1 Laboratory for Vertebrate Body Plan, Center for Developmental Biology (CDB),
RIKEN Kobe, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0046, Japan
2 Laboratory for Animal Resources and Genetic Engineering, Center for
Developmental Biology (CDB), RIKEN Kobe, 2-2-3 Minatojima-minamimachi,
Chuo-ku, Kobe 650-0046, Japan
* Author for correspondence (e-mail saizawa{at}cdb.riken.jp)
Accepted 8 April 2004
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SUMMARY |
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Key words: Otx2, Otx1, Enhancer, Forebrain, Midbrain, Anterior neuroectoderm, WNT signaling, Mouse
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Introduction |
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Otx2, Six3 and Rpx/Hesx1 are the genes expressed earliest
in the anterior neuroectoderm induced by anterior visceral endoderm and
anterior mesendoderm (Ang et al.,
1994; Thomas and Beddington,
1996
; Rhinn et al.,
1998
; Kimura et al.,
2000
). The initial morphological landmark in this anterior neural
plate is the preotic sulcus, which corresponds to the future boundary between
rhombomere 2 and 3; the sulcus becomes apparent around the one-somite stage.
In the neural plate rostral to the sulcus, Otx2 expression covers the
entire future forebrain and midbrain, though its caudal limit is initially not
distinct. Within the Otx2-positive region, Otx1 expression
begins at around the two-somite stage
(Simeone et al., 1992
;
Simeone et al., 1993
;
Suda et al., 1997
).
Gbx2 expression in the region rostrally adjacent to the preotic
sulcus, which corresponds to the future metencephalon, becomes apparent at
around the three- to four-somite stage
(Bouillet et al., 1995
;
Wassarman et al., 1997
).
Otx2 and Gbx2 expression initially overlap, but they become
segregated by the six-somite stage, when the boundary between midbrain and
hindbrain or ithmus is formed (Broccoli et
al., 1999
; Millet et al.,
1999
). Also at around the three- to four-somite stage,
Emx2 and Pax6 expression begins in laterocaudal forebrain
primordium (Suda et al., 2001
;
Inoue et al., 2000
). At this
stage, their caudal limits nearly coincide, but later Emx2 expression
is not found in the dorsal thalamus or pretectum, where Pax6 is
expressed. Pax6 expression initially overlaps with Pax2
expression caudally, but at around the six- to eight-somite stage its caudal
margin coincides with the boundary between the diencephalon and mesencephalon
(Araki and Nakamura, 1999
;
Schwarz et al., 1999
;
Matsunaga et al., 2000
).
Initially at the three-somite stage, Pax2 expression covers the
future entire midbrain (Rowitch and
McMahon, 1995
; Suda et al.,
2001
); however, as development proceeds it retracts caudally and
is eventually found in caudal mesencephalon and metencephalon. Six3
and Irx3 expressions also initially overlap, but by the six-somite
stage they become segregated in avian. The anterior margin of the
Irx3 expression is delineated by zona limitans interthalamica (ZLTH)
(Oliver et al., 1995
;
Bosse et al., 1997
;
Kobayashi et al., 2002
). It
has been suggested that the ZLTH divides the anterior neuroectoderm into
rostral and caudal halves that differentially respond to FGF8 and SHH
signaling (Crossley et al.,
1996
; Shimamura and
Rubenstein, 1997
; Martinez et
al., 1999
; Kobayashi et al.,
2002
). The isthmus and anterior neural ridge act as local
organizers in midbrain and forebrain development, respectively. Fgf8
expression is faint and broad at the three-somite stage, but it focuses at
isthmus by the six-somite stage (Crossley
and Martin, 1995
; Suda et al.,
1997
). Fgf8 expression also occurs in the anterior neural
ridge at around the four-somite stage
(Crossley and Martin, 1995
;
Shimamura and Rubenstein,
1997
; Tian et al.,
2002
). Thus, the initial regionalization of the brain occurs at
around the three- to six-somite stage.
The results of analyses of
Otx1+/Otx2+/
mutants suggested that Otx2 and Otx1 cooperate in the
regionalization of the rostral brain
(Acampora et al., 1997;
Suda et al., 1996
;
Suda et al., 1997
;
Suda et al., 2001
). Owing to
the occurrence of earlier visceral defects in
Otx2/ mutants, however, this cooperation
could not be examined in the Otx2 homozygous mutant state.
Furthermore,
Otx1+/Otx2+/
mutants are postnatally lethal, and Otx1/
Otx2+/ phenotype could also not be examined.
Otx1/ mutants do not exhibit marked defects
in forebrain or midbrain development (Suda
et al., 1996
). These situations obscured the onset and the extent
of cooperation between Otx1 and Otx2 in rostral brain
development.
Kurokawa et al. (Kurokawa et al.,
2004) analyzed the enhancers of Otx2 expression in
anterior neuroectoderm at 95 to 80 kb. The activity of the AN
enhancer ceased by E8.5 in anterior neuroectoderm. In the present study, we
analyzed enhancers of Otx2 expression in forebrain and midbrain; two
distinct enhancers were identified at 75 kb 5' upstream and 115 kb
3' downstream. Their activities were absent in the entire forebrain and
midbrain at E8.0 (two- to four-somite stage), but were found at E8.5 (6 to 8
somite stage). Thus, the transition of the activities from the AN enhancer to
FM enhancer occurs at a stage crucial to rostral brain regionalization.
Studies of mutants lacking each of these enhancers demonstrated that they, in fact, regulate Otx2 expression in vivo. Moreover, analysis of these mutants revealed that Otx2 expression under FM and FM2 enhancers cooperates with Otx1 in the development of the mesencephalon and diencephalon. The affected regions developed at E8.0 and were subsequently lost later than E8.5. The results of this investigation also suggested several upstream factors that are involved in the regulation of Otx2 expression in forebrain and midbrain. Finally, the phylogenetic significance of enhancer organization was discussed on the basis of available genomic information.
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Materials and methods |
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Genomic clones of the Otx2 locus of mouse and other animals
A mouse Otx2 genomic BAC clone 391F17 here referred to as BAC #2
was isolated from a C57BL/6 BAC library (Research Genetics). This was
subdivided into fragments of about 10 to 15 kb in length by Sau3AI partial
digestion and subcloned into BamHI site of pBluescript SK ().
These clones were aligned as shown in Fig.
4A by walking. A 3 kb human genomic DNA in
Fig. 5B, part c was amplified
by PCR with 5'-ACTAGTTCTCAAAGTGTCCACTAAGCCGCT-3' and
5'-CCATGCACCTGGGAAGCCCTAAAAAGATCA-3' as primers from RPCI11-1085N6
(BACPAC resources) human OTX2 BAC clone. A 0.6 kb zebrafish genomic
DNA containing domain ß in Fig.
8A, part b was amplified with
5'-GCTGGAATTGCTCTGGTCTTTTTC-3' and
5'-CCAACTCTAAAATCTAACATCACG-3' as primers from zebrafish genomic
DNAs.
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Generation of enhancer mutant mice
A neomycin resistance gene with Pgk1 promoter and SV40 polyadenylation
signal was flanked by loxP sequences. This cassette (Neo) was replaced with
the ApaI/HindIII 1314 bp or BsmI/BamHI 995
bp region to delete the FM or FM2 enhancer, respectively. Homologous
recombinant ES clones were isolated with TT2 ES cells, as described previously
(Matsuo et al., 1995). These
were identified by PCR with 5'-ATCGCCTTCTTGACGAGTTCTTCTG-3' in the
neo gene as the 5' side primer and the following 3' side
primers: 5'-CATAAGACCATGAGTTTAGTTCACAGC-3' and
5'-CTGAACACACAATACTCCTCAGCTGG-3' for the FM and FM2 targeting
vectors, respectively. Recombinants were confirmed by Southern blot analyses
as described (Matsuo et al.,
1995
). Two mutant mouse lines were generated from independent
homologous recombinant ES clones for each enhancer mutation. The genotype of
each mutant mouse or embryo was routinely determined by PCR using tail or yolk
sac specimens. Sense primers employed to detect the wild-type allele were
5'-GAGTGGCTTCTGTCTTTCCATTCCAC-3' (FM) and
5'TTGTCAACCTCCTCTTTGAAGAGCC-3' (FM2);
5'-ATCGCCTTCTTGACGAGTTCTTCTG-3' (Neo) served as the primer to
detect the mutant allele. The antisense primers were
5'-GAGCATGCTGCATCTCTGAAATACAC-3' (FM) and
5'-AAGACTCTGTCATTGGGTGTGTTGC-3' (FM2). The deletion of the
neo insert by Cremediated loxp recombination was
accomplished by mating Otx2+/
AN mice with
Lefty-Cre mice (Yamamoto et al.,
2001
).
RNA in situ hybridization
The probes used in this study were: Emx2
(Yoshida et al., 1997),
Otx2 (Matsuo et al.,
1995
), Fgf8 (Crossley
and Martin, 1995
) and Pax6
(Walther and Gruss, 1991
).
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Results |
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To define the FM enhancer in the #13 fragment at 70 kb to 80 kb 5' upstream, the 10 kb region was divided into XhoI/ApaI 2.0 kb (XA2kb), ApaI/HindIII 1.4 kb (AH1.4kb), HindIII/XbaI 2.0 kb (HXb2kb) and XbaI/ClaI 3.0 kb (XbC3kb) fragments (Fig. 2A). The ApaI/HindIII 1.4 kb fragment exhibited full FM activity in a transient transgenic assay at E10.5. This finding was confirmed by the generation of permanent transgenic lines (Fig. 2C, part a). ß-gal expression in the forebrain and midbrain was detected at E8.5 with this fragment. The caudal limit of ß-gal expression occurred at the boundary between the midbrain and the hindbrain.
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Targeted disruption of Otx2 FM enhancer activity
In order to confirm that Otx2 expression in forebrain and midbrain
is governed by the FM enhancer after E8.5, a mutant,
Otx2FM/
FM, in which the
ApaI/HindIII 1.4 kb region was replaced with a cassette
encoding a neomycinresistant gene, was generated
(Fig. 3A). In the homozygous
enhancer mutant, Otx2 expression was present, but diminished, at E9.5
(data not shown). Forebrain and midbrain development in this mutant also
appeared normal (3B, parts a-d), suggesting that another enhancer of
Otx2 expression in forebrain/midbrain most probably exists.
|
BAC #1 displayed no transcriptional activity in posterior mesencephalon at
E9.5 (see Fig. 1B, part c in
the preceding paper), while the #13 fragment did exhibit this activity. As a
result, the presence of a silencer in the BAC #1 was initially hypothesized.
However, we could establish only one BAC #1/lacZ transgenic line. The
fact that the mesencephalon was affected in
Otx2FM/ mutants demonstrates rather that the
FM enhancer in the ApaI/HindIII 1.4 kb fragment is active in
midbrain in vivo.
Search for the second enhancer in the 3' side of the coding region
Enhancer mutants indicated the presence of another enhancer of
Otx2 expression in forebrain/midbrain. To investigate this, a search
was conducted for the second enhancer in the 3' region of the
Otx2 gene. A BAC transgenic mouse, which harbored a genomic DNA
sequence that spanned 50 kb to +120 kb 3' downstream, was
generated (BAC #2; Fig. 4A).
The 3' BAC#2/lacZ transgene was prepared by homologous
recombination in E. coli (Yang et
al., 1997) between BAC#2 and a lacZ construct in which
the lacZ gene was placed inframe at the translational start site and
flanked by 1.8 kb 5' and 2.5 kb 3' sequences. Two
3'BAC#2/lacZ transgenic lines were produced, neither
of which displayed activity in epiblast or anterior neuroectoderm. At a later
point, these lines exhibited activities in forebrain, midbrain, eyes and nose
(Fig. 4B, part a).
The Otx2 3' genomic region was then divided into 14 fragments, and the enhancer activity of each fragment was assayed via generation of permanent transgenic lines, as indicated in the parentheses in Fig. 4A. No activity was detected in epiblast or anterior neuroectoderm with any of these fragments. The #17 and #20 fragments showed activity in nasal placode (Fig. 4B, parts b,c). However, the enhancer of expression in E9.5 forebrain and midbrain was mapped to the #29 fragment, which locates at +110 kb to +120 kb 3' downstream. This fragment displayed faint activity at E7.75 in the future mid/hindbrain boundary region (Fig. 4B, part d). Furthermore, activity was evident in future dorsal areas of the prospective mid/hindbrain boundary region at E8.5 (Fig. 4B, part e). At E9.5, ß-gal expression directed by #29 covered the entire mesencephalon (Fig. 4B, parts f,g). ß-gal expression in mesencephalon and diencephalon declined at E10.5 (Fig. 4B, parts h,i). However, intensive ß-gal expression continued in the dorsocaudal telencephalon. With the exception of this aspect, the #29 fragment did not exhibit activity in telencephalon, as was true of the FM enhancer. The enhancer in the #29 fragment was designated as the FM2 enhancer.
After E9.5 the endogenous Otx2 expression in the dorsal
telencephalon is found in its medial aspect
(Fig. 1B, parts k,l,n-p). At
E10.5, the AN, FM and FM2 enhancers all had differential activities in this
region, the significance of which remains for future studies
(Fig. 1B, parts m,q;
Fig. 4B, part i)
(Kurokawa et al., 2004).
3' forebrain/midbrain enhancer
The #29 10 kb region was divided into MluI/SalI 2.5 kb
(MS2.5kb), SalI/SpeI 2.5 kb (SS2.5kb),
SpeI/BamHI 2.8 kb (SB2.8kb) and
BamHI/HindIII 1.2 kb (BH1.2kb) fragments to characterize the
FM2 enhancer (Fig. 5A). FM2
enhancer activity was inherited by the SpeI/BamHI 2.8 kb
fragment (Fig. 5B, part a). The
BamHI/HindIII fragment exhibited activity in the cortical
area (Fig. 5B, part b);
however, this activity was not observed with the BAC #2 or the #29 fragment.
Moreover, no endogenous Otx2 expression was detected in the cortex
(Fig. 1Bi-l, n-p). Based on
these, no further analysis was conducted on the BamHI/HindIII fragment.
In the SpeI/BamHI 2.8 kb fragment, the 5' 700 bp region is well conserved between mouse and human with sequence identity of 81%; however, no region homologous to the 3' 2.1 kb region (NCR) exists in the human or Xenopus genome. Sequence identity of the NCR region between mouse and rat is 83%. Unexpectedly, FM2 enhancer activity was present in this NCR region (Fig. 5A). Subsequently, the NCR was divided into HincII 1.3 kb and BsmI/BamHI 1 kb (BB1.0kb) fragments. The BB 1.0 kb subfragment retained enhancer activity of expression in forebrain and midbrain. Moreover, its sequence identity with the rat counterpart region was 83%. This fragment, however, exhibited intense ectopic activity in the trunk dorsal root ganglion (DRG). This finding was confirmed via generation of permanent transgenic lines (Fig. 5B, part d). Expression in DRG was faint with the SB 2.8 kb fragment (arrowhead in Fig. 5B, part a) and moderately increased with NCR. ß-Gal expression was observed at E8.5 in midbrain with the BB 1.0 kb fragment to an extent similar to that of the #29 fragment (Fig. 4B, part e). Expression persisted until E10.5, after which it gradually decreased.
Further deletion of the BsmI/HincII 127 bp region at the 5' end of the BB 1.0 kb fragment drastically reduced enhancer activity (HB 0.9 kb; Fig. 5A and 5B, part e), although residual activity remained in midbrain. Thus, the 5' region is essential for FM2 activity. This region displays 91% sequence identity between mouse and rat and includes three TCF/LEF binding sites, all of which are conserved in the rat Otx2 locus (Fig. 5C). Mutations in all three TCF/LEF binding sites nearly abolished the enhancer activity of the mouse BB 1.0 kb fragment (Fig. 5B, part f). However, the BP 200 bp fragment (Fig. 5A) containing BsmI/HincII 127 bp failed to capture FM2 activity, but directed disorganized expression (Fig. 5B, part g).
It has been shown that enhancer activity can be conserved in spite of the
high divergence of the sequences overall if the core sequences for
transcription are conserved (Flint et al.,
2001; Müller et al.,
2002
). NCR demonstrating FM2 activity occurs between the domains
conserved in the human genome (blue boxes in
Fig. 5A). This region in the
human genome (human 3.0 kb) was isolated by PCR employing conserved sequences
(indicated by P in blue boxes of Fig.
5C) and was then tested for enhancer activity. No consensus
sequences for TCF binding sites exist in the human 3.0 kb, nor was any FM
activity detected with human 3.0 kb (Fig.
5B, part c).
Targeted disruption of FM2 enhancer activity
In order to confirm that Otx2 expression in forebrain and midbrain
is also governed by the FM2 enhancer later than E8.5, a mutant was generated
in which the BsmI/BamH1 1.0 kb region was replaced with a
cassette encoding a neomycin-resistant gene
(Fig. 6A). The homozygous
mutant, Otx2FM2/
FM2, demonstrated normal
forebrain and midbrain development (Fig.
6B, parts a,b). However, the mutant in which one Otx2
allele displayed deletion of the FM2 enhancer and in which the other allele
was null, Otx2
FM2/, exhibited defects in
midbrain and forebrain development (Fig.
6B, parts c,d) similar to those observed in
Otx2
FM/
(Fig. 3B). Marker analyses
indicated that the defects in the Otx2
FM2/
mutant were milder (Fig. 6C)
than those in the Otx2
FM/
(Fig. 3C). Reductions of the
Emx2-negative, Pax6-positive region and
Pax6-negative, Otx2-positive regions were minimal, and the
anterior shift and expansion of the Fgf8-positive isthmus was less
pronounced.
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Based on these comparative genomic analyses, the existence of enhancer activity in domain ß of zebrafish Otx2 was determined. A 0.6 kb zebrafish Otx2 region covering the 170 bp (Fig. 8B) in the middle was isolated by PCR. Using the 1.8 kb promoter, this 0.6 kb region clearly directed ß-gal expression in mouse forebrain and midbrain (Fig. 8A, part b).
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Discussion |
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Enhancers of Otx2 expression in forebrain and midbrain
The separation of the enhancer of Otx2 expression in forebrain and
midbrain later than E8.5 from the AN enhancer was unexpected. Otx2
expression initially covers the entire anterior neuroectoderm, but is
subsequently lost in the most anterior region corresponding to the dorsal
telencephalon. This change can be explained readily by the transition from
Otx2 expression under the AN enhancer to that under the FM and FM2
enhancers. ß-gal expression under the AN enhancer is evident at E8.0, but
not at E8.5. FM and FM2 enhancer activities are not found at E8.0 over the
entire anterior neuroectoderm, but become apparent at E8.5. ß-Gal
expression is sensitive and stable, usually persisting longer than the
expression of endogenous gene products. However, the AN enhancer must retain
the activity at the stage when the Emx2 expression takes place
(three-somite stage) as demonstrated by
Otx2AN/
ANEmx2/
mutants (Kurokawa et al.,
2004
). The timing of the switch from the AN enhancer to FM/FM2
enhancers corresponds to the stage critical for the regionalization of the
rostral brain (see Introduction).
More unexpected was the presence of two distinct enhancers, FM and FM2, of
Otx2 expression in forebrain and midbrain. Not only are the overall
sequences not conserved between FM and FM2 enhancers, there are no apparent
conserved sequences between them except for TCF/LEF-binding sites. The overall
sequences in the FM enhancer region are conserved in mouse, human and
Xenopus. The FM2 enhancer, which is not conserved in human or
Xenopus, is most probably unique to the rodent. This raises questions
for future studies regarding whether human OTX2 functions without the
second FM2 enhancer or whether the human OTX2 gene acquired the
second FM enhancer independently. Does the second FM enhancer exist in avian
or Xenopus? Moreover, it is possible that the putative second EP/AN
enhancer that failed to be identified by Kurokawa et al.
(Kurokawa et al., 2004) is
also unique to the rodent.
Organization of Otx2 enhancers
Analysis of Otx homologues in lamprey suggests that the ancestral
vertebrate originated with a single copy of the Otx gene and that
divergence into the Otx2 and Otx1 lineages occurred in the
gnathostome lineage (Ueki et al.,
1998; Williams and Holland,
1998
; Tomsa and Langeland,
1999
; Germot et al.,
2001
). In the tetrapod lineage, Otx2 is thought to have
retained its ancestral roles. By contrast, Otx1 appears to have been
co-opted for more complex functions associated with vertebrate brains
(Acampora et al., 1996
;
Acampora et al., 1999
;
Suda et al., 1999
;
Morsli et al., 1999
;
Mazan et al., 2000
). However,
the specialization of Otx2 and Otx1 functions appears quite
divergent in the lineage leading to the extant teleosts
(Li et al., 1994
;
Mori et al., 1994
;
Mercier et al., 1995
). In
light of the essential roles of Otx in head development, the question
of how the organizations of Otx enhancers in each vertebrate are
related is one of keen interest. The mouse Otx1 locus has no regions
homologous to the mouse Otx2 enhancers identified.
In the genomic region surrounding Otx2-coding region, 22 domains
are conserved in mouse, human and Xenopus. The organization of these
domains is also conserved among these animals
(Kurokawa et al., 2004), and
probably throughout all tetrapods. Furthermore, nine and five of these domains
are conserved in FrOtx2a and zebrafish Otx2, respectively,
in the same array (Fig. 8A). To
consider these Otx2-cis elements in evolutional terms, Otx2
genome information is desired for coelacanth/lungfish, shark and lamprey. With
the exception of the FM2 enhancer, all Otx2 enhancers identified
correspond to one of these 22 domains; enhancer activities were confirmed in
six domains in mouse in the present and accompanying study
(Kurokawa et al., 2004
). Many
other domains may also exhibit enhancer activities at later stages.
Visceral endoderm is unique to mammals; the neural crest cells responsible
for generation of cephalic mesenchyme arose with vertebrate evolution.
Moreover, mesendoderm is common to chordates
(Satou et al., 2001). In
Xenopus Otx2 and zebrafish Otx2, the regions homologous to
the mouse non-ectodermal enhancers (domain
) also exist near the coding
region; it remains for future studies to determine whether the domains retain
these non-ectodermal enhancer activities. By contrast, non-ectodermal
enhancers occur at about 3' 15 kb downstream in FrOtx2a, and
overall sequences are not conserved
(Kimura et al., 1997
)
(C.K.-Y., I.M. and S.A., unpublished).
EP and AN enhancer regions are not conserved in either zebrafish or
pufferfish (see Kurokawa et al.,
2004). The lack of conservation of the AN enhancer region in these
fish genomes was particularly unexpected. Otx2 alone in tetrapod and
all Otx genes in zebrafish are expressed in the anterior
neuroectoderm. By contrast, the FM enhancer region (domain ß) is deeply
conserved in gnathostome Otx2. Obviously, discussion of the
phylogenetic significance of the non-conservation of the EP/AN enhancer region
and the conservation of the FM region requires the identification of the
second mouse EP/AN enhancer. Nevertheless, the differences in Otx2
expression in anterior neuroectoderm in teleost and tetrapod is notable. In
tetrapods, Otx2 is initially expressed in the entire neuroectoderm
(under AN enhancer) and subsequently the expression is lost in the anterior
region, which corresponds to the dorsal telencephalon (under FM enhancer). In
zebrafish, Otx2 is not expressed in the most anterior part of the
neuroectoderm, even at the earliest phase
(Li et al., 1994
;
Mori et al., 1994
). It is
tempting to speculate that in fish the FM enhancer might function also as the
AN enhancer. A comprehensive analysis of Otx enhancers in fish is
awaited (Kimura et al., 1997
;
Kimura-Yoshida et al.,
2004
).
Otx2 functions in forebrain/midbrain and upstream regulators
After an analysis of
Otx1+/Otx2+/
double mutants, we reported previously that Otx2 cooperates with
Otx1 for midbrain development
(Suda et al., 1997). The
cooperation, however, could not be examined in
Otx1/Otx2+/,
Otx1+/Otx2/
or Otx1/Otx2/
mutants because of earlier visceral endoderm defects in
Otx2/ mutants and postnatal lethality of
Otx1+/Otx2+/
mutants. This situation obscured the onset and the extent of the cooperation
between Otx1 and Otx2 in rostral brain development
(Acampora et al., 1997
;
Suda et al., 1997
). With the
AN and FM/FM2 enhancers and their mutants, we now propose that the initial
brain regionalization occurs around the three-somite stage under the AN
enhancer, at a stage when FM and FM2 enhancers are still inactive (see
Kurokawa et al., 2004
).
Metencephalic, mesencephalic, diencephalic and telencephalic regions
differentiate at this stage; however, these regions are not yet determined
when Otx2 expression under FM and FM2 enhancers takes place. In the
most severe
Otx1/Otx2
FM/
FM
mutants, which lacked most of mesencephalon and diencephalon at E10.5, their
primordia or the Otx2-, Emx2- and Pax2-positive
regions developed normally at the 5 somite stage.
Even in the most severe
Otx1/Otx2FM/
FM
mutants Emx2-positive telencephalon developed normally. Apparently,
the cooperation between Otx1 and Otx2 under FM and FM2
enhancers does not participate in telencephalon development. This is
consistent with the lack of Otx2 expression under these enhancers in
dorsal telencephalon and of Otx1 expression in ventral telencephalon
(Frantz et al., 1994
).
Moreover, an analysis of the mutants suggests that the future telencephalic
region has already differentiated when Otx1 cooperates with
Otx2 under FM and FM2 enhancers. However, the precise anterior limit
of the regions affected by the loss of the cooperation remains to be
determined.
In Otx2FM/ and
Otx2
FM2/ mutants, Fgf8-positive
isthmus expanded at E10.5 and cerebellum was enlarged at E15.5 as in
Otx1+/ Otx2+/ mutants
(Suda et al., 1997
). In a
series of double mutants in Fig.
7A, the lower the Otx dose and the more severe the
phenotype, the more expanded was the Fgf8-positive ithmus. Most
probably, mesencephalic and diencephalic regions that initially differentiated
at the three-somite stage were secondarily transformed into metencephalon in
these mutants (Acampora et al.,
1997
; Suda et al.,
1997
). Interestingly, however, Fgf8-positive isthmus was
lost in the most severe
Otx1/Otx2
FM/
FM
mutant (Fig. 7B, part c). It
has been reported that the induction of the Fgf8 expression in
isthmus is independent of Otx2; rather, that Otx2, along
with Gbx2, refines the Fgf8 expression
(Li and Joyner, 2001
;
Martinez-Barbera et al., 2001
;
Ye et al., 2001
). However,
these are observations made in the presence of Otx1 expression.
Moreover, Fgf8 expression is also lost in the anterior neural ridge
of Otx1/Otx2
FM/
FM
mutant. The details of the double mutant defects warrant examination in future
studies.
The double mutants in Fig.
7A also indicate that OTX dose dependence differs regionally, i.e.
the posterior mesencephalon requires elevated OTX for development. The onset
of FM and FM2 enhancer activities, at around E8.5, coincides with the stage
when broad, faint Fgf8 expression contracts and the boundary between
the midbrain and the hindbrain or the isthmus is established. A higher OTX
dose might be required in the more posterior portion of the forebrain/midbrain
to suppress posteriorizing signals from isthmus and/or anterior hindbrain
(Martinez et al., 1999). The
caudal end of the activities of both FM and FM2 enhancers coincides with the
midbrain/hindbrain boundary. An attempt to identify the sequences that
delineate the expression at the boundary is currently under way.
The identification of direct upstream regulators of Otx2
expression at each site and at each stage is also an objective of our enhancer
analysis. Potential TCF/LEF binding sites are essential in both FM and FM2
enhancers, whereas a putative OTX-binding site is additionally required in the
FM enhancer. These sites in the FM enhancer are conserved in mouse, human,
Xenopus, pufferfish and zebrafish; one TCF/LEF-binding site is
converted into a potential SOX-binding site in the fish. It is probable that
Otx2 expression under the FM enhancer is initiated by OTX2 directed
by the AN enhancer. Subsequently Otx2 expression under the FM and FM2
enhancers may be regulated by the WNT and TCF cascade. Wnt and
Tcf/Lef expressions appear in dorsal forebrain/midbrain following
E8.5 and in the cortical hem following E10
(Parr et al., 1993;
Galceran et al., 2000
;
Lee et al., 2000
).
This investigation, however, presents only a starting point in terms of the
elucidation of many unknown factors that directly regulate these enhancers. AN
enhancer analysis by Kurokawa et al.
(Kurokawa et al., 2004) has
been most successful in narrowing the focus of the essential, sufficient
region; however, this essential region is still 90 bp long. The limited size
of mouse anterior neuroectoderm also limits the biochemical identification of
factors that bind to the 90 bp sequence. Full FM activity is harbored in the
ApaI/HindIII 1.4 kb fragment; a 364 bp region within this
fragment was essential to the activity and could not be shortened further. The
FM region is also present in zebrafish Otx2, FrOtx2a and
FrOtx2b, where the TCTAATTAAAAWGGATA sequence is conserved, the
significance of which remains for future studies. The
BsmI/BamH1 1.0 kb region displayed full FM2 enhancer
activity. Within this region, BsmI/HincII127bp was essential
for FM2 activity; however, the 127 bp fragment alone did not exhibit this
activity, and neither did the 3.0 kb FM2 region in the human OTX2
locus. Moreover, the EP enhancer required multiple domains exceeding 2.3 kb.
Enhancers that regulate expression in vivo during development may commonly
exhibit such complexity, meaning that novel strategies will be required to
identify critical upstream regulators with such enhancers.
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ACKNOWLEDGMENTS |
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REFERENCES |
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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.[Medline]
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., Tuorto, F., Barone, P., Perera,
M., Choo, D., Wu. D., Corte, G. and Simeone, A.
(1999). Differential transcriptional control as the major
molecular event in generating Otx1/ and
Otx2/ divergent phenotypes.
Development 126,1417
-1426.
Ang, S.-L., Conlon, R. A., Jin, O. and Rossant, J.
(1994). Positive and negative signals from mesoderm regulate the
expression of mouse Otx2 in ectoderm explants.
Development 120,2979
-2989.
Araki, I. and Nakamura, H. (1999). Engrailed
defines the position of dorsal di-mesencephalic boundary by repressing
diencephalic fate. Development
126,5127
-5135.
Bosse, A., Zulch, A., Becker, M. B., Torres, M., Gomez-Skarmeta, J. L., Modolell, J. and Gruss, P. (1997). Identification of the vertebrate Iroquois homeobox gene family with overlapping expression during early development of the nervous system. Mech. Dev. 69,169 -181.[CrossRef][Medline]
Bouillet, P., Chazaud, C., Oulad-Abdelghani, M., Dolle, P. and Chambon, P. (1995). Sequence and expression pattern of the Stra7 (Gbx-2) homeobox-containing gene induced by retinoic acid in P19 embryonic carcinoma cells. Dev. Dyn. 204,372 -382.[Medline]
Brannon, M., Gomperts, M., Sumoy, L., Moon, R. T. and Kimelman,
D. (1997). A ß-catenin/XTcf-3 complex binds to the
siamois promoter to regulate dorsal axis specification in Xenopus.
Genes Dev. 11,2359
-2370.
Broccoli, V., Boncinelli, E. and Wurst, W. (1999). The caudal limit of Otx2 expression positions the isthmic organizer. Nature 401,164 -168.[CrossRef][Medline]
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, S. and Martin, G. R. (1996). Midbrain development induced by FGF8 in the chick embryo. Nature 380,66 -68.[CrossRef][Medline]
Czerny, T. and Busslinger, M. (1995). DNA-binding and transcription properties of Pax-6, Three amino acids in the paired domain are responsible for the different sequence recognition of Pax-6 and BSAP (Pax-5). Mol. Cell. Biol. 15,2858 -2871.[Abstract]
Flint, J., Tufarelli, C., Peden, J., Clark, K., Daniels, R. J.,
Hardison, R., Miller, W., Philipsen, S., Tan-Un, K. C., McMorrow et
al. (2001). Comparative genome analysis delimits a
chromosomal domain and identifies key regulatory elements in the
globin cluster. Hum. Mol. Genet.
10,371
-382.
Frantz, G. D., Weimann, J. M., Levin, M. E. and McConnell, S. K. (1994). Otx1 and Otx2 define layers and regions in developing cerebral cortex and cerebellum. J. Neurosci. 14,5725 -5740.[Abstract]
Galceran, J., Miyashita-Lin, E. M., Devaney, E., Rubenstein, J.
L. R. and Grosschedl, R. (2000). Hippocampus
development and generation of dentate gyrus granule cells is regulated by
LEF1. Development 127,469
-482.
Germot, A., Lecointre, G., Plouhinec, J.-L., le Mentec, C.,
Girardot, F. and Mazan, S. (2001). Structural
evolution of Otx genes in craniates. Mol. Biol. Evol.
18,1668
-1678.
Inoue, T., Nakamura, S. and Osumi, N. (2000). Fate mapping of the mouse prosencephalic neural plate. Dev. Biol. 219,373 -383.[CrossRef][Medline]
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 suppression posteriorizing signals. Dev. Biol. 225,304 -321.[CrossRef][Medline]
Kimura-Yoshida, C., Kitajima, K., Oda-Ishii, I., Tian, E.,
Suzuki, M., Yamamoto, M., Suzuki, T., Kobayashi, M., Aizawa, S. and
Matsuo, I. (2004). Characterization of the pufferfish
Otx2 cis-regulators reveals evolutionarily conserved genetic
mechanisms for the vertebrate head specification.
Development 131,57
-71.
Kobayashi, D., Kobayashi, M., Matsumoto, K., Ogura, T.,
Nakafuku, M. and Shimamura, K. (2002). Early subdivisions in
the neural plate define distinct competence for inductive signals.
Development 129,83
-93.
Kurokawa, D., Takasaki, N., Kiyonari, H., Nakayama, R.,
Kimura-Yoshida, C., Matsuo, I. and Aizawa, S. (2004).
Regulation of Otx2 expression and its functions in mouse epiblasts
and anterior neuroectoderm. Development
131,3307
-3317.
Lee, S. M. K., Tole, S., Grove, E. and McMahon, A. P.
(2000). A local Wnt-3a signal is required for development of the
mammalian hippocampus. Development
127,457
-467.
Li, J. Y. H. and Joyner, A. L. (2001). Otx2 and Gbx2 are required for refinement and not induction of mid-hindbrain gene expression. Development 128,4979 -4991.[Medline]
Li, Y., Allende, M. L., Finkelstein, R. and Weinberg, E. S. (1994). Expression of two zebrafish orthodenticle-related genes in the embryonic brain. Mech. Dev. 48,229 -244.[CrossRef][Medline]
Mao, C.-A., Gan, L. and Klein, W. H. (1994). Multiple Otx binding site required for expression of the Strongyrocentrotus purpuratus Spec2a gene. Dev. Biol. 165,229 -242.[CrossRef][Medline]
Martinez, S., Crossley, P. H., Cobos, I., Rubenstein, J. L. R.
and Martin, G. R. (1999). FGF8 induces formation of an
ectopic isthmic organizer and isthmocerebellar development via a repressive
effect on Otx2 expression. Development
126,1189
-1200.
Martinez-Barbera, J. P., Signore, M., Boyl, P. P., Puelles, E.,
Acampora, D., Gogoi, R., Schubert, F., Lumsden, A. and Simeone, A.
(2001). Regionalisation of anterior neuroectoderm and its
competence in responding to forebrain and midbrain inducing activities depend
on mutual antagonism between OTX2 and GBX2.
Development 128,4789
-4800.
Matsunaga, E., Araki, I. and Nakamura, H.
(2000). Pax6 defines the dimesencephalic 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.[Abstract]
Mazan, S., Jaillard, D., Baratte, B. and Janvier, P. (2000). Otx1 genecontrolled morphogenesis of the horizontal semicircular canal and the origin of the gnathostome characteristics. Evol. Dev. 2,186 -193.[CrossRef][Medline]
Mercier, P., Simeone, A., Cotelli, F. and Boncinelli, E. (1995). Expression pattern of two otx genes suggests a role in specifying anterior body structures in zebrafish. Int. J. Dev. Biol. 39,559 -573.[Medline]
Mertin, S., McDowall, S. G. and Harley, V. R.
(1999). The DNA-binding specificity of SOX9 and other SOX
proteins. Nucleic Acids Res.
27,1359
-1364.
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.[CrossRef][Medline]
Mori, H., Miyazaki, Y., Morita, T., Nitta, H. and Mishina, M. (1994). Different spatio-temporal expression of three otx homeoprotein transcripts during zebrafish embryogenesis. Mol. Brain Res. 27,221 -231.[Medline]
Morsli, H., Tuorto, F., Choo, D., Postiglione, M. P., Simeone,
A. and Wu, D. K. (1999). Otx1 and Otx2 activities are
required for the normal development of the inner ear.
Development 126,2335
-2343.
Müller, F., Blader, P. and Strähle, U. (2002). Search for enhancers, teleost models in comparative genomic and transgenic analysis of cis regulatory elements. BioEssays. 24,564 -572.[CrossRef][Medline]
Oliver, G., Mailhos, A., Wehr, R., Copeland, N. G., Jenkins, N.
A. 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. A., 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.
Rhinn, M., Dierich, A., Shawlot, W., Behringer, R. R., le Meur,
M. and Ang, S.-L. (1998). Sequential roles for Otx2 in
visceral endoderm and neuroectoderm for forebrain and midbrain 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. 52, 3-8.[CrossRef][Medline]
Satou, Y., Imai, K. S. and Satoh, N. (2001). Early embryonic expression of a LIM-homeobox gene Cs-lhx3 is downstream of ß-catenin and responsible for the endoderm differentiation in Ciona savignyi embryos. Development 128,3559 -3570.[Medline]
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.[CrossRef][Medline]
Shimamura, K. and Rubenstein, J. L. (1997).
Inductive interactions direct early regionalization of the mouse forebrain.
Development 124,2709
-2718.
Simeone, A., Acampora, D., Gulisano, M., Stornaiuolo, A. and Boncinelli, E. (1992). Nested expression domains of four homeobox genes in the developing rostral brain. Nature 358,687 -690.[CrossRef][Medline]
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 in the gastrulating mouse embryo. EMBO J. 12,2735 -2747.[Abstract]
Suda, Y., Matsuo, I., Kuratani, S. and Aizawa, S.
(1996). Otx1 function overlap with Otx2 in development of mouse
forebrain and midbrain. Genes Cells
1,1031
-1044.
Suda, Y., Matsuo, I. and Aizawa, S. (1997). Cooperative between Otx1 and Otx2 genes in developmental patterning of rostral brain. Mech. Dev. 69,125 -141.[CrossRef][Medline]
Suda, Y., Nakabayashi, J., Matsuo, I. and Aizawa, S.
(1999). Functional equivalency between Otx2 and Otx1 in
development of the rostral head. Development
126,743
-757.
Suda, Y., Hossain, Z. M., Kobayashi, C., Hatano, O., Yoshida,
M., Matsuo, I. and Aizawa, S. (2001). Emx2 directs the
development of diencephalon in cooperation with Otx2.
Development 128,2433
-2450.
Tian, E., Kimura, C., Takeda, N., Aizawa, S. and Matsuo, I. (2002). Otx2 is required to respond to signals from anterior neural ridge for forebrain specification. Dev. Biol. 242,204 -223.[CrossRef][Medline]
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.[Medline]
Tomsa, J. M. and Langeland, J. A. (1999). Otx expression during lamprey embryogenesis provides insights into the evolution of the vertebrate head and jaw. Dev. Biol. 207, 26-37.[CrossRef][Medline]
Ueki, T., Kuratani, S., Hirano, S. and Aizawa, S. (1998). Otx cognates in a lamprey, Lampetra Japonica. Dev. Genes Evol. 208,223 -228.[CrossRef][Medline]
Walther, C. and Gruss, P. (1991). Pax-6, a murine paired box gene, is expressed in the developing CNS. Development 113,1435 -1449.[Abstract]
Wassarman, K. M., Lewandoski, M., Campbell, K., Joyner, A.
L., Rubenstein, J. L. R., 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.
Williams, N. A. and Holland, P. W. H. (1998). Gene and domain duplication in the chordate Otx gene family, Insights from amphioxus Otx. Mol. Biol. Evol. 15,600 -607.[Abstract]
Yamamoto, M., Meno, C., Sakai, Y., Shiratori, H., Mochida, K.,
Ikawa, Y., Saijoh, Y. and Hamada, H. (2001). The
transcription factor FoxH1 (FAST) mediates Nodal signaling during
anterior-posterior patterning and node formation in the mouse.
Genes Dev. 15,1242
-1256.
Yang, X. W., Model, P. and Heintz, N. (1997). Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat. Biotech. 15,859 -865.[Medline]
Ye, W., Bouchard, M., Stone, D., Liu, X., Vella, F., Lee, J., Nakamura, H., Ang, S.-L., Busslinger, M. and Rosenthal, A. (2001). Distinct regulators control the expression of the mid-hindbrain organaizer signal FGF8. Nat. Neurosci. 4,1175 -1181.[CrossRef][Medline]
Yoshida, M., Suda, Y., Matsuo, I., Miyamoto, N., Takeda, N.,
Kuratani, S. and Aizawa, S. (1997). Emx1 and Emx2 functions
in development of dorsal telencephalon. Development
124,101
-111.
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