of a wild-typeallele was detected using two primers located in deleted portions ofthe first and the second intron (sense primer : 5′-GTCACTGA-GAAACTGCTCCC-3′; antisense primer : 5′-GTCTCTACATCTGC-CCTACC-3′). 30 cycles (denaturation : 1 minute, 95°C, annealing :1 minute, 65°C; elongation : 30 seconds, 74°C) were performed andthe amplified products, respectively 429 bp and 223 bp long, wereseparated by 2% agarose gel electrophoresis.

HistologyMid-day of the day of the vaginal plug was considered as 0.5 dpc inthe timing of embryos. When removed from the decidua, embryoswere staged according to Ang and Rossant (1993) and genotyped asdescribed above, after DNA extraction from the yolk sac or from theembryo itself after photographing. For in situ hybridizations, up to 7.5dpc, the decidua were sectioned and the genotypes were determinedby hybridizations using probes located in the lacZ sequence (lacZprobe) and in the deleted part of the Otx2 gene (Otx2-del probe). Forwax sections, embryos were fixed in 4% PFA at 0°C for 10-30minutes depending on the stage, dehydrated in ethanol and transferredto 100% xylene. After clearing, they were incubated in a mixture of50% xylene/50% paraplast at 60°C for 30 minutes-1.0 hour, followedby three 1.0 hour incubations at 60°C in paraplast, mounted andsectioned (12 µm). The sections were collected on gelatin-coatedslides, dewaxed, rehydrated and stained with phloxin or thionin.

In situ hybridization of sections and whole-mounthybridizationWhole-mount hybridizations of 7.5 dpc embryos were carried outfollowing the protocol described by Wilkinson, 1992, using digoxy-genin-UTP labelled single-stranded RNA probes. In situ hybridiza-tions on sections of embryos were carried out as described byWilkinson and Green, 1990, using [35S]CTP labelled single-strandedRNA probes.

The following probes were used in the in situ hybridization exper-iments : Otx2, Otx1, Evx1, Emx2 and cripto probes were previouslydescribed (Simeone et al., 1992b, 1993; Dono et al., 1993); the Hoxb-1probe is a PCR-fragment spanning the region between nucleotides 911and 1263 of the sequence reported in Frohman et al., 1990; the Shhprobe is a PCR-fragment spanning the region between aminoacid 258and the stop codon (Echelard et al., 1993); the Brachyury probe cor-responds to the one described in Herrmann (1991); the Krox-20 probeis a PCR-fragment spanning the region between the aminoacids 178and 350 of the sequence reported in Chavrier et al. (1989); the En-2probe is a gift from R. M. Alvarado-Mallart.

In addition, we used two probes to genotype sectioned embryos andto follow the lacZ transcripts, consisting respectively of a 0.8 kb

3281Otx2 and neuroectoderm specification

EcoRV-SacI fragment located in the lacZ coding sequence (lacZprobe) of the PGN vector and of a 0.15 kb fragment contained in theportion of the Otx2 cDNA deleted in the mutated allele (Otx2-delprobe) and including aminoacids 1 to 33 of the Otx2 coding sequence.

Histochemical stainingFor β-galactosidase staining, embryos were dissected in PBS, fixedon ice in 4% formaldehyde, 0.2% glutaraldehyde for 5-20 minutes,depending on their size. After 3 washes in PBS (phosphate-bufferedsaline) for 5 minutes, the embryos were incubated at 37°C for 4 hoursto overnight, in 2 mM MgCl2, 4 mM K3Fe(CN)6, 4 mM K4Fe(CN)6,1 mg/ml X-Gal in PBS. Embryos were then washed in PBS, pho-tographed and embedded in paraffin and sectioned as described above.

RESULTS

Disruption of an Otx2 allele in mice by homologousrecombination in ES cellsConstruction of the targeting vectorOne allele of the Otx2 gene was inactivated by homologousrecombination in ES cells. A 6.5 kb SmaI fragment, locatedapproximately 220 bp upstream from the Otx2 translationinitiation methionine, and a 1.2 kb NsiI fragment, spanning the3′ terminal 500 bp of the second intron and the 5′ terminal 300bp of the third exon, were inserted in the same orientation, intothe polylinker of the pGN vector (Le Mouellic et al., 1990,1992). In the resulting targeting vector, pGN31, these twofragments flank both a neomycin resistance gene, and the lacZcoding sequence fused to regions of the Otx2 endogenous locuslying 213 pb upstream from the translation initiation site (Fig.1a). Homologous recombination events therefore result in thedeletion of a large part of the Otx2 coding sequence, includingmost of the homeodomain, and its replacement by the lacZreporter sequence. Since the transcription initiation site of theOtx2 gene was mapped at position −684 (A. S., unpublished

including most of the homeodomain (fifth line). The transcription initiati700 pb upstream from the Otx2 translation initiation site (thick arrow). Vdetect homologous recombination events. Thin lines (first and last lines)probes external to the vector or in the neomycin gene (black bars above tmutated (3.0 kb) allele. N, NsiI; S, SmaI; H, HindIII. (b) Southern blot anusing a probe external to the mutagenesis vector. (c) Genotyping of hetefragments specific for the wild-type (223 bp) or the mutated allele (429 b

data), the lacZ reporter gene should be transcribed as acomposite RNA, consisting of 471 bp of the Otx2 mRNA 5′terminal sequence, fused to the bacterial lacZ coding sequence.

Characterization of recombined ES cell lines andgeneration of Otx2+/− miceThe targeting plasmid, pGN31, was linearized and electropo-rated into HM-1 ES cells. Neomycin-resistant clones contain-ing a disrupted Otx2 allele were selected by PCR analysis usingtwo primers, respectively specific for the targeting vector(located downstream from the neomycin resistance gene in thepGN vector) and for the Otx2 locus (located in the genomicsequence, downstream from the Otx2 homologous 3′ DNAfragment cloned in pGN31) (Fig. 1a). From 250 independentES clones analyzed, 13 produced the expected 1.6 kb bandindicative of a disrupted Otx2 allele (data not shown). Thepresence of a recombined Otx2 allele in these 13 clones wasfurther verified by Southern blot analysis. Genomic DNAs,digested with HindIII and probed with an Otx2 fragmentexternal to the targeting vector, showed the expected patternof two positive bands, 5.3 kb and 3.0 kb long, correspondingto the wild-type and recombined loci respectively (Fig. 1b).Two clones had additional integration events, as showed byhybridization with a probe from the neomycin resistancecoding sequence (data not shown).

Cells from three ES clones showing a correct recombinationevent in the Otx2 locus were injected into C57BL/6 blastocystsand generated chimeras transmitting the mutation to theiroffspring (Fig. 1c).

lacZ expression in Otx2+/− embryoslacZ expression in heterozygous mice was examined through-out embryogenesis, both at the protein level, by histochemicallocalization of β-galactosidase activity, and at the mRNA level,by in situ hybridization of histological sections (Fig. 2).

Fig. 1. Inactivation of the mouseOtx2 locus by homologousrecombination. (a) The cDNAstructure (second line) is shownabove the restriction map of thewild-type locus (third line), openboxes and broken linescorresponding to exons andintrons respectively. Thehomeodomain is shaded. In thetargeting vector (fourth line), tworegions homologous to genomicDNA (hatched boxes) flank alacZ reporter gene and theneomycin resistance gene (blackboxes), in the same orientation(indicated by a thin arrow belowthe coding sequences).Homologous recombinationresults in the deletion of 2.8 kb

on site of Otx2 was mapped in 10.5 dpc embryos and is located aboutertical arrowheads point to the positions of the PCR primers used to

show HindIII fragments detected by Southern blot analysis usinghe HindIII fragments) and corresponding to the wild-type (5.3 kb) oralysis of a targeted cell line (+/−) or of wild-type (wt) HM-1 ES cells

rozygous (+/−) or wild-type (wt) mice by PCR amplification ofp) with allele-specific primers.


In pregastrulating embryos, by 5.75-6.0 dpc, lacZ and Otx2mRNAs were evenly distributed throughout the epiblast andthe embryonic visceral endoderm of heterozygous and wild-type embryos respectively (Fig. 2c,d). At the protein level,however, the β-galactosidase staining appeared slightly fainterin the embryonic ectoderm than in the embryonic endodermlayer and various degrees of mosaïcism could be observed inthe epiblast (Fig. 2a,b).

From early to mid-streak stages, lacZ mRNA accumulatedboth in the ectoderm and the external layer of heterozygousembryos with a progressive anteriorization (Fig. 2h). Thispattern corresponds to the one previously reported for Otx2mRNA in wild-type embryos, as shown in Fig. 2i. Bycontrast, a clear difference could be observed between thedomains of expression of the β-galactosidase activity, repre-sentative of the protein accumulation and of the correspond-


Fig. 2. lacZ and Otx2 expression in Otx2+/− embryos. (a-d) 5.75 dpcembryos. lacZ staining of whole-mount embryos (a) or sections (b)show the presence of the β-galactosidase activity both in the visceralembryonic endoderm and in the embryonic ectoderm. Somemosaïcism is observed especially in the ectoderm layer (b). In situhybridization of sections show that lacZ (c) and Otx2 (d) transcriptsdisplay identical patterns of expression. (e-j) 6.5-7.0 dpc embryos.(f) A 6.5 dpc lacZ-stained Otx2+/− embryo. As shown on sections of 6.5dpc (g) or 7.0 dpc (e, j) lacZ-stained embryos, the β-galactosidaseactivity is almost completely restricted to the external layer. In situhybridization of sections at 6.5 dpc show that the lacZ (h) or Otx2 (i)transcripts are equally distributed in the external layer and in theectoderm. (k-n) 7.75 dpc embryos. (k) A whole-mount lacZ-stainedembryo at the head-fold stage. Sections of lacZ-stained embryos (l)and in situ hybridization with a lacZ (m) or Otx2 (n) probe show thatthe β-galactosidase activity, and both the lacZ and Otx2 transcriptsdisplay identical patterns of expression, in the anterior part of allthree germ layers. (o-q) 16-20 somites stage. In a whole-mount 9.25dpc embryo (o), the lacZ-staining is mainly restricted to the headregion but is also detectable in the foregut (arrow in o). (p-q) Sagittalsections of a lacZ-stained 9.25 dpc embryo, with arrowheadspointing to a strong site of labelling including the thyroid rudiment(p) or to the notochord (q). (r) Whole-mount 10.5 dpc lacZ-stainedembryo. The limit between midbrain and hindbrain regions isindicated by a thin arrow, an arrowhead points to the labelling liningthe oral cavity. Scale bar : 50 µm, (a-j); 100 µm (k-n, p, q); 250 µm(o); 500 µm (r); ee, embryonic ectoderm; ve, visceral embryonicendoderm ; en, endomesoderm; mes, mesoderm; ne, presumptiveanterior neuroectoderm; nt, notochord; tr, thyroid rudiment.

Table 1. Frequency of genotypes resulting from Otx2heterozygous matings

Genotypes

Stage +/+ +/− −/−8.5-9.5 dpc 26 (27%) 50 (51%) 22 (22%)Newborn 13 (34%) 25 (66%) 0 (0%)

Table 2. Frequency of genotypes resulting from the matingof an Otx2+/− male and a wild-type B6/D2 female

Genotypes

Stage +/+ +/−Weaning 66 (69%) 30 (31%)

ing transcript. The β-galactosidase activity displayed a strongsignal essentially confined to the external layer and wasalmost undetectable in the ectoderm layer at 6.5, 6.75, 7.25dpc, whatever the detection protocol used (Fig. 2e,f,g,j),despite the presence of lacZ mRNA. However, like Otx2 andlacZ mRNAs, the enzymatic activity in the external layer wasprogressively displaced to the anterior part of the embryo(Fig. 2e,f,g and j).

From the headfold stage (7.75 dpc) onwards, the discrep-ancy between the domains of expression of the β-galactosidaseactivity and the lacZ messenger RNA was no longer observedin heterozygous embryos. At the headfold stage, the patternsof hybridization of wild-type or heterozygous embryos withOtx2 or lacZ probes, respectively, were perfectly superimpos-able (Fig. 2m,n), and also corresponded to the distribution ofthe β-galactosidase activity, which was detectable in all threegerm layers and restricted to the most anterior one-third of theembryos (Fig. 2k,l). In the midline, a strong staining was alsopresent at the oral plate level, immediately adjacent to theembryonic-extraembryonic junction (Fig. 2l), in line with theexpression of Otx2 in the foregut at later stages. This patternclosely reflects the pattern of Otx2 transcription, previouslydescribed by in situ hybridization (A. S., unpublished data;Ang and Rossant, 1994; Simeone et al., 1992a, 1993).

At the 16-20 somites stage (Fig. 2o), the β-galactosidaseactivity was mainly restricted to the forebrain and midbrainregions, but additionally labeled regions included the foregut,the first branchial arch, the thyroid rudiment, the anterior halfof the notochord and the rostral-most head ectoderm includingthe olfactory placodes (Fig. 2p,q). A sharp posterior limit oflacZ expression in the rostral neural tube became visible by10.5 dpc (arrow in Fig. 2r). At this stage, the regions showingβ-galactosidase activity included the entire forebrain and

midbrain, except the regions of the optic chiasma and the opticrecess. A clear labelling was also detectable in the developingeye and in the epidermal layer lining the oral cavity. Thispattern of expression again corresponds to the one previouslyreported for Otx2 mRNA (Simeone et al., 1992a, 1993).

Embryonic lethality of the Otx2−/− phenotypeMatings between Otx2+/− mice failed to produce progeny pos-sessing two disrupted alleles, indicating the embryoniclethality of the homozygous mutant phenotype (Table 1).

Moreover, when B6/D2 wild-type females were mated toOtx2+/− males, the percentage of heterozygotes that reachedweaning among the progeny did not exceed 35% (Table 2).However, the segregation of the mutated allele showed aMendelian distribution in 9.5 dpc embryos and at birth (Table1) and no obvious abnormalities were noted among heterozy-gous embryos up to 12.5 dpc. This suggests that a dominanteffect of the mutation, probably depending on the genetic back-ground, affects the survival of some heterozygous animalsduring the first weeks following birth. This heterozygousphenotype was not further analyzed.

Phenotype of Otx2−/− mice at 8.5-9.5 dpcThe morphology of homozygous mutant embryos was firstexamined at 8.5-9.5 dpc. At 8.5 dpc, all Otx2−/− embryosshowed a strong reduction in size and marked morphologicalabnormalities compared to their heterozygous or wild-typecounterparts (Fig. 3a). As immediately apparent, the pheno-types observed displayed variations, possibly due to the mixedgenetic background of the progenitors.

Mesodermal derivativesAlthough at 9.5 dpc, Otx2−/− embryos displayed variable phe-notypes, two main groups could be distinguished. Embryosshowing the strongest phenotype (Figs 3b,d-f, 4g) had a com-pletely abnormal body plan, with no more than 10 somites(Figs 3d, 4g). At 9.5 dpc, embryos showing a less severephenotype (Figs 3c, 4a,c) were delayed in their developmentand resembled 8.5 dpc embryos. They displayed a number ofsomites ranging between 10 and 13, often irregular in shapeand disorganized (Fig. 4a,c).

The heart was present, even in the strongest phenotypes, butmarkedly reduced in size (Fig. 3b,c). Although the presence ofthe prechordal plate was difficult to assess, due to the absenceof specific markers, the notochord was recognized in all cases


Fig. 3. Morphology of 8.5 dpcand 9.5 dpc Otx2-/− embryos.(a) Comparison of Otx2+/− orOtx2−/− whole-mount lacZ-stained embryos at 8.5 dpc. (b-f) Whole-mount Otx2−/−

embryos at 9.5 dpc. (b)Side-view and (d) dorsal viewof an Otx2−/− embryo showingthe strongest phenotype. Theheart is indicated by anarrowhead. Somites are clearlyvisible in d. (c) Whole-mountOtx2−/− embryo at 9.5 dpcshowing a less severephenotype. The heart isindicated by an arrowhead. (e-f) Enlarged views of the headregion of Otx2−/− embryosshowing the strongestphenotype. An arrowheadpoints to the anterior limit ofthe neural tube, either at thepresumptivehindbrain/midbrain junction(e), or at the hindbrain level(f). h, heart. Scale bar: 100 µm.

both by morphological criteria, in transverse sections of Otx2−/− embryos (Fig. 4f), and by its hybridization to the Shhprobe (Fig. 4i; Echelard et al., 1993).

Gut developmentThe gut was formed in all Otx2−/− embryos analyzed (Fig. 4fand 4i) and showed the expected hybridization signal with the Shh probe. However, a lacZ signal was never detected alongthe foregut of 8.5-9.5 dpc Otx2−/− embryos (data not shown).

Non-neural head structuresNon-neural anterior structures (optic vesicles, branchialarches), normally visible at 8.5 dpc (6-9 somites stage), werenot individualized, even in the weaker phenotype of Otx2−/−

embryos (Fig. 3b,c). The presence of olfactory placodes,normally present at 9.0 dpc (8-10 somites stage), was notrevealed, either by histological observations, or by in situhybridization with Otx1 or Emx2 probes (data not shown). The

absence of these structures is unlikely to result merely from adelay in development and probably represents specific featuresof the Otx2−/− phenotype.

Morphology and antero-posterior patterning of theneural tubeIn all Otx2−/− embryos isolated at 9.5 dpc, the neural tube wasrecognizable and morphologically normal at the spinal cordlevel (Fig. 3d-f). At this axial level, the tube was closed andthe floor plate cells were differentiated as assessed by theirpositive hybridization signal with the Shh probe (Fig. 4i).

At the hindbrain level, the morphology of the neural tubewas variable. In the majority of embryos showing the strongestphenotype (Fig. 3b), the neural tube proceeded anteriorly to therostralmost somites but did not show the typical hindbrain mor-phology, thus resembling the spinal cord (Fig. 3d,e). However,a minority (less than 20%) of these highly affected embryosshowed a hindbrain-like structure at the level of the anterior-


Fig. 4. RNA in situhybridization of 9.5 dpcOtx2−/− embryos. (a,c,f,g)Bright-field pictures of b, d-e, i and h, respectively. (b)A sagittal section of anembryo displaying a mildphenotype hybridized toEmx2. Only the posteriordomain of expression ofEmx2 is detected(arrowheads). (d,e) Sagittalsections of the embryoshown in Fig. 3c, hybridizedto Hoxb-1 and Krox20probes, respectively.Arrowheads in d and eindicate rhombomere 4 (d),or rhombomeres 3 and 5 (e).(h) Sagittal section of anembryo showing thestrongest phenotype (Fig.3b), hybridized to a Hoxb-1probe. (i) Transverse sectionof an embryo showing thestrongest phenotype (Fig.3b) hybridized to Shh. Thethin arrow points to thenotochord, the arrowheadpoints to the gut. nt,notochord : g, gut; fp, floorplate. Scale bar, 100 µm

most somite, but the neural tube was abruptly interruptedimmediately anterior to the hindbrain/spinal cord junction (Fig.3f). No evidence of hindbrain segmentation was obtained ineither of these two types of embryos, which displayed a Hoxb-1transcription pattern reminiscent of that reported for wild-typeembryos at 8.0 dpc, with two broad domains of expressionflanking the segmented mesoderm (Fig. 4g,h). By contrast,embryos displaying a less severe phenotype (Fig. 3c) showedan almost normal hindbrain morphology. A clear evidence forthe presence of the hindbrain and its segmented organizationwas also obtained by in situ hybridization (Fig. 4c-e). At thestage analyzed, in wild-type embryos, Krox20 is activated intwo transverse stripes, corresponding to rhombomeres 3 and 5(Wilkinson et al., 1989), whereas expression of Hoxb-1 isrestricted to rhombomere 4 (Frohman et al., 1990). This patternof hybridization of Hoxb-1 and Krox20 was clearly recogniz-able on the sections shown in Fig. 4d,e, respectively. However,the distance between the rostral end of the embryos and theanterior border of Krox20 and Hoxb-1 expression domains wasstrongly reduced, relative to wild-type mice (Fig. 4d,e).Beyond the presumptive hindbrain level, the neuroectodermwas either abruptly interrupted (Fig. 4c) or replaced by a thinlayer of epidermal cells in the head region (Fig. 4a).

To delineate more precisely the missing part of the neuraltube, the expression pattern of several anterior or posteriormarkers, normally expressed at this stage in heterozygous or

wild-type embryos, was analyzed by in situ hybridization ofsections from the embryos shown in Fig. 4. At 9.5 dpc, Otx1,En-2 and Emx2 expression domains define specific territoriesin the developing midbrain and forebrain (Davis and Joyner,1988; Davis et al., 1988; Simeone et al., 1992a,b). No positivehybridization signal was obtained with any of these probes inthe head region of the Otx2−/− embryos analyzed, even afteroverexposure of the hybridized sections (data not shown). Aposterior domain of expression of Emx2 has also been reportedfor wild-type embryos at this stage, including the coelomicepithelium covering the mesonephric vesicle and the final partof the mesenteric attachment. Although these structures weredifficult to unambiguously identify in Otx2−/− mutants, a clearsignal was apparent at the corresponding level in one of theweakest Otx2 embryonic phenotype (Fig. 4b). Finally, no lacZsignal could be detected in the head region of any Otx2−/− 9.5dpc embryo.

Otx2−/− phenotype during gastrulationBefore the onset of gastrulation, defined by the appearance ofmesoderm and primitive streak, no morphological differencecould be reliably detected between homozygous, wild-type orheterozygous embryos.

Morphology of Otx2−/− mutantsUp to the mid-streak stage, Otx2−/− embryos did not show


Fig. 5. Morphology, lacZ and goosecoid expression of Otx2−/− mutant embryos at early streak stage. (a) Whole-mount β-galactosidase-stained6.5 dpc Otx2−/− embryo. The β-galactosidase activity is restricted to the distal half of the embryonic part. (b) Sagittal section of the embryoshown in a. The staining is only detectable in the external layer containing endomesoderm cells. (c-e) Sagittal sections of a 6.5 dpc Otx2−/−

embryo; (c), bright field, (d,e) dark-field pictures; (d,e) hybridized to the Otx2-del and lacZ probes, respectively. The embryo shown ishomozygous, as proved by the absence of signal with the Otx2-del probe. The lacZ transcript (e) shows a distribution identical to the onedisplayed by the β-galactosidase activity in b. (f-k) Sagittal sections of a 6.5 dpc Otx2+/− embryo (f,g), a 6.8 dpc Otx2+/− embryo (h,i) and a 6.5dpc Otx2−/− embryo (j,k). (f,h,j) Bright-field pictures, (g,i,k) corresponding sections hybridized with a goosecoid probe. While Otx2+/− embryosshow the expected signal (g,i), no signal is detected in the Otx2−/− embryo (k). ee, embryonic ectoderm; en, endomesoderm; mes, mesoderm.Scale bar, 50 µm.

strong morphological defects either in shape or in size (Fig.5a). Histological sections revealed the presence of theprimitive streak elongating from the embryonic-extraembry-onic junction and of mesodermal cells, intercalating posteriorlybetween the ectoderm and the external layer (Fig. 5b,c).

By contrast, Otx2−/− embryos showed strong abnormalitiesat the late streak stage (Fig. 6a). The amnion was not clearlyindividualized, making the embryonic-extraembryonicjunction difficult to delineate precisely (Fig. 6b). Whileextraembryonic structures appeared almost normal, with aclearly discernable chorion and allantoïs (Fig. 6b), the pre-sumptive embryonic part (containing the ectoderm) wasmarkedly reduced in size and appeared as a sphere, linked tothe proximal region by a more or less severe constriction. Asshown by histological sections (Fig. 6b,c,f,i,k), mesodermalcells were present, intercalating between the ectoderm and theexternal layer and also accumulating in a disorganized fashionat the level of the constriction. The ectoderm itself was circu-larized and entirely contained in the spherical, distal part of theembryo.

lacZ expression in Otx2−/− mutantsAt the early streak stage, analysis of the β-galactosidaseactivity, which, as shown for Otx2+/− embryos, is detectedalmost exclusively in the external layer, pointed to two maindifferences between heterozygous and homozygous mutantembryos.

First, the domain of lacZ expression appeared clearly alteredboth in the ectoderm and in the external layer. The lacZ tran-scription domain, which shows an anterior localizationextending from the distal tip to the embryonic/extraembryonicjunction in Otx2+/− embryos (Fig. 2h), remained confined tothe distal third to half of the external layer, without clear ante-riorization in 6.5-6.75 dpc Otx2−/− embryos (Fig. 5e). The lacZstaining showed the same pattern in the external layer (Fig.5a,b). Likewise, in the embryonic ectoderm, lacZ transcriptswere not distributed from the distal tip to the junction betweenembryonic and extraembryonic ectoderm as observed inOtx2+/− embryos (Fig. 2h), but remained confined to the distalpart of the ectoderm, adjacent to the β-galactosidase-express-ing external layer (Fig. 5e).

Second, in Otx2−/− embryos, the level of lacZ mRNA tran-scription in the ectoderm layer was markedly reduced relativeto heterozygous embryos at the early streak stage (compareFigs 5e and 2h), and became almost undetectable at the latestreak stage (compare Figs 6d and 2m). By contrast, the levelof lacZ expression, both at the RNA and protein level,remained high in the external layer of 6.5 dpc Otx2−/− embryos(Fig. 5a,b,e) and a limited domain of strong expression wasalso still present in the presumptive anterior part of the externallayer of 7.5 dpc embryos (Fig. 6c,d).

Expression of gastrulation markersAt 7.5 dpc, a strong expression of Brachyury (Herrmann, 1991)


Fig. 6. Otx2−/− phenotype at 7.5 dpc. (a) β-galactosidase activity in a whole-mount lacZ-stained 7.5 dpc Otx2−/− embryo. An arrow points to theconstriction observed in these embryos. (b) Sagittal section, showing the circularized ectoderm and the disorganization of the formingmesoderm. The amnion is not clearly individualized but the allantois and chorion are clearly visible. (c) Sagittal section of a lacZ-stained Otx2−/−

embryo, showing that the labelling is restricted to the external layer. (d) Sagittal section of an Otx2−/− embryo hybridized to the lacZ probe. Thelabelling is clear in the external mesendoderm layer but absent from the embryonic ectoderm. (e) Whole-mount hybridization of an Otx2−/−

embryo with a Brachyury probe, showing the extent of the primitive streak. (f-l) RNA in situ hybridizations of sagittal sections of Otx2-/− 7.5dpc embryos. (f,i,k) Bright-field pictures corresponding to the dark-field pictures shown in g,h,j,l, respectively. (g,h,j,l) Hybridized respectivelyto Brachyury, Evx1, Hoxb-1 and cripto probes. ee, embryonic ectoderm; al, allantois; ch, chorion; en, endomesoderm. Scale bar : 100 µm.

extending up to the distal tip of the embryos was observed,suggesting that the primitive streak was formed in Otx2−/−

animals (Fig. 6e,g). At this stage, two other markers of the pos-teriorly forming mesoderm, Hoxb-1 (Frohman et al., 1990) and

cripto (Dono et al., 1993), were also transcribed at high levelsalong the primitive streak, as expected (Fig. 6j,l). Evx1 isnormally expressed in wild-type embryos in all three germlayers along the primitive streak, with a progressive decrease


in the level of expression from the proximal to the distal partof mid- to late-streak embryos (Dush and Martin, 1992).Despite the strong abnormalities observed in mutant embryos,the main characteristics of this pattern of Evx1 expression wereunaffected in Otx2−/− embryos (Fig. 6h). In several cases,however, an anterior displacement of some Evx1, Hoxb-1,cripto and Brachyury-expressing mesoderm cells wasobserved, in line with the disorganization of mesoderm alsonoticed in histological sections (data not shown, Fig. 6j).

By contrast, in the ectoderm, the respective anterior limitsof expression of the different posterior probes were alwaysrespected. Hoxb-1, cripto or Evx1 were never expressed inanterior regions of the ectoderm in Otx2−/− 7.5 dpc embryos.

The expression of goosecoid, which is transiently expressedin 6.5 to 6.9 dpc wild-type embryos (Blum et al., 1992; Faustet al., 1995; Conlon et al., 1994) was also tested by in situhybridization. As previously reported, in Otx2+/− or wild-typeembryos, goosecoid transcripts were detected in the visceralembryonic endoderm and at the proximal posterior region ofthe ectoderm at 6.5 dpc (Fig. 5f,g), and in the anterior part ofthe primitive streak at 6.9 dpc (Fig. 5h,i). By contrast, hybrid-ization signals remained either undetectable or stronglyreduced in Otx2−/− embryos from the same litters (Fig. 5j,k).Likewise, at 7.5 dpc, Shh expression was observed in themidline mesoderm of the head process in Otx2+/− or wild-typeembryos but could not be detected in their Otx2−/− counterparts(data not shown).

DISCUSSION

Otx2−/− embryos are characterized by the absence offorebrain and midbrain regions The inactivation of Otx2, a murine homolog of the Drosophilaorthodenticle homeodomain gene, resulted in the deletion ofanterior structures. In all homozygous embryos tested, anteriorparts of the neural tube, corresponding to the presumptiveforebrain and midbrain, were deleted. At 9.5 dpc, this deletionwas clearly visible on histological sections and was furtherassessed by the absence of hybridization signal with a numberof markers specific for the corresponding regions. The absenceof other ectodermal derivatives, like the optic lens placodesand the olfactory placodes, was also consistent with thedeletion of forebrain neuroectoderm, since the ventral anteriorforebrain and the optic vesicles are required to induce theolfactory placodes and the lens placodes, respectively. The roleof Otx2 appears therefore similar to that of the Drosophila Otdgene which was shown to function as a ‘gap gene’, since itsmutation results in the deletion of specific head segments(Cohen and Jürgens, 1990; Finkelstein and Perrimon, 1990,1991).

By contrast, regions of the neural tube fated to the spinalcord were always present. At the hindbrain level, the pheno-types observed were more complex, suggesting a variableexpressivity of the mutation. In embryos showing the strongestphenotypes, the typical hindbrain morphology was notobserved and evidence of segmentation was never obtained.However, in weaker Otx2−/− phenotypes, the hindbrain regionwas present and showed clear evidence of anteroposterior seg-mentation, based on the identification of rhombomeres 3, 4 and5 with Krox20 or Hoxb-1 probes. We also detected the

presence of floor plate cells by hybridization with a Sonichedgehog probe, which indicated some dorsoventral pattern-ing. However, we could never detect En-2 expression in thefirst rhombomere at 9.5 dpc, which suggests that anteriormostregions of the hindbrain were also either missing, orimproperly specified.

Regionalisation and specification of the embryonicectoderm during gastrulation are defective in Otx2−/−

embryosBy the end of gastrulation, Otx2 is transcribed in the pre-sumptive anterior neuroectoderm in mouse, chick and Xenopus(Simeone et al., 1993; Bally-Cuif et al., 1995; Pannese et al.,1995). The absence of lacZ transcripts in the ectoderm of 7.5dpc Otx2−/− embryos therefore suggests that, at this stage,anterior neuroectoderm was not properly specified. However,expression of posterior neural markers was never observed inanterior regions of the ectoderm layer, also suggesting thatanterior neuroectoderm did not acquire an alternative (moreposterior) identity, but failed to be induced. At the early streakstage, cell lineage studies in mouse have shown that a rostro-caudal pattern is already established in the epiblast, in whichcells fated to anterior neuroectoderm are localized at the distalanterior third of the embryonic ectoderm (Lawson andPedersen, 1992; Quinlan et al., 1995). The abnormal distribu-tion of ectodermal cells expressing lacZ mRNA, observed in6.5 dpc Otx2−/− embryos, together with the strongly reducedlevel of transcription relative to their Otx2+/− counterparts,shows that the regionalisation of the epiblast was alreadyaffected at this stage.

The requirement for an inductive signal to maintain orinduce Otx2 transcription in the ectoderm layer is supported byseveral lines of evidence. In vitro, isolated explants of mouseectoderm become committed to express Otx2 in a cellautonomous fashion by the mid-streak stage. At earlier stages,such explants are unable to stably express Otx2 and a signalfrom mesendoderm (which consists, in this in vitro study, inboth the mesoderm and external layer from 7.5 dpc embryos)is required to maintain ectodermal Otx2 transcription (Ang etal., 1994). The Otx2 expression pattern in Xenopus gastrulaeand exogastrulae also suggests that Otx2 transcription in thepresumptive anterior neuroectoderm is dependent upon a directcontact with the underlying mesendoderm (Pannese et al.,1995). Consistent with these data, lacZ expression in Otx2−/−

gastrulating embryos was strongly maintained in the externallayer, but was almost undetectable in the embryonic ectoderm,suggesting that an inductive signal required to maintain, orinduce, Otx2 expression in the ectoderm layer, but not in theexternal layer, could not take place in the absence of a func-tional Otx2 allele.

Alterations of the node in Otx2−/− mutantsThe anterior end of the mouse primitive streak, or node, of 7.5dpc embryos, is able to induce neural structures in transplan-tation experiments and thus appears as a functional equivalentof the chick Hensen’s node or Xenopus organizer (Beddington,1994). At early stages of gastrulation, its fate map is alsolargely similar to those described in avians and amphibians: inparticular, endomesoderm cells, derived from regions locatedat the anterior end of the primitive streak, contribute to defin-itive endoderm, prechordal and chordal mesoderm (for nomen-


clature of embryonic cells in early mouse gastrulae: seeLawson and Pedersen, 1992, p. 3-36). Cells in the midlineectoderm of the neural tube are also derived from the nodeectoderm (Lawson and Pedersen, 1992; Sulik et al., 1994).

In Otx2−/− embryos, the node structure could not be recog-nized at the end of gastrulation and its formation was alteredas early as the early streak stage, as shown by the absence orstrong reduction of goosecoid expression. Similarly Shhexpression was delayed. However, the precise cell populationinvolved in the anterior neuroectoderm specification remainsunidentified. In the external layer of Otx2−/− embryos, theidentity of lacZ-expressing embryonic cells cannot be defini-tively established for lack of early cellular markers. Accord-ingly, an endomesoderm subpopulation deriving from the nodecould be either missing or unproperly specified. Our data arealso consistent with grafting experiments performed in chicksince at stage HH4, sectors of Hensen’s node able to induceneural structures expressing anterior markers are characterizedby the presence of progenitor cells to chordal and prechordalmesoderm, gut endoderm and neural tissue (Selleck and Stern,1991; Storey et al., 1995).

In Otx2−/− embryos, the absence of anterior neuroectodermtherefore appears related to defects observed as early as theearly streak stage, which provides genetic evidence that itsspecification occurs at early gastrulation. This timing is con-sistent with grafting experiments performed in chick, showingthat later than stage HH4, Hensen’s node transplants becomeunable to induce anterior neural structures (Storey et al., 1992).Furthermore, the inactivation in mouse of another homeoboxgene Lim-1, expressed in mesodermal derivatives, includingthe node region at the onset of gastrulation and prechordalmesoderm at the headfold stage, has been shown to result in aphenotype clearly related to the one displayed by Otx2−/−

embryos (Shawlot and Behringer, 1995). Lim1−/− embryosshow the same absence of forebrain and midbrain regions andabnormalities are also apparent in the node region shortly afterthe onset of gastrulation, as shown by the altered location ofthe subpopulation of cells expressing goosecoid. However, incontrast to Lim1−/− embryos, Otx2−/− embryos present severedefects in the trunk. We also never noticed the formation ofany secondary axis in Otx2−/− mutants. The genetic interactionsbetween these genes will have to be investigated by codetec-tion at the single cell level in the primitive streak and noderegion.

Anteriorization of lacZ expression domain in theembryonic external layer is defective in Otx2−/−

embryosFate mapping studies have shown that, during gastrulation, thevisceral embryonic endodermal cells are progressivelyreplaced by a complex population of cells, including definitiveendoderm cells that will contribute to the gut (Lawson andPedersen, 1987), prechordal and chordal mesoderm cells. Thedynamics of this process remain largely unknown. However,lineage analysis after single cell labelling experiments haveshown that cells located in the external embryonic layeranteriorly to the node are progressively displaced anteriorlyduring gastrulation (Lawson and Pedersen, 1987, 1992). Suchmorphogenetic movements are strikingly similar, and couldcontribute, to the observed anteriorization of the lacZexpression domain in the Otx2+/− embryos. Furthermore,

whereas the lacZ transcripts appeared uniformly distributed inthe visceral embryonic endoderm layer, the anteriorization ofthe lacZ expression domain was clearly visible by the earlystreak stage. This suggests that cell displacements in theexternal embryonic layer might be initiated at, or even before,the onset of gastrulation. In line with this hypothesis, fatemapping studies of chick embryos have shown that several celltypes, including presumptive gut endoderm, head process andnotochord cells, converge to the posterior midline and then tothe centre of the blastoderm, where the node will eventuallyform, even before primitive streak formation (Hatada andStern, 1994). However, the dynamic pattern of lacZ expressionin the external embryonic layer might also result, at least inpart, from genetic mechanisms that maintain the Otx2expression anteriorly and repress it posteriorly.

In the external layer, the anteriorization of lacZ-expressingcells was impeded in Otx2−/− mutant embryos as soon as theearly streak stage. These data suggest that Otx2-positive cellslocated in the external embryonic layer are involved in thegenetic cascade of regulatory events leading to the establish-ment of anteroposterior polarity.

Post-transcriptional regulation of lacZ expression inectoderm during gastrulationThe patterns of lacZ and Otx2 transcription in Otx2+/− embryosappeared perfectly superimposable throughout embryogenesis,showing a progressive anteriorization in both the ectoderm andthe external layer during gastrulation. By contrast, the β-galac-tosidase activity remained almost undetectable specifically inthe ectoderm of gastrulating Otx2+/− embryos, even though thecorresponding RNA was clearly present. Instability of theenzyme activity in such a stage- and tissue-specific fashion isunlikely since β-galactosidase activity can be detected in theectoderm of other transgenic gastrulating embryos (Lallemandand Brûlet, 1990). The observed repression might reflect mech-anisms of post-transcriptional regulation exerted on the Otx2locus specifically in the ectoderm, up to 7.25 dpc. This repres-sion might be exerted on β-galactosidase, but not Otx2, proteinaccumulation, due to the deletion of part of the 5′ leadersequence or of the introns in the lacZ transcript. Alternatively,the regulation exerted on the lacZ gene might reflect a post-transcriptional mechanism also controlling Otx2 protein accu-mulation and possibly involving cis-acting elements in the 5′leader portion of Otx2 mRNA, which is present in the lacZfusion transcript. Translational repression mechanisms duringearly embryogenesis are known to play a crucial role in thespecification of the anteroposterior embryonic polarity inseveral species (reviewed in Kimble, 1994). The possibility ofsuch a regulation in the case of Otx2 will have to be confirmed.

Philippe Brûlet thanks Dr Kristie Lawson for very helpful discus-sion and Dr D.W. Melton for the gift of the HM-1 ES cells; Dr R.M.Alvarado-Mallart for the gift of the En-2 probe; Dr G. Persico for thegift of the cripto probe; Prof F. Jacob, Drs H. Le Mouellic and J.Shellard for their critical comments on the manuscript; Mrs M.Compain and C. Sengmany for secretarial and technical assistance.D. A. was the recipient of an EMBO long term fellowship while inP. B. laboratory. This work was supported by grants from the CentreNational de la Recherche Scientifique, the Association Françaisecontre les Myopathies, the Association pour la Recherche sur leCancer, the Institut National de la Santé et de la Recherche Médicale,the Ligue Nationale contre le Cancer, the Groupement de Recherches


et d’Etudes sur le Génome as well as from the Italian Association forCancer Research and the Italian Telethon Program.

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(Accepted 27 June 1995)

forebrain and midbrain regions are deleted in otx2 …...3280 furthermore, in avians,...

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