1 Head Organizer Project, Vertebrate Body Plan Group, RIKEN Center for
Developmental Biology, 2-2-3 Minatojima Minami Cho, Chuou-Ku, Kobe, Hyougo
650-0047, Japan
2 Department of Morphogenesis, Institute of Molecular Embryology and Genetics,
Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
3 Division of Transgenic Technology, Center for Animal Resources and
Development, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
4 The Center for Tsukuba Advanced Research Alliance and Institute of Basic
Medical Sciences, University of Tsukuba, Tsukuba 305-8577, Japan
5 Nutrition Division, National Research Institute of Aquaculture, Fisheries
Research Agency, Nansei-cho, Watarai-gun, Mie 516-0193, Japan
6 Animal Resources and Genetic Engineering Team and Vertebrate Body Plan Group,
RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minami Cho, Chuou-Ku,
Kobe, Hyougo 650-0047, Japan
* Author for correspondence (e-mail: isao{at}cdb.riken.go.jp)
Accepted 23 September 2003
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SUMMARY |
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Key words: Homeobox, Otx2, Pufferfish, Gene regulation, cis-region, Brain development, Evolution, Local signaling center
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Introduction |
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The vertebrate head is a complex structure, which is primarily composed of
the skull, specific sense organs, muscles, glands, skin and the brain, which
can be further subdivided into distinct and complex domains. Therefore,
vertebrate head development requires highly coordinated processes. An
understanding of the genetic mechanisms that control regional specification
and morphogenesis of the embryonic rostral brain has only recently come to
light; homeobox genes, Fgf, Wnt and other signaling molecules play an
important role in these processes (reviewed by
Rubenstein et al., 1998).
Among these genes, Otx2, a paired-type homeobox gene, has
been isolated as a mouse cognate of orthodenticle in
Drosophila (Finkelstein and
Perrimon, 1991
; Simeone et
al., 1992
). The protein sequence of the homeodomains, and their
patterns of expression, have all been highly conserved during evolution of the
animal kingdom. Notably, mouse Otx2 expression occurs earliest in the
inner cell mass at the blastocyst stage, and in the visceral endoderm and
epiblast at the prestreak stage (Acampora
et al., 1995
; Ang et al.,
1996
; Kimura et al.,
2000
; Kimura et al.,
2001
). During midstreak stages, the expression domain is
restricted to the anterior region in all three germ layers corresponding to
the presumptive rostral head (Simeone et
al., 1993
; Ang et al.,
1994
). At the pharyngula stage, Otx2 expression is
present in developing sense organs, such as the eyes, olfactory epithelium and
the inner ear, as well as in the prospective forebrain and midbrain regions
(Simeone et al., 1993
;
Mallamaci et al., 1996
).
Proper regulation and function of this gene is crucial, as the mutation of
Otx2 leads to defects in the rostral head in several developmental
processes (Acampora et al.,
1995; Acampora et al.,
1997
; Matsuo et al.,
1995
; Ang et al.,
1996
; Suda et al.,
1996
; Suda et al.,
1997
; Suda et al.,
2001
; Tian et al.,
2002
; Hide et al.,
2002
). However, very little is known about how Otx2 gene
expression is dynamically regulated. The expression of homeobox genes is
regulated primarily at the level of transcription. Accordingly, cis-elements
and trans-factors that govern spatiotemporal Otx2 expression may
provide valuable insight into the genetic pathways that control
anteroposterior axis formation, patterning of the head, and specification of
the forebrain and midbrain. Previously, we identified cis-regulatory regions
in the mouse Otx2 5'-flanking region, which are mainly
responsible for the expression patterns in the head mesenchyme, anterior
visceral endoderm and mesendoderm (Kimura
et al., 1997
; Kimura et al.,
2000
). However, we were not able to detect any cis-elements
governing embryonic expression in the forebrain, midbrain and sense organs
within the 80 kb mouse Otx2 locus (65 kb to +15 kb) (C.K.-Y.
and I.M., unpublished). This finding suggests that cis-elements of mouse
Otx2 expression must lie outside of this surveyed 80 kb region.
To unravel the difficulty of identifying Otx2 cis-regulatory
regions, we took advantage of the compact genome of the pufferfish Fugu
rubripes (Fugu), which displays a genome of 365 Mb, which is
approximately eight times smaller than the human and mouse genomes
(Brenner et al., 1993;
Aparicio et al., 2002
). The
pufferfish genome has a similar number of genes and this fish shares a common
body plan for specialized functions in higher vertebrates; consequently, it
provides an ideal system with which to investigate conserved mechanisms of
Otx2 gene regulation. Coincidentally, it has been shown that several
regulatory cis-elements have been functionally conserved between mammals and
pufferfish (Marshall et al.,
1994
; Pöpperl et al.,
1995
; Aparicio et al.,
1995
; Kimura et al.,
1997
; Camacho-Hubner et al.,
2000
; Brenner et al.,
2002
).
In the present study, the Fugu Otx2 (Fotx2) gene was cloned. In addition, endogenous Fotx2 expression was analyzed during Fugu embryogenesis. Subsequently, cis-acting regulators throughout the entire Fotx2 genomic locus were surveyed by employing transgenic mice. Many distinct cis-regulators were identified, which directed temporally and spatially specific expression, mimicking endogenous Otx2 expression. Additionally, these cis-regions possessed the ability to drive transgene expression in zebrafish embryos and contained evolutionarily conserved sequence elements. These results indicate that developmental domain-specific multiple cis-regulators control highly coordinated Otx2 expression during vertebrate rostral head development.
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Materials and methods |
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Expression analysis of Fotx2 by RT-PCR and in situ hybridization
Wild pufferfish embryos were maintained at 18°C, and sacrificed at
subsequent developmental stages for RNA preparation and in situ hybridization
as described (Suzuki et al.,
2002). Primers for Fotx2 expression,
5'-TTACGCGCGCCCAGTTAGACGTTTTGGAGG-3' and
5'-GACGCCGGGGACTGGTTCAGATGGCTTGTG-3', afforded a 586 bp product.
Primers for ß-actin expression, 5'-GGCACCGCTGCCTCCTC-3' and
5'-GCCAGGATGGAGCCTCC-3', yielded a 359 bp product. Whole-mount in
situ hybridization was conducted according to the method of Jowett and Lettice
(Jowett and Lettice,
1994
).
Transgene construction of the Fotx2 gene
All constructs used in this study were generated using standard molecular
cloning techniques (Sambrook and Russell,
2001). Generation of construct F0placZ was described
previously, as construct #11 (Kimura et
al., 1997
). Construct F1placZ was generated by insertion
of a 5' 4.0 kb EcoRV-NspV genomic fragment, blunted by
T4 DNA polymerase, into a SmaI site of construct F0placZ.
Construct F2placZ was produced by insertion of a 5' 2.5 kb
EcoRV-BamHI genomic fragment, blunted by T4 DNA polymerase,
into a SmaI site of construct F0placZ. Construct
F3placZ was generated by cloning of a 5'
SacII-BamHI genomic fragment into the SacII and
BglII sites of construct F0placZ. Construct F4placZ
was generated by insertion of a 5' 4.0 kb NotI-BamHI
genomic fragment into NotI and BglII sites of construct
F0placZ. Construct F5placZ was generated by insertion of a
5' 11.0 kb BamHI-BamHI genomic fragment into a
BglII site of construct F0placZ. Construct F6placZ
was generated by insertion of a 5' 1.5 kb NotI-BamHI
genomic fragment into NotI and BglII sites of construct
F0placZ. Construct F7placZ was produced by insertion of a
3' 2.5 kb BstEII-BamHI genomic fragment blunted by T4
DNA polymerase into a SmaI site of construct F0placZ.
Construct F8placZ was generated by insertion of a 3' 8.0 kb
XbaI-BamHI genomic fragment into XbaI and
BglII sites of construct F0placZ. F9placZ was generated by
insertion of a 3' 5.0 kb XbaI-BamHI genomic fragment
into the XbaI and BglII sites of construct F0placZ.
Construct F10placZ was produced by insertion of a 3' 9.0 kb
BamHI-NotI genomic fragment into NotI and
BglII sites of construct F0placZ. Construct
F11placZ was generated by insertion of a 3' 5.5 kb
NotI-BamHI fragment into NotI and BamHI
sites of construct F0placZ. Construct F12placZ was generated
by insertion of a 3' 6.3 kb BamHI-BamHI fragment into
a BglII site of construct F0placZ. Construct
F13placZ was produced by insertion of a 4.8 kb
NotI-BamHI fragment into NotI and BamHI
sites of construct F0placZ.
In order to construct the F3hsplacZ vector, a 2.4 kb
SmaI-EcoRV fragment of hsp-lacZ
(Kothary et al., 1989) was
first inserted into SmaI and EcoRV sites of construct
pBSlacZIII (Kimura et al.,
1997
), yielding the pBShsplacZ vector. Construct
F3hsplacZ was then generated by cloning of a 5'
SacII-BamHI genomic fragment into the SacII and
BglII sites of construct pBShsplacZ. In order to construct
F3pGFP and F8pGFP transgenes, the sequence of the
translational start site of the lacZ gene in F3placZ and
F8placZ was converted into a NcoI linker sequence by
PCR-based mutagenesis, yielding F3placZ(Nco) and
F8placZ(Nco), respectively. Subsequently, a 740 bp
NcoI-EcoRI fragment of the plasmid pEGFP (Clontech)
was inserted between the NcoI and EcoRI sites of the
F3placZ(Nco) and F8placZ(Nco) vectors, respectively.
Construct E6/7dplacZ was produced by insertion of three genomic
fragments, the 660 bp NotI-ClaI (blunted), the 960 bp
BsgI (blunted)-SphI (blunted) and the 916 bp SapI
(blunted)-BamHI, into NotI and BamHI sites of
construct F0placZ.
Production and genotyping of transgenic mice
Transgenic mice were generated by microinjection of fertilized eggs from
CD-1, as described by Hogan et al. (Hogan
et al., 1994). Transgenic mouse embryos were identified by PCR
analysis as previously described (Kimura
et al., 1997
).
ß-Gal staining and histological analysis of embryos
ß-Gal staining was conducted as described previously
(Kimura et al., 1997). For
whole-mount views of 10.5 dpc to 13.5 dpc embryos, specimens were washed with
PBS and dehydrated in graded ethanol. Specimens were then cleared in 1:2
benzyl alcohol/benzoate as described (Eng
et al., 2001
). For histological analysis, embryos were embedded in
paraplast. Serial sections (10 µm) were produced and stained with
Eosin.
GFP reporter analysis using zebrafish embryos
Zebrafish embryos were obtained by natural mating
(Westerfield, 1995) and were
staged accordingly (Kimmel et al.,
1995
). F3pGFP and F8pGFP constructs were
linealized by digestion with SacII and SalI, respectively.
Digested DNA was resuspended in water and injected into the blastomere of
early one-cell stage embryos as described
(Kobayashi et al., 2001
).
Embryos were fixed in PBS containing 4% paraformaldehyde, and examined under
GFP-plus filters on a MZFLIII microscope (Leica) equipped with a C5810 chilled
CCD camera (Hamamatsu Photonics).
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Results |
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Expression of the Otx2 gene during pufferfish development
In order to investigate Fotx2 expression during development,
RT-PCR analysis was performed in wild-type Fugu embryos. The
developmental stage of Fugu is substantially similar to that of other
teleosts, such as zebrafish (data not shown). Fotx2 mRNA expression
was not detected at 22 hours postfertilization (hpf), which is equivalent to
the dome stage in zebrafish. However, it was observed initially as early as 30
hpf, equivalent to the shield stage in zebrafish
(Fig. 2A). These data indicate
that Fotx2 mRNA is not present in the oocyte and, furthermore, that
it is zygotically transcribed following midblastula transition. Subsequently,
Fotx2 transcripts were significantly upregulated around 54 hpf, an
early somites stage (Fig. 2A).
These transcripts were detected throughout the segmentation and pharyngula
periods; additionally, transcripts were detected at 172 hpf when embryos had
been hatched (Fig. 2A).
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Multiple and long-distance cis-regions combinatorially regulate the Fotx2 locus in transgenic mice
In order to understand the cis-regulatory mechanism of Fotx2 gene
expression, the activity of reporter gene constructs harboring its genomic DNA
were surveyed. First, five lambda phage contigs of genomic DNA from
30.5 kb to +38.5 kb (Fig.
3) were cloned and mapped. These contigs largely cover the entire
Fotx2 genomic locus (see Discussion). Next, 13 genomic fragments were
tested for cis-activity via generation of transgenic mouse embryos carrying
the reporter lacZ gene (Figs
3,
4). Transgene expression
analysis was transiently conducted at 10.5 dpc; seven genomic fragments
directing lacZ expression were identified (Figs
3,
4). All thirteen constructs
contained a 2.4 kb Fotx2 promoter, which displays no cis-activity
during embryogenesis (Fig. 3;
F0placZ) (Kimura et al.,
1997). For seven of the 13 DNA fragments tested, lacZ
expression occurred in spatial and temporal patterns that mimicked a subset of
the normal Otx2 expression pattern, with the exception of the F4
cis-region. This fine correlation suggests that these cis-regions can function
independently of one another, and that their spacing relative to the promoter
is not crucial for expression.
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lacZ expression governed by F3placZ carrying the
17 to 8.9 kb region was observed initially in the restricted
domain of the anterior neural plate at 8.5 dpc
(Fig. 5A,B). At 9.5 dpc,
lacZ expression was upregulated in the prospective dorsocaudal
telencephalon and dorsal diencephalon (Fig.
5C). By 10.5 dpc, lacZ expression was further restricted
in the diencephalic roof and dorsocaudal telencephalon
(Fig. 5D,H). Histological
analysis revealed that lacZ expression was present in the most caudal
and medial aspects of the dorsal telencephalon, surrounding the lamina
terminalis (Fig. 5E,F). At the
level of the diencephalon, lacZ expression, which was evident in the
most dorsal neural tube, occurred as two stripes excluding the dorsal-most
roof (Fig. 5G). At 12.5 dpc,
cis-activity remained in structures that developed into the choroid plexus;
however, activity was not apparent in the cortical hem
(Fig. 5I-L). In actuality,
expression governed by the F3 cis-region appears to be closely related to Bmp
and noggin expression (Fig. 5O)
(Furuta et al., 1997;
Hebert et al., 2002
). This
activity remained at 13.5 dpc (Fig.
5M,N).
In order to assess whether the F3 fragment can drive similar expression
patterns for a heterogeneous promoter, the mouse hsp68 promoter,
which is widely used in transgenic mice, was employed in place of the
pufferfish Otx2 promoter (Kothary
et al., 1989). Subsequently, we found that the F3 fragment was
also able to direct expression under the control of the hsp68
promoter in the diencephalic roof and the dorsocaudal telencephalon at 10.5
dpc (n=4/5; F3hsp68lacZ;
Fig. 5P). This finding
indicates that lacZ expression patterns are induced primarily by the
F3 cis-region, and are not due to the Fotx2 promoter activity
itself.
lacZ activity driven by F8placZ, carrying the +2.5 to
+10.5 kb region, was detected initially in the dorsal diencephalon and in
restricted areas of the dorsocaudal telencephalon at 9.5 dpc
(Fig. 6A,B). Subsequently, at
10.5 dpc, lacZ expression was upregulated in the dorsal diencephalon,
including in the prospective epithalamus and the mediocaudal telencephalon
(Fig. 6C,D). Further
histological examination demonstrated that lacZ activity was present
in the dorsomedial portion of the caudal telencephalon, corresponding to the
edge of the embryonic cerebral cortex, which is referred to as the cortical
hem (Fig. 6E-G)
(Grove et al., 1998). From 9.5
to 10.5 dpc, the expression domains governed by the F8 cis-region appear to
display partial overlap with those governed by the F3 cis-region. Later, at
12.5 and 13.5 dpc, lacZ activity was detected in both the
diencephalon and the telencephalon, including the cortical hem; however,
activity was absent in the choroid plexus, which is directed by
F3placZ as described (Fig.
6H-K, Fig. 5). The
expression pattern generated by the F8 cis-acting region appears to be similar
to that of Wnt3a (Fig.
6L) (Grove et al.,
1998
; Lee et al.,
2000
).
lacZ expression governed by the F5 (29.0 to 18.0 kb) cis-region was observed initially in the ventral portion of the diencephalon at 9.5 dpc (Fig. 7A). By 10.5 dpc, lacZ expression was detected in the ZLI, the ventral diencephalon and the lateral portion of the mesencephalon, with a longitudinal direction as described (see Fig. 7B,C). Further histological examination indicated activity, at 11.5 dpc, in the lateral mesencephalon, ZLI, and ventral portion of P2 and P3 diencephalon, including in the retromammillary region (Fig. 7D-G). Even at 13.5 dpc, lacZ expression remained in these structures (Fig. 7H,I).
At 9.5 dpc, lacZ expression mediated by the F11 (22.8 to
26.5 kb) cis-region was detected initially in the dorsal portion of the
mesencephalic neuroepithelium, corresponding to the mesencephalic trigeminal
ganglions, oculomotor nerves and a rostral aspect of trigeminal nerves
(Fig. 8A). The active domains
were extended both rostrally and caudally by 10.5 dpc in these structures
(Fig. 8B, D-G). Additionally,
expression was also evident in the dorsal region of the pretectum
(Fig. 8B). With respect to
branches of the trigeminal nerve, the F11 region drives expression exclusively
in the opthalmic and maxillar branches, but not in the mandibular branch
(Fig. 8B). Precise histological
examination indicated that lacZ expression was present throughout the
trigeminal ganglions and dorsal mesencephalic neural tube at 10.5 dpc
(Fig. 8D-G). Subsequently, most
lacZ expression in cranial nerves was diminished considerably by 11.5
dpc (Fig. 8C). Consequently,
lacZ activity covered the dorsal pretectum, and the inferior and
superior collicullus, by 13.5 dpc (Fig.
8H-J). Moreover, lacZ expression was observed
consistently, from 9.5 dpc, in ventral domains of the neural tube at the level
of the hindbrain, where Otx2 protein is also localized
(Fig. 8A-C,E-G)
(Mallamaci et al., 1996).
Additionally, lacZ expression at the spinal cord level was observed
ectopically in the transgenic embryos (Fig.
8A,B).
F12 (+26.5 to +32.8 kb) cis-regulatory activity was detected initially in the rostral first branchial groove and nasal portions as early as 9.5 dpc (Fig. 9A). lacZ activity in the retina commenced by 10.5 dpc (Fig. 4B,C). Subsequently, at 11.5 dpc, lacZ activity in the caudal portion of the mandibular arch extends to the cranial portion of the hyoid arch; moreover, activity was evident in both epithelium and mesenchyme, which corresponds to the auricular hillocks (Fig. 9E). This activity remained detectable in the external acoustic meatus of the ears at 12.5 dpc (Fig. 9F-I). Close histological examination revealed the presence of lacZ activity in the olfactory epithelium, as well as the in the outer layer of the optic cup corresponding to the prospective pigment layer of the retina at 12.5 dpc (Fig. 9J,K). Moreover, lacZ activity was observed in the developing inner ear, cochlea and saccule (Fig. 9H,I).
These expression patterns indicate that the aforementioned cis-regulators, with the exception of F4, mediate Otx2 expression in spatiotemporally distinct domains during rostral head development. Many cis-regulatory regions of genes have been shown to govern overlapping expression domains in a redundant manner; however, the seven cis-regulators currently identified displayed no redundant expression patterns (Figs 4, 5, 6, 7, 8, 9, 10). Furthermore, we concluded that the Fotx2 cis-regions function in a domain-specific fashion, rather than in a developmental stage-specific fashion. Their activities most likely depend on the expression of position-specific transactivators, which are expressed over time, spanning several subdomains within a given lineage.
Fotx2 enhancers can drive transgene expression in zebrafish embryos
In order to test whether the cis-regulators identified here can direct
transgene expression in fish, cis-activity in zebrafish embryos was
investigated. Among the seven cis-regulators, F3 and F8 regions faithfully
govern expression in distinct domains of the dorsal diencephalon and
mediocaudal telencephalon (Figs
4,
5,
6,
10). Thus, the activity of
these two cis-regulatory regions was transiently examined in transgenic
zebrafish. All constructs were tested via injection of DNA into zygotes;
subsequently, the resulting mosaic zebrafish embryos were analyzed for GFP
activity. First, the Fugu Otx2 promoter fused with the EGFP vector
(pGFP) alone afforded no GFP signals throughout zebrafish
embryogenesis (data not shown; n=0/74). The F3pGFP
construct, which harbors the 17 to 8.9 kb genomic fragment fused
with the pGFP vector, was initially expressed specifically at the
anterior ectoderm, and in the posterior portion of zebrafish embryos
ectopically from 80% epiboly onward (Fig.
11A). At early somites stage, GFP activity was observed
specifically in the prospective forebrain region
(Fig. 11B). Subsequently, at
the 18 somites stage, GFP activity was consistently detected in the
diencephalon and the telencephalon (Fig.
11C,D; n=38/51). At the later pharyngula, 32 hpf, stage,
GFP activity remained in evidence in the dorsal diencephalon
(Fig. 11E).
In contrast to the F3pGFP transgene, F8pGFP, which contains the 17 to 8.9 kb genomic fragment fused with the pGFP vector, was initially expressed from 12 to 14 hpf, and from the 6 to 10 somites stage (data not shown). Subsequently, GFP activity directed by F8pGFP was detected consistently in the diencephalon and in the telencephalon at 18 hpf (18 somites stage) (Fig. 11F; n=20/69). Later, at 30 hpf, an early pharyngula stage, F8pGFP expression remained in the telencephalon and the diencephalon (Fig. 11G). These expression patterns indicated that the activity of F3pGFP consistently occurs earlier, and is stronger and more widely distributed, than that of F8pGFP, suggesting that each fragment mediates distinct expression in the forebrain regions of the transient transgenic zebrafish embryos (Figs 5, 6, 11). These data indicate that the spatiotemporal expression governed by these two cis-regions is conserved between transgenic mouse and zebrafish, which suggests that an evolutionarily conserved mechanism controls these two cis-regions of Otx2 gene.
Identification and characterization of sequence elements conserved between mouse and pufferfish
Recent reports of large-scale sequence analysis, combined with transgenic
mouse approaches, reveal that evolutionarily conserved non-coding genomic
sequence elements tend to control gene expression
(Hardison, 2000). Thus, in
order to identify highly conserved, putative regulatory sequence elements, a
comprehensive comparative analysis of the intergenic regions flanking the
mouse and pufferfish Otx2 genes was performed by PipMaker analysis
(Schwartz et al., 2000
).
Consequently, we identified eight highly conserved sequence elements within
the pufferfish cis-regulators (Fig.
12). The F4, F9 and F12 cis-regions possess conserved elements,
E4, E5 and E8, respectively. The F5 cis-region possesses three conserved
elements, E1, E2 and E3. F11 contains two conserved regions, E6 and E7. In
addition, the corresponding eight conserved elements in mouse were located in
an order similar to that of pufferfish; however, these elements were within an
approximately eightfold extended region up to 550 kb. These data further
support the notion that genetic mechanisms of Otx2 gene regulation
are evolutionarily conserved among vertebrates. Moreover, as homologous
regulatory elements present in different species represent direct descendants
of a common ancestral regulatory element, individual cis-acting regions appear
to have developed their distinct regulatory elements independently.
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Discussion |
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Fugu genome as a model system for the identification of cis-regulators
Fugu has the most compact genome among vertebrates. Comparative
genomic analysis of human, mouse and pufferfish genome sequences identifies
the coding region of the genome. However, the function of non-coding regions
for gene expression remains elusive. The compact nature of the pufferfish
genome is primarily due to the smaller size of the non-coding region; on
average there is one gene per 10 kb in the pufferfish genome
(Aparicio et al., 2002).
Therefore, the Fugu genome may provide an ideal system with which to
analyse the function of non-coding regions; in particular, to analyse those
regions with a pivotal role in gene expression. In the present investigation,
in excess of 69 kb of the entire Fotx2 locus (the Otx2 locus
occupies
700 kb in the mouse genome) was examined. Coincidentally,
cis-regulatory activities were not observed within the 70 kb spanning from
50 to +20 kb of the mouse Otx2 locus, with the exception of
the 5' 1.8 kb promoter sequence (C.K.-Y. and I.M., unpublished). It is
evident from these findings that cis-regions regulating Otx2
expression are scattered over extremely large genomic distances from the
coding region.
Coincidentally, genes immediately upstream and downstream of Fotx2 include the Brain secretory protein Sec10 gene located at 38 kb, and the pellino 2 gene located at +43 kb (http://fugu.hgmp.mrc.ac.uk/). Comparison with the mouse Otx2 locus reveals that these immediate gene orders are conserved between pufferfish and mouse (http://www.ensembl.org/Mus_musculus/). By contrast, the mouse ortholog Brain secretory protein Sec10 gene is located approximately 350 kb, and pellino 2 is located approximately +500 kb, downstream of the mouse Otx2 locus. These genome sequence data indicate that the Otx2 locus is an extremely gene-bare region when compared with the average of one gene per 10 kb in the pufferfish genome. Together, these suggest that the 69 kb Fugu sequence from 30.5 to +38.5 kb may encompass most of the cis-regulatory regions of the Fugu Otx2 locus.
Consistently, the Fugu cis-acting regions we identified possess sequence elements that are highly conserved with those that are located within the 550 kb region of the mouse Otx2 locus (Fig. 12). Moreover, the conserved elements E6 and E7 were shown to be crucial for F11 cis activity (Fig. 12D). Curiously, most cis-regulators identified in the Fotx2 locus do not direct redundant expression patterns, which implies that cis-regulatory regions for redundant or overlapping expression may be exclusively abolished in the pufferfish so as to reduce its genome to as small a size as possible.
We have described pufferfish Otx2 gene expression during wild
Fugu embryogenesis. Developmental patterns of the Fugu
teleost are essentially identical with those of other teleosts such as
zebrafish and medaka. Accordingly, Fotx2 expression appears to be
substantially similar to zebrafish Otx2 expression
(Li et al., 1994); briefly, a
common aspect of vertebrate Otx2 gene expression in anterior region
of the body is shared. Additionally, we have also identified multiple
pufferfish cis-acting regions in transient transgenic zebrafish embryos. This
finding implies that the cis-elements and the trans-acting factors, which are
involved in Otx2 expression, had fully evolved in the common ancestor
of teleosts and mammals, and have been conserved during 400 million years of
evolution.
Expression domains by Fotx2 cis-regulators coincide with local signaling centers of the neuroectoderm
The F3, F5 and F8 cis-regulatory regions identified in this study direct
transgene expression that nearly coincides with local signaling centers in the
rostral brain, as defined by the expression Bmps, Shh and Wnts, respectively
(Figs 4,
5,
6,
7,
10). The F3 cis-acting region
governed expression in the most medial and caudal aspects of the
telencephalon, and in the roof of the diencephalon, coincident with the
prospective site of choroid plexus development (Figs
5,
10). The expression domains
afforded by the F3 cis-region are strikingly colocalized with Bmps, noggin,
Msx1 and transthyretin (Ttr)
(Furuta et al., 1997). Indeed,
Otx2 is expressed in the dorsocaudal telencephalon and in the dorsal
diencphalon, including in the prospective choroid plexus and the `cortical
hem' (see below) in mouse embryos (Simeone
et al., 1993
; Boncinelli et
al., 1993
; Stoykova et al.,
1996
). In order to establish whether the F3 cis-region is
controlled directly by Bmp signaling, experiments were performed involving
neural plate explants with BMP beads as described
(Furuta et al., 1997
). The
neuroectoderm was isolated at the level of the forebrain from F3placZ
transgenic embryos at 10.5 dpc. Recombinant BMP2-coated beads were
transplanted into these forebrain explants, and the explants were then
cultured for 24 hours. However, no ectopic lacZ expression induced by
the BMP2-coated beads was detected (C.K.-Y. and I.M., unpublished). These data
suggest that the F3 cis-regulator may not be regulated by BMP signaling.
Alternatively, the Otx2 expression from this F3 cis-regulator might
participate in the expression of Bmp molecules in the prospective choroid
plexus. By contrast, the F8 cis-acting region directed transgene expression in
the mediocaudal telencephalon where Wnt molecules are co-expressed, defining a
zone termed `cortical hem' (Figs
6,
10)
(Parr et al., 1993
;
Grove et al., 1998
).
Therefore, Wnt signals might control the Otx2 expression mediated by
the F8 cis-region in the cortical hem and dorsal telencephalon. Additional
transgenic zebrafish studies have indicated that F3 and F8 cis-regions
directed considerably conserved expression in the forebrain
(Fig. 11). Concomitant with
this finding, zebrafish Wnt8b is expressed in the dorsal forebrain
(Kelly et al., 1995
). These
data suggest that local signaling centers play an essential role in forebrain
development in zebrafish embryos and are evolutionarily conserved among
vertebrates.
The F5 cis-acting region directs expression in the ZLI, and in ventral
portions of the diencephalon and the lateral mesencephalon that may be related
to longitudinal columns termed `midbrain arcs' (Figs
7,
10)
(Agarwala et al., 2001;
Sanders et al., 2002
).
Notably, mouse Otx2 mRNA and Otx2 protein are expressed in the ZLI,
and in the ventral diencephalon and mesencephalon in mouse embryos
(Simeone et al., 1993
;
Mallamaci et al., 1996
;
Stoykova et al., 1996
).
Importantly, expression in the ZLI, ventral diencephalon and midbrain arcs are
proposed to be closely related to Shh signaling, which is essential for the
dorsoventral patterning of the ventral neural tube. Therefore, these
expression patterns suggest the possibility that Shh signaling controls the
Otx2 expression mediated by the F5 cis-acting region.
Fotx2 cis-regulators direct expression in the developing organs
lacZ activity directed by the F9, F11 and F12 cis-regions appears
to be closely related to the developing organs. Notably, it has been suggested
that Otx2 is required for the development and specification of these
specific organs. The F9 cis-region governs expression in the cephalic
mesenchyme (Kimura et al.,
1997). Consistently, Otx2 is expressed in the cephalic
mesenchyme, and functions in the formation of premandibular and mandibular
skulls (Matsuo et al., 1995
;
Kimura et al., 1997
;
Hide et al., 2002
). The F11
region governs expression in the dorsal pretectum and mesencephalon, including
in the posterior commissure, the mesencephalic trigeminal neurons, the
oculomotor nerve, the first and second branches of the trigeminal nerve, and
the trigeminal ganglions (Figs
8,
10). In actuality,
Otx2 mRNA is expressed in the dorsal pretectum and mesencephalon, in
the trigeminal ganglions and opthalmic branch
(Simeone et al., 1993
;
Ang et al., 1994
)
(Fig. 8M). Furthermore, as
Otx2 heterozygousmutant mice display anomalies of these neurons and
cranial nerves, Otx2 may be required for the proper development of
these tissues cell-autonomously (Matsuo et
al., 1995
). The F12 cis-region drives expression in the developing
sense organs, the olfactory epithelium, the pigment layer of the retina, and
the developing inner and outer ears. Otx2 expression is consistently
found in these developing sense organs
(Simeone et al., 1993
;
Mallamaci et al., 1996
;
Morsli et al., 1999
)
(Fig. 8K). Moreover,
Otx2 is required for normal development of the inner ear and pigment
epithelium, cooperatively with the Otx1 gene
(Morsli et al., 1999
;
Cantos et al., 2000
;
Martinez-Morales et al.,
2001
).
Genetic control of Otx2 expression during head development
We have surveyed the entire pufferfish Otx2 locus; furthermore, we
observed the regulation of Otx2 expression by multiple, yet distinct,
cis-acting regions. The regulatory mechanism of this gene is much more complex
than previously anticipated. It is certain that these regions transactivate
Otx2 expression independently. One possible assumption regarding the
presence of such long-distance and multiple cis-acting regions entails the
ability of each cis-region to transactivate Otx2 expression upon
adjacent cis-elements without mutual interference. Current reports have shown
that Hox genes are clustered into complexes; moreover, their positions within
the cluster correlate with their time of expression and with the position of
the AP boundaries of their expression domains
(Duboule and Morata, 1994).
Similarly, ß-globin gene clusters are controlled in order of
their expression during ontogeny
(Grosveld, 1999
). In the
present study, Fotx2 cis-regulators are clustered into the 70 kb
genomic sequence of the Fotx2 locus; however, their positions
relative to the coding region, and the order of the 5' to the 3'
side, do not correlate with their time of expression, or with the expression
domains along the anteroposterior or dorsoventral axis. This observation
suggests that these cis-regulators may direct multiple promoters located
within this genomic locus. Consistent with this notion, a novel gene, annoted
as NM172600, which is located over +150 kb from the mouse Otx2 coding
region, is a homolog of Fugu Q9NX78
(Fig. 12B), and is expressed
in the rostral brain, in which Otx2 expression occurs during
embryogenesis (C.K.-Y. and I.M., unpublished). This finding supports our
assumption that genes located over 500 kb from this locus may be
coincidentally controlled by these distant cis-regulators. Curiously, the
corresponding conserved sequence elements in mouse are also located in an
order similar to that in pufferfish; however, these elements are within an
approximately eightfold extended region, up to 550 kb
(Fig. 12). Thus, we propose
that, during evolution in the common ancestor of teleosts and mammals, the
Otx2 promoter might have acquired multiple region-specific
cis-regulators in order to control the highly coordinated processes of rostral
head development. Consequently, the Otx2 homeobox gene could play
multiple and pivotal roles in rostal head development by means of these
cis-elements. Investigation of the mechanism of non-vertebrate Otx2
gene expression is of keen interest
(Oda-Ishii and Saiga,
2003
).
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ACKNOWLEDGMENTS |
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