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, Emx2, Enhancer, Epiblasts, Anterior neuroectoderm, Mouse
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Introduction |
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Mutant studies in mouse have suggested that Otx2 plays essential
roles in head development (Acampora et al.,
1995; Acampora et al.,
1997
; Acampora et al.,
1998
; Ang et al.,
1996
; Matsuo et al.,
1995
; Rhinn et al.,
1998
; Rhinn et al.,
1999
; Suda et al.,
1996
; Suda et al.,
1997
; Suda et al.,
1999
; Suda et al.,
2001
; Tian et al.,
2002
; Kimura et al.,
1997
; Kimura et al.,
2000
; Kimura et al.,
2001
). However, owing to its expression in multiple sites, it has
not to date been possible to distinguish clearly the functions of
Otx2 in each site. In addition, information is scarce regarding the
upstream factors that regulate Otx2 expression at each developmental
site. In light of the essential roles played by Otx genes in head development,
it will be important to develop an understanding of how mechanisms governing
the regulation of Otx expression in each vertebrate are related and
altered.
Previously, we have identified mouse enhancers of Otx2 expression
in visceral endoderm (VE), definitive anterior mesendoderm (AME) and cephalic
neural crest cells or mesenchyme (CM); the sites located proximal to the
Otx2-coding region (Kimura et
al., 1997; Kimura et al.,
2000
). However, no ectodermal enhancers were present in this
proximal region. Enhancers of Otx2 expression in epiblast, anterior
neuroectoderm and forebrain/midbrain were then sought by BAC transgenesis, and
indications were that they existed within the 170 kb 5' upstream
(A of the ATG translation start codon is taken as +1 bp). In the present
study, we have analyzed the enhancers of Otx2 expression in epiblast
(EP) and anterior neuroectoderm (AN); analysis of the enhancers of
Otx2 expression in forebrain and midbrain is described in an
accompanying paper (Kurokawa et al.,
2004
). EP and AN enhancers were identified at about 90 kb 5'
upstream. The EP activity was found in the inner cell mass of the blastocyst
stage embryos and the entire epiblast of egg cylinder stage embryos. AN
enhancer activity was detected initially at E7.0 and had ceased by E8.5. The
EP enhancer included multiple required domains over 2.3 kb, and the AN
enhancer was an essential component of the EP enhancer. Mutants lacking the AN
enhancer were generated to demonstrate that these cis-sequences do indeed
regulate Otx2 expression in EP and AN, but additional EP and AN
enhancers must exist outside of the 170 kb to +120 kb range.
Otx2 expression under the AN enhancer was essential to maintaining
the anterior neuroectoderm once induced. Furthermore, Otx2 under the
AN enhancer cooperated with Emx2 in diencephalon development.
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Materials and methods |
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PCR mutagenesis
Utilizing the PCR-based overlap extension method
(Kucharczuk et al., 1999),
each 15 bp block of EB165 bp AN core enhancer (#1-#11 in
Fig. 4B) was replaced with the
linker sequence 5'-AGACTGGATCCTGTG-3'. Putative transcription
factor binding sites in each enhancer were also mutated by the transverse
substitution of nucleotides using this approach.
|
Generation of AN enhancer knockout mice
A neomycin resistance gene with Pgk1 promoter and SV40 polyadenylation
signal was flanked by loxP sequences. This cassette (Neo) was replaced with
the SpeI/BglII 559 bp region, which harbors the AN enhancer.
Lengths of the homologous regions were 8.0 kb and 4.0 kb at the 5' and
3' sides of the neo cassette, respectively. Diphtheria toxin A
fragment gene with MC1 promoter was used for the negative selection of
homologous recombinants as described (Yagi
et al., 1993b). The targeting vector was linearized with
SalI and electroporated into TT2 ES cells
(Yagi et al., 1993a
).
Homologous recombinants were identified as those that generate 4.0 kb products
by PCR with 5'-ATCGCCTTCTTGACGAGTTCTTCTG-3' in the neo
gene as the 5' side primer and
5'-GATCCTCCTGCCTCTGCGTCAATAGC-3' as the 3' side primer; the
3' primer is located outside of the 3' side 4kb homologous region
in Otx2 locus. The recombinants were confirmed by Southern blot
analyses as described (Matsuo et al.,
1995
). Two mutant mouse strains were generated from independent
homologous recombinant ES clones. The genotype of mutant mouse or embryo was
routinely determined by PCR using tail or yolk sac specimens. Wild-type allele
was detected with the sense primer of the
5'-CTGCAGATGCTTGGGCTTTCTGCAGC-3' and the antisense primer of the
5'-GGGGTCTTCATGAGTTTCTGTTGCAC-3'. The mutant allele was identified
with the sense primer of 5'-ATCGCCTTCTTGACGAGTTCTTCTG-3' in the
neo gene and the antisense primer. The deletion of the neo
insert by Cre-mediated loxp recombination was accomplished by mating
Otx2+/
AN mice with Lefty-Cre mice
(Yamamoto et al., 2001
).
Histological analysis
Mouse embryos were fixed overnight with Bouin's fixative solution at room
temperature. Specimens were subsequently dehydrated and embedded in paraplast.
Serial sections (10 µm) were prepared and stained with Hematoxylin and
Eosin or with 0.1% Cresyl Violet.
RNA in situ hybridization
Whole-mount and section in situ hybridization was performed using
digoxigenin probes, as described by Suda et al.
(Suda et al., 2001). The
probes used in this study were for the genes: brachyury/T
(Herrmann, 1991
),
Fgf8 (Crossley and Martin,
1995
), Cer1 (Belo et
al., 1997
), Dlx1
(Bulfone et al., 1993
)
Emx2 (Yoshida et al.,
1997
), Gbx2 (Bulfone
et al., 1993
), Lhx1/Lim1
(Fujii et al., 1994
),
Otx2 (Matsuo et al.,
1995
), Pax6 (Walther
and Gruss, 1991
), Six3
(Oliver et al., 1995
) and
Tcf4 (Korinek et al.,
1998
).
Quantitative RT-PCR
RNA isolation and reverse transcription PCR was performed according to
Kimura et al. (Kimura et al.,
2001). Primer sets used were as follows: Otx2,
5'-TCTTATCTAAAGCAACCGCCTTACGCAGTC-3' and
5'-GCACCCTGGATTCTGGCAAGTTGATTTTCA-3'; glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), 5'-TGTCATCAACGGGAAGCCCA-3' and
5'-TTGTCATGGATGACCTTGGC-3'.
Animal housing
Animals were housed in environmentally controlled rooms in accordance with
the Institute of Physical and Chemical Research (RIKEN) guidelines for animal
experiments.
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Results |
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The 5'BAC#1/lacZ transgenic mouse line captured
ß-gal expression in E6.5 epiblast, E7.75 anterior neuroectoderm and E9.5
forebrain and anterior midbrain (Fig.
1B, parts a-c). ß-Gal expression was not detected in
posterior midbrain (Fig. 1B,
part c). Subsequently, the Otx2 genomic region encompassing
170 kb to 30 kb was systematically dissected into 15 fragments
with partial overlaps at both ends to determine the locations of regulatory
elements (Fig. 2A). All
enhancer analyses described below were conducted using the mouse Otx2
1.8 kb promoter. Expression by this promoter
(Fig. 1B, part d) is indicated
by arrows in each figure. For several DNA fragments, enhancer activities were
also assayed using Fugu Otx2 promoter
(Kimura et al., 1997) or
hsp68 promoter. For example, an EcoRV/BglII 2.3kb
fragment capable of driving lacZ expression in epiblast with the 1.8
kb promoter (Fig. 3B, parts
b-d) exhibited the activity, though weak, using the hsp68 promoter
(Fig. 3B, parts e,f). An
enhancer in the #13 fragment (Fig.
2A) that had the transcriptional activity in forebrain and
midbrain with the 1.8 kb promoter showed a similar activity with the
Fugu promoter. By contrast, a SpeI 6.2 kb fragment
(Fig. 3A) that exhibited
activity in the anterior neuroectoderm did not show the same activity with the
Fugu promoter, and exhibited low activity in the ectoderm and ectopic
activity in extra-embryonic ectoderm with the hsp68 promoter (data
not shown). Moreover, a EcoRV/BglII 2.3 kb fragment
(Fig. 3A) exhibited similar
activities in the anterior neuroectoderm with the hsp68 promoter and
mouse 1.8 kb promoter (Fig. 3B,
parts g,h). Thus, the enhancer activity of several DNA fragments described
below is promoter dependent. The details of the promoter dependence, however,
remain a topic for future studies. Note that, in the present paper, the term
`enhancer' is used in the broad sense of `cis-regulatory sequence'.
|
The activity of the #12 fragment was initially noted in entire epiblast; at E6.5 it was absent in the posterior region (Fig. 2B, parts a,b). At E7.75, ß-gal expression was restricted to the anterior neuroectoderm, which corresponds to the presumptive forebrain and midbrain (Fig. 2B, parts c,d); this activity was lost by E8.5 (Fig. 2B, part e). After E9.0, the fragment exhibited activity in the most dorsomedial aspect of the telencephalon and in the eyes (Fig. 2B, part f). From this activity profile, the enhancer in the #12 fragment was designated as the EP/AN (epiblast/anterior neuroectoderm) enhancer.
The expression of ß-gal in the epiblast at the Otx2 locus is
noteworthy. Endogenous Otx2 is expressed in epiblast. However,
ß-gal expression is not observed in the epiblast of
Otx2+/lacZ embryos
(Fig. 2B, part g)
(Boyl et al., 2001;
Courtois et al., 2003
). By
contrast, ß-gal expression was evident in epiblast with the BAC
#1/lacZ transgene (Fig.
1B, part a) and with the lacZ transgene under the #12
fragment (Fig. 2B, parts a,b),
respectively. The #12 fragment also drove ß-gal expression in epiblast
under the Otx2+/ background (data not shown); the
discrepancy is not due to the Otx2 dose effect. Translational control
has been suggested in the expression of transgenes knocked-in into
Otx2 locus (Boyl et al.,
2001
). However, in both the lacZ knock-in mutation into
the Otx2 locus and the random lacZ transgenesis in the
enhancer analysis, the lacZ gene was fused to the Otx2
5' untranslated region and SV40 3'UTR in exactly the same
manner. The cause of these variations of ß-gal expression in epiblast is
unclear.
Epiblast enhancer
The 15 kb #12 fragment was subdivided by SpeI digestion
(Fig. 3A) in order to define
the EP/AN enhancer. The enhancer activity of each subfragment was determined
by transient transgenic assay at E6.5 and E7.75. However, none of these
SpeI fragments drove ß-gal expression in epiblast at E6.5.
Subsequently, the #12 fragment was re-subdivided into five fragments so that
each SpeI site was preserved (Fig.
3A). In this subdivision, ß-gal expression in epiblast was
driven by the EcoRV/BglII 2.3 kb fragment
(Fig. 3A), which was confirmed
by generation of permanent transgenic lines. Activity of the
EcoRV/BglII 2.3 kb fragment was initially found in the inner
cell mass of blastocysts at E3.5 (Fig.
3B, part a) and in the entire epiblast at E5.5
(Fig. 3B, part b), and
gradually declined in the posterior region with the progress of gastrulation
in a manner similar to endogenous Otx2 expression
(Fig. 3B, parts c-f). At the
headfold stage the fragment exhibited the activity in anterior neuroectoderm
(Fig. 3B, parts g,h).
Unexpectedly, however, when the EcoRV/BglII 2.3 kb fragment was subdivided further, fragments that lacked any of the following sequences lost their activity: (1) EcoRV/HindIII 0.95 kb, (2) ScaI/NspI 0.6 kb, (3) NspI/EcoT22I 0.57 kb or (4) EcoT22I/BglII 165 bp (Fig. 3A). Thus, all of these domains were essential for enhancer activity in epiblast. Regions homologous to the EcoRV/BglII 2.3 kb fragment exist at similar positions relative to the Otx2 coding region in the rat, human and Xenopus (Silurana) tropicalis (hereafter, with regard to the genome information, `Xenopus' refers to this species) genomes (see Fig. 7). Regions displaying greater than 80% sequence identity over more than 100 bp are present throughout the EcoRV/BglII 2.3 kb fragment (Fig. 3A).
Anterior neuroectoderm enhancer
In contrast to the epiblast enhancer, the regulatory sequences of
Otx2 expression in anterior neuroectoderm were present in the
SpeI 6.2 kb fragment (Fig.
3A). This fragment was successively dissected as shown in
Fig. 4A, and the transient
transgenic assay at E7.75 revealed the existence of activity exclusively in
the SpeI/BglII 559 bp. With this fragment ß-gal
expression in the anterior neuroectoderm was detected initially at E7.0 and
ceased by E8.5. The caudal boundary of ß-gal expression was not distinct
near the preotic sulcus (Fig.
4C, part a; data not shown).
The SpeI/BglII 559 bp fragment was further dissected, and
finally the EcoT22I/BglII 165 bp fragment was found to
retain the enhancer activity; this was confirmed by generation of permanent
transgenic lines (Fig. 4C, part
a). A region exhibiting 92% identity to this 165 bp fragment is found about
5' 97 kb upstream in the human Otx2 locus
(Fig. 4B; see
Fig. 7A). The 5' part of
the 165 bp fragment includes the sequences TGGCGACTGAC and TGGGCGCTGGC, which
correspond to each other with the exception of two bases. Furthermore, these
sequences are conserved in the human counterpart. Five tandem repeats of these
sequences, however, did not direct ß-gal expression in anterior
neuroectoderm (Fig. 4A). In the
165 bp fragment, potential OCT/HOX (Herr
and Cleary, 1995) and SOX (van
Beest et al., 2000
) binding sites exist; however, AN activity was
unaffected by mutations in these sites.
A linker-scanner approach was then employed to define the core elements.
Each 15 bp sequence of the EcoT22I/BglII 165 bp fragment was replaced
with the BamHI linker (Fig.
4B) (Kucharczuk et al.,
1999). Replacement of any of the six 5' side 15 bp blocks
(#6-#11) affected ß-gal expression in the anterior neuroectoderm at
E7.75. Such replacements in three of these blocks (mt#6, mt#7 and mt#10)
essentially abolished the activity (Fig.
4C, part b), whereas mutations in #8, #9 and #11 demonstrated
faint ß-gal expression (Fig.
4C, part c) at the frequencies indicated in the parentheses of
Fig. 4B (asterisks). In
addition, when 75 bp sequences corresponding to #11-7 were duplicated in
tandem and combined with 1.8 k-lacZ, the transgene exhibited
significant ß-gal expression in E7.75 anterior neuroectoderm
(Fig. 4C, part d). A region
homologous to EcoT22I/BglII 165 bp is present about 5' 44 kb
upstream in the Xenopus Otx2 locus
(Fig. 7, Fig. 4B), displaying sequence
identity of 77%. Identity is 87% in the #11-#6 90 bp sequences; however, no
homologous region is present in the zebrafish or pufferfish genomes. A 1.6 kb
Xenopus DNA region covering this domain was then isolated by PCR, and
its enhancer activity was tested using the mouse 1.8 kb promoter. The 1.6 kb
region clearly directed ß-gal expression in mouse anterior neuroectoderm
(Fig. 4C, part e)
Targeted disruption of Otx2 epiblast enhancer activity
The analysis described above indicated that the
SpeI/BglII 559 bp region was essential for expression in
epiblast, though not, by itself, sufficient. This fragment is located 90
kb away from the coding region, which raised a question regarding its
involvement in the regulation of Otx2 expression in vivo. In order to
examine this point, mutant mice (Otx2
AN/
AN)
were generated in which the SpeI/BglII 559 bp was replaced
with a cassette encoding a neomycin-resistant gene
(Fig. 5A).
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In mammals, the anteroposterior axis is first generated along the
distoproximal axis. Prior to primitive streak formation, distal visceral
endoderm cells move to the future anterior and proximal epiblast cells to the
future posterior. This axis rotation does not occur in
Otx2/ mutants
(Kimura et al., 2000).
However, axis rotation proceeded normally in
Otx2
AN/
AN mutants as well as in
Otx2
AN/ embryos, in which one Otx2
allele lacks the SpeI/BglII 500 bp and the other allele is
null, as indicated by cerberus-like expression in the anterior
visceral endoderm (Fig. 5C,
part a) and T expression in the primitive streak
(Fig. 5C, part b) at E6.5. This
observation is consistent with our previous conclusion that
Otx2-positive distal visceral endoderm cells, but not
Otx2-positive epiblast, are responsible for the axis rotation defect
in Otx2/ mutants
(Kimura et al., 2000
).
Targeted disruption of Otx2 anterior neuroectoderm enhancer activity
The SpeI/BglII 559 bp was essential and sufficient for
driving Otx2 expression in anterior neuroectoderm. In
Otx2AN/
AN mutants, however, no defects were
found in the forebrain or midbrain. Otx2 expression was reduced but
not eliminated in E7.5 Otx2
AN/
AN mutants
(data not shown). Subsequently, Otx2 levels were reduced further via
the generation of Otx2
AN/ mutants. At E12.5
the Otx2
AN/ mutant phenotype was variable.
In milder cases forebrain was present though greatly deformed; in severe cases
both forebrain and midbrain were lost (Fig.
6A, part b). At E7.5 Six3-positive anterior neuroectoderm
was induced in all Otx2
AN/ mutants examined
(Fig. 6B, part b). At E8.5,
however, Six3-positive region was reduced
(Fig. 6B, part d), and
concomitantly Fgf8- or Gbx2-negative anterior neuroectoderm
was reduced but still present at E8.5 (Fig.
6B, parts f,h). The phenotype became variable at E9.5, and in
severe mutants Gbx2-negative forebrain/midbrain and
Emx2-positive forebrain were entirely missing
(Fig. 6B, parts j, l). Thus,
the SpeI/BglII 559 bp located 90 kb 5' upstream
clearly regulates Otx2 expression in anterior neuroectoderm; however,
another enhancer must also exist.
|
Search for the second EP/AN enhancer
Enhancer mutants indicated the presence of another enhancer of
Otx2 expression in epiblast and anterior neuroectoderm. Consequently,
a search was conducted for the second enhancer in the 3' region of the
Otx2 gene. BAC#2 transgenic mice, which harbor a genomic DNA region
spanning 50 kb to +120 kb 3' downstream, were generated
(Fig. 7)
(Kurokawa et al., 2004).
However, this region displayed no activity in epiblast or anterior
neuroectoderm.
Mouse Otx2 locates at chromosome14 19 cM; the nearest genes are
encoded 350 kb upstream on the 5' side and
150 kb downstream
on the 3' side (Fig. 7).
More than 100 highly conserved domains (sequence identity greater than 80%
over more than 100 bp) exist in this 500 kb span among mouse, rat and human.
Of these domains, 22 are also conserved in the Xenopus genome.
Fig. 7 depicts the locations of
these domains in the mouse, human and Xenopus Otx2 loci. The
organization is identical in mouse and human. The contiguous mapping is
partial in the Xenopus Otx2 locus; however, domains
-
,
and
locate in identical order in a more compact fashion.
The EP/AN enhancer region identified above was one of the 22 domains
conserved, . We next sought a second EP/AN enhancer among domains
-o,
and
, which exist outside of the regions analyzed
with BAC#1 and #2. Depending on the sizes of the conserved regions, the
lengths of genome DNAs isolated by PCR were 3.1, 3.3, 2.8, 3.5, 1.6, 1.9, 0.7
and 0.7 kb for
,
, µ,
,
, o,
and
domains, respectively. Subsequently, sequences were confirmed and enhancer
activity was determined with the 1.8 kb promoter by transient transgenic
assay. None of these domains, however, exhibited enhancer activity in E6.5
epiblast or E7.75 anterior neuroectoderm.
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Discussion |
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Otx2 epiblast and anterior neuroectoderm enhancers
Otx2 expression is differentially regulated in each germ layer.
The regulation of these expressions by different enhancers could be reasonably
expected; the existence of distinct VE, AME, CM and ectoderm enhancers was
anticipated. In mouse, Otx2 expression in epiblast and anterior
neuroectoderm is continuous. However, Otx2 expression in chick and in
Xenopus is consistent with discrete EP and ANE enhancers. In avian,
following the disappearance of Otx2 expression in epiblast,
expression occurs transiently in Hensen's node and in the anterior primitive
streak exclusively and appears in the anterior neuroectoderm subsequently
(Bally-Cuif et al., 1995;
Kablar et al., 1996
). AN
enhancer activity begins around E7.0 and ceases by E8.5. Of note is that the
EP enhancer also directed the expression in inner cell mass of
blastocysts.
Generally, the identification of enhancers is conducted solely by random
transgenesis. This study unequivocally demonstrated the necessity of mutating
the enhancer in vivo to determine whether and how the enhancer is responsible
for endogenous gene expression. The EP/AN enhancer mutants indicated that
another enhancer exists for Otx2 expression in epiblast and anterior
neuroectoderm. However, the second enhancer was not present within the
3' 120 kb region covered by BAC #2. The overall sequences in EP and AN
enhancer region are well conserved in mouse, human and Xenopus. Then
the second EP/AN enhancers were searched for among domains conserved in mouse,
human and Xenopus, outside of the 290 kb region covered by BAC#1 and
BAC#2. However, no EP/AN activity was detected in any of these domains. The
second enhancer might exist much farther away over the adjacent genes. At the
same time, the possibility remains that the second EP/AN enhancer exists
uniquely in rodent outside of the 290 kb genomic region, as the second
enhancer of Otx2 expression in forebrain and midbrain, FM2, is not
conserved in human or Xenopus, and is most probably unique to the
rodent (Kurokawa et al.,
2004).
The EP enhancer appears to consist of multiple domains, and the AN enhancer
is a component of the EP enhancer, the significance of which remains to be
addressed in future studies. EP and AN enhancer regions are conserved in human
and Xenopus, a Xotx2 1.6 kb region covering the AN enhancer
region exhibited the enhancer activity in mouse anterior neuroectoderm. The
EP/AN region is, however, not conserved in zebrafish or pufferfish. In
zebrafish blastoderm Otx1, but not Otx2, is expressed. Fish
blastoderm may be a product independent of the amniote epiblast. However, the
lack of conservation of the AN enhancer region in fish genome was unexpected.
Otx2 alone in tetrapod and all Otx genes in zebrafish are expressed
in the anterior neuroectoderm. It is possible that the ancient AN enhancer
widely diverged between the lineages to the extant teleost and tetrapod to
cope with the divergence in anterior neuroectoderm development, and so the
molecular machinery involved in its development might be extremely varied
between teleost and tetrapod (Kurokawa et
al., 2004). Discussion of the phylogenetic significance of
enhancer organization obviously requires the identification of the second
EP/AN enhancer. In addition, a comprehensive enhancer analysis must be
performed in fish Otx genes (Kimura et
al., 1997
; Kimura-Yoshida et
al., 2004
).
Otx2 functions in anterior neuroectoderm
The generation of enhancer mutants to allow the dissection of Otx2
functions in each site at each step of head development was a primary
objective of enhancer analysis in the present investigation. The presence of
two enhancers of Otx2 expression in anterior neuroectoderm
complicated the analysis. Nevertheless,
Otx2AN/ mutants clearly demonstrated that
Otx2 expression under the AN enhancer functioned to maintain the
anterior neuroectoderm once induced. This was originally suggested by analyses
of Otx1 knock-in mutants into Otx2 locus
(Otx2Otx1/Otx1) and a hypomorphic Otx2 mutant
(Otx2frt-neo/frt-neo)
(Suda et al., 1999
;
Li and Joyner, 2001
;
Martinez-Barbera et al., 2001
;
Tian et al., 2002
). However,
in these mutants the role of Otx2 expression in anterior mesendoderm
in the loss of the anterior neuroectoderm could not be ruled out.
Studies of Otx2+/Emx2/
mutants have previously suggested that Otx2 cooperates with
Emx2 in the development of the diencephalic region
(Suda et al., 2001). However,
the onset of the defects was not clear.
Otx2
AN/
ANEmx2/
phenotypes were quite similar to
Otx2+/Emx2/ phenotypes; at
E12.5 ventral thalamus, dorsal thalamus and anterior part of the pretectum
were lost, but the commissural region of the pretectum had developed. No
marker analyses were made, but histologically archipallium was not apparent,
while neopallium and ganglionic eminences developed. Mesencephalon was largely
normal, while cerebellum was enlarged in the double mutants. Emx2
expression takes place in the laterocaudal forebrain primordium around the
three-somite stage (E8.0) when the AN enhancer is active. Furthermore,
ß-gal expression under the AN enhancer was not detected at E8.5;
ß-gal is stable and usually persists longer than endogenous gene
products. Thus, we propose that the diencephalic region may be induced around
the three-somite stage by cooperation between Emx2 and Otx2
under the AN enhancer. With loss of this cooperation, the structures that
derive from the Emx2-positive region at the three-somite stage may be
lost.
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ACKNOWLEDGMENTS |
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