The vertebrate brain is among the most complex biological structures of which the organization remains unclear. Increasing numbers of studies have accumulated on the molecular basis of midbrain/hindbrain development, yet relatively little is known about forebrain organization. Nested expression among Otx and Emx genes has implicated their roles in rostral brain regionalization, but single mutant phenotypes of these genes have not provided sufficient information. In order to genetically determine the interaction between Emx and Otx genes in forebrain development, we have examined Emx2−/−Otx2+/− double mutants and Emx2 knock-in mutants into the Otx2 locus (Otx2+/Emx2). Emx2−/−Otx2+/− double mutants did not develop diencephalic structures such as ventral thalamus, dorsal thalamus/epithalamus and anterior pretectum. The defects were attributed to the loss of the Emx2-positive region at the three- to four-somite stage, when its expression occurs in the laterocaudal forebrain primordia. Ventral structures such as the hypothalamus, mammillary region and tegmentum developed normally. Moreover, dorsally the posterior pretectum and posterior commissure were also present in the double mutants. In contrast, Otx2+/Emx2 knock-in mutants displayed the majority of these diencephalic structures; however, the posterior pretectum and posterior commissure were specifically absent. Consequently, development of the dorsal and ventral thalamus and anterior pretectum requires cooperation between Emx2 and Otx2, whereas Emx2 expression is incompatible with development of the commissural region of the pretectum.

INTRODUCTION

Initial regionalization of the neural plate along its anterior-posterior axis occurs concurrent with, or shortly after, neural induction. Recent studies have suggested that anterior neuroectoderm is induced by permissive and instructive signals from anterior visceral endoderm, anterior definitive mesendoderm and others (Ang et al., 1996; Thomas and Beddington, 1996; Beddington and Robertson, 1998; Beddington and Robertson, 1999; Shawlot et al., 1999; Tam and Steiner, 1999; Kimura et al., 2000). Otx2, Rpx/Hesx1 and Six3 are the genes expressed in the early anterior neuroectoderm after induction (Suda et al., 1999; Martinez-Barbera et al., 2000a; Martinez-Barbera et al., 2000b; Wallis and Muenke, 2000). The initial morphological landmark of regionalization in the anterior neuroectoderm is the preotic sulcus, which corresponds to the future boundary between rhombomeres r2 and r3. This landmark becomes apparent around the three-somite stage. No molecular devices are known in the establishment of the boundary or initial development of r1 and r2. Rostral to the preotic sulcus, the midbrain/hindbrain junction (or isthmus) establishes molecularly around the six-somite stage (Crossley and Martin, 1995; Millet et al., 1999; Suda et al., 1999) and morphologically around 9.0 days post coitus (dpc). A growing number of molecular studies have been conducted on developmental patterning of the midbrain and the establishment of the isthmus. Otx2 and Otx1, Pax2 and Pax5, and En1 and En2 cooperate in midbrain development (Hanks et al., 1995; Acampora et al., 1997; Acampora et al., 1998; Acampora et al., 1999; Suda et al., 1996; Suda et al., 1997; Suda et al., 1999; Urbanek et al., 1997; Schwarz et al., 1999). Wnt1 functions to maintain the midbrain, possibly via the regulation of En expression (McMahon et al., 1992; Danielian and McMahon, 1996). The isthmus is established where Otx2 and Gbx2 meet (Wassarman et al., 1997; Broccoli et al., 1999; Hidalgo-Sanchez et al., 1999; Millet et al., 1999; Simeone, 2000), and plays an essential role in the rostrocaudal patterning of the midbrain. A loop of mutual interactions between Fgf8, En1, En2, Pax2, Pax5 and Wnt1 genes has been suggested in the isthmus (Balley-Cuif and Wassef, 1995; Joyner, 1996). Fibroblast growth factor (FGF8) is currently the sole factor demonstrating midbrain patterning activity of the isthmus (Crossley et al., 1996; Lee et al., 1997; Martinez et al., 1999).

In contrast, the mechanisms that delineate the telencephalon and diencephalon are poorly understood. The forebrain or primary procencephalon segregates into the diencephalon and secondary procencephalon, from which alar plates protrude to generate the telencephalon. The boundary between the mesencephalon and diencephalon has been proposed to occur around the ten-somite stage, at the point where Pax6 expression segregates from that of Pax2 and En1 (Araki and Nakamura, 1999; Schwarz et al., 1999; Matsunaga et al., 2000). The posterior commissure, which is formed near 10.5 dpc, is the morphological landmark of its dorsal boundary. Zona limitans interthalamica (zlth), which divides the ventral and dorsal thalamus, is formed earlier. It occurs molecularly near the five-somite stage and morphologically at 10.0 dpc, approximately where axial mesoderm is subdivided into the prechordal plate and notochord (Figdor and Stern, 1993; Shimamura et al., 1995; Inoue et al., 2000). The diencephalic region caudal to zlth is compatible with transformation into a mesencephalic phenotype by ectopic transplantation of isthmus/FGF8-soaked beads and by ectopic En expression (Crossley et al., 1996; Araki and Nakamura, 1999; Martinez et al., 1999). Sonic hedgehog (SHH) is expressed by zlth (Erickson et al., 1995), and it has been suggested that zlth functions not only as a barrier to restrict cell mixing and the spread of pattern information, but also as a source of morphogenetic information or as a local organizing center (Balley-Cuif and Wassef, 1995).

A number of genes are now known to be expressed in specific domains of the forebrain, and a neuromeric organization of the forebrain has been proposed (Bulfone et al., 1993; Shimamura et al., 1995; Shimamura et al., 1997). Otx2, Otx1, Emx2 and Emx1, mouse cognates of Drosophila head gap genes, otd and ems, are among these genes. Otx2 expression occurs throughout the anterior neuroectoderm during the initial phase of its induction. At 10.5-12.5 dpc, Otx2 expression regresses in the dorsal telencephalon corresponding to the presumptive cerebral cortex (Simeone et al., 1993; Mallmaci et al., 1996). Otx1 expression is evident around the 1 somite stage. At 10.25-12.5 dpc, Otx1 expression covers the region from the cerebral cortex to the midbrain (Simeone et al., 1993). Emx2 expression is evident around the three-somite stage. At 10.5-12.5 dpc, Emx2 expression ranges from a portion of the subcortical domain in the telencephalon to the diencephalon rostral to zlth (Simeone et al., 1992a; Shimamura et al., 1997; Mallamaci et al., 1998). Emx1 expression occurs in the cortical region around 9.5 dpc. As a result, Otx2, Otx1, Emx2 and Emx1 genes constitute a ‘nested’ expression, and the roles of these genes in brain regionalization have been suggested (Simeone et al., 1992b).

However, the phenotype of each single mutant of these genes has not been informative with respect to their roles in rostral brain regionalization. Otx2−/− mutants fail to develop a rostral head by the loss of earlier Otx2 functions in visceral endoderm (Acampora et al., 1995; Matsuo et al., 1995; Ang et al., 1996; Rhinn et al., 1998; Kimura et al., 2000). Defects are apparent exclusively in the archipallium in Emx2−/− mutants (Pellegrini et al., 1996; Yoshida et al., 1997). Otx1−/− and Emx1−/− single mutants exhibit only subtle defects (Suda et al., 1996; Acampora et al., 1996; Qiu et al., 1996; Yoshida et al., 1997). In contrast, data from Otx1 and Otx2 double heterozygous mutants (Otx1+/−Otx2+/−) indicate that Otx2 and Otx1 cooperate in developing mesencephalon and posterior diencephalon at the time of brain regionalization (Acampora et al., 1997; Suda et al., 1997).

In the present study, we have generated Emx2 homozygous and Otx2 heterozygous double mutants (Emx2−/−Otx2+/−), and Emx2 knock-in mutants into the Otx2 locus (Otx2+/Emx2), in order to genetically determine the interaction between Emx2 and Otx2 genes in forebrain development. Otx2−/− homozygous mutants exhibit defects caused by the lack of early Otx2 function, as described above. Consequently, interactions between the two genes in brain regionalization could not be evaluated in the Emx2−/−Otx2−/− double homozygous mutant state. Nevertheless, Emx2−/−Otx2+/− double mutants demonstrate that these two genes indeed play crucial roles in the development of the ventral and dorsal thalamus, and anterior pretectum (precommissural region of pretectum) (Martinez and Puelles, 2000). Emx2 ectopic expression by its knock-in into the Otx2 locus results in a phenotype complementary to the double mutants; Emx2 expression must be suppressed for the development of the posterior pretectum (commissural region of the pretectum).

MATERIALS AND METHODS

Construction of knock-in vector

Emx2 (762 bp) cDNA lacking 3′UTR was isolated from a 11.5 dpc mouse cDNA library. The neomycin resistance gene (neor), which has no polyadenylation signal and is driven by the Pgk1 promoter, was flanked by loxP sequences (see Fig. 6A). These sequences (Emx2-loxp/neo/loxp) were inserted into the NlaIII site at the initial codon of the Otx2 gene. The in-frame ligation was confirmed by nucleotide sequencing. Lengths of the homologous regions were 6.9 kb and 4.1 kb at the 5′ and 3′ sides of the insert (Emx2-loxp/neo/loxp), respectively, in the targeting vector. The diphtheria toxin-A fragment (DT-A) gene, driven by the MC1 promoter, was used for negative selection of homologous recombinants as described (Yagi et al., 1993b). (The details of vector construction can be provided upon request.)

Generation of mutant mice

TT2 ES cells (Yagi et al., 1993a) were cultured, electroporated with SalI linearized targeting vector and selected against G418 as described (Nada et al., 1993; Yagi et al., 1993b). Homologous recombinants were assessed by long-PCR with a sense primer, p1 (5′-ATCGCCTTCTTGACGAGTTCTTCTG), in the neor gene and an antisense primer, p2 (5′-CTTATAATCCAAGCAATCAGTGGTTGAG), in the Otx2 genome located downstream of the 3′ end of the homologous region in the targeting vector (see Fig. 6A). Homologous recombinants were obtained at a frequency of 115 of 120 G418 resistant clones. Recombinants were confirmed by Southern blot hybridization using probes in the neor gene and in the third exon of the Otx2 genome (SphI/HindIII fragment in Fig. 6A). Chimeric mice were obtained through injection of targeted TT2 cells into eight-cell stage embryos, as described (Yagi et al., 1993b). Chimeras were crossed with Cre females, which expressed Cre in their zygotes (Sakai and Miyazaki, 1997), in order to generate F1 heterozygotes. Otx2 and Emx2 heterozygotes were obtained as described (Matsuo et al., 1995; Suda et al., 1996; Yoshida et al., 1997). The genetic background of animals used in the present study is noted in Results. Mice were housed in environmentally controlled rooms of the Laboratory Animal Research Center of Kumamoto University under University guidelines for animal and recombinant DNA experiments.

Genotyping of mice

Genotypes of newborn mice and embryos were routinely determined by PCR analyses. Confirmation, when necessary, was by Southern blot analyses. Genomic DNAs used in the analyses were prepared from tails or yolk sacs. In PCR analysis, knock-in alleles, which retained or lost the neor gene, were detected as the 2.1 and 0.7 kb PCR products, respectively. A sense primer, p3 (5′-CCGAGAGTTTCCTTTTGCACAACGC), was placed in the Emx2 cDNA and an antisense primer, p4 (5′-TGTGGCACTCGGCAGTTTGGTAGC), was in the first exon of the Otx2 genome (see Fig. 6A). Genotypes of Otx2 and Emx2 knockout alleles were identified as described previously (Matsuo et al., 1995; Suda et al., 1996; Yoshida et al., 1997).

Histological analysis

Mouse embryos were fixed with Bouin’s fixative solution at room temperature for 18-24 hours. Specimens were subsequently dehydrated and embedded in paraplast. Serial sections (8 μm) were prepared and stained with Hematoxylin and Eosin or with 0.1% Cresyl Violet (Sigma).

RNA probes and in situ hybridization

Embryos were dissected in PBS and fixed overnight at 4°C in 4% paraformaldehyde (PFA) in PBS. Specimens were gradually dehydrated in methanol/PBT (PBS containing 0.1% Tween-20) up to 100% methanol and stored at −20°C. The protocol for in situ hybridization of embryos was as described previously(Wilkinson,1993). Single-stranded digoxigenin-UTP-labeled (Boehringer Mannheim) RNA probes were used. The probes were as described for Wnt1 and Wnt7b (McMahon and Bradley,1990), BF1 (Foxg1 – Mouse Genome Informatics) (Tao and Lai, 1992), En1 and En2 (Davis and Joyner,1988), Pax2 (Dressler et al., 1990), Pax5 (Asano and Gruss, 1992), Pax6 (Walther and Gruss, 1991), Dlx1 (Bulfone et al., 1993), Fgf8 (Crossley and Martin,1995), Otx1 and Otx2 (Matsuo et al., 1995), Gbx2 (Bulfone et al., 1993), Six3 (Oliver et al., 1995), Tcf4 (Korinek et al.,1998), ephrin-A2 (Efna1 – Mouse Genome Informatics) (Flenniken et al., 1996), Mek4 (Cheng and Flanagan, 1994), Lim1 (Lhx – Mouse Genome Informatics) (Fujii et al., 1994), Ebf1 and Ebf3 (Garel et al., 1997), and COUP-TFI (Nr2f1 – Mouse Genome Informatics) (Qiu et al., 1994). A 3′UTR probe was used as described (Yoshida et al., 1997) in order to detect endogenous Emx2 expression in knock-in mutants. A cDNA probe was used for the detection of both endogenous and ectopic Emx2 expression.

Immunohistochemistry

The peptide of 50 amino acid residues (EMX2-C50) from the C-terminal of mouse EMX2 protein was chemically synthesized. The rabbit anti-EMX2-C50 antiserum was obtained with the peptide as described (Tanaka et al., 1991). The antiserum was absorbed with mouse liver powder and used at the dilution of 1:2000 in PBS containing 1% goat serum. Paraffin sections of embryos were prepared as described (Gurdon et al., 1976; Mallamaci et al., 1996). Sections were deparafinized in xylene. Subsequently, specimens were rehydrated and incubated with 4% blocking serum for 1 hour, followed by incubation with antibodies. EMX2 expression was detected with rabbit anti-mouse EMX2-C50 antiserum and the secondary antibody (biotinylated goat anti-rabbit IgG) at 1:200 dilution. Posterior commissure neurons were detected with anti-mouse GAP43 monoclonal antibody (Sigma) at a dilution of 1:2000 and horseradish peroxidase-labeled secondary antibody (goat anti-mouse IgG, ZYMED, USA) at a dilution of 1:500. Chromogenic staining was effected according to the protocol supplied by the manufacturer (VECTASTAIN Elite ABC kit, Vector Laboratories).

RESULTS

Morphological features of double mutant brain

Genetic background influences the phenotype of Otx2 heterozygous mutants (Matsuo et al., 1995). Our ES cells, TT2, were derived from an F1 embryo between C57BL/6 and CBA mice. Otx2 is expressed in cephalic neural crest cells (Kimura et al., 1997), and F1Otx2 heterozygotes obtained from crosses of chimeras with C57BL/6 females exhibit craniofacial defects, yielding few fertile heterozygotes. F1 heterozygotes that resulted from mating chimeras with CBA females did not possess the defects. Consequently, Otx2 heterozygotes have been maintained by crosses with CBA mice. Accordingly, Emx2 heterozygotes employed in this study were obtained via crosses of chimeras with CBA females and have been maintained by crosses with CBA mice (Yoshida et al., 1997; Miyamoto et al., 1997). The F5 and F3 generations of Otx2 and Emx2 heterozygotes, respectively, were the source for the present analysis.

Double heterozygotes (Emx2+/−Otx2+/−) were obtained at Mendelian ratio by crosses between Emx2+/− and Otx2+/− single heterozygotes. These double heterozygotes were subsequently mated with Emx2+/− single heterozygotes so as to generate Emx2−/− homozygous and Otx2+/− heterozygous (Emx2−/−Otx2+/−) double mutants (hereafter referred to as double mutants). The double mutants were obtained at Mendelian ratio at 15.5 dpc; however, none survived beyond 16.5 dpc. Morphologically, defects were not apparent in Otx2+/− mutants of this pedigree (Matsuo et al., 1995; Fig. 1). Emx2−/− mutants exhibited defects solely in the medial pallium, as previously reported (Yoshida et al., 1997; Fig. 1). Emx2+/−Otx2+/− double heterozygotes displayed defects in forebrain structures similar to, but significantly milder than, Emx2−/−Otx2+/− double mutants.

The 15.5 dpc Emx2−/−Otx2+/− double mutant brain demonstrated severe defects in the dorsal forebrain (Fig. 1A-H). In the telencephalon, cerebral hemispheres were present, but greatly diminished. The medial region of the cerebral cortex, which juxtaposes to the choroidal roof, is normally the territory of archicortex structures, i.e. the hippocampus (the dentate gyrus and CA fields) and para-hippocampal subicular cortex. The choroidal roof was expanded in the double mutants. Furthermore, no histological signs denoting archipallium structures could be identified; the defects were substantially more severe than those in Emx2−/− single mutants (Yoshida et al., 1997; Fig. 1). The choroid plexus did not develop in the third ventricle. In the cerebral cortex that was present laterally, no axonal fasciculation of the corpus callosum, anterior commissure, fornix or fimbria was in evidence. Cortical layers were poorly differentiated and laminar organization was not apparent; that is, the cortical plate was thin and sparse, and boundaries between the cortical plate and other layers could not be distinguished (data not shown). Defects were not observed in anterior structures, including the septum, but lateral and medial ganglionic eminences were hyperplastic.

The dorsal and ventral thalamus and pretectum were not apparent in Emx2−/−Otx2+/− double mutants. The hypothalamus, mammillary region and hypophysis were relatively normal. The neurohypophysis was normal, but the adenohypophysis was irregularly shaped (data not shown). Zlth, which divides the ventral and dorsal thalamus, or the habenulopeduncular tract separating the pretectum and dorsal thalamus, were not observed in the double mutants. The posterior commissure typically occurs between the pretectum and tectum. The commissure was located proximal to the sulcus telodiencephalicus in the double mutants (Fig. 1D); moreover, only traces of structures that may have corresponded to the posterior pretectum were evident. The posterior commissure was reduced in size and poorly fasciculated. Additionally, the anterior boundary of the mesencephalon was poorly differentiated. The tectum was greatly enlarged occupying the original epithalamus and pretectum regions; in this midbrain, the darkly stained ventricular proliferating field was expanded, whereas the differentiating field was narrowed. The tegmentum developed normally.

Histological analysis was subsequently conducted at 12.5 dpc upon near completion of rapid cell proliferation in the telencephalon. In Emx2−/−Otx2+/− double mutants, the telencephalon was small and diminished, particularly in the dorsomedial aspect (Fig. 1I-L). Consequently, the telencephalic roof was enlarged and exposed. Evagination of the medial telencephalic pallium beyond the sulcus telodiencephalicus was poor and the hippocampal region did not develop in the double mutants (Fig. 1M-P). Ganglionic eminence and the mesencephalon were not hyperplastic at this stage. Neither the ventral nor dorsal thalamus was apparent; however, a commissural structure was present that may have corresponded to the posterior pretectum. Ventral structures, possibly corresponding to the hypothalamus, mammillary region and tegmentum, were evident. Isthmic constriction was observed, however, it was shifted rostrally (Fig. 1P).

Wild-type olfactory bulb primordium exhibits mitral and tufted cell layers at 12.5 dpc. The double mutants displayed a very small olfactory bulb that lacked the layered structure. Histologically tufted cells were present, but mitral cell layers were not (data not shown). Olfactory neurons did not project to the olfactory bulb; rather, the nerve fivers were tangled outside the bulb. Several defects also occurred in the eyes. Lenses were irregularly shaped and the outer/inner layers of the retina were hyperplastic (data not shown). Consequently, Otx2 and Emx2 appear to cooperate in several steps of forebrain development. These genes interact in corticogenesis, archipallium/roof development and the formation of sensory organs, the details of which will be reported elsewhere. This study focuses on the roles of Emx2 and Otx2 in diencephalon development.

Molecular characterization of diencephalic defects

At 11.5 dpc, several genes are expressed region specifically in the diencephalon. Affected structures were confirmed with these molecular markers; expression of each marker used is schematically summarized in Fig. 9B. Tcf4 is strongly expressed in the dorsal thalamus and pretectum, whereas expression in the ventral thalamus is weak (Fig. 2A; Cho and Dressler, 1998; Korinek et al., 1998). The double mutants lost the Tcf4-weak ventral thalamus and the majority of Tcf4-intense structures (Fig. 2B); however, traces of structures displaying intense Tcf4 expression were in evidence. The posterior commissure, which originates from the posterior pretectum (Mastick et al., 1997), developed in the double mutants. Consequently, this Tcf4-positive structure in the double mutants most probably corresponds to the posterior pretectum. The posterior pretectum normally expresses Lim1 (Barnes et al., 1994; Fujii et al., 1994; Mastick et al., 1997). Indeed, Lim1 expression was typically present in the double mutants (Fig. 2C,D). Wild-type embryos display Dlx1 expression in the ventral thalamus, entopedunucular area, hypothalamic cell cord and ganglionic eminence (Fig. 2E; Bulfone et al., 1993; Eisenstat et al., 1999). Dlx1-positive ganglionic eminence, hypothalamic cell cord and entopedunucular area were present in the double mutants; however, the ventral thalamus was absent (Fig. 2F), as indicated by Tcf4 expression. Gbx2 is expressed in the dorsal thalamus and ganglionic eminence (Fig. 2G; Bulfone et al., 1993). Expression was present in the ganglionic eminence; however, Gbx2-positive dorsal thalamus was absent in the double mutants (Fig. 2H). Ebf1 is expressed in the anterior pretectum, anterior tectum and tegmentum (Fig. 2I; Garel et al., 1997). Double mutants exhibited the expression in the anterior tectum and tegmentum, but these mutants lacked Ebf1-positive anterior pretectum (Fig. 2J).

Pax6 is expressed in the telencephalon and diencephalon. At 11.5 dpc, it is strong in the ventral thalamus, posterior pretectum and tegmentum (Fig. 2K; Walther and Gruss, 1991). The strong expression in the basal posterior pretectum and midbrain tegmentum was retained, but all other Pax6 expression was lost in the double mutants (Fig. 2L). Otx1 is expressed spanning the telencephalon, diencephalon and mesencephalon. The most intense Otx1 expression is observed in the anterior pretectum, tegmentum and a boundary region along zlth (Fig. 2M; Simeone et al., 1993; Boncinelli et al., 1993). Double mutants lacked the Otx1-rich anterior pretectum and zlth region (Fig. 2N).

Morphologically, the mesencephalon was normal in the 12.5 dpc double mutants. It is possible, however, that the diencephalic defects affected rostrocaudal regionalization in the mesencephalon. Mek4 is expressed in a gradient in wild-type embryos. High levels of Mek4 occur in the anterior tectum, whereas low levels occur caudally (Fig. 2O; Cheng and Flanagan, 1994). In contrast, ephrin-A2 expression is high in caudal mesencephalon and low in anterior mesencephalon (Fig. 2Q; Flenniken et al., 1996; Feldheim et al., 1998; Feldheim et al., 2000). The double mutants exhibited normal patterns for Mek4 and ephrin-A2 expression (Fig. 2P,R).

Marker analyses, in concert with morphological features, indicate that the precommissural region of the pretectum and dorsal and ventral thalamus do not develop in double mutants. The commissural region of the pretectum and the ventral structures were present, including the hypothalamus, mammillary region and tegmentum.

Molecular characterization of defects at earlier stages

No diencephalic structures are subdivided morphologically at 9.5 dpc; nor are any region-specific molecular markers expressed in the diencephalon. At this stage, telencephalic vesicle formation begins and BF1 is expressed throughout the entire telencephalic neuroepithelium with the exceptions of the most medial and caudal regions adjacent to the choroidal roof and diencephalon (Tao and Lai, 1992). In the 9.5 dpc double mutants, the BF1-positive region was largely normal (Fig. 3A,B). Normally Otx2 is expressed in the forebrain and midbrain with the caudal limit at the mid/hindbrain junction (Simeone et al., 1993; Matsuo et al., 1995). In comparison with Otx2+/− single heterozygotes, Otx2 expression from the remaining allele was greatly reduced in Emx2−/−Otx2+/− double mutants, particularly in the region caudal to the sulcus telodiencephalicus (Fig. 3C,D). The caudal limit shifted rostrally and expression was restricted to the more dorsal region.

Pax6 expression is found over the telencephalon and diencephalon at 9.5 dpc in wild-type embryos (Fig. 3E; Walther and Gruss, 1991; Stoykova et al., 1996). Pax6 expression was scarcely observed in double mutants, however, its expression in the eyes was retained (Fig. 3F). Wnt7b expression was present in the double mutant telencephalon, but the mutants lost a Wnt7b-positive diencephalon (data not shown, compare Fig. 3F with Fig. 7G). Fgf8 expression in the anterior neural ridge was normally present, but the roof at the position of the telodiencephalicus or the roof of ventral thalamus was absent (Fig. 3M,N).

Wnt1 is expressed in the dorsal midline over the caudal diencephalon and mesencephalon, and in a characteristic lateral stripe at the isthmus in 9.5 dpc wild-type embryos (Fig. 3G; Parr et al., 1993). Double mutants demonstrated a reduced Wnt1-positive region in the roof as well as a decrease in the level of Wnt1 expression laterally. The isthmic stripe was formed, but it was shifted rostrally. In wild-type embryos, caudally to the isthmus, the anterior metencephalon, r1 and r2, does not express Wnt1 (Fig. 3G; McMahon et al., 1992; Echelard et al., 1994). This Wnt1-negative region was expanded in the double mutants (Fig. 3H).

En2 is expressed in a gradient over the caudal mesencephalon and r1 at 9.5 dpc in wild-type embryos (Fig. 3I; Davis and Joyner, 1988; Davis et al., 1988). The double mutants exhibited expanded En2 expression rostral to the isthmic constriction, and the gradient is less distinct (Fig. 3J). Concomitantly, the region between the anterior terminus of En2 expression and the sulcus telodiencephalicus was greatly diminished. Moreover, the En2-positive region caudal to the isthmic constriction and the region between the posterior terminus of En2 expression and the otic vesicle appeared enlarged. Normally Pax5 is also expressed in the mesencephalon and r1 (Fig. 3K; Asano and Gruss, 1992; Urbanek et al., 1994). The Pax5-positive region both rostral and caudal to the isthmic constriction was enlarged, while the negative region extending up to the sulcus telodiencephalicus was greatly reduced in double mutants (Fig. 3L). The region between the caudal end of Pax5 expression and the otic vesicle appeared enlarged. Isthmus also expresses Fgf8 and Gbx2 (Fig. 3M,O; Bulfone et al., 1993; Crossley and Martin, 1995; Mahmood et al., 1995). These expressions were shifted anteriorly in the double mutants (Fig. 3N,P). In addition, the distance between the Fgf8- and Gbx2-positive isthmic stripe and otic vesicles appeared increased by the double mutation.

Marker analyses at 9.5 dpc are consistent with the defects at 11.5 dpc. Telencephalon and mesencephalon formation is fairly normal; however, the major region of the diencephalon does not develop and Pax6 expression in the forebrain is lost in Emx2−/−Otx2+/− mutants. Thus, the onset of the defects precedes this stage. The expansion of the anterior hindbrain was notable.

Onset of double mutant defects

Marker analyses were next performed at the six-somite stage, the point at which the initial brain regionalization is completed. The caudal limit of Otx2 expression becomes distinct at the isthmus and the territory of the future forebrain and midbrain is established by this stage (Fig. 4A; Acampora et al., 1995; Rubenstein and Shimamura, 1998). Double mutants displayed a greatly reduced Otx2-positive region (Fig. 4B). Pax6 expression normally occurs in the forebrain, as well as in the laterocaudal component of the incipient optic cup, and the hindbrain and spinal cord (Fig. 4C; Walther and Gruss, 1991). Pax6 expression was unchanged in the hindbrain and spinal cord, and remained evident in the optic cup in double mutants (Fig. 4D); however, it was scarcely observed in the forebrain.

The Wnt1 expression was already reduced and shifted anteriorly in the double mutants at the six-somite stage (Fig. 4E,F; McMahon et al., 1992; Bally-Cuif et al., 1995). In addition, the Wnt1-negative anterior hindbrain, corresponding to future r1 and r2, exhibited expansion. In wild-type brain, the En1- and Pax5-positive regions, which initially cover the entire prospective mesencephalon (compare with Fig. 8G,I), have completed regression to the caudal portion by this stage (Fig. 4G,I; Joyner, 1996). Double mutants demonstrated somewhat expansion of the positive domains accompanied by an anterior shift (Fig. 4H,J). Fgf8 expression in the midbrain-hindbrain boundary in wild-type embryos begins to be confined to the future isthmus at the six-somite stage (Fig. 4K; Suda et al., 1997). However, this event was less distinct in the double mutants (Fig. 4L). Six3 expression is observed in the most rostral neuroectoderm at the six-somite stage in wild-type embryos (Fig. 4M; Oliver et al., 1995). Double mutants exhibited normal Six3 expression (Fig. 4M,N). Consequently, the diencephalon corresponding to the future ventral thalamus, dorsal thalamus and anterior pretectum is probably already lost at this point. Additionally, anterior hindbrain expansion, the loss of Pax6 expression, reductions in Otx2 and Wnt1 expression, and the elevation of En1 and Pax5 expression were also evident at the six-somite stage in Emx2−/−Otx2+/− mutants.

Otx2 is expressed in the anterior neuroectoderm from the earliest stage of its induction (Fig. 5A-D; Acampora et al., 1995). As a result, the onset of the double mutant defects should closely correlate with the onset of Emx2 expression. Emx2 was not detectable at the two-somite stage, but at the three-somite stage, it occurred in the laterocaudal forebrain primordia (Fig. 5E,F). This Emx2 expression increases and expands rostrally to the future dorsal aspect by the five-somite stage (Fig. 5G,H; Shimamura et al., 1997). Pax2 expression, which corresponds to the future midbrain, also occurs around the three-somite stage in the caudal portion of the Otx2-positive region (Fig. 5I,J; Rowitch and McMahon, 1995). At this stage, the anterior limit of Pax2 expression appears to overlap with the posterior limit of Emx2 expression; moreover, its caudal limit extends beyond the caudal limit of Otx2 expression. At the four-somite stage, Pax2 expression extends caudally into the preotic sulcus at the most lateral (future dorsal) edge; however, in the medial (future lateral and ventral) region, expression terminates more rostrally (Fig. 5K,L). This laterally Pax2-positive and medially Pax2-negative region corresponds to future r1 and/or r2. Gbx2 is expressed in this preotic as well as the otic region at the 3-4 somite stage (Fig. 5W). Pax6 expression occurs at the four-somite stage, and its caudal limit nearly coincides with the caudal limit of Emx2 expression (Shimamura et al., 1997; Inoue et al., 2000).

The defect was evident at the three-somite stage upon evaluation of the double mutant phenotype via Otx2 expression (Fig. 5M,N). The Otx2-positive domain was reduced both rostrocaudally and lateromedially (future dorsoventrally). The rostrocaudal reduction of the Otx2-positive domain was more apparent at the four-somite stage (Fig. 5O,P). Pax2 expression was more diffuse and expanded rostrally in double mutants at the three-somite stage (Fig. 5Q,R). At the four-somite stage, the caudal limit of Pax2 expression in the medial region had shifted rostrally. Furthermore, the laterally Pax2-positive and medially Pax2-negative region had expanded (Fig. 5S,T), and the anterior Pax2-negative domain was greatly reduced (Fig. 5U,V). Concomitantly, the anterior limit of the Gbx2 expression in the preotic region shifted rostrally (Fig. 5X). Thus, it appears most likely that the Emx2-positive domain failed to develop, the Pax2-positive domain underwent a rostral shift and the Gbx2-positive region anterior to preotic sulcus expanded by the four-somite stage in the Emx2−/−Otx2+/− double mutants (see Fig. 9A). Wnt1 expression was faint, whereas Pax6 expression was not detected in double mutants at the four-somite stage as in later stages (data not shown).

Ectopic Emx2 expression in Emx2-negative diencephalon and mesencephalon

At 10.5 dpc, Emx2 is typically expressed rostrally to zlth. Emx2 expression is not observed in the dorsal thalamus, pretectum or tectum (Simeone et al., 1992a; Shimamura, et al., 1997; Yoshida et al., 1997). Emx2 functions in the development of diencephalic structures were also examined by its ectopic expression in these endogenously Emx2-negative regions under the Otx2 heterozygous state. For this purpose the Otx2 gene was replaced with Emx2 cDNA by homologous recombination in TT2 ES cells, as described in Fig. 6A. The neo-resistant gene directed by the phosphoglycerate kinase 1 (Pgk1) promoter for the selection of the recombinants was flanked with loxP sequences. To exclude possible effects of the Pgk1 promoter on the Otx2 transcriptional machinery, the gene was deleted by crossing chimeras derived from the homologous recombinants with transgenic females expressing Cre in their zygotes (Fig. 6B; Sakai and Miyazaki, 1997). F2 embryos derived from these F1 heterozygotes (Otx2+/Emx2) expressed Emx2 in the dorsal thalamus, pretectum and tectum, as expected (Fig. 6C-H).

The ventral and dorsal thalamus, tectum, hypothalamus, mammillary region and tegmentum were morphologically normal in these 12.5 dpc Otx2+/Emx2 mutants (Fig. 7A,B). A structure similar to the precommissural region of the pretectum was also evident, but the commissural region of the pretectum was not apparent.

Endogenous Emx2 and Emx1 were expressed typically in the 10.5 dpc telencephalon of knock-in mutants (Fig. 7C-F; Simeone et al., 1992a; Yoshida et al., 1997). Wnt7b is normally expressed in the 9.5 dpc forebrain, however, expression does not occur in its most posterior aspect (Fig. 7G; Parr et al., 1993). This Wnt7b-negative caudal diencephalon was nearly lost in knock-in mutants (Fig. 7H). Tcf4-intense diencephalon existed in the 10.5 dpc mutants; however, the domain was narrowed and its posterior aspect was not distinctly delineated, as in wild-type embryos (Fig. 7I,J). Gbx2-positive dorsal thalamus and Ebf1-positive anterior pretectum and anterior tectum were present despite the lack of a Ebf1-negative posterior pretectum in the 12.5 dpc knock-in mutants (Fig. 7K,L; data not shown; compare with Fig. 2I). Pax6 expression in the 10.5 dpc forebrain was largely normal, although its caudal-most expression, typically corresponding to the posterior pretectum, was not sharply delineated (Fig. 7M,N). At 12.5 dpc, a Pax6-intense ventral thalamus was present, but the posterior pretectum was not observed (Fig. 7O,P). Ventral thalamus development was also confirmed with Dlx1 (data not shown, compare with Fig. 2E). The Lim1-positive tegmentum was present, whereas the Lim1-positive posterior pretectum was not found in the 10.5 dpc knock-in mutants (Fig. 7Q,R). Anti-GAP43 antibody specifically detects the posterior commissure (Fig. 7S; Matsunaga et al., 2000). A GAP43-positive structure was not identified in 12.5 dpc Otx2+/Emx2 mutants (Fig. 7T).

Among gene activity dorsally spanning the caudal diencephalon and mesencepalon,Wnt1 and Ebf3 (Garel et al., 1997) expression were decreased at 10.5 dpc (Fig. 8A-D). COUP-TFI expression (Qiu et al., 1994) was unchanged (Fig. 8E-F), whereas En1 and Pax5 expression was strongly enhanced (Fig. 8G-J). Knock-in mutants, however, displayed normal patterns of Mek4 and ephrin-A2 expression (data not shown; compare with Fig. 2O,Q). Isthmus formation typically occurs with normal Fgf8 expression. Fgf8 expression in the anterior neural ridge and the roof at the position of the telodiencephalicus was also normally present (Fig. 8K,L).

Marker analyses, in conjunction with morphological observations, indicate that the telencephalon, ventral and dorsal thalamus, anterior pretectum and mesencephalon developed in the knock-in mutants. However, the commissural region of the pretectum specifically failed to develop as a consequence of ectopic Emx2 expression.

DISCUSSION

Emx2 cooperates with Otx2 at the onset of its expression to generate the territory of the future diencephalon

In contrast to the hindbrain, the forebrain has long been thought not to possess neuromeric organization. However, a prosomeric model that postulates forebrain neuromeric organization has been presented on the basis of region-specific gene expression and other evidence. It is now generally believed to divide the caudal forebrain along the rostrocaudal axis into p1 pretectum, p2 dorsal thalamus/epithalamus and p3 ventral thalamus (Bulfone et al., 1993; Figdor and Stern, 1993; Puelles and Rubenstein, 1993; Rubenstein et al., 1994; Shimamura et al., 1995). Adjacent to the boundary between the tectum and p1 pretectum lies the posterior commissure; between p1 and p2 dorsal thalamus, the habenulopeduncular tract is found; between p2 and p3 ventral thalamus, the zona limitans interthalamica develops, respectively. The ventral and dorsal thalamus, epithalamus and anterior pretectum were lost in Emx2−/−Otx2+/− mutants (Fig. 9B). Consequently, Emx2 and Otx2 play crucial roles in diencephalon development. No other such genes are known.

Otx2 and Emx2 are co-expressed in a variety of stages and sites during brain development. The double mutant defects at 15.5 dpc may reflect each of these Otx2 and Emx2 functions. Defects in the formation of the diencephalon, however, appear to reside within their functions at the three-somite stage when Emx2 expression first occurs (Fig. 9). Otx2 and Gbx2 are among the genes known to be first expressed in the neuroectoderm. Otx2 expression hallmarks the anterior neuroectoderm which corresponds to the future forebrain and midbrain. In contrast, Gbx2 marks the posterior neuroectoderm (Wassarman et al., 1997); at the three- to four-somite stage its expression is found in preotic and otic regions. Pax2 and En1 expression occurs nearly simultaneously at the three-somite stage in the entire prospective midbrain (Rowitch and McMahon, 1995), probably as a result of signals such as Fgf4 from the anterior notochord (Shamim et al., 1999). Their rostral limits likely overlap with Emx2 expression. Wnt1 expression begins at this stage in a similar manner with respect to En1 (Bally-Cuif et al., 1995). Onset of Pax6 expression is somewhat late around the four-somite stage. Moreover, the caudal limits of Pax6 and Emx2 expression appear to coincide with one another at this stage (Shimamura et al., 1997; Inoue et al., 2000).

At the three-somite stage, double mutants displayed an Otx2-positive region which was reduced rostrocaudally. Additionally, the Pax2-positive domain was diffuse and extended in a rostral manner. By the four-somite stage, the anterior Pax2-negative region was reduced, whereas the caudal limit of Pax2 and the anterior limit of the Gbx2 expression shifted rostrally; the Gbx2-positive region rostral to preotic sulcus expanded (Fig. 9A). It is quite simple to interpret the phenotype as a consequence of the loss of the Emx2-positive domain in double mutants and the shift of the Pax2-positive domain to the anterior Otx2-positive/Emx2-negative domain.

Pax6 expression was not induced in double mutants. Apparently, Emx2 and Otx2 are located upstream of Pax6 in development of the diencephalon. Sey mutants, which possess a null mutation in the Pax6 gene, suggested that Pax6 is important for correct dorsoventral and anteroposterior patterning in the diencephalon (Grindley et al., 1997; Stoykova et al., 1997; Warren and Price, 1997). Each region of the diencephalon, ventral thalamus, dorsal thalamus and pretectum, however, emerges in sey/sey mutants. Consequently, Emx2 and Otx2 regulate other genes with respect to diencephalon development. Wnt1 expression also decreased at the initial stage in double mutants. Emx-binding sites exist in the cis regulatory region of the Wnt1 gene (Iler et al., 1995). Wingless and engrailed are hypothesized to be targets of the head gap genes otd and ems in Drosophila (Royet and Finkelstein, 1995). However, Wnt1 mutants as well as En1 and En2 mutants develop diencephalic structures (Joyner et al., 1991; McMahon et al., 1992; Millen et al., 1994; Wurst et al., 1994).

The 15.5 dpc defects in the neocortex are apparently due to later Emx2 and Otx2 functions in corticogenesis, as suggested by BF1 expression at 9.5 dpc; however failure of archipallium formation in the double mutants may also reside within the Emx2 functions at the three-somite stage (Fig. 9). In the prosomeric model, the archipallium belongs to the p4 structures anterior to p3 ventral thalamus (Rubenstein et al., 1998). Wild-type embryos demonstrate a rostral extension of the lateral (future dorsal) portion of the anterior-most Emx2-positive region (Fig. 5C,D; Fig. 9A). This extension may include the presumptive dorsomedial region of the forebrain or archipallium (Rubenstein and Shimamura, 1998). The Otx2-positive region was reduced not only rostrocaudally but also lateromedially (future dorsoventrally) in double mutants at the three-somite stage (Fig. 5G,H; Fig. 9B). The detailed analysis of archipallium defects in double mutants will be reported elsewhere.

Does the neuroectoderm anterior to the preotic sulcus constitute a unit?

The expansion of the anterior hindbrain in 9.5 dpc Emx2−/−Otx2+/− double mutants is puzzling. The region between the otic vesicle and isthmus, indicated by Fgf8 and Gbx2 expression, appeared expanded. The region between the otic vesicle and the caudal terminus of En2 and Pax5 expression was enlarged; their expression in r1 was also expanded. The Wnt1-negative region in the anterior hindbrain increased at 9.5 dpc and the six-somite stage. Consequently the expanded region may correspond to r1 and r2. The r1/2 also enlarges in Otx1+/−Otx2+/− double heterozygotes that fail to develop a midbrain (Suda et al., 1997). We speculated that suppressive signals originating within the midbrain might be necessary for correct r1/2 development; however, such signals from the diencephalon are very unlikely.

Of note is the expansion of the laterally Pax2-positive (but medially Pax2-negative region) or the Gbx2-positive region anterior to the preotic sulcus in double mutants at the four-somite stage (Fig. 5S,T,X; Fig. 9). This event is most probably brought about by the loss of the Emx2-positive region and anterior shift of Pax2-positive region. Morphologically, the anterior neuroectoderm is initially demarcated by the preotic sulcus, which is apparent around the three-somite stage and includes future r1 and r2. Unfortunately, no molecular markers are available specific to the prospective r1/2 territory at early stages. As a result, more detailed analyses are necessary in future studies in order to determine the direct correlation of the defect at the four-somite stage with r1/2 expansion at 9.5 dpc.

The loss of anterior hindbrain in Gbx2-null mutants accompanies a posterior expansion of the Otx2-positive midbrain (Wassarman et al., 1997; Miller et al., 1999). En1, En2, Pax2, Pax5 or Wnt1 mutants fail to develop midbrain without expansion of r1/2. These midbrain genes, however, may function in midbrain development after the establishment of its territory (McMahon et al., 1992; Schwarz et al., 1999; Liu and Joyner, 2001). It is tempting to speculate that the region anterior to the preotic sulcus initially develops as a unit and that the anterior hindbrain expands to compensate when more anterior structures are lost before their subdivision into forebrain, midbrain and the anterior hindbrain. Clarification of the development of the preotic sulcus and r1/r2 might be important regarding investigations of rostral brain regionalization.

Emx2 and Otx2 direct the development of the dorsal thalamus and anterior pretectum

The posterior limit of Pax6 expression and the anterior limit of En1 and Pax2 expression are known to overlap at the six- to eight-somite stages in chicken. However, the expression of these genes segregates by the 10 somite stage (Matsunaga et al., 2000). The interaction between Pax6, En1 and Pax2 at this stage is believed to establish the boundary between the diencephalon and mesencephalon (Araki and Nakamura, 1999; Schwarz et al., 1999; Matsunaga et al., 2000). In the absence of Pax6 expression, however, the posterior pretectum and tectum were regionalized in Emx2−/−Otx2+/− double mutants; the present study does not exclude the role of Pax6 in the fine tuning of the boundary. After segregation from the Pax6-positive region, En1, En2 and Pax2 expression regress caudally (Dressler et al., 1990). At 10.5 dpc, the caudal limit of Emx2 expression is in zlth (Simeone et al., 1992a; Shimamura et al., 1997), whereas that of Pax6 occurs in the boundary between the diencephalon and mesencephalon (Walther and Gruss, 1995). En1 and En2, and Pax2 and Pax5 are expressed in the caudal mesencephalon (Davis and Joyner, 1988; Dressler et al., 1990; Asano and Gruss, 1992; Millet et al., 1999). These observations raise the question of why the Pax6-positive, Emx2-negative anterior pretectum and dorsal thalamus are lost in Emx2−/−Otx2+/− double mutants.

A cell lineage analysis has revealed that restriction in cell movement between regions anterior and posterior to the presumptive zlth occurs immediately after the onset of Pax6 expression at the four-somite stage (Inoue et al., 2000). At this juncture, the prospective pretectum and dorsal thalamus regions are diminutive. Consequently, two refined analyses are necessary to assess the origin of dorsal thalamus and pretectum cells. The caudal limit of Emx2 expression must first be determined by expression analysis at the single cell level. This process will ascertain the distinction between its coincidence with the caudal limit of Pax6 expression or the presence of Emx2-negative and Pax6-positive cells in this region already at the four-somite stage. The analysis should demonstrate the sequence of growth of the Emx2-negative, Pax6-positive diencephalon. Secondly, cell lineage analysis by genetic approach is necessary in order to determine the fate of Emx2-positive cells at the three- to four-somite stage. These studies are in progress; however, we speculate that the Emx2-positive domain at the three-somite stage includes cells of the future dorsal thalamus and anterior pretectum, where Emx2 expression is subsequently lost. Their development is compatible with Emx2 expression as demonstrated by the Otx2+/Emx2 knock-in mutation. Of course, it can not be excluded that a population of Emx2-negative and Pax6-positive cells at the four-somite stage generates the dorsal thalamus and anterior pretectum, depending on signals from Emx2-positive ventral thalamus.

Emx2 expression is not compatible with development of the commissural region of the pretectum

This study distinguished the developmental nature of the anterior and posterior pretectum. The anterior pretectum was lost in the double mutants, while the Lim1-positive commissural region of the pretectum developed (Fig. 9C). In contrast, formation of not only the entire endogenously Emx2-positive region, but also of the Emx2-negative dorsal thalamus, anterior pretectum and tectum, was compatible with ectopic Emx2 expression by the Otx2+/Emx2 knock-in mutation. Specifically, the development of the commissural region of the pretectum was not allowed by ectopic Emx2 expression. Unfortunately, molecular markers for posterior pretectum were unavailable at earlier stages. As a result, analyses of defect onset is left to future investigations. The cellular origin of the posterior pretectum is the most significant aspect to be determined.

The tract of the posterior commissure forms just rostral to the caudal border of the Pax6-positive domain. Although few posterior commissure neurons express Pax6 at 10.5 dpc, the point at which the posterior commissure can be identified, the neurons are thought to originate from Pax6-positive pretectum cells (Mastick et al., 1997). Schwartz et al. argue that Pax6 is sufficient and necessary for the development of the commissure on the basis of its loss in their sey/sey mutants and its subsequent recovery by Pax6-transgenesis under a Pax2 enhancer (Schwartz et al., 1999). Pax6 expression was lost in Emx2−/−Otx2+/− double mutants, however, the posterior commissure was present despite blurring. In the sey/sey mutants reported, the commissure was either completely absent (Stoykova et al., 1996; Warren and Price, 1997), lacking in dorsal axons (Mastick et al., 1997) or present but smaller than normal (Grindley et al., 1997).

Curiously, Wnt1 expression decreased and En1, En2, Pax2 and Pax5 expression were enhanced by both double and knock-in mutations. However, the midbrain was normal in size at 12.5 dpc and displayed the normal rostrocaudal pattern of Mek4 and ephrin-A2 expression in both mutants. The anterior tectum developed as indicated by Ebf1, as well as Mek4 expression. The causes and significance of changes in the expression of several midbrain genes, in conjunction with possible roles of the commissural region of the pretectum or diencephalon with respect to later midbrain development, including its hyperplasia at 15.5 dpc (Fig. 1), require further study.

Interaction between Emx2 and Otx2

This study examined genetically the existence of interaction between Emx2 and Otx2 genes. It indicated that these two genes indeed interact in diencephalon development. Otx1 also participates in diencephalon formation, as demonstrated by Otx1/Otx2 double mutation (Acampora et al., 1997; Suda et al., 1997). Consequently Otx2 interacts with both Emx2 and Otx1 in the development of the diencephalon. In addition, both Emx2/Otx2 and Otx1/Otx2 double mutants indicated the gene dose-dependent nature of the interactions between these genes. Each head subdomain appears to require differing otd and ems levels in Drosophila (Royet and Finkelstein, 1995). However, the question regarding the specifics of how Emx2 interacts with Otx2 remains unanswered. Both Otx1 and Otx2 gene products (OTX2 and OTX1) exhibit paired-like homeodomains; interaction between OTX2 and OTX1 through these homeodomains is probable. Furthermore, the homeodomains of Emx1 and Emx2 gene products (EMX1 and EMX2) belong to HEX class. The HEX homeodomain is too distant to allow homeodomain-homeodomain interactions with the paired-like homeodomains, however, our preliminary studies have suggested a direct protein-protein interaction between OTX2 and EMX2 proteins, possibly through homeodomain and non-homeodomain regions (Nakano et al., 2000). Alternatively, these genes might bind to each recognition site. Synergistic binding would activate the expression of common target genes.

The role of EMX2 in diencephalon development indicated by Emx2−/−Otx2+/− double mutants, however, must reconcile with the fact that the diencephalon develops normally in Emx2−/− single mutants. Otx2 expression is not restricted to the diencephalon. Several explanations are possible; however, it is quite simple to hypothesize that there exists a gene that complements Emx2 and regulates diencephalon development through gene dose-dependent interactions with Emx2, Otx2 and Otx1. Emx1 would be a primary candidate for this unidentified gene. However, Emx1 becomes expressed later at 9.5 dpc and not in the early diencephalon. Two genes, Vax1 and Vax2, are also known that locate very closely to Emx2 and Emx1 genes in chromosomes 19 and 6, respectively. They possess similar homeodomains (Hallonet et al., 1998; Ohsaki et al., 1999), but their expression does not overlap with Emx2 expression in the forebrain. Neither the Emx1, Vax1 or Vax2 gene was ectopically induced in the diencephalon of Emx2 single mutants at the four-somite stage (data not shown). The Not class of genes also exhibits homeodomains similar to Emx2 (Stein and Kessel, 1995). Chicken Cnot1 is expressed in prospective forebrain at the early neurula stage and later in the diencephalon. A Not2 cognate, floating head, is essential for the development of the epiphysial region in zebrafish (Masai et al., 1997). However, no mouse Not homologues have been identified. The identification of a gene that complements Emx2 using Emx2 single mutants at the three- to four-somite stage is eagerly awaited, in addition to the examination of other possibilities.

Fig. 1.

Morphological features of Emx2−/−Otx2+/− double mutant brains. (A,E,I,M) Wild-type, (B,F,J,N) Otx2+/−, (C,G,K,O) Emx2−/− and (D,H,L,P) Emx2−/−Otx2+/− embryos. (A-D) Sagittal sections stained with Cresyl Violet, (E-H) frontal sections stained with Cresyl Violet and (M-P) sagittal sections stained with Hematoxylin and Eosin. (I-L) Dorsal views of embryos. (A-H) 15.5 dpc, (I-P) 12.5 dpc. Arrows in A-D indicate the position of posterior commissure; circles in I-L indicate cerebral hemispheres. cb, cerebellum; cp, choroid plexus; Di, diencephalon; dt, dorsal thalamus; ept, epithalamus; hip, hippocampus; hp, hypophysis; ht, hypothalamus; ic, inferior coliculus; lge, lateral ganglionic eminence; ma, mammillary region; Me, mesencephalon; met, metencephalon; mge, medial ganglionic eminence; ncx, neocortex; ob, olfactory bulb; opc, optic chiasma; pt, pretectum; s, septum; spc, superior coliculus; Te, telencephalon; tg, tegmentum; vt, ventral thalamus; 3v, the third ventricle. Scale bars: 500 μm.

Fig. 1.

Morphological features of Emx2−/−Otx2+/− double mutant brains. (A,E,I,M) Wild-type, (B,F,J,N) Otx2+/−, (C,G,K,O) Emx2−/− and (D,H,L,P) Emx2−/−Otx2+/− embryos. (A-D) Sagittal sections stained with Cresyl Violet, (E-H) frontal sections stained with Cresyl Violet and (M-P) sagittal sections stained with Hematoxylin and Eosin. (I-L) Dorsal views of embryos. (A-H) 15.5 dpc, (I-P) 12.5 dpc. Arrows in A-D indicate the position of posterior commissure; circles in I-L indicate cerebral hemispheres. cb, cerebellum; cp, choroid plexus; Di, diencephalon; dt, dorsal thalamus; ept, epithalamus; hip, hippocampus; hp, hypophysis; ht, hypothalamus; ic, inferior coliculus; lge, lateral ganglionic eminence; ma, mammillary region; Me, mesencephalon; met, metencephalon; mge, medial ganglionic eminence; ncx, neocortex; ob, olfactory bulb; opc, optic chiasma; pt, pretectum; s, septum; spc, superior coliculus; Te, telencephalon; tg, tegmentum; vt, ventral thalamus; 3v, the third ventricle. Scale bars: 500 μm.

Fig. 2.

Characterization of diencephalic defects with molecular markers. (A,C,E,G,I,K,M,O,Q) Wild-type and (B,D,F,H,J,L,N,P,R) Emx2−/−Otx2+/− embryos. (A,B) Tcf4, (C,D) Lim1, (E,F) Dlx1, (G,H) Gbx2, (I,J) Ebf1, (K,L) Pax6, (M,N) Otx1, (O,P) Mek4 and (Q,R) ephrin-A2 expressions. (C,D) Whole-mount hybridization of 10.5 dpc embryos; the rest are in situ hybridization of sagittal sections of 11.5 dpc embryos. apt, anterior pretectum; at, anterior tectum; dt, dorsal thalamus; ge, ganglionic eminence; ht, hypothalamus; ppt, posterior pretectum; pt, pretectum; tg, tegmentum; vt, ventral thalamus; zl, zona limitans. Scale bars: 125 μm.

Fig. 2.

Characterization of diencephalic defects with molecular markers. (A,C,E,G,I,K,M,O,Q) Wild-type and (B,D,F,H,J,L,N,P,R) Emx2−/−Otx2+/− embryos. (A,B) Tcf4, (C,D) Lim1, (E,F) Dlx1, (G,H) Gbx2, (I,J) Ebf1, (K,L) Pax6, (M,N) Otx1, (O,P) Mek4 and (Q,R) ephrin-A2 expressions. (C,D) Whole-mount hybridization of 10.5 dpc embryos; the rest are in situ hybridization of sagittal sections of 11.5 dpc embryos. apt, anterior pretectum; at, anterior tectum; dt, dorsal thalamus; ge, ganglionic eminence; ht, hypothalamus; ppt, posterior pretectum; pt, pretectum; tg, tegmentum; vt, ventral thalamus; zl, zona limitans. Scale bars: 125 μm.

Fig. 3.

Characterization of double mutant defects with molecular markers at 9.5 dpc. (A,E,G,I,K,M,O) Wild-type, (C) Otx2+/− and (B,D,F,H,J,L,N,P) Emx2−/−Otx2+/− embryos. (A,B) BF1, (C,D) Otx2, (E,F) Pax6, (G,H) Wnt1, (I,J) En2, (K,L) Pax5, (M,N) Fgf8 and (O,P) Gbx2 expression. Arrows and arrowheads indicate the positions of the telodiencephalicus and isthmus, respectively. Double arrows in G,H indicate the anterior terminus (r3) of Wnt1 expression in the roof of spinal cord, and asterisks in E,F denote eyes. ov, otic vesicle. Scale bars: 250 μm.

Fig. 3.

Characterization of double mutant defects with molecular markers at 9.5 dpc. (A,E,G,I,K,M,O) Wild-type, (C) Otx2+/− and (B,D,F,H,J,L,N,P) Emx2−/−Otx2+/− embryos. (A,B) BF1, (C,D) Otx2, (E,F) Pax6, (G,H) Wnt1, (I,J) En2, (K,L) Pax5, (M,N) Fgf8 and (O,P) Gbx2 expression. Arrows and arrowheads indicate the positions of the telodiencephalicus and isthmus, respectively. Double arrows in G,H indicate the anterior terminus (r3) of Wnt1 expression in the roof of spinal cord, and asterisks in E,F denote eyes. ov, otic vesicle. Scale bars: 250 μm.

Fig. 4.

Characterization of double mutant defects with molecular markers at six-somite stage. (A) Otx2+/−, (C,E,G,I,K,M) wild-type and (B,D,F,H,J,L,N) Emx2−/−Otx2+/− embryos. (A,B) Otx2, (C,D) Pax6, (E,F) Wnt1, (G,H) En1, (I,J) Pax5, (K,L) Fgf8 and (M,N) Six3 expression. The Wnt1-negative r1/2 is indicated by arrowheads in E,F. Arrow in K indicates the concentration of the Fgf8 expression into future isthmus at this stage. Asterisks indicate the optic cup. Scale bars: 150 μm.

Fig. 4.

Characterization of double mutant defects with molecular markers at six-somite stage. (A) Otx2+/−, (C,E,G,I,K,M) wild-type and (B,D,F,H,J,L,N) Emx2−/−Otx2+/− embryos. (A,B) Otx2, (C,D) Pax6, (E,F) Wnt1, (G,H) En1, (I,J) Pax5, (K,L) Fgf8 and (M,N) Six3 expression. The Wnt1-negative r1/2 is indicated by arrowheads in E,F. Arrow in K indicates the concentration of the Fgf8 expression into future isthmus at this stage. Asterisks indicate the optic cup. Scale bars: 150 μm.

Fig. 5.

Onset of double mutant defects. (A-D,M-P) Otx2, (E-H) Emx2, (I-L,Q-V) Pax2 and Gbx2 (W,X) expression at the three- (A,B,E,F,I,J,M,N,Q,R) and four- (C,D,G,H,K,L,O,P,S-X) somite stages. (A-L,U,W) Wild-type embryos and (M-T,V,X) Emx2−/−Otx2+/− embryos. (A,C,E,G,I,K,M,O,Q,S,W,X) Lateral views; (B,D,F,H,J,L,N,P,R,T) dorsal views; and (U,V) frontal views. Arrows denote the posterior limit and arrowheads the anterior limit of expressions. In S,T, arrows and double arrows indicate the posterior limits of expression in the most medial (future ventral) and lateral (future dorsal) points, respectively; the double arrows coincide with preotic sulcus. Broken lines in U,V indicate the anterior end of the neural plate. In W,X, arrows indicate the anterior limit of the expression, single arrowheads preotic sulcus and double arrowheads otic sulcus. Scale bars: 150 μm.

Fig. 5.

Onset of double mutant defects. (A-D,M-P) Otx2, (E-H) Emx2, (I-L,Q-V) Pax2 and Gbx2 (W,X) expression at the three- (A,B,E,F,I,J,M,N,Q,R) and four- (C,D,G,H,K,L,O,P,S-X) somite stages. (A-L,U,W) Wild-type embryos and (M-T,V,X) Emx2−/−Otx2+/− embryos. (A,C,E,G,I,K,M,O,Q,S,W,X) Lateral views; (B,D,F,H,J,L,N,P,R,T) dorsal views; and (U,V) frontal views. Arrows denote the posterior limit and arrowheads the anterior limit of expressions. In S,T, arrows and double arrows indicate the posterior limits of expression in the most medial (future ventral) and lateral (future dorsal) points, respectively; the double arrows coincide with preotic sulcus. Broken lines in U,V indicate the anterior end of the neural plate. In W,X, arrows indicate the anterior limit of the expression, single arrowheads preotic sulcus and double arrowheads otic sulcus. Scale bars: 150 μm.

Fig. 6.

Targeted knock-in of Emx2 cDNA into the Otx2 locus. (A) Diagrammatic representation of knock-in strategy. Thick lines represent Otx2 genomic sequences; thin lines, pBluescript sequences; black boxes, Otx2 coding exons; black triangles, loxP sequences. White boxes denoted as Em, neor and DT-A indicate Emx2 cDNA lacking 3′UTR, neomycin-resistant gene lacking polyadenylation signal driven by Pgk1 promoter and diphtheria toxin-A fragment gene with MCI promoter, respectively. S is the probe for Southern blotting used to identify homologous recombinant ES cells shown in B; p1 and p2 are PCR primers used in the detection of the homologous recombinants in ES cells, and p3 and p4 are PCR primers used in the detection of the deletion of the neor cassette in F1 heterozygotes. H, HindIII; N, NotI; Nl, NlaIII; S, SalI; Sm, SmaI; Sp, SphI. (B) The top panel displays an example of Southern blotting used to identify homologous recombinants in ES cells; the bottom panel displays an example of the PCR genotyping for the deletion of neor cassette in F1 offspring obtained by mating between male chimera and Cre females. (C,D) RNA expressions from both endogenous and knocked-in Emx2 at 10.5 dpc in a wild-type embryo (C) and a knock-in mutant (D). Emx2 is expressed in the caudal diencephalon and mesencephalon where endogenous Emx2 is absent. (E,F) EMX2 protein expressions at 10.5 dpc in a wild-type (E) and a knock-in embryo (F). Consistent with the ectopic mRNA expression, EMX2 protein is detected in the caudal diencephalon and mesencephalon. The endogenous Otx2 expression and thus the knocked-in Emx2 expression is somewhat weaker in the midbrain than in the forebrain at this stage. (G,H) Enlarged views of E,F (respectively) at the telodiencephalicus level indicated by arrows. Arrowheads indicate the isthmus. Scale bars: 125 μm.

Fig. 6.

Targeted knock-in of Emx2 cDNA into the Otx2 locus. (A) Diagrammatic representation of knock-in strategy. Thick lines represent Otx2 genomic sequences; thin lines, pBluescript sequences; black boxes, Otx2 coding exons; black triangles, loxP sequences. White boxes denoted as Em, neor and DT-A indicate Emx2 cDNA lacking 3′UTR, neomycin-resistant gene lacking polyadenylation signal driven by Pgk1 promoter and diphtheria toxin-A fragment gene with MCI promoter, respectively. S is the probe for Southern blotting used to identify homologous recombinant ES cells shown in B; p1 and p2 are PCR primers used in the detection of the homologous recombinants in ES cells, and p3 and p4 are PCR primers used in the detection of the deletion of the neor cassette in F1 heterozygotes. H, HindIII; N, NotI; Nl, NlaIII; S, SalI; Sm, SmaI; Sp, SphI. (B) The top panel displays an example of Southern blotting used to identify homologous recombinants in ES cells; the bottom panel displays an example of the PCR genotyping for the deletion of neor cassette in F1 offspring obtained by mating between male chimera and Cre females. (C,D) RNA expressions from both endogenous and knocked-in Emx2 at 10.5 dpc in a wild-type embryo (C) and a knock-in mutant (D). Emx2 is expressed in the caudal diencephalon and mesencephalon where endogenous Emx2 is absent. (E,F) EMX2 protein expressions at 10.5 dpc in a wild-type (E) and a knock-in embryo (F). Consistent with the ectopic mRNA expression, EMX2 protein is detected in the caudal diencephalon and mesencephalon. The endogenous Otx2 expression and thus the knocked-in Emx2 expression is somewhat weaker in the midbrain than in the forebrain at this stage. (G,H) Enlarged views of E,F (respectively) at the telodiencephalicus level indicated by arrows. Arrowheads indicate the isthmus. Scale bars: 125 μm.

Fig. 7.

Forebrain phenotype in knock-in mutants; loss of commissural region of pretectum. (A,B) Parasagittal sections of 12.5 dpc wild-type (A) and knock-in embryos (B) stained with Hematoxylin and Eosin. 1, ventral thalamus; 2, dorsal thalamus; 3, anterior pretectum; and 4, posterior pretectum. The area of low cell density indicated as 4 in A is not apparent in the knock-in mutant (B). (C-R) Analyses with forebrain markers in wild type (C,E,G,I,K,M,O,Q) and in knock-in mutants (D,F,H,J,L,N,P,R). Endogenous Emx2 (C,D), Emx1 (E,F), Wnt7b (G, H), Tcf4 (I, J), Gbx2 (K,L), Pax6 (M-P), Lim1 (Q,R) expression in whole-mount (C-J,M,N,Q,R) and in midsagittal (K,L,O,P) views at 9.5 dpc (G,H), 10.5 dpc (C-F,I,J,M,N,Q,R) and 12.5 dpc (K,L,O,P). (S,T) Midsagittal views of anti-GAP43 staining of 12.5 dpc wild-type and knock-in embryos, respectively. Arrows in G-J,M,N indicate the caudal limits of the expressions, arrowheads in G,H indicate the anterior end of the midbrain, arrowheads in O,R indicate expression in the posterior pretectum and in the tegmentum, respectively. Asterisks in S indicate the anti-GAP43-positive posterior commissure. Scale bars: 250 μm.

Fig. 7.

Forebrain phenotype in knock-in mutants; loss of commissural region of pretectum. (A,B) Parasagittal sections of 12.5 dpc wild-type (A) and knock-in embryos (B) stained with Hematoxylin and Eosin. 1, ventral thalamus; 2, dorsal thalamus; 3, anterior pretectum; and 4, posterior pretectum. The area of low cell density indicated as 4 in A is not apparent in the knock-in mutant (B). (C-R) Analyses with forebrain markers in wild type (C,E,G,I,K,M,O,Q) and in knock-in mutants (D,F,H,J,L,N,P,R). Endogenous Emx2 (C,D), Emx1 (E,F), Wnt7b (G, H), Tcf4 (I, J), Gbx2 (K,L), Pax6 (M-P), Lim1 (Q,R) expression in whole-mount (C-J,M,N,Q,R) and in midsagittal (K,L,O,P) views at 9.5 dpc (G,H), 10.5 dpc (C-F,I,J,M,N,Q,R) and 12.5 dpc (K,L,O,P). (S,T) Midsagittal views of anti-GAP43 staining of 12.5 dpc wild-type and knock-in embryos, respectively. Arrows in G-J,M,N indicate the caudal limits of the expressions, arrowheads in G,H indicate the anterior end of the midbrain, arrowheads in O,R indicate expression in the posterior pretectum and in the tegmentum, respectively. Asterisks in S indicate the anti-GAP43-positive posterior commissure. Scale bars: 250 μm.

Fig. 8.

Midbrain phenotype in knock-in mutants. (A,C,E,G,I,K) Wild-type and (B,D,F,H,J,L) knock-in mutant embryos. (A,B) Wnt1, (C,D) Ebf3, (E,F) COUP-TFI, (G,H) En1, (I,J) Pax5 and (K,L) Fgf8 expression at 9.5 dpc (K,L) and 10.5 dpc (A-J). Scale bars: 125 μm.

Fig. 8.

Midbrain phenotype in knock-in mutants. (A,C,E,G,I,K) Wild-type and (B,D,F,H,J,L) knock-in mutant embryos. (A,B) Wnt1, (C,D) Ebf3, (E,F) COUP-TFI, (G,H) En1, (I,J) Pax5 and (K,L) Fgf8 expression at 9.5 dpc (K,L) and 10.5 dpc (A-J). Scale bars: 125 μm.

Fig. 9.

An interpretation of Emx2−/−Otx2+/− double and Otx2+/Emx2 knock-in mutant defects. (A) The onset of double mutant defects at the four-somite stage. Emx2-positive domain shown in green horizontal lines was lost in the double mutants. Consequently Otx2-positive region shown in red oblique lines was reduced both rostrocaudally and lateromedially. Moreover, the Pax2-positive area shown in blue oblique lines shifted rostrally, resulting in the reduction of anterior Otx2-positive and Pax2-negative area. Concomitantly Gbx2-positive area rostral to preotic sulcus (POS), painted in yellow, expanded. (B) A prosomeric view of affected region in double and knock-in mutants. P3 ventral thalamus, P2 dorsal thalamus and rostral P1 anterior pretectum (shown in red) were lost by the defects at the three- to four-somite stage in double mutants. P4 archipallium shown in purple was probably also lost by the defects at the same stage, though no detailed analysis was conducted in the present study. The r1 and r2 shown in yellow expand to compensate for the loss of the anterior structures in the double mutants. Posterior pretectum shown in blue develops in double mutants, but is specifically lost in knock-in mutants. Emx2, Otx2, Otx1, Pax6, Tcf4, Lim1, Dlx1, Gbx2, Ebf1 and GAP43 give their dorsal expression at 11.5dpc. Intense and weak expression are shown by thick and thin lines, respectively, in Otx1, Pax6 and Tcf4. Fgf8, Gbx2, Wnt1, En2 and Pax5 give the expression at 9.5 dpc. ACX, archicortex; APT, anterior pretectum; DT, dorsal thalamus; EMT, eminentia thalami; ET, epithalamus; HPT, habenulopeduncular tract; HT, hypothalamus; Is, isthmus; M, midbrain; MA, mammillary area; ov, otic vesicle; PC, posterior commissure; POS, preotic sulcus; PPT, posterior pretectum; RM, retro mammillary area; SPV, suprapreoptic paraventricular area; TG, tegmentum; TU, tuberal hypothalamus; VT, ventral thalamus; Zlth, zonalimitans interthalamica. Red lines indicate the boundary between alar and basal plates.

Fig. 9.

An interpretation of Emx2−/−Otx2+/− double and Otx2+/Emx2 knock-in mutant defects. (A) The onset of double mutant defects at the four-somite stage. Emx2-positive domain shown in green horizontal lines was lost in the double mutants. Consequently Otx2-positive region shown in red oblique lines was reduced both rostrocaudally and lateromedially. Moreover, the Pax2-positive area shown in blue oblique lines shifted rostrally, resulting in the reduction of anterior Otx2-positive and Pax2-negative area. Concomitantly Gbx2-positive area rostral to preotic sulcus (POS), painted in yellow, expanded. (B) A prosomeric view of affected region in double and knock-in mutants. P3 ventral thalamus, P2 dorsal thalamus and rostral P1 anterior pretectum (shown in red) were lost by the defects at the three- to four-somite stage in double mutants. P4 archipallium shown in purple was probably also lost by the defects at the same stage, though no detailed analysis was conducted in the present study. The r1 and r2 shown in yellow expand to compensate for the loss of the anterior structures in the double mutants. Posterior pretectum shown in blue develops in double mutants, but is specifically lost in knock-in mutants. Emx2, Otx2, Otx1, Pax6, Tcf4, Lim1, Dlx1, Gbx2, Ebf1 and GAP43 give their dorsal expression at 11.5dpc. Intense and weak expression are shown by thick and thin lines, respectively, in Otx1, Pax6 and Tcf4. Fgf8, Gbx2, Wnt1, En2 and Pax5 give the expression at 9.5 dpc. ACX, archicortex; APT, anterior pretectum; DT, dorsal thalamus; EMT, eminentia thalami; ET, epithalamus; HPT, habenulopeduncular tract; HT, hypothalamus; Is, isthmus; M, midbrain; MA, mammillary area; ov, otic vesicle; PC, posterior commissure; POS, preotic sulcus; PPT, posterior pretectum; RM, retro mammillary area; SPV, suprapreoptic paraventricular area; TG, tegmentum; TU, tuberal hypothalamus; VT, ventral thalamus; Zlth, zonalimitans interthalamica. Red lines indicate the boundary between alar and basal plates.

Acknowledgements

We thank Mr Naoki Takeda (Division of Transgenic Technology, Center for Animal Resources and Development, Kumamoto University) for production of chimeric mice. We are indebted to Drs A. P. McMahon, G. Martin, A. Joyner, P. Gruss, E. Lai, J. L. R. Rubenstein, H. Clevers, M. Taira, J. G. Flanagan, D. G. Wilkinson, P. Charnay and M.-J. Tsai for in situ hybridization probes. We thank Dr J. Miyazaki for providing the Cre mice. We are also grateful to the Laboratory Animal Research Center of Kumamoto University for housing of the mice. This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (Specially Promoted Research); the Science and Technology Agency, CREST, JST; and the Ministry of Public Welfare, Japan.

References

Acampora, D., Mazan, S., Lallemand, Y., Avantaggiato, V., Maury, M., Simeone, A. and Brulet, P. (
1995
). Forebrain and midbrain regions are deleted in Otx2−/− mutants due to a defective anterior neuroectoderm specification during gastrulation.
Development
121
,
3279
-3290.
Acampora, D., Mazan, S., Avantaggiato, V., Barone, P., Tuorto, F., Lallemand, Y., Brulet, P. and Simeone, A. (
1996
). Epilepsy and brain abnormalities in mice lacking the Otx1 gene.
Nat. Genet
.
14
,
218
-222.
Acampora, D., Avantaggiato, V., Tuorto, F. and Simeone, A. (
1997
). Genetic control of brain morphogenesis through Otx gene dosage requirement.
Development
124
,
3639
-3650.
Acampora, D., Avantaggiato, V., Tuotro, F., Briata, P., Corte, G. and Simeone, A. (
1998
). Visceral endoderm-restricted translation of Otx1 mediates recovery of Otx2 requirements for specification of anterior neural plate and normal gastrulation.
Development
125
,
5091
-5104.
Acampora, D., Avantaggiato, V., Tuorto, F., Barone, P., Perera, M., Choo, D., Wu, D., Corte, G. and Shimeone, A. (
1999
). Differential transcriptional control as the major molecular event in gene Otx1−/− and Otx2−/− divergent phenotypes.
Development
126
,
1417
-1426.
Ang, S.-L., Jin, O., Rhinn, M., Daorigle, N., Stevenson, L. and Rossant, J. (
1996
). A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain.
Development
122
,
243
-252.
Araki, I. and Nakamura, H. (
1999
). Engrailed defines the position of dorsal di-mesencephalic boundary by repressing diencephalic fate.
Development
126
,
5127
-5135.
Asano, M. and Gruss, P. (
1992
). Pax-5 is expressed at the midbrain-hindbrain boundary during mouse development.
Mech. Dev
.
39
,
29
-39.
Bally-Cuif, L., Cholley, B. and Wassef, M. (
1995
). Involvement of Wnt-1 in the formation of the mes/metencephalic boundary.
Mech. Dev
.
53
,
23
-34.
Bally-Cuif, L. and Wassef, M. (
1995
). Determination events in the nervous system of the vertebrate embryo.
Curr. Opin. Genet. Dev
.
5
,
450
-458.
Barnes, J. D., Crosby, J. L., Jones, C. M., Wright, C. V. E. and Hogan, B. L. M. (
1994
). Embryonic expression of Lim-1, the mouse homolog of Xenopus Xlim-1, suggests a role in lateral mesoderm differentiation and neurogenesis.
Dev. Biol
.
161
,
168
-178.
Beddington, R. S. P. and Robertson, E. J. (
1998
). Anterior patterning in mouse.
Trends Genet
.
14
,
277
-284.
Beddington, R. S. P. and Robertson, E. J. (
1999
). Axis development and early asymmetry in mammals.
Cell
96
,
195
-209.
Boncinelli, S., Gulisano, M. and Broccoli, V. (
1993
). Emx and Otx homeobox genes in the developing mouse brain.
J. Neurobiol
.
24
,
1356
-1366.
Broccoli, V., Boncinelli, E. and Wurst, W. (
1999
). The caudal limit of Otx2 expression positions the isthmic organizer.
Nature
401
,
164
-168.
Bulfone, A., Puelles, L., Porteus, M. H., Frohman, M. A., Martin, G. R. and Rubenstein, J. L. R. (
1993
). Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2, and Wnt-3 in the embryonic day12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries.
J. Neurosci
.
13
,
3155
-3172.
Cheng, H.-J. and Flanagan, J. G. (
1994
). Identification and cloning of ELF-1, a developmentally expressed ligand for Mek4 and Sek receptor tyrosine kinases.
Cell
79
,
157
-168.
Cho, E. A. and Dressler, G. R. (
1998
). TCF-4 binds beta-catenin and is expressed in distinct regions of the embryonic brain and limbs.
Mech. Dev
.
77
,
9
-18.
Crossley, P. H. and Martin, G. R. (
1995
). The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo.
Development
121
,
439
-451.
Crossley, P. H., Martinez, Z. and Martin, G. R. (
1996
). Midbrain development induced by FGF8 in the chick embryo.
Nature
380
,
66
-68.
Danielian, P. S. and McMahon, A. P. (
1996
). Engrailed-1 as a target of the Wnt-1 signaling pathway in vertebrate midbrain development.
Nature
383
,
332
-334.
Davis, C. A. and Joyner, A. L. (
1988
). Expression patterns of the homeo box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge during mouse development.
Genes Dev
.
2
,
1736
-1744.
Davis, C. A., Noble-Topham, S. E., Rossant, J. and Joyner, A. L. (
1988
). Expression of the homeo box-containing gene En-2 delineates a specific region of the developing mouse brain.
Genes Dev
.
2
,
361
-371.
Dressler, G. R., Deutsch, U., Chowdhury, K., Nornes, H. O. and Gruss, P. (
1990
). Pax2, a new paired-box-containing gene and its expression in the developing excretory system.
Development
109
,
787
-795.
Echelard, Y., Vassileva, G. and McMahon, A. P. (
1994
). Cis-acting regulatory sequences governing Wnt-1 expression in the developing mouse CNS.
Development
120
,
2213
-2224.
Eisenstat, D. D., Liu, J. K., Mione, M., Zhong, W., Yu, G., Anderson, S. T., Ghattas, I., Puelles, L. and Rubenstwin, J. L. R. (
1999
). DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation.
J. Comp. Neurol
.
414
,
217
-237.
Ericson, J., Muhr, J., Placzek, M., Lints, T., Jessell, T. M. and Edlund, T. (
1995
). Sonic Hedgehog induces the differentiation of ventral forebrain neurons: A common signal for ventral patterning within the neural tube.
Cell
81
,
747
-756.
Feldheim, D. A., Vanderhaeghen, P., Hansen, M. J., Frisen, J., Lu, Q., Barbacid, M. and Flanagan, J. G. (
1998
). Topographic guidance labels in a sensory projection to the forebrain.
Neuron
21
,
1303
-1313.
Feldheim, D. A., Kim, Y.-I., bergemann, A. D., Frisen, J., Barbacid, M. and Flanagan, J. G. (
2000
). Genetic analysis of ephrin-A2 and ephrin-A5 shows their requirement in multiple aspects of retinocollicular mapping.
Neuron
25
,
563
-574.
Figdor, M. and Stern, C. D. (
1993
). Segmental organization of embryonic diencephalon.
Nature
363
,
630
-634.
Flenniken, A. M., Gale, N. W., Yancopoulos, G. D. and Wilkinson, D. G. (
1996
). Distinct and overlapping expression patterns of ligands for Eph-related receptor tyrosine kinases during mouse embryogenesis.
Dev. Biol
.
179
,
382
-401.
Fujii, T., Pichel, J. G., Taira, M., Toyama, R. and Dawid, I. B. (
1994
). Expression patterns of the murine LIM classs homeobox gene lim1 in the developing brain and excretory system.
Dev. Biol
.
199
,
73
-83.
Garel, S., Marin, F., Mattei, M.-G., Vesque, C., Vincent, A and Charnay, P. (
1997
). Family of Ebf/Olf-1-related genes potentially involved in neuronal differentiation and regional specification in the central nervous system.
Dev. Dyn
.
210
,
191
-205.
Gurdon, J. B., Partington, G. A. and De Robertis, E. M. (
1976
). Injected nuclei in frog oocytes: RNA synthesis and protein exchange.
J. Embryol. Exp. Morphol
.
36
,
541
-553.
Grindley, J. C., Hargett, L. K., Hill, R. E., Ross, A. and Horgan, B. L. M. (
1997
). Disruption of Pax6 function in homozygous for the Pax6Sey-1Neu mutation produces abnormalities in the early development and regionalization of the diencephalon.
Mech. Dev
.
64
,
111
-126.
Hallonet, M. E. R., Hollemann, T., Wehr, R., Jenkins, N. A., Copeland, N. G., Pieler, T. and Gruss, P. (
1998
). Vax1 is a novel homeobox-containing gene expressed in the developing anterior ventral forebrain.
Development
125
,
2599
-2610.
Hanks, W., Wurst, W., Anson-Cartwright, L., Suerbach, A. B. and Joyner, A. L. (
1995
). Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2.
Science
269
,
679
-682.
Hidalgo-Sanchez, M., Simeone, A. and Alvarado-Mallart, R. (
1999
).Fgf8 and Gbx2 induction concomitant with Otx2 repression is correlated with midbrain-hindbrain fate of caudal prosencephalon.
Development
126
,
3191
-3203.
Iler, N., Rowitch, D. H., Echelad, Y., McMahon, A. P. and Abate-Shen, C. (
1995
). A single homeodomain binding site restricts spatial expression of Wnt-1 in the developing brain.
Mech. Dev
.
53
,
87
-96.
Inoue, T., Nakamura, S. and Osumi, N. (
2000
). Fate mapping of the mouse prosencephalic neural plate.
Dev. Biol
.
219
,
373
-383.
Joyner, A. L., Herrup, K., Auerbach, A., Davis, C. A. and Rossant, J. (
1991
). Subtle cerebellar phenotype in mice homozygous for a targeted deletion of the En-2 homeobox.
Science
251
,
1239
-1243.
Joyner, A. L. (
1996
). Engrailed, Wnt and Pax genes regulate midbrain-hindbrain development.
Trends Genet
.
12
,
15
-20.
Kimura, C., Takeda, N., Suzuki, M., Oshimura, M., Aizawa, S. and Matsuo, I. (
1997
). Cis-acting elements conserved between mouse and pufferfish Otx2 genes govern the expression in mesencephalic neural crest cells.
Development
124
,
3929
-3941.
Kimura, C., Yoshinaga, K., Tian, E., Suzuki, M., Aizawa, S. and Matsuo, I. (
2000
). Visceral endoderm mediates forebrain development by suppressing posteriorizing signals.
Dev. Biol
.
225
,
304
-321.
Korinek, V., Barker, N., Willert, K., Molenar, M., Roose, J., Wagenaar, G., Markman, M., Lamers, W., Drestree, O. and Clevers, H. (
1998
). Two members of the Tcf family implicated in Wnt/β-catenin signaling during embryogenesis in the mouse.
Mol. Cell. Biol
.
18
,
1248
-1256.
Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (
1997
). Evidence that FGF8 signalling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain.
Development
124
,
959
-969.
Liu, A. and Joyner, A. L. (
2001
). EN and GBX2 play essential roles downstream of FGF8 in patterning the mouse mid/hindbrain region.
Development
128
,
181
-191.
Mahmood, R., Bresnick, J., Hornbruch, A., Mahony, C., Morton, N., Colquhou, K., Martin, P., Lumsden, A., Dickson, C. and Mason, I. (
1995
). A role for FGF-8 in the initiation and maintenance of vertebrate limb but outgrowth.
Curr. Biol
.
5
,
797
-806.
Mallamaci, A., DiBlas, E., Brista, P., Boncinelli, E. and Corte, G. (
1996
). Otx2 homeoprotein in developing central nervous system and migratory cells of the olfactory area.
Mech. Dev
.
58
,
165
-178.
Mallamaci, A., Iannone, R., Briata, P., Pintonello, L., Mercurio, S., Boncinelli, E. and Corte, G. (
1998
). Emx2 protein in the developing mouse brain and olfactory area.
Mech. Dev
.
77
,
165
-172.
Martinez, S., Crossley, P. H., Cobos, I., Rubenstein, J. L. R. and Martin, G. R. (
1999
). FGF8 induces formation of an ectopic isthmus organizer and isthmocerebellar development via a repressive effect on Otx2 expression.
Development
126
,
1189
-1200.
Martinez, S. and Puelles, L. (
2000
). Neurogenetic compartments of the mouse diencephalon and some characteristic gene expression patterns. In Mouse Brain Development:; Resuts and Problems in Cell Differentiation. Vol. 30 (ed. M. Goffinet and P. Rakic), pp. 91-106. New York: Springer Life Sciences.
Martinez-Barbera, J. P., Rodriguez, T. A. and Beddington, R. S. (
2000
a). The homeobox gene Hesx1 is required in the anterior neural ectoderm for normal forebrain formation.
Dev. Biol
.
223
,
422
-430.
Martinez-Barbera, J. P., Clements, M., Thomas, P., Rodriguez, T., Meloy, D., Kioussis, D. and Beddington, R. S. (
2000
b). The homeobox gene Hex is required in definitive endodermal tissues for normal forebrain, liver and thyroid formation.
Development
127
,
2433
-2445.
Masai, I., Heisenberg, C. P., Barth, K. A., Macdonald, R., Adamek, S. and Wilson, S. W. (
1997
). Floating head and masterblind regulate neuronal patterning in the roof of the forebrain.
Neuron
18
,
43
-57.
Mastick, G. S., Davis, N. M., Andrews, G. L. and Easter, S. S., Jr (
1997
). Pax-6 functions in boundary formation and axon guidance in the embryonic mouse forebrain.
Development
124
,
1985
-1997.
Matsunaga, E., Araki, I. and Nakamura, H. (
2000
). Pax defines the di-mesencephalic boundary by repressing En1 and Pax2.
Development
127
,
2357
-2365.
Matsuo, I., Kuratani, S., Kimura, C., Takeda, N. and Aizawa, S. (
1995
). Mouse Otx2 functions in the formation and patterning of rostral head.
Genes Dev
.
9
,
2646
-2658.
McMahon, A. P. and Bradley, A. (
1990
). The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain.
Cell
62
,
1073
-1085.
McMahon, A. P., Loyner, A. L., Bradley, A. and McMahon, J. A. (
1992
). The midbrain-hindbrain phenotype of Wnt-1−/− mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum.
Cell
69
,
581
-595.
Millen, K. J., Wurst, W., Herrup, K. and Joyner, A. (
1994
). Abnormal embryonic cerebellar development and patterning of postnatal foliation in two mouse Engrailed-2 mutants.
Development
120
,
695
-706.
Millet, S., Campbell, K., Epstein, D. J., Losos, K., Harris, E. and Joyner, A. L. (
1999
). A role for Gbx2 in repression of Otx2 and positioning the mid/hindbrain organizer.
Nature
401
,
161
-164.
Miyamoto, N., Yoshida, M., Kuratani, S., Matsuo, I. and Aizawa, S. (
1997
). Defects of urogenital development in mice lacking Emx2.
Development
124
,
1653
-1664.
Nada, S., Yagi, T., Takeda, N., Tokunaga, H., Nakagawa, Y., Ikawa, Y., Okada, M. and Aizawa, S. (
1993
). Constitutive activation of Src family kinases in mouse embryos that lack Csk.
Cell
73
,
1125
-1135.
Nakano, T., Murata, T., Matsuo, I. and Aizawa, S. (
2000
). OTX2 directly interacts with lim1 and HNF-3β.
Biochem. Biophys. Res. Commun
.
267
,
64
-70.
Ohsaki, K., Morimitsu, T., Ishida, Y., Kominami, R. and Takahashi, N. (
1999
). Expression of the Vax family homeobox genes suggests multiple roles in eye development.
Genes Cells
4
,
267
-276.
Oliver, G., Mailhos, A., Wehr, R., Copeland, G. N., Jenkins, A. N. and Gruss, P. (
1995
). Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development.
Development
121
,
4045
-4055.
Parr, B. P., Shea, M. J., Vassileva, G. and McMahon, A. P. (
1993
). Mouse Wnt genes exhibit discrete domains of expression in the early embryonic CNS and limb buds.
Development
119
,
247
-261.
Pellegrini, M., Mansouri, A., Simeone, A., Boncinelli, E. and Gruss, P. (
1996
). Dentate gyrus formation requires Emx2.
Development
122
,
3893
-3898.
Puelles, L. and Rubenstein, J. L. R. (
1993
). Expression pattern of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization.
Trends Neurosci
.
16
,
472
-479.
Qiu, Y., Cooney, A. J., Kuratani, S., DeMayo, F. J., Tsai, S. Y. and Tsai, M.-J. (
1994
). Spatiotemporal expression patterns of chicken ovalbumin upstream promoter-transcription factors in the developing mouse central nervous system: evidence for a role in segmental patterning of the diencephalon.
Proc. Natl. Acad. Sci. USA
91
,
4451
-4455.
Qiu, M., Anderson, S., Chen, S., Meneses, J. J., Hevner, R., Kuwana, E., Pedersen, R. A. and Rubenstein, J. L. R. (
1996
). Mutation of the Emx-1 homeobox gene disrupts the corpus callosum.
Dev. Biol
.
178
,
174
-178.
Rhinn, M., Dierich, A., Shawlot, W., Behringer, R. R., Meur, M. L. and Ang, S.-L. (
1998
). Sequential roles for Otx2 in visceral endoderm and neuroectoderm for forebrain and midbraimn induction and specification.
Development
125
,
845
-856.
Rowitch, D. H. and McMahon, A. P. (
1995
). Pax-2 expression in the murine neural plate precedes and encompasses the expression domains of Wnt-1 and En-1.
Mech. Dev
.
38
,
3
-8.
Royet, J. and Finkelstein, R. (
1995
). Pattern formation in Drosophila head development: the role of the orthodenticle homeobox gene.
Development
121
,
3561
-3572.
Rubenstein, J. L. R., Martinez, S., Shimamura, K. and Puelles, L. (
1994
). The embryonic vertebrate forebrain: the Prosomeric model.
Science
266
,
578
-580.
Rubenstein, J. L. R. and Shimamura, K. (
1998
). Regionalization of the prosencencephalic neural plate.
Annu. Rev. Neurosci
.
21
,
445
-477.
Rubenstein, J. L. R., Shimamura, K., Martinez, S. and Puelles, L. (
1998
). Regionalization of the prosencephalic neural plate.
Annu. Rev. Neurosci
.
21
,
445
-477.
Sakai, K. and Miyazaki, J. (
1997
). A transgenic mouse line that retains Cre recombinase activity in mature oocytes irrespective of the cre transgene transmission.
Biochem. Biophys. Res. Commun
.
237
,
318
-324.
Schwarz, M., Alvarez-Bolado, G., Dressler, G., Urbanek, P., Busslinger, M. and Gruss, P. (
1999
). Pax2/5 and Pax6 subdivide the early neural tube into three domains.
Mech. Dev
.
82
,
29
-39.
Shamim, H., Mahmood, R., Logan, C., Doherty, P., Lumsden, A. and Mason, I. (
1999
). Sequential roles for Fgf4, En1 and Fgf8 in specification and regionalisation of the midbrain.
Development
126
,
945
-959.
Shawlot, W., Wakamia, M., Kwan, K. M., Kania, A., Jessell, T. M. and Behringer, R. R. (
1999
). Lim1 is required in both primitive streak-derivatived tissues and visceral endoderm for head formation in the mouse.
Development
126
,
4925
-4932.
Shimamura, K., Hartigan, D., Martinez, S., Puelles, L. and Rubenstein, J. L. R. (
1995
). Longitudinal organization of the anterior neural plate and neural tube.
Development
121
,
3923
-3933.
Shimamura, K., Martinez, S., Puelles, L. and Rubenstein, J. L. R. (
1997
). Patterns of gene expression in the neural plate and neural tube subdivide the embryonic forebrain into transverse and longitudinal domains.
Dev. Neurosci
.
19
,
88
-96.
Simeone, A., Gulisano, M., Acampora, D., Stornaiuolo, A., Rambaldi, M. and Boncinelli, E. (
1992
a). Two vertebrate homeobox genes related to the Drosophila empty spiracles gene are expressed in the embryonic cerebral cortex.
EMBO J
.
11
,
2541
-2550.
Simeone, A., Acampora, D., Gulisano, M., Stornaiuolo, A. and Boncinelli, E. (
1992
b). Nested expression domains of four homeobox genes in developing rostral brain.
Nature
358
,
687
-690.
Simeone, A., Acampora, D., Mallamaci, A., Stornaiuolo, A., D’Apice, M. R., Nigro, V. and Boncinelli, E. (
1993
). A vertebrate gene related to orthodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm of the gastrulating mouse embryo.
EMBO J
.
12
,
2735
-2747.
Simeone, A. (
2000
). Positioning the isthmic organizer. Where Otx2 and Gbx2 meet.
Trend Genet
.
16
,
237
-239.
Stein, S. and Kessel, M. (
1995
). A homeobox gene involved in node, notochord and neural plate formation of chick embryos.
Mech. Dev
.
49
,
37
-48.
Stoykova, A., Fritssch, R., Walther, C. and Gruss, P. (
1996
). Forebrain patterning defects in small eye mutant mice.
Development
122
,
3453
-3465.
Stoykova, A., Gotz, M., Gruss, P. and Price, D. J. (
1997
). Pax6-dependent regulation of adhesive patterning, R-cadherin expression and boundary formation in developing forebrain.
Development
124
,
3765
-3777.
Suda, Y., Matsuo, I., Kuratani, S. and Aizawa, S. (
1996
). Otx1 function overlaps with Otx2 in development of mouse forebrain and midbrain.
Genes Cells
1
,
1031
-1044.
Suda, Y., Matsuo, I. and Aizawa, S. (
1997
). Cooperation between Otx1 and Otx2 genes in developmental patterning of rostral brain.
Mech. Dev
.
69
,
125
-141.
Suda, Y., Nakabayashi, J., Matsuo, I. and Aizawa, S. (
1999
). Functional equivalency between Otx2 and Otx1 in development of the rostral head.
Development
124
,
743
-757.
Tam, P. P. L. and Steiner, K. A. (
1999
). Anterior patterning by synergistic activity of the early gastrula organizer and the anterior germ layer tissues of the mouse embryo.
Development
126
,
5171
-5179.
Tanaka, S., Nomizu, M and Kurosumi, K. (
1991
). Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in the rat pituitary.
J. Histochem. Cytochem
.
39
,
809
-821.
Tao, W. and Lai, E. (
1992
). Telencephalon-restricted expression of BF-1, a new member of the HNF-3/fork head gene family, in the developing rat brain.
Neuron
8
,
957
-966.
Thomas, P. and Beddington, R. (
1996
). Anterior primitive endoderm may be responsible for patterning the anterior neural plate in the mouse embryo.
Curr. Biol
.
6
,
1487
-1496.
Urbanek, P., Wang, Z., Fetla, I., Wagner, E. F. and Busslinger, M. (
1994
). Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP.
Cell
79
,
901
-912.
Urbanek, P., Fetka, I., Heisler, M. H. and Busslinger, M. (
1997
). Cooperation of Pax2 and Pax5 in midbrain and cerebellum development.
Proc. Natl. Acad. Sci. USA
94
,
5703
-5708.
Wallis, D. and Muenke, M. (
2000
). Mutation in holoprosencephaly.
Hum. Mutat
.
16
,
99
-108.
Walther, C. and Gruss, P. (
1991
). Pax-6, a murine paired box gene, is expressed in_the developing CNS.
Development
113
,
1435
-1449.
Warren, N. and Price, D. J. (
1997
). Roles of Pax-6 in murine diencephalic development.
Development
124
,
1573
-1582.
Wassarman. K. M., Lewandoski, M., Campbell, K., Joyner, A. L., Rubenstein, J. L., Martinez, S. and Martin, G. R. (
1997
). Specification of the anterior hindbrain and establishment of a normal mid/hindbrain organizer is dependent on Gbx2 gene function.
Development
124
,
2923
-2934.
Wilkinson, D. G. (
1993
). In situ hybridization. In Essential Developmental Biology: A Practical Approach (ed. C. D. Stern and P. W. H. Holland), pp. 257-274. Oxford: IRL Press.
Wurst, W., Auerbach, A. B. and Joyner, A. L. (
1994
). Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum.
Development
120
,
2065
-2075.
Yagi, T., Tokunaga, T., Furuta, Y., Nada, S., Yoshida, M., Tsukada, T., Saga, Y., Takeda, N., Ikawa, Y. and Aizawa, S. (
1993
a). A novel ES cell line, TT2, with high germline-differentiating potency.
Anal. Biochem
.
214
,
70
-76.
Yagi, T., Nada, S., Watanabe, N., Tamemoto, H., Kohmura, N., Ikawa, Y. and Aizawa, S. (
1993
b). A novel negative selection for homologous recombinants using diphtheria toxin A fragment gene.
Anal. Biochem
.
214
,
77
-86.
Yoshida, M., Suda, Y., Matsuo, I., Miyamoto, N., Takeda, N., Kuratani, S. and Aizawa, S. (
1997
). Emx1 and Emx2 functions in development of dorsal mtelencephalon.
Development
124
,
101
-111.