otx2 is required to respond to signals from anterior ... · otx2 is required to respond to signals...

Developmental Biology 242, 204–223 (2002)doi:10.1006/dbio.2001.0531, available online at http://www.idealibrary.com on

Otx2 Is Required to Respond to Signals fromAnterior Neural Ridge for Forebrain Specification

E Tian,* Chiharu Kimura,* ,† Naoki Takeda,‡ Shinichi Aizawa,* ,† ,1

and Isao Matsuo* ,†*Department of Morphogenesis, Institute of Molecular Embryology and Genetics (IMEG), and†Vertebrate Body Plan Group, RIKEN Center for Developmental Biology, and ‡Divisionof Transgenic Technology, Center for Animal Resources and Development (CARD),Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan

Previous analysis employing chimeric and transgenic rescue experiments has suggested that Otx2 is required in theneuroectoderm for development of the forebrain region. In order to elucidate the precise role of Otx2 in forebraindevelopment, we attempted to generate an allelic series of Otx2 mutations by Flp- and Cre-mediated recombination for theproduction of conditional knock-out mice. Unexpectedly, the neo-cassette insertion created a hypomorphic Otx2 allele;consequently, the phenotype of compound mutant embryos carrying both a hypomorphic and a null allele (Otx2frt-neo/2) wasanalyzed. Otx2frt-neo/2 mutant mice died at birth, displaying rostral head malformations. Molecular marker analysisdemonstrated that Otx2frt-neo/2 mutant embryos appeared to undergo anterior–posterior axis generation and induction ofanterior neuroectoderm normally; however, these mutants subsequently failed to correctly specify the forebrain region. Asthe rostral margin of the neural plate, termed the anterior neural ridge (ANR), plays crucial roles with respect to neural platespecification, we examined expression of molecular markers for the ANR and the neural plate; moreover, neural plateexplant studies were performed. Analyses revealed that telencephalic gene expression did not occur in mutant embryos dueto defects of the neural plate; however, the mutant ANR bore normal induction activity on gene expression. These resultsfurther suggest that Otx2 dosage may be crucial in the neural plate with respect to response to inductive signals primarilyfrom the ANR for forebrain specification. © 2002 Elsevier Science (USA)

Key Words: forebrain; neural plate; anterior neural ridge; regionalization; hypomorphic allele; Otx2.

INTRODUCTION

Anterior–posterior (A-P) patterning of the vertebrate cen-tral nervous system (CNS) begins prior to and duringgastrulation (Beddingtion and Robertson, 1999). The initialprocess is mediated by signals from the anterior visceralendoderm (AVE) and primitive streak-derived structuressuch as the anterior definitive endoderm (ADE) in mouse(Ang and Rossant, 1993; Beddington and Robertson, 1999;Kimura et al., 2000). Later, at neural plate stages, localsignaling centers play crucial roles in refining the identityof neuroectoderm at specific A-P positions (Rubenstein etal., 1998; Wilson and Rubenstein, 2000). Two local signal-ing centers have been identified. These regions are com-

1
To whom correspondence should be addressed. Fax: 181-96-373-6586. E-mail: [email protected].
204

prised of cells at the rostral margin of the neural plate, e.g.,the anterior neural ridge (ANR), and the mid/hindbrainjunction, also referred to as the isthmus (Joyner, 1996;Shimamura and Rubenstein, 1997; Houart et al., 1998;Joyner et al., 2000). The ANR is involved in the de-velopment of forebrain, whereas the isthmus functions inthe development of midbrain and cerebellar structures,respectively.

The molecular mechanisms underlying inductive activ-ity of the ANR and patterning of the forebrain have beenshown by tissue explants and genetic studies (reviewed inRubenstein et al., 1998; Wilson and Rubenstein, 2000). TheANR is a morphologically defined structure located at thejunction of the anterior neural plate and the nonneuralectoderm (Couly and Le Douarin, 1988; Eagleson et al.,1995). Notably, removal of the ANR results in loss ofexpression of the telencephalic gene BF-1; moreover, addi-
tional ANR can induce ectopic BF-1 expression in mouse
0012-1606/02 $35.00© 2002 Elsevier Science (USA)

All rights reserved.

205Otx2 Functions in Forebrain Specification

FIG. 1. Generation of an allelic series of Otx2 mutations. (A) Strategy for production of an allelic series of mutations at the Otx2 locus.Schematic representation of the Otx2 wild-type allele (a), the targeting vector (b), the Otx2frt-neo allele (c), the Otx2flox allele (d), and the

Dpro-ex1 frt-neo
Otx2 allele (e), respectively. The Otx2 allele is obtained by the standard method of homologous recombination (c). Miceheterozygous for the Otx2frt-neo allele are crossed with Flpe transgenic mice to generate animals bearing the Otx2flox allele (d), in which the
© 2002 Elsevier Science (USA). All rights reserved.

206 Tian et al.

neural plate explants (Shimamura and Rubenstein, 1997).Similarly, in zebrafish, telencephalic gene expression isreduced or absent when cells at the margin of the neuralplate, referred to as row 1 cells, are ablated at the gastrulastage. Additionally, these cells can induce telencephalicgene expression when transplanted to more caudal regionsof the neural plate (Houart et al., 1998). Thus, signals fromthe ANR are crucial for telencephalic development. Fur-thermore, Fgf8 expression in the ANR is suggestive of animportant role for Fgf8 in forebrain patterning. Indeed,FGF8b-soaked beads can restore BF-1 expression in explantsdenuded of the ANR (Shimamura and Rubenstein, 1997),and inhibitors of Fgf function reduce BF-1 expression inneural plate (Ye et al., 1998). Telencephalon formation canproceed in the absence of Fgf8 in zebrafish and mice(Shanmugalingam et al., 2000; Wilson and Rubenstein,2000); however, Fgf8 is not the sole factor for the inductionof telencephalon. This observation implies that other sig-naling molecules may be involved in this inductive process(Shanmugalingam et al., 2000; Wilson and Rubenstein,2000). On the other hand, the genetic mechanism governingcompetence in the prosencephalic neural plate with respectto signals from the ANR remains to be determined.

Otx2 has been shown to be essential in forebrain devel-opment; the entire forebrain and midbrain fail to develop inOtx2 homozygous mutant embryos (Acampora et al., 1995;Matsuo et al., 1995; Ang et al., 1996). Chimeric and geneticstudies have indicated that Otx2 is required at an earlierstage prior to and during gastrulation for the formation ofAVE (Rhinn et al., 1998; Acampora et al., 1998; Kimura etal., 2000). Nevertheless, analysis of chimeric embryos,Otx1 knock-in mutation, and transgenic rescue experi-ments have suggested that Otx2 is also required in theneuroectoderm for forebrain development (Acampora et al.,1998; Rhinn et al., 1998, 1999; Suda et al., 1999; Kimura etal., 2000). Additionally, Otx2 functions in the formation ofmesencephalic territory by determining the position of theisthmic organizing center cooperating with Otx1 (Suda et

neo-cassette is excised. Heterozygotes for the Otx2flox allele are fuOtx2Dpro-ex1 allele (e), in which the Otx2 promoter and first coding ewhereas the thin line represents pBluescript sequences. Filled boxedenote loxP and frt sites, respectively. Neor, neomycin-resistant gentoxin A fragment gene with MC1 promoter. Restriction enzyme siSm, SmaI; X, XhoI. Probes A, B, C, and D are used for Southern blidentification of the different Otx2 alleles by PCR analysis. (B)analysis. (a) Genomic DNAs from wild-type and mutant mice wecorrect 39 recombination. The 5.6- and 3-kb bands represent wilhybridized with probe B for the neo-cassette identification. Thehybridized with probe C for the correct 59 recombination and theaddition to the 20-kb band of a wild-type allele. (d) Digested with NOtx2flox (2 kb), and Otx2Dpro-ex1 (5 kb) alleles, as well as an 8-kb band(a) PCR amplification employing a combination of primers F1, Nwild-type alleles, respectively. (b) Primers P1 and P2 amplified a 22
59 upstream in the mutant allele and the absence of the loxP site in the wproduct is amplified by primers F1 and R1. (d) Primers P1 and R1 are u
© 2002 Elsevier Science (USA

al., 1996, 1997; Acampora et al., 1997). Furthermore, Otx2is essential for diencephalon development in cooperationwith Emx2 (Suda et al., 2001). However, the molecularmechanism by which Otx2 functions in forebrain develop-ment remains unclear.

In order to elucidate the precise role of Otx2 in forebrainspecification at the neural plate stage, we have attempted togenerate an allelic series of Otx2 mutations through Flp-and Cre-mediated recombination for the production ofconditional knock-out mice. In the course of the generationof various alleles, we found that the neo-cassette insertioncreates a hypomorphic Otx2 allele. The phenotype of themutant embryo carrying both a hypomorphic allele and anull allele was analyzed. This investigation revealed thatmutant embryos failed to form the forebrain region cor-rectly. Analyses employing molecular markers and neuralplate explant studies have indicated that Otx2 may berequired to respond to inductive signals primarily from theANR with respect to forebrain specification.

MATERIALS AND METHODS

Construction of the Targeting Vector

Construction of the targeting vector was effected via initiallysubcloning a 3-kb EcoRI–SphI fragment of the mouse Otx2 39homologous region into modified pBluescriptII with a DT-A frag-ment (Yagi et al., 1993). Subsequently, the PGKneopA-cassette(neo-cassette), flanked by frt sites and including a loxP site insertedin 59 fashion relative to the frt site, was inserted into 59 of the Otx239 region adjacent to the NheI and EcoRI sites. Following neo-cassette insertion, a 2.5-kb BamHI–NheI fragment, containingOtx2 promoter and the first coding exon, was inserted 59 of theneo-cassette. Finally, a SmaI–BamHI 5.2-kb 59 Otx2 region possess-ing a loxP site at the 39 end was inserted 59 homologous of the Otx2promoter. The two frt sequences were generated by annealing thetwo synthetic oligo DNA strands (59-CCGGATCCGAAGTTCCT-ATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCA-CTGCAGGG-39 and 59-CCCTGCAGTGAAGTTCCTATACTTT-

r crossed with Cre transgenic mice to generate mice carrying theare deleted. Horizontal thick lines represent Otx2 genomic DNA,resent Otx2 coding exons. Filled arrowheads and open arrowheadsth the PGK promoter and polyadenylation signal; DT-A, diphtheria, BamHI; H, HindIII; E, EcoRI; N, NheI; NI, NotI; S, SphI; SI, SalI;entification. P1, P2, F1, N1, and R1 show primers utilized for theification and characterization of mutant allele by Southern blotgested with HindIII and hybridized with probe A to examine thee and targeted alleles, respectively. (b) Digested with XhoI and

b band represents targeted allele. (c) Digested with BamHI andence of the first loxP site. Recombinants generate a 7-kb band inand hybridized with probe D for identification of Otx2frt-neo (4 kb),ild-type allele. (C) PCR analysis of genotyping for different alleles.d R1 generates a 550- and 360-bp product for the Otx2frt-neo andd 160-bp product, indicating the presence of the loxP site located

flox

rthexons repe wi

tes: Bot idIdentre did-typ8-k

presheI

of w1, an0- an

ild-type allele, respectively. (c) In the Otx2 allele, a 560-bp PCRsed to detect the Otx2Dpro-ex1 allele (300 bp).

). All rights reserved.


CTAGAGAATAGGAACTTCGGAATAGGAACTTCGGATCCGG),and by PCR amplification (primers: 59-GCCAAGCTTGAAGTTC-CTATTCCGAAGTTC-39 and 59-CCGGTACCGAATTCTGAA-GTTCCTATACTTTCTAG-39), respectively. The two loxP se-

FIG. 2. The Otx2frt-neo allele produces aberrant Otx2 transcripts byN2 utilizing cDNAs from wild-type (left) and Otx2frt-neo/frt-neo homomutant indicate that the neo-cassette contains a splice acceptor. (b)types of aberrant mRNA transcripts (see below). (c) RT-PCR analwild-type mRNA transcripts, whereas the 200-bp products correspothe level of wild-type mRNA expression is downregulated. (B) (a) Scoding exons; an open box represents neo-cassette. Filled and openprimers used in RT-PCR analysis. (b, c) Schematic diagrams represstructures were determined by sequence analysis of RT-PCR produwere also evident in Otx2frt-neo/frt-neo mutant embryos at 6.5- and 7.5

quences were obtained from pBS246 plasmid (GIBCO BRL) by PCR


amplification (primers: 59-CGGGATCCTGATATCGGCTGGA-CGTAAACTCCTCTT-39 and 59-AAAGATCTCGATCATATTC-AATAACCCTT-39, and 59-AGAGATCTAATAACTTCGTATA-GCATA-39 and 59-CGGGATCCTTAATATAACTTCGTATA-39,

eo-cassette insertion. (A) (a) RT-PCR analysis with primers E1 andus mutant embryo (right) at 8.5 dpc. The 800-bp products in theCR analysis with primers N1 and E3 indicates the presence of two

with primers E1, N0, and E3. The 272-bp products correspond toOtx2 and neo hybrid mRNA, respectively. In the mutant embryo,atic diagram of the Otx2frt-neo allele. Shaded boxes represent Otx2heads denote loxP and frt sites, respectively. The arrows indicateg exon structures of mRNAs produced from this allele. The exonsterisks indicate the stop codon of neo. Aberrant Otx2 transcriptsstages (data not shown).

the nzygoRT-Pysisnd tochemarrowentincts. A-dpc

respectively).


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208 Tian et al.

Generation and Genotyping of Mice Carryingthe Different Otx2 Alleles

The targeting vector was linearized with SalI and introducedinto TT2 embryonic stem (ES) cells via electroporation as describedelsewhere (Yagi et al., 1993). G418-resistant ES clones were iden-tified by PCR and confirmed by Southern blot analysis as described

FIG. 3. Morphological features of Otx2frt-neo/2 mutant embryos.Otx2frt-neo/2 (D) embryos at 18.5 dpc on the CBA genetic background,in the CBA background (B, C). Otx2frt-neo/2 mutants fail to develappropriate formation of ventral portions of the cranial region (D).(F, H) in the CBA background at 18.5 dpc. Ventral views of the whH) are shown. Most of the skull elements are formed, although slsagittal sections (K, L) of wild-type (I, K) and Otx2frt-neo/2 (J, L) embrmutant embryos (J, L). The isthmic constriction (arrows in I, K) idevelops normally in the mutant embryo (J, L). Abbreviations: asexoccipital; m, mesencephalon; mt, metencephalon; mx, maxillar;

below. Two independent homologous recombinant clones were


injected into morula-stage embryos to generate chimera mice. Malechimeras were mated with CBA females in order to produceheterozygous mutants. These heterozygotes were crossed with Flpetransgenic mice carrying the CAG-Flpe transgene (provided by Dr.F. Stewart) in order to delete the neo-cassette, resulting in theproduction of Otx2flox heterozygous mice (Fig. 1A). The flox micecan be inverted into Dpro-ex1 heterozygous mice, which are

) Lateral view of wild-type (A), Otx21/2 (B), Otx2frt-neo/1 (C), andectively. Otx21/2 and Otx2frt-neo/1 show no noticeable abnormalitiesrsal and rostral regions of the head; however, they do undergo

l morphologies of wild-type (E, G) and Otx2frt-neo/2 mutant embryosount skull without mandibles (E, F) and separated mandibles (G,

y distorted, in Otx2frt-neo/2 mutant embryos. Lateral view (I, J) andt 10.5 dpc. The telencephalon and diencephalon are not formed inclearly observed in mutants (J, L). The first branchial arch (ba1)

phenoid; bs, basisphenoid; bo, basioccipital; d, diencephalon; eo,alatine; pm, premaxillar; sy, symphysis; t, telencephalon.

(A–Dresp

op doSkulole-mightlyos as not

characterized by deletion of the Otx2 promoter and first coding


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210 Tian et al.

exon, through mating with Cre transgenic mice (Fig. 1A; Lewan-doski et al., 1997). Transgenic mice were generated by microinjec-tion of DNA into fertilized eggs as described (Hogan et al., 1994).These mutant phenotypes were analyzed mainly in the CBAgenetic background. Genotypes of mutant mice were performedroutinely by PCR (Fig. 1C) and confirmed by Southern blot (Fig.1B). F1 (59-GTATTTTCCTTGCTACCAAACTGCCGAGTG-39),N1 (59-TCGTGCTTTACGGTATCGCCGCTCCCGATT-39), R1(59-CTGGAGGGAAGCCACACCTCTAAGGATTAA-39), P1 (59-TAG-AGGGTATTTAAATAAGGAACGACTGGG-39), and P2 (59-GTC-CTTCTAAGAGAACTGGATTGTTAACGA-39) primers were usedfor PCR identification. Four kinds of probes were employed forSouthern blot. HindIII-digested genomic DNAs were hybridizedwith Probe A (SphI–HindIII-digested 230-bp fragment of the Otx2third coding exon). XhoI-digested genomic DNAs were hybridizedwith Probe B (BamHI–PstI-digested 650-bp fragment of the neogene). BamHI-digested genomic DNAs were hybridized with ProbeC (EcoRI–EcoRV-digested 600-bp fragment of the Otx2 59 region).NheI-digested genomic DNAs were hybridized with Probe D(EcoRI–KpnI-digested 750-bp fragment of the Otx2 first intron).

Reverse Transcription (RT)-PCR Analysis

Total RNA was isolated from wild-type and Otx2frt-neo/frt-neo mu-tant embryos at 6.5, 7.5, and 8.5 dpc stages with TRIZOL reagent(GIBCO BRL), respectively. First-strand cDNA synthesis was per-formed with oligo-dT primers and Superscript kit (GIBCO BRL).The cDNA was utilized as a PCR substrate by standard protocols.The PCR primers E1 (59-TATGGACTTGCTGCATCCCTCCG-TGGGCTA-39), E3 (59-TGGCAGGCCTCACTTTGTTCTGAC-CTCCAT-39), N0 (59-TTGACAAAAAGAACCGGGCGCCCCTGC-GCT-39), N1 (59-TCGTGCTTTACGGTATCGCCGCTCCCGATT-39),and N2 (59-AATCGGGAGCGGCGATACCGTAAAGCACGA-39)were used.

Histological and Skeletal Analysis

Embryos were fixed in Bouin’s fixative, dehydrated, embedded inparaffin, and sectioned at 8 mm. Sections were then dewaxed,rehydrated, and stained with hematoxylin–eosin solution. Skeletalpreparation and staining were conducted as described previously(Kelly et al., 1983; Matsuo et al., 1995).

Whole-Mount RNA in Situ Hybridization

Embryos were dissected in PBS and fixed in 4% para-formaldehyde/PBS at 4°C, followed by gradual dehydration inmethanol/PBS containing 0.1% Tween 20. Specimens were storedin 100% methanol at 220°C. Whole-mount in situ hybridizationwas performed by digoxigenin-labeled riboprobes (BoehringerMannheim) as described (Wilkinson, 1993). The following probeswere employed: Otx1 (Suda et al., 1996), Otx2 (Matsuo et al., 1995),En2 (Davis and Joyner, 1988), Fgf8 (Crossley and Martin, 1995),Gbx2 (Bulfone et al., 1993), BF-1 (Foxg1; Tao and Lai, 1992), Emx2(Yoshida et al., 1997), Six3 (Oliver et al., 1995), Pax6 (Stoykova andGruss, 1994), Nkx2.1 (Kimura et al., 1996), mdkk-1 (Glinka et al.,1998), Cer-l (Belo et al., 1997), Foxa2 (Sasaki and Hogan, 1993),Cripto (Ding et al., 1998), Shh (Echelard et al., 1993), Foxd4
(Kaestner et al., 1995), and Brachyury/T (Herrmann, 1991).

Explant Culture and FGF8b Bead ImplantationMice were mated and fertilization was assumed to occur at the

midpoint of the dark cycle. Zero- to five-somite embryos werecollected in chilled Hank’s solution. The cephalic region wasisolated from the embryos and digested with 2.5–3.5% pancreatinand 0.5–0.7% trypsin in MgCl2- and CaCl2-depleted Hank’s solu-tion, depending on embryonic stages. Germ layer separation wasachieved with tungsten needles (Shimamura and Rubenstein,1997).

The neural plate from zero- to two-somite wild-type embryoswas isolated exclusively (see Fig. 7, Exp. 1; Shimamura and Ruben-stein, 1997). The anterior axial mesendoderm, which consists ofthe prechordal plate and anterior portion of the head process fromwild-type or mutant embryos at the zero- to two-somite stage, wastransplanted to the wild-type neural plate, respectively (see Fig. 7,Exps. 2 and 3). These explants were cultured on a nuclepore filter(Whatman no. 110614) floating in DMEM supplemented with 20%fetal bovine serum and 13 nonessential amino acids in a CO2

incubator at 37°C for 24 h (Shimamura and Rubenstein, 1997;Kimura et al., 2000).

Heparin acrylic beads (Sigma) were rinsed in PBS several times.Approximately 50 beads were soaked in 5 ml 0.2 mg/ml FGF8brecombinant protein solution (R&D) overnight at 4°C (Liu et al.,1999). The beads were rinsed in PBS prior to use. Control beadswere soaked in 50 mg/ml BSA–PBS in an identical manner. The leftside of the ANR was removed from the wild-type neural plate at thethree- to five-somite stages (see Fig. 9, Exp. 1). The ANR fromwild-type or mutant embryos at the three- to five-somite stage wastransplanted to the wild-type neural plate in the region where theleft side of the ANR had been ablated (see Fig. 9, Exp. 2). The intactmutant neural plate at the three- to five-somite stages was isolatedexclusively (see Fig. 9, Exp. 3). The ANR from wild-type embryos atthe three- to five-somite stages was transplanted to the intactmutant neural plate (see Fig. 9, Exp. 4). A single heparin acrylicbead soaked in BSA or FGF8b was implanted to the left side of thewild-type neural plate, where the ANR had been excised unilater-ally (see Fig. 9, Exp. 5), or to the intact mutant neural plate (see Fig.9, Exp. 6). These neural plate explants were cultured as describedabove.

RESULTS

Generation of an Allelic Series of Otx2 MutantsTo generate a series of Otx2 mutant alleles, a targeting

vector was designed in which the Otx2 promoter and firstcoding exon region were flanked with two loxP sites;additionally, a PGKneopA (neo-) cassette flanked by two frtsites was inserted into the Otx2 first intron (Fig. 1A). Weplanned to excise the neo-cassette in the first intron byFlp-mediated recombination and in the first coding exon byCre-mediated recombination; the latter should eliminateOtx2 function since this exon encodes the translationalstart site (Fig. 1A). Following electroporation in TT2 EScells, 8 of 182 G418-resistant ES clones were isolated ashomologous recombinants by PCR and finally confirmed bySouthern blot (Fig. 1B). Chimeric mice and their mutantoffspring were generated from two independent recombi-nant ES clones (E90 and E92). No phenotypic difference was
evident between these two mutant lines (see below). Het-


erozygous offspring of chimera bearing the Otx2frt-neo allelewas viable and fertile; offspring exhibited no noticeablephenotype (see Figs. 3A and 3C). However, Otx2frt-neo/frt-neo

homozygous mutants died at birth or by weaning (data notshown). Otx2frt-neo/1 mice were mated to CAG-Flpe trans-genic animals, which ubiquitously express Flpe, a variantFlp recombinase possessing enhanced thermostability, inorder to remove the neo-cassette (Buchholz et al., 1998).The mutant progeny carrying the transgene effectivelyinherited the recombined allele (Otx2flox allele) (Figs. 1B and1C). Subsequently, Otx2Dpro-ex1/1 heterozygous mutants wereobtained by mating with b-actin-cre mice, which ubiqui-tously express Cre under the human b-actin promo-ter (Lewandoski et al., 1997). Otx2flox/1, Otx2flox/flox, andOtx2Dpro-ex1/1 mutant mice thus produced were fertile andshowed no noticeable phenotype in the CBA genetic back-ground. Otx2frt-neo/1 mice were then crossed with Otx21/2

mutant mice (Matsuo et al., 1995), which carry a null allele,resulting in offspring containing both a frt-neo and a nullallele. Unexpectedly, all of the Otx2frt-neo/2 mutant fetusesexhibited dramatic head malformations, such as excen-cephaly and acephaly, at 18.5 dpc (see Fig. 3D; and data notshown). In contrast, Otx2flox/2 fetuses were viable and dem-onstrated no apparent abnormalities, suggesting that theOtx2flox allele functions as a wild-type Otx2 allele.

Neo-Cassette Insertion Creates a HypomorphicOtx2 Allele

Neo-cassette was supplied as a selectable marker toisolate homologous recombinant clones; however, it hasbeen reported to contain potential cryptic splice sites thatmay interfere with gene expression (Carmeliet et al., 1996;Meyers et al., 1998; Nagy et al., 1998). To examine whetherthe presence of the neo-cassette in the intron affected Otx2gene expression by aberrant splicing, RT-PCR analysis wasperformed by using primers from Otx2 and neo sequences(Fig. 2A). Indeed, the neo-cassette included both a crypticsplice acceptor and a splice donor (Fig. 2). Sequence analysisof RT-PCR products indicated that the splice acceptor islocated 5 bp upstream of the neo translation start site,whereas the splice donor is located 15 bp downstream of theneo TGA stop codon (Fig. 2B). In addition, two types of neofusion transcripts were produced: one contained the Otx2

FIG. 5. Molecular analysis of neural markers at 8.5 dpc. Emx2 (A,(M, N) expression by whole-mount in situ hybridization in wild-typat 8.5 dpc. Emx2 expression is detected in the prospective dorsal fomutant embryo (B). Although Six3 expression, a marker for theexpression domain is significantly reduced (D). Pax6 expression, a m(F). Otx1 expression, a marker for prospective forebrain and midbris shifted to the anterior side (I, J); however, the En2-negative doma(J, arrowheads). Gbx2 expression domain, a marker for anterior mete
(L). Shh expression, a marker for the ventral midline of neural tube anembryo (N).

second exon, which encodes homeodomain, whereas thesecond lacked this exon as a consequence of aberrantsplicing (Fig. 2B). Therefore, Otx2frt-neo/frt-neo homozygousembryos exhibited three kinds of transcripts: wild-typenormal transcripts and the two types of Otx2-neo fusionmRNA transcripts (Fig. 2). Nucleotide sequences of twoaberrant transcripts predicted that neither produced normalOtx2 protein; transcripts contained only 33 amino acids inthe N-terminal peptides of Otx2 in fusion with all neoprotein products (Fig. 2B). Furthermore, semiquantitativePCR analysis indicated that the expression level ofwild-type mRNA transcripts was significantly reduced inOtx2frt-neo/frt-neo mutant embryos (Fig. 2A). Analysis of Otx2expression by in situ hybridization using the third exon asa probe demonstrated that neo-cassette insertion did notdirect ectopic Otx2 mRNA expression in Otx2frt-neo/2 mu-tant embryos (data not shown).

Defects in Otx2frt-neo/2 mutants were more severe thanthose in Otx2frt-neo/frt-neo and Otx21/2 mutants; however, de-fects were milder than those observed in Otx22/2 mutants(Fig. 3; and data not shown). This finding demonstrated thatthe frt-neo allele was not functioning normally. Analysis ofmRNA expression and phenotype, taken together, indicatedthat Otx2frt-neo is a hypomorphic allele; moreover, these dataindicated that the defects in mutant mice carrying theOtx2frt-neo allele are due to a reduction in the level offunctional Otx2 gene expression brought about by thepresence of the neo-cassette in the first intron of the Otx2gene. As described below, detailed analysis of the defectscaused by the hypomorphic allele has provided new insightinto Otx2 function regarding the mechanism of forebraindevelopment.

Forebrain Development Was Arrested in Otx2frt-neo/2

Hypomorphic Mutant Embryos

Previously, we reported that mice heterozygous for theOtx2 mutation display a craniofacial malformation whichis mainly characterized as the loss of the lower jaw depend-ing on the genetic background of the C57BL/6 strain (Mat-suo et al., 1995). The majority of skull elements at the levelof premandibular and distal portions of the mandibularregions were lost or severely affected by this mutation(Matsuo et al., 1995). However, 18.5 dpc Otx2frt-neo/2 mu-

ix3 (C, D), Pax6 (E, F), Otx1 (G, H), En2 (I, J), Gbx2 (K, L), and ShhC, E, G, I, K, M) and Otx2frt-neo/2 mutant (B, D, F, H, J, L, N) embryosin of the wild-type embryo (A); however, it is not observed in theanterior neuroectoderm (C), is present in mutant embryos, the

r for the prospective diencephalon (E), is not evident in the mutant), also occurs in the mutant embryo (H). The En2-positive regionstill present in the anterior neuroectoderm of the mutant embryohalon (K), is expanded and shifted anteriorly in the mutant embryo

frt-neo/2

B), Se (A,rebramostarke

ain (Gin isncep

d axial mesendoderm (M), is unchanged in the Otx2 mutant


212 Tian et al.


214 Tian et al.

tants properly developed upper and lower jaws (Fig. 3D).Skeletal analysis also indicated that most of the skullelements in premandibular and distal components of themandibular regions were present and developed althoughthe morphology of these structures was distorted (Figs.3E–3H). These structures are derived from mesencephalicneural crest cells (Couly et al., 1993); therefore, the neuralcrest at the level of mesencephalon appeared to be formedappropriately in Otx2frt-neo/2 mutant embryos.

Otx2frt-neo/2 mutant mice at 18.5 dpc did not developrostral brain and eyes (Fig. 3D). We next examined earlierdefects in the mutant brain (Figs. 3I–3L). At 10.5 dpc, therostral aspect of the mutant brain appeared to be truncatedwith failure in the neural tube closure (Figs. 3I and 3J).Histological examination revealed the complete absence ofthe structures of the telencephalic vesicles and diencepha-lon (Figs. 3K and 3L). Additionally, the mesencephalon androstral portion of metencephalic regions, including theisthmus, also appeared to be malformed in the mutantembryos (Figs. 3K and 3L). These morphological data indi-cate that the majority of the rostral brain failed to developnormally in Otx2frt-neo/2 embryos at 10.5 dpc.

Expression of neuroectoderm markers by whole-mount insitu hybridization at 9.5 dpc was subsequently analyzed inorder to define rostral brain abnormalities in Otx2frt-neo/2

embryos (Fig. 4). Expression of molecular markers for fore-brain and isthmus were absent or severely affected inOtx2frt-neo/2 mutant embryos. Normally, BF-1 is expressed inmost of the telencephalic region (Fig. 4A; Tao and Lai,1992); however, its expression in this region was not de-tected in Otx2frt-neo/2 mutant embryos (Fig. 4B). Emx2 is alsoexpressed in dorsal telencephalon and diencephalons ofwild-type 9.5 dpc embryos (Fig. 4C). Emx2 expression wasabsent in Otx2frt-neo/2 hypomorphic mutants (Fig. 4D). Atthis stage, Six3 is expressed in the anterior forebrain regionand eyes in wild type (Fig. 4E; Oliver et al., 1995); however,its expression was undetectable in mutant embryos (Fig.4F). Normally, Nkx2.1 is expressed in ventral forebrain (Fig.4G; Kimura et al., 1996); however, Nkx2.1 expression wasnot observed in mutant embryos (Fig. 4H). Fgf8 is expressedin the commissural plate of telencephalon and in the dorsaldiencephalon and isthmic constriction in wild type (Fig.4I; Heikinheimo et al., 1994; Ohuchi et al., 1994). InOtx2frt-neo/2 mutant embryos, Fgf8 expression in these re-gions was not detected; rather, Fgf8 was expressed through-out the entire rostral region, including nonneural ectoderm

FIG. 6. Normal development of AVE and ADE in Otx2frt-neo/2 mutaembryos (B, D, F, H, J, L, N, P, R). (A–H) 6.5 dpc; (M, N) 7.5 dpc, anposterior to the right (except I and J, which are frontal views).distinguishable from the wild-type embryo (A–D). Expression of pprimitive streak (p) in both wild-type and mutant embryos (E–Happropriately in mutant embryos at 7.8 dpc (I, J). Expression of m

frt-neo/2
affected in Otx2 mutants at 7.5–7.8 dpc (K–N). Expression of Six3 (at 7.8 dpc (P, R). Abbreviation: pp, prechordal plate.

(Fig. 4J). Engrailed-2 (En2) expression, which covers themidbrain and anterior hindbrain at 9.5 dpc in wild type (Fig.4K; Davis and Joyner, 1988), underwent an anterior shift inOtx2frt-neo/2 hypomorphic embryos (Fig. 4L).

To determine the initial defects in brain formation,expression of neuroectoderm markers at 8.5 dpc was furtheranalyzed (Fig. 5). We found that prosencephalon was notspecified correctly in Otx2frt-neo/2 embryos. At this stage,Emx2 expression, a marker for the prospective telencepha-lon and dorsal diencephalon, was not detected in Otx2frt-neo/2

mutant embryos (Figs. 5A and 5B). Expression of Six3, theearliest anterior ectoderm marker, was observed; however,expression was significantly reduced in mutant embryos(Figs. 5C and 5D). At 8.5 dpc, Pax6 expression, evident inthe wild-type forebrain (Fig. 5E; Stoykova and Gruss 1994;Shimamura et al., 1997), was absent in mutant embryos(Fig. 5F). Nkx2.1 expression was observed in neither mutantembryo at this stage (data not shown). Otx1, which isexpressed in the prospective forebrain and midbrain in wildtype (Fig. 5G; Simeone et al., 1993), was shifted to theanterior side in mutant embryos (Fig. 5H). En2 expression, amarker for mesencephalon and anterior metencephalon(Fig. 5I), also underwent a shift to the anterior side inmutant embryos (Fig. 5J). Similarly, the domain of Gbx2expression, a marker for anterior metencephalon (Fig. 5K;Bouillet et al., 1995), was shifted anteriorly in mutantembryos (Fig. 5L). It is noteworthy that an En2-negativeregion was present in the most anterior region of themutant neuroectoderm (Fig. 5J), suggesting the occurrenceof anterior structures rostral to mesencephalon in mutantembryos. In contrast to these neuroectoderm markers alongthe A-P axis, Sonic hedgehog (Shh), a marker for the ventralmidline of the neural tube and axial mesendoderm (Ech-elard et al., 1993), was unaffected in mutant embryos (Figs.5M and 5N). Analysis of these aforementioned markerssuggests that, in Otx2frt-neo/2 hypomorphic mutants, theprospective forebrain region marked by Six3 and Otx1expression is formed; however, the forebrain is not specifiedcorrectly as judged by the markers for the prospectivetelencephalon and diencephalon (see Discussion).

Otx2frt-neo/2 Hypomorphic Mutants Form AVE andADE Normally

Induction of the anterior neuroectoderm has been pro-posed to result from signals emanating from AVE prior to

bryos. Wild-type (A, C, E, G, I, K, M, O, Q) and Otx2frt-neo/2 mutant, O–R) 7.8 dpc. The direction of embryo is anterior to the left andand mdkk-1 expression in the mutant AVE at 6.5 dpc are not

ior markers Cripto and T is present in the posterior ectoderm orxa2 expression is evident in the head process (hp) and node (n)1 and Cer-l in the prechordal plate and ADE, respectively, is not

nt emd (I–LCer-loster). Fodkk-

O) and Foxd4 (Q) in the ANE is unchanged in the mutant embryos



and in ADE and/or axial mesendoderm after gastrulation(Ang and Rossant, 1993; Beddington and Robertston, 1999;Shawlot et al., 1999; Camus et al., 2000; Kimura et al.,2000; Wilson and Rubenstein 2000; Kiecker and Niehrs,2001). To examine whether the failure in forebrain specifi-cation is a consequence of defects in these tissues, expres-sion of molecular markers was analyzed (Fig. 6). We foundthat marker expression was unchanged in Otx2frt-neo/2 mu-tant embryos (Fig. 6). In Otx22/2 mutant embryos, expres-sion of AVE markers such as Cer-l and Lim1 occurred;however, expression remained in the distal visceralendoderm even at 6.5 dpc; that is, expression does not shiftto the anterior side (Acampora et al., 1998; Kimura et al.,2000). In contrast, mdkk-1 expression (Pearce et al., 1999) isspecifically lost in Otx22/2 mutant visceral endoderm(Zakin et al., 2000; Kimura et al., 2001; Perea-Gomez et al.,2001). In the epiblast of Otx22/2 mutants, the posteriormarkers, T and Cripto (Ding et al., 1998; Herrmann, 1991),were expanded in the proximal ectoderm (Kimura et al.,2000). Thus, generation of the A-P axis fails in Otx22/2

mutant embryos. In Otx2frt-neo/2 mutant embryos, however,expression of both Cer-l and mdkk-1 was detected in theAVE (Figs. 6A–6D). Moreover, Cripto and T expres-sions were observed normally in the posterior epiblast inOtx2frt-neo/2 mutants (Figs. 6E–6H).

At the headfold stage, Foxa2 expression, a marker foraxial mesendoderm (Fig. 6I; Sasaki and Hogan, 1993), wasunaffected in mutant embryos (Fig. 6J). At this stage,mdkk-1, a marker for prechordal plate and the lateral regionof ADE (Fig. 6K; Kiecker and Niehrs, 2001), and Cer-l, amarker for ADE (Fig. 6M; Belo et al., 1997), were alsopresent in mutant embryos, which is a normal occurrence(Figs. 6L and 6N). In addition, expression of Six3 and Foxd4(previously referred to as fkh-2), the earliest anterior neuro-ectoderm markers (Oliver et al., 1995; Kaestner et al., 1995),were also unaffected in mutant embryos at 7.8 dpc (Figs.6O–6R), supporting that the prospective forebrain region isonce formed appropriately in Otx2frt-neo/2 mutant embryos.These results suggest that Otx2frt-neo/2 hypomorphic mutantembryos can establish the A-P axis and form ADE andanterior axial mesendoderm tissues normally.

The Anterior Axial Mesendoderm of Otx2frt-neo/2

Hypomorphic Mutants Functions Normally

Notably, the anterior axial mesendoderm has been shownto induce and maintain Nkx2.1 expression in the neuroec-toderm (Ericson et al., 1995; Shimamura and Rubenstein,1997; Pabst et al., 2000). Indeed, Nkx2.1 expression was notevident in Otx2frt-neo/2 hypomorphic mutant embryos at 8.5-and 9.5-dpc stages (Fig. 4F; and data not shown). Thus, inorder to investigate whether the inductive signals producedin the mutant anterior axial mesendoderm are defective,neural plate explant analysis was performed (Fig. 7). Asreported previously, the anterior axial mesendoderm frag-ment, which consists of the prechordal plate and the
anterior portion of the head process from wild-type embryos

at zero- to two-somite stage, could induce Nkx2.1 expres-sion in neural plate explants from wild-type embryos at theidentical stage, followed by culture for 24 h (Shimamuraand Rubenstein, 1997; Figs. 7B and 7C). Unexpectedly, theanterior axial mesendoderm from mutant embryos was alsoable to induce Nkx2.1 expression transplanted on wild-typeneural plate explants (Fig. 7D; n 5 5). This result indicatesthat the mutant anterior axial mesendoderm is capable ofnormal induction of Nkx2.1 expression. Furthermore, thesefindings suggest that failure in the forebrain specification ofmutant embryos does not result from defects of the axialmesendoderm. Furthermore, additional explant analysiswas conducted in order to establish whether mutant ADE isable to induce and/or maintain Otx2 expression trans-planted to the wild-type epiblast (Ang et al., 1994). As weexpected, mutant ADE could induce and/or maintain Otx2expression in the wild-type epiblast (n 5 3; data not shown).These explant studies suggest that failure in forebrainspecification of Otx2frt-neo/2 mutant embryos is not a conse-quence of defects in either the ADE or the anterior axialmesendoderm; rather, failure is due to later and/or otherdevelopmental events.

BF-1 Expression Is Not Initiated in theNeuroectoderm of Mutant Embryos

Several reports have indicated that the ANR functions asa local signaling center essential for forebrain specification(Shimamura and Rubenstein, 1997; Houart et al., 1998).Fgf8 is expressed in the ANR and later in the commissuralplate of telencephalon and, indeed, can induce and/or main-tain BF-1 expression (Shimamura and Rubenstein, 1997).

In order to investigate the possibility that mutant defectsare due to the failure in forebrain specification by the ANR,expression of markers for the ANR and prosencephalicneural plate was analyzed at early somite stages (Fig. 8). Wefound that BF-1 and Fgf8 expression of the nonneuralectoderm occurred properly; however, expression of BF-1and Fgf8 was absent in the neural plate of Otx2frt-neo/2

mutants (Fig. 8). Normally, BF-1 expression is initiallypresent in nonneural ectoderm adjacent to the anterior edgeof the neural plate around the two- to three-somite stage(data not shown). Subsequently, BF-1 expression begins inthe rostral margin of the ectoderm after the five-somitestage in wild type (Fig. 8A; Shimamura and Rubenstein,1997). In mutant embryos, BF-1 expression was detected inthe ANR at this stage, as was the case with wild type (Fig.8B). At the ensuing eight-somite stage, neuroectodermalexpression was induced in wild-type embryos (Fig. 8C);however, induction was not evident in Otx2frt-neo/2 mutantembryos (Fig. 8D). Even at the 12-somite stage, BF-1 expres-sion remained undetectable in the neuroectoderm of mu-tant embryos (Figs. 8E and 8F). Similarly, Emx2 expressionwas initiated in the laterocaudal neural plate at the three-somite stage (Shimamura et al., 1995; and data not shown).However, no Emx2 expression occurred in mutant embryos,
even at the eight-somite stage (Figs. 8G and 8H).

(C; n

216 Tian et al.

Normally, Fgf8 expression commences in the nonneuralectoderm around the four-somite stage and in neuroecto-derm following the six-somite stage (Shimamura andRubenstein, 1997; and data not shown). In mutant embryosat the eight-somite stage, however, Fgf8 expression was notobserved in the neural plate; on the other hand, normal Fgf8expression was displayed in nonneural ectoderm (Figs.8I–8L). At the subsequent 12-somite stage, Fgf8 expressionin the prospective commissural plate remained undetect-able in mutant embryos (Figs. 8M and 8N). Thus, in mutantembryos, expression of the telencephalic genes BF-1, Emx2,and Fgf8 was not initiated in neuroectoderm, althoughexpression of these genes began normally in nonneuralectoderm.

Otx2frt-neo/2 Mutant Neural Plate Cannot Respondto the Signals from the ANR

To investigate another possibility that the mutant ANR

FIG. 7. The anterior axial mesendoderm of Otx2frt-neo/2 embryorepresentation of the neural plate and anterior axial mesendodermstage, respectively, and the explant experiments (Exps. 1–3). (B–Dfollowing a 24-h culture of neural plates. In neural plate explant oevident (B; n 5 6). The anterior axial mesendoderm from wild-typewild-type neural plate.

retains the ability to induce telencephalic markers while


the mutant neural plate loses competence to the inductivesignals from the ANR, neural plate explant assays wereconducted (Fig. 9; Shimamura and Rubenstein, 1997). Wild-type anterior neural plate explants lose BF-1 expressionupon excision of the ANR at the three- to five-somite stagesfollowed by culture for 24 h (Fig. 9B; n 5 9; Shimamura andRubenstein, 1997). Initially, we examined whether theANR fragment from mutant embryos (ANRmut) can restoreBF-1 expression in wild-type neural plate explants in amanner similar to the ANR fragment from wild type(ANRWT) (Fig. 9C; n 5 10). Indeed, the ANRmut was able toinduce BF-1 expression transplanted on the wild-type ex-plants (Fig. 9D; n 5 14). In order to investigate the compe-tence of the mutant neural plate, the ANRWT was trans-planted on the mutant neural plate (Figs. 9E and 9F). TheANRWT, however, was unable to induce BF-1 expression(Fig. 9F; n 5 7). Further, a FGF8b bead was transplanted onthe mutant neural plate; a FGF8b-bead is demonstrated tobe sufficient for BF-1 induction in the neural plate (Figs. 9G

ctions normally in neural plate explant assays. (A) Schematicld-type and Otx2frt-neo/2 mutant embryos at the zero- to two-somitehole-mount in situ hybridization analysis of Nkx2.1 expressioned from wild-type embryos exclusively, Nkx2.1 expression is not5 8) or mutant embryos (D) can induce Nkx2.1 expression in the

s funof wi

) Wbtain

and 9H; n 5 14; Shimamura and Rubenstein, 1997). A



FIG. 8. Defects of neural plate in the Otx2frt-neo/2 hypomorphic mutant embryos. BF-1 (A–F), Emx2 (G, H), and Fgf8 (I–N) expression bywhole-mount in situ hybridization at the 5-somite (A, B), 8-somite (C, D, G–L), and 12-somite (E, F, M, N) stages in wild-type (A, C, E, G, I, K,M) and Otx2frt-neo/2 mutant embryos (B, D, F, H, J, L, N). (K, L) Sagittal sections of embryos shown in (I) and (J), respectively. All whole-mountembryos are lateral views. At the five-somite stage, BF-1 expression occurs in the nonneural ectoderm (arrowhead) of the wild-type embryo butnot in the neuroectoderm (A). BF-1 expression is not changed in the nonneural ectoderm of the mutant embryo at the five-somite stage (B,arrowhead). BF-1 expression is found in the neuroectoderm (arrow) and nonneural ectoderm (arrowhead) in the wild-type embryo at theeight-somite stage (C). In the mutant embryo, BF-1 expression is detected solely in the nonneural ectoderm (D, arrowhead). At the 12-somitestage, BF-1 expression is evident throughout the prospective telencephalic region of wild type (E, arrow). At this stage, BF-1 expression is notinduced in the neural ectoderm; however, it remains apparent in the nonneural ectoderm of mutant embryos (F, arrowhead). At the eight-somitestage, Emx2 expression is present in the neuroectoderm in the wild type (G). However, no Emx2 expression occurs in the mutant embryo at thisstage (H). At the eight-somite stage, Fgf8 expression is observed in the neuroectoderm (arrow) and nonneural ectoderm (arrowhead) of thewild-type embryo (I and K); in contrast, Fgf8 expression occurs in the nonneural ectoderm (arrowheads) but not in the neuroectoderm in mutantembryos (J, L). At the 12-somite stage, Fgf8 expression is present in the prospective telencephalic commissural plate in the wild-type embryo (M,
arrow). In the mutant embryo, expression of Fgf8 is not initiated in the neuroectoderm; however, it remains evident in the nonneural ectoderm(N, arrowhead). Abbreviations: be, branchial ectoderm; fg, foregut; is, isthmic constriction.

218 Tian et al.

FGF8b bead, however, could not induce BF-1 expression inthe mutant neural plate (Fig. 9I; n 5 11). Therefore, failurein forebrain specification of mutant embryos results fromthe defects of the neural plate, but not of the ANR. Thisfinding suggests that Otx2 may be required in the neuralplate for proper response to inductive signals produced inthe ANR.

DISCUSSION

Generation of Otx2 Hypomorphic Mutation

We were able to generate a hypomorphic allele of Otx2through gene targeting capable of neo-cassette insertioninto the intron. Intriguingly, the Otx2frt-neo/2 hypomorphicmutant embryos displayed a unique phenotype, whichpermitted normal A-P axis generation and gastrulation.These mutants failed to specify the forebrain region, how-ever. These results provide new insights into the roles ofOtx2 with respect to forebrain specification. RT-PCR anal-ysis demonstrated that normal Otx2 mRNA expression ofthe hypomorphic allele was significantly reduced as thiscassette contains cryptic mRNA splice sites and directsaberrant splicing patterns (Fig. 2). Predicted protein prod-ucts consist of only 33 amino acids of N-terminal residuesof Otx2 fused with an entire neo protein resulting from theaberrant splicing. As Otx2frt-neo/1 hypomorphic mutant micedevelop normally, it is unlikely that neo-fused productsexert dominant-negative effects. Thus, the forebrain defectsobserved in the mutant embryos may be due to the subcriti-cal thresholds of Otx2 expression in the anterior neuroec-toderm. Recently, neo-cassette insertion into the intron hasbeen shown to create a hypomorphic mutation by targetingof other genes (Meyers et al., 1998; Nagy et al., 1998; Gageet al., 1999; Lowe et al., 2001). Early embryonic lethalityresulting from complete loss-of-function mutations rendersmilder alleles valuable in the elucidation of roles at laterdevelopmental stages; consequently, this approach mayprovide an applicable method for extensive genetic analysisof genes of interest.

Otx2 Functions in the Neuroectoderm to InduceForebrain Gene Expression

Our detailed analysis of Otx2frt-neo/2 hypomorphic mutantembryos provides evidence that Otx2 is required in theneural plate to induce telencephalic gene expression forforebrain specification. Previous studies have suggestedthat Otx2 expression in AVE can direct the induction ofanterior neuroectoderm but is not sufficient to form theforebrain region correctly (Acampora et al., 1998; Suda etal., 1999; Kimura et al., 2000). Moreover, it was suggestedthat Otx2 expression in the neuroectoderm is essential forforebrain development, based on investigations involvingmouse chimeras containing Otx22/2 and wild-type cells(Rhinn et al., 1998, 1999). Consistent with the aforemen-
tioned reports, it appears that the anterior neural plate

marked by Six3, Foxd4, and Otx1 expression can be formedonce in Otx2frt-neo/2 hypomorphic mutant embryos normally(Figs. 5 and 6). Notably, Six3 demarcates the most anteriorneural plate (Oliver et al., 1995). Overexpression of Six3leads to enlarged forebrain, whereas the dominant activatorform of Six3 results in the reduction of forebrain in ze-brafish (Kobayashi et al., 1998, 2001). This observationsuggests that Six3 may define the forebrain territory.

Recently, we found the cis-regulatory region of Otx2,which governs transgene expression in AVE and ADE butnot in anterior neuroectoderm within endogenous Otx2expression (Kimura et al., 2000). Transgenic rescue studieswith this cis-regulatory region have demonstrated thatOtx2 expression in the anterior neural plate is not requiredfor induction of Six3 expression (Kimura et al., 2000).Furthermore, the fact that Otx22/2 cells express Six3 basedon chimeric studies (Rhinn et al., 1999), in concert with theaforementioned findings regarding Otx2, supports the pos-sibility that Otx2 expression in neural plate may notfunction in an essential capacity in formation of the pro-spective forebrain territory. Consistently, marker expres-sion of AVE, primitive streak, ADE, and anterior axialmesendoderm, which have been proposed to play importantroles in forebrain development in mouse (Beddington andRobertson, 1999; Shawlot et al., 1999; Camus et al., 2000),was not affected during gastrulation in Otx2frt-neo/2 mutantembryos (Figs. 5N, 6, and 7). Indeed, the ADE fromOtx2frt-neo/2 mutant embryos was able to induce or maintainOtx2 expression transplanted on the epiblast from wild-type embryos at early streak stage. Moreover, the mutantanterior axial mesendoderm could induce Nkx2.1 expres-sion transplanted on wild-type neural plate explants, sug-gesting that the mutant anterior axial mesendoderm func-tions normally (Fig. 7). These data indicate that AVE, ADE,and anterior axial mesendoderm form appropriately in mu-tant embryos; furthermore, these results support the tran-sient formation of the prospective prosencephalic neuralplate in mutant embryos.

In Otx2frt-neo/2 mutant embryos, however, subsequentforebrain region marked by BF-1, Emx2, Nkx2.1, and Pax6expression was not formed correctly (Figs. 4, 5, and 8).Notably, loss-of-function analysis revealed that BF-1 isessential in the development of the cerebral hemispheres(Xuan et al., 1995). Emx2 functions in the formation ofdorsal telencephalon and diencephalon (Yoshida et al.,1997; Suda et al., 2001). Small eye mutation analyses havedemonstrated that Pax6 is required for the development andregionalization of diencephalon (Stoykova et al., 1996;Warren and Price, 1997; Grindley et al., 1997). Furthermore,overexpression experiments suggest that Pax6 also definesthe dien–mesencephalic boundary via repression of En1 andPax2 (Matsunaga et al., 2000). Nkx2.1 is critical to ventralforebrain development (Kimura et al., 1996; Sussel et al.,1999). Therefore, Otx2 may be involved in forebrain devel-opment through direct or indirect transactivation of theseforebrain genes crucial for subsequent forebrain specifica-
tion. On the other hand, expression of the mesencephalic


marker, En2, was not altered; however, the expressiondomain underwent an anterior shift in mutant embryos(Figs. 4 and 5). This result is supported by the previousreport that Otx2 expression in the neural plate may not berequired for the initiation, but rather the maintenance, ofEn2 expression indirectly (Rhinn et al., 1999). It is notewor-thy that En2 was not expressed in the most anterior portionof mutant neuroectoderm (Figs. 4L and 5J), suggesting thepresence of the prospective prosencephalic neural plate inmutant embryos. In conjunction, these observations sug-gest that A-P axis development and induction of the pro-spective prosencephalic neural plate appear to have beenundergone normally once; however, at subsequent stages,the forebrain region failed to be specified in Otx2frt-neo/2

mutant embryos.

Otx2 May Define the Competence of ProsencephalicNeural Plate to Inductive Signals from the ANR

Expression of forebrain markers such as BF-1 and Emx2was not induced in Otx2frt-neo/2 mutant embryos from itsinitial manifestation (Fig. 8). Recently, the ANR in mouseor the first row of cells in zebrafish has been proposed tofunction as a local signaling center necessary for forebrainspecification (Shimamura and Rubenstein, 1997; Houart etal., 1998). Removal of the ANR results in the loss ofexpression of the telencephalic gene, BF-1; additional ANRcan induce ectopic BF-1 expression in mouse neural plateexplants (Shimamura and Rubenstein, 1997). To assess thecause of the Otx2frt-neo/2 mutant defects, molecular markerswere examined for ANR and prosencephalic neural plateduring early somite stage in mutant embryos. At thisjuncture, molecular defects of Otx2frt-neo/2 mutant embryosbeginning in the anterior neural plate were detected by theeight-somite stage at the time of forebrain regionalization;expression of BF-1, Fgf8, and Emx2 was not induced in theneuroectoderm of mutant embryos, whereas expression ofFgf8 and BF-1 was not affected in nonneural ectoderm (Fig.8). It is open to question as to whether the ANR or theneural plate is defective for the inductive interaction be-tween the ANR and the neural plate in mutant embryos.

In order to address this issue, neural plate explant studieswere performed. These experiments demonstrated that themutant ANR was able to induce BF-1 expression whentransplanted on the wild-type neural plate; in contrast,neither wild-type ANR nor a FGF8b bead, which mimicsthe properties of the ANR, induce BF-1 expression inmutant neural plate (Fig. 9). This evidence suggests thatmutant embryos can form ANR bearing normal inductionactivity; however, mutants are unable to respond to signalsof induction produced in the ANR. Concomitantly, Otx2 isexpressed in the anterior neuroectoderm but not in theANR of wild-type embryos at the five-somite stage (data notshown). Thus, it is likely that, due to the incompetence ofmutant neural plate, expression of BF-1 and Emx2 genes,which are crucial for telencephalon development (Xuan et
al., 1995; Yoshida et al., 1997), cannot be initiated in

Otx2frt-neo/2 mutant embryos. Consequently, mutant neuralplate might result in the loss of the entire forebrain regionat later stages. More interestingly, this study suggests thatOtx2 may be involved downstream of the signals from theANR, i.e., Fgf signaling, to mediate the induction of telen-cephalic genes for forebrain regionalization. Otx2 is atranscription activating factor (Simeone et al., 1993); there-fore, it is likely that Otx2 transactivates telencephalicgenes as a target directly or indirectly in response to Fgfsignals. It is notable that Otx22/2 cells in the forebrainregion subsequently underwent apoptosis in chimeric em-bryos (Rhinn et al., 1999). Apoptosis in these cells may beinduced as a result of the incompetence of mutant neuralplate with respect to growth or differentiation signals (i.e.,Fgfs produced by the ANR for forebrain specification).Additional molecular analysis is required in order to deter-mine which point(s) of the pathway is/are regulated byOtx2 in the prosencephalic neural plate.

Recently available evidence suggests the existence ofdifferential competence within the neuroectoderm to in-ductive signals produced by the ANR or isthmic organizer(Shimamura and Rubenstein, 1997; Rubenstein et al., 1998;Rubenstein and Beachy, 1998). For example, Fgf8 inducesBF-1 expression in the prospective telencephalic neuralplate, whereas in more caudal regions of the neural plate,Fgf8 induces En2 or Gbx2 expression. Thus, such distinctneuroectodermal gene expression at differing positionsalong the A-P axis within the neural plate is due to intrinsicdifferences in competence to the signals. (Shimamura andRubenstein, 1997; Rubenstein et al., 1998). Otx2frt-neo/2 mu-tant embryos do not express BF-1; however, expression ofEn2 and Gbx2 is normal (Figs. 4, 5, and 8). Indeed, FGF8bbeads induced neither BF-1 nor En-2 expression in theprospective prosencephalic region of mutant neural plates(Fig. 9; and data not shown), implying that the competenceof prosencephalic neural plate may not be transformed tothat of the mesencephalic neural plate; rather, prosence-phalic competence may be lost exclusively in thisOtx2frt-neo/2 mutant embryo.

Additional transplant and genetic studies are necessary inorder to identify the components involved in the differen-tial competence of the anterior neural plate. Nevertheless,given the arguments stated above, we believe that Otx2most likely involves the competence of the prosencephalicneural plate with regard to signals produced mainly in theANR.

Otx2 Is Involved in Critical Processesfor Forebrain Development

In mouse, the A-P axis is initially generated in aproximal–distal (P-D) direction around 5.5 dpc; subse-quently, a process of axis rotation occurs prior to primitivestreak formation (Beddington and Robertson, 1999). As aresult of this process, the expression of genes that mark thedistal visceral endoderm shifts to the anterior visceral
endoderm, whereas markers expressed in the proximal

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220 Tian et al.



epiblast shift to the posterior aspect, where the primitivestreak is to be formed (Beddington and Robertson, 1999). Ithas been suggested that Otx2 possesses a crucial role in A-Paxis formation in P-D orientation from its initial manifes-tation in cooperation with Cripto in epiblast (Kimura et al.,2001) and the subsequent autonomous anterior movementof distal visceral endoderm cells (Kimura et al., 2000). TheAVE thus formed does not induce anterior neuroectodermdirectly; rather, AVE mediates forebrain development byrepressing posteriorizing signals (Tam and Steiner, 1999;Klingensmith et al., 1999; Kimura et al., 2000; Stern, 2001).Moreover, Otx2 is also essential to this suppression(Kimura et al., 2000).

At subsequent mid-to-late-streak stages, ADE derivedfrom the primitive streak also plays an important role in thecorrect patterning of neuroectoderm (Ang and Rossant,1993; Ang et al., 1994). Requirement of ADE in forebraindevelopment is also suggested by germ-layer explant stud-ies, surgical removal experiments, and chimeric analysis ofLim1 mutant embryos (Ang et al., 1994; Shawlot et al.,1999; Camus et al., 2000). Thus, ADE may function in theestablishment or maintenance of anterior territory that isinitiated by AVE (Shawlot et al., 1999; Kimura et al., 2000).However, the precise role of Otx2 expression in ADEremains to be determined.

Within the anterior neuroectoderm, the neural plate istransversely subdivided into forebrain, midbrain, and hind-brain regions, each with characteristic morphological andmolecular properties. Two local signaling centers, ANR andmid/hindbrain junction, termed the isthmus, play crucialroles in the refinement of the identity within neuroecto-derm along the A-P axis (Joyner, 1996; Shimamura andRubenstein, 1997; Rubenstein et al., 1998). Otx2 interactsin a repressive manner with Fgf8 and Gbx2 for the correctpositioning of the isthmic organizing center (Acampora etal., 1997; Suda et al., 1997; Liu et al., 1999; Martinez et al.,1999; Garda et al., 2001; Liu and Joyner, 2001). In thepresent investigation, we found that Otx2 is also essentialfor the longitudinal subdivision of the forebrain by definingthe competence of the neural plate to signals from theANR.

Further elucidation of Otx2 function in processes of brainspecification and regionalization will be resolved throughconditional knock-out experiments. These studies can beperformed via the mating of mice carrying the Otx2flox

allele, which we have generated, with mice that express Creunder the control of appropriate tissue-specific regulatoryregions.

ACKNOWLEDGMENTS

We are grateful to Drs. R. Beddington, E. M. De Robertis, M.Frohman, P. Gruss, B. G. Herrmann, B. Hogan, A. Joyner, K.Kaestner, S. Kimura, E. Lai, M. Lewandoski, G. Martin, A. McMa-hon, C. Niehrs, M. Shen, and F. Stewart for gifts of plasmids; Drs.K. Shimamura and M. Nakafuku for discussions; and K. Kitajima
for her assistance. We are also grateful to the Division of Microbi-

ology and Genetics, Center for Animal Resources and Develop-ment, Kumamoto University, for housing the mice. E.T. is aninternational student supported by the Ministry of Education,Culture, Sports Science and Technology, Japan. C.K. is supportedby a special postdoctoral researchers program. This work wassupported in part by Grants-in-Aid from the Ministry of Education,Culture, Sports Science and Technology, Japan.

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Received for publication August 7, 2001
Accepted November 1, 2001
Published online January 8, 2002


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