1 Department of Morphogenesis, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University, Japan
2 CREST, JST, Japan
3 Vertebrate Body Plan Group, RIKEN Center for Developmental Biology, 2-2-1 Honjo, Kumamoto-860-0811, Japan
4 Department of Anatomy, Nara Medical University, 840 Saijo-machi, Kashihara, Nara-634-8521, Japan*These authors contributed equally to this work.
Author for correspondence (e-mail: saizawa{at}gpo.kumamoto-u.ac.jp)
Accepted April 11, 2001
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SUMMARY |
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Key words: Emx, Otx, Diencephalon, Forebrain development, Mouse
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INTRODUCTION |
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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).
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MATERIALS AND METHODS |
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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 Bouins 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).
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RESULTS |
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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.
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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).
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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.
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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.
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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.
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DISCUSSION |
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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.
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