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First published online 5 December 2007
doi: 10.1242/dev.009548
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Department of Biology, University of Virginia, Charlottesville, VA 22904, USA.
* Author for correspondence (e-mail: rmg9p{at}virginia.edu)
Accepted 23 October 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Competence, Genomics, Induction, Lens, Notch, Xenopus
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Putative enhancers are predicted in silico within entire
genomes as clusters of transcription factor-binding motifs and/or intergenic
sequences conserved among related species. Especially in vertebrates, enhancer
activities are often mapped to conserved non-coding elements associated with
developmental regulatory genes (Woolfe et
al., 2005).
From this perspective, we have investigated mechanisms of lens induction in
Xenopus, where essential tools for genomics-based cis-regulatory
analysis, i.e. the whole genome sequence
(http://genome.jgi-psf.org/Xentr4/Xentr4.home.html)
and a highly efficient transgenesis technique, have become recently available
(Offield et al., 2000;
Smith, 2005). The relatively
large evolutionary distance between Xenopus tropicalis and mammalian
genomes (350 million years) allows sequence comparison to highlight the most
conserved elements involved in vertebrate genomic regulatory networks
(Muller et al., 2002).
Embryological studies have shown that lens induction is a stepwise process
that begins when a broad domain of the animal cap ectoderm acquires a
lens-forming competence at mid-gastrula stages
(Grainger, 1992). This
lens-competent field is subsequently narrowed down to the non-neural ectoderm
surrounding the anterior margin of the newly formed neural plate. Accompanying
this field-restriction process, a lens-forming bias is established in this
region by planar signals provided by the adjacent anterior neural plate. This
biased region corresponds to (or includes) the part of the non-neural ectoderm
termed pre-placodal ectoderm, which will give rise to the lens, otic,
olfactory and adenohypophyseal placodes at later stages. After neural tube
formation, only the lateral part of the lens-biased ectoderm makes contact
with the developing optic vesicle and at this stage determination occurs,
followed several hours later by differentiation into lens tissue (lens placode
and, subsequently, the lens vesicle).
This stepwise commitment process is likely to be mediated, at least in
part, by several transcription factor genes that exhibit distinct but
overlapping expression patterns during the course of lens-field specification
(Ogino and Yasuda, 2000).
Otx2, the earliest of these genes, exhibits expression from the end
of gastrulation in the pre-placodal ectoderm and the adjacent anterior neural
plate (Blitz and Cho, 1995;
Zygar et al., 1998).
Pax6 and Six3 show more-restricted expression in the
lens-field within the pre-placodal ectoderm
(Zhou et al., 2000;
Zygar et al., 1998). After
neural tube formation, their expression is followed by activation of
lens-specific transcription factor genes such as MafB, L-maf and
Pitx3, exclusively in the presumptive lens ectoderm (PLE) overlying
the optic vesicle (Ishibashi and Yasuda,
2001; Pommereit et al.,
2001).
The expression of Otx2, Pax6 and Six3 implies their
involvement in the establishment of lens bias, and recent significant progress
made by mouse genetic studies supports this view
(Lang, 2004). However, little
is known about how their activities are sequentially integrated to narrow down
the lens-field, and how signaling from the optic vesicle is involved in this
process. To address this question, we chose to study the regulation of a
forkhead-family gene, Lens1, which is the earliest of the genes that
are expressed primarily in the PLE overlying the optic vesicle
(Kenyon et al., 1999), and
which is activated at the time when commitment to the lens fate occurs
(Grainger, 1992). Synteny
analysis using Metazome 1.1
(http://www.metazome.net/)
showed that the X. tropicalis Lens1 locus is orthologous to mouse
Foxe3 and we therefore refer to it as Xenopus FoxE3 in this
study. Analyses of mouse mutants have shown that Foxe3 is essential
for lens epithelial proliferation and lens vesicle closure
(Blixt et al., 2000;
Brownell et al., 2000).
Using in vivo and in silico approaches, we identify and characterize an
enhancer of X. tropicalis FoxE3 responsible for its PLE-specific
expression, and demonstrate that Xenopus-mammalian genome comparison
is a powerful strategy for prediction and for further detailed analysis of
vertebrate cis-regulatory elements. A `co-transgenesis' assay, in which
separate enhancer and reporter element constructs are co-injected, was also
developed to facilitate the rapid survey of possible enhancer activities of
the predicted cis-regulatory elements. Our analysis has led to the first
recognition that Notch signaling (Lai,
2004) is a lens-inducing signal, and revealed a role for Otx2
acting in concert with Notch signaling to specify the lens-field. The data
presented here reveal one of the first molecular mechanisms found to underlie
stepwise determination of the lens, as well as suggesting a general mechanism
for how organ progenitor cells are segregated within a broader `zone of
responsiveness' during vertebrate development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) in X. tropicalis genome assembly 4.1 (scaffold 1:
2902082-2903173,
http://genome.jgi-psf.org/Xentr4/Xentr4.home.html).
We assigned the transcription start site (+1) to the 5' end of the
putative 5' untranslated region (UTR) (scaffold 1: 2901906-2902081),
which was predicted from its homology to the 5' UTR of X. laevis
FoxE3 (94% identity), to number the flanking sequence. βGFP was
generated by introducing the chicken β-actin basal promoter (-55
to +53) excised from pβLuc (Ogino and
Yasuda, 1998) into pBSSK+EGFP. A series of base-substitution
mutant constructs (mt1-mt9) were generated from Xt462-βGFP using the
Quikchange Site-Directed Mutagenesis Kit (Stratagene) with the following
primers and their complements (mutated sequences are underlined): mt1, 5'-CAAGGAGAGTGAAATGAGAGGTACCATGTTTTCATCATCCG-3';
mt2, 5'-GAGTGAAATGAGATAATCCATCGATTCATCATCCGTAGGCC-3';
mt3, 5'-CTCTTTTCACAAGCCATGGTACGTACTTTATTAGGCTGAGC-3';
mt4, 5'-CATGGGCCGTACTTTATTAGGTACCGCAGTTCTGGGCCTGTAAG-3';
mt5, 5'-ATGCAGAATGGCAGAAACCGGTAGGCCCAGTACATTTTCC-3';
mt6, 5'-CAACATCAGATTTTCCTACATCTAGAGTGCAGAAATCCCACAC-3';
mt7, 5'-CTACAGATAGAGTGCAGAAATCTAGAACATGTCCAAATCTGTTAACATC-3';
mt8, 5'-GCAGAAATCCCACACATGTGGCCATCTGTTAACATCTGACATG-3';
mt9, 5'-CCACACATGTCCAAATCGATTAACATCTGACATGAAGTC3'.
Otx-Su(H)-βGFP, mtOtx-Su(H)-βGFP and Otx-mtSu(H)-βGFP were generated by introducing the following double-stranded oligonucleotides into βGFP (Otx- and Su(H)-binding sequences are underlined; linker sequences are shown in lowercase): Otx-Su(H), 5'-ctagaGGGATTAGAGTTCCCACACGGGATTAAATTTCCCACGGAGGATTAGGGTTCCCACAAGGGATTAGATTTCCCACg-3'; mtOtx-Su(H), 5'-ctagaGGCGGTAGAGTTCCCACACGGCGGTAAATTTCCCACGGAGCGGTAGGGTTCCCACAAGGCGGTAGATTTCCCACg-3';
Otx-mtSu(H), 5'-ctagaGGGATTAGAGTTCGGTCACGGGATTAAATTTCGGTCGGAGGATTAGGGTTCGGTCAAGGGATTAGATTTCGGTCg-3'.
Details of other reporter constructs are given in the Results. pGEM-XFoxE3
was generated by cloning the coding sequence of X. laevis FoxE3 into
the pGEM-T Easy vector (Promega). pCS2+GR-Otx2-En was generated by introducing
the coding sequence of a GR ligand-binding domain isolated from
pCS2+GR-Su(H)VP16 (Rones et al.,
2000) into the 5' end of the Otx2 coding sequence of
pCS2+Otx2-En (Gammill and Sive,
2001).
Xenopus transgenesis
Transgenic embryos were generated by a sperm nuclear transplantation method
(Kroll and Amaya, 1996), and
their GFP expression was detected by in situ hybridization
(Sive et al., 2000) for
maximum sensitivity. The fraction of embryos that developed normally until
scoring stages (stages 22-24) (Nieuwkoop
and Faber, 1967) varied between 10-20% of total injected embryos
depending on egg quality. However, the frequency of reporter gene expression
within the group of normal embryos was fairly constant. In the case of three
independent assays using Xt462-βGFP, the average fraction of embryos with
PLE-specific GFP expression in a normal group was 21±1.7%. This
construct was injected in parallel with a series of its mutant constructs
(mt1-mt9) as a control to monitor transgenesis efficiency. For
co-transgenesis, the 462 bp enhancer fragment of Xenopus FoxE3,
amplified by PCR, was mixed with a β-actin promoter-GFP cassette
excised from βGFP in a molar ratio of 4:1 and directly used for
transgenesis.
In situ hybridization and RNA injections
In situ hybridization analyses of Delta1, Delta2, Otx2,
1-crystallin and Rx were performed as described
(Blitz and Cho, 1995;
Chitnis et al., 1995;
Jen et al., 1997;
Mathers et al., 1997;
Offield et al., 2000). The
antisense probes for GFP, FoxE3, Serrate1 and Notch2 were
generated using pBSSK+EGFP, pGEM-XFoxE3, pBS-XSerrate1 (a gift from Dr C.
Kintner, The Salk Institute for Biological Studies, La Jolla, CA) and an EST
clone (NCBI accession BX855333), respectively.
Capped mRNAs for microinjections were transcribed from pCS2+GR-Su(H)VP16,
pCS2+GR-Su(H)DBM (Rones et al.,
2000), pCS2+NICD (Chitnis et
al., 1995), pCS2+XOtx2-GR
(Gammill and Sive, 1997),
pCS2+GR-Otx2-En, pCS2+XOtx2 (Blitz and Cho,
1995), pCS2+XDelta1, pCS2+XDelta1Stu
(Chitnis et al., 1995),
pCS2+XDelta2, pCS2+XDelta2Tr
(Jen et al., 1997), pCS2+EGFP
and pCS2+nlacZ. For lacZ staining, magenta-gal was used as
the substrate (Rones et al.,
2000).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This
expression was closely recapitulated by GFP expression in transgenic X.
laevis embryos generated with a reporter construct carrying a 10.6 kb
upstream sequence of X. tropicalis FoxE3
(Fig. 1B, -10.6kGFP; see Fig.
S1 in the supplementary material). Sequences responsible for PLE-specific
expression were narrowed down by a series of promoter deletion assays
(Fig. 1C-G), which identified a
901 bp element (-6313 to -5413) as the one that essentially recapitulates the
activity of the -10.6 kb promoter when linked to the basal promoter region
(-640 to +118) (Fig. 1G).
We also approached the same question from a bioinformatic perspective. To
examine a possible relationship between conserved non-coding elements
distributed around vertebrate FoxE3 loci and the functionally
identified 901 bp enhancer, we aligned a 
60 kb sequence of the human
FOXE3 locus with orthologous mouse, chicken and X.
tropicalis sequences using a genome alignment program, MultiPipmaker
(http://pipmaker.bx.psu.edu/pipmaker/)
(Fig. 2A). In this alignment,
the percent identity plot (pip) shows both the position in the human sequence
and the degree of similarity for each aligning segment between the human and
other sequences. The pip in the human-mouse alignment
(Fig. 2A, top row) indicates
extensive sequence conservation between these two species. However, the pip in
the human-chicken and the human-Xenopus alignments
(Fig. 2A, second and third
rows) indicates that only one region is conserved in all four species besides
the coding region (Fig. 2A, red
box). This conserved region corresponds to the 462 bp sequence between -6159
and -5698 in the X. tropicalis FoxE3 locus, and is included in the
901 bp element (-6313 to -5413) described above.
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); a
Su(H) (also known as CBF1/RBP-J
/Lag-1)-binding motif for Notch
signaling (Tun et al., 1994);
and a Tcf3/Lef1-binding motif for canonical Wnt signaling
(Eastman and Grosschedl, 1999).
Other predicted motifs are two Otx-binding motifs
(Gan et al., 1995), two Fox
motifs (Kaufmann et al., 1995)
and a GATA motif (Ko and Engel,
1993).
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Transgenic embryos generated with a construct in which the 462 bp
Xenopus element was replaced with the orthologous 423 bp element of
mouse Foxe3 (-3529 to -3107) used for the phylogenetic footprinting
(Fig. 2B) exhibited GFP
expression that was indistinguishable from that driven by the Xenopus
element (compare Fig. S2A with B in the supplementary material), suggesting
that the sum of the discontinuous stretches of short conserved sequences
identified by the phylogenetic footprinting is sufficient to account for the
expression. To evaluate the role of each short conserved sequence, we
introduced base-substitution mutations individually into all of the eight
putative transcription factor-binding motifs mapped there, and into one of the
conserved motifs with no similarity to known transcription factor-binding
motifs (indicated as Factor X motif in Fig.
2B). The mutant constructs were generated from Xt462-βGFP,
which drove PLE-specific expression in 21% of the generated embryos in
transgenic assays as described (Fig.
3A and `wt' in Fig.
3D). None of the mutations led to additional ectopic expression,
but the percentage of embryos with PLE-specific expression was decreased to
different extents (Fig. 3D).
The most striking result was obtained with the construct carrying a mutation
in the Su(H) motif (Fig. 3D,
mt7), which completely abolished the expression in all cases except one
(n=70). Even in this one positive case, the expression was very faint
(not shown). The mutation of the 3'-most Otx motif and the mutation of
the unknown Factor X motif decreased the positive cases to 6% and 8%,
respectively (Fig. 3D, mt5 and
mt4). Mutation of the 5'-most Fox motif, Smad1 motif, or GATA motif,
somewhat decreased the positive cases, to 12%
(Fig. 3D. mt2, mt3 and mt6).
Mutation of the 5' Otx motif, Tcf3/Lef1 motif, or 3' Fox motif did
not significantly reduce the percentage of positive cases
(Fig. 3D, mt1, mt8 and mt9). By
2 test
(http://www.graphpad.com/quickcalcs/chisquared1.cfm),
the percentage of positive cases in the wild type and the Su(H), 3'-most
Otx, or unknown Factor X mutant constructs are significantly different
(P<0.0001, P=0.0006 and P=0.0018, respectively),
whereas the differences observed in other cases are not significant
(P>0.05). These results show that the Su(H), 3'-most Otx,
and unknown Factor X motifs are essential for the enhancer activity, whereas
other motifs might serve to boost its level and/or are involved in the
regulation at different developmental stages.
Regulation of FoxE3 expression and lens placode formation by Notch signaling
Gel retardation assays showed direct binding of Xenopus Su(H)
protein to the putative Su(H) motif identified in the FoxE3 enhancer
in vitro, and chromatin immunoprecipitation experiments confirmed in vivo
binding of the Su(H) protein to the enhancer (see Fig. S3A and Fig. S4A in the
supplementary material). Su(H) is ubiquitously expressed and activates
transcription only when it forms a nuclear complex with the intracellular
domain of Notch receptor (NICD) that is translocated from the cytoplasm upon
activation by a ligand (Fig.
7A) (Lai, 2004).
We found that a Xenopus homolog of mammalian Notch2, newly
identified in this study, is expressed during the course of lens formation in
the pre-placodal ectoderm, PLE and developing lens vesicle
(Fig. 4A-B' and see Fig.
S5 in the supplementary material).
|
),
Delta2 (Jen et al.,
1997) and Serrate1
(Kiyota et al., 2001) have
been identified in Xenopus. To assess the involvement of Notch
ligands in FoxE3 regulation, their expression was compared with that
of FoxE3 during the course of lens-field formation. In neural
plate-stage embryos, neither Delta1 nor Delta2 is expressed
in the presumptive retina fields that will give rise to optic vesicles
(Fig. 4C,F)
(Chitnis et al., 1995).
Interestingly, Delta1 expression in the anterior neural ridge is
adjacent to FoxE3 expression in the pre-placodal ectoderm at this
stage (Bourguignon et al.,
1998; Kenyon et al.,
1999) (compare Fig.
4C with I), where it may act as an early signal involved in
FoxE3 expression. Accompanying neural tube formation, both Delta
genes exhibit strong upregulation in the developing optic vesicle
(Fig. 4D-E',G-H'),
which is followed by FoxE3 expression in the PLE
(Fig. 4J-K')
(Kenyon et al., 1999). The
cells expressing Delta genes are located in the outermost region of the optic
vesicle and make contact with the overlying PLE cells expressing
FoxE3 (Fig.
4E',H',K'). Serrate1 is not expressed
in these tissues at the neurula or early tailbud stages, but later is
expressed in the developing lens placode as reported (not shown)
(Kiyota et al., 2001).
To examine possible roles of the Delta genes in FoxE3 regulation,
we blocked Delta1 and Delta2 activities using their dominant-negative forms,
Delta1Stu and Delta2Tr, respectively
(Chitnis et al., 1995;
Jen et al., 1997). mRNA
encoding either Delta1Stu, Delta2Tr or GFP was injected
along with a lineage tracer, nlacZ mRNA (nuclear lacZ, 50
pg), into one dorsal blastomere of eight-cell stage X. laevis
embryos. The injected embryos were fixed at the early tailbud stages (stages
22-24), and stained for lacZ expression to trace distribution of the
injected mRNAs. Only the embryos that showed lacZ staining in the
optic vesicle were subjected to in situ hybridization with a FoxE3
probe. Control injections using GFP mRNA did not have any significant effects
on FoxE3 expression (n=55). Embryos injected with
Delta1Stu mRNA exhibited loss or severe reduction of FoxE3
expression on the injected side (100%, n=22), but this phenotype
appeared to be associated with head abnormalities caused by the expression of
this construct (not shown). Although the effect seen here is consistent with a
role for Delta1 in lens formation, we did not study Delta1 further because of
the complexity in interpreting the cause of lens defects in light of the head
abnormalities seen in these experiments. By contrast, injection of
Delta2Tr mRNA led to a very specific phenotype: FoxE3
expression was lost or severely reduced (75%, n=32,
Fig. 5A) in the PLE overlying
the optic vesicle stained for lacZ, but the optic vesicle itself,
which was marked by expression of the retina-specific homeobox gene
Rx (Mathers et al.,
1997), did not show any detectable abnormalities (100%,
n=37, Fig. 5B).
In addition to FoxE3, we examined expression of a lens
differentiation marker, 1-crystallin
(Offield et al., 2000), by in
situ hybridization, to investigate late phenotypes of embryos expressing
Delta2Tr. At late tailbud stages (stages 29/30), the lens placode
of the uninjected sides showed clear 1-crystallin expression
(Fig. 5D), but the
1-crystallin-positive cells on the injected sides formed a
tiny cell mass or were absent (71%, n=42,
Fig. 5C,I). Expression analysis
of Rx showed that the optic vesicle of the injected sides still had
no significant defects, at least through these stages (100%, n=47,
Fig. 5F-H). The downregulation
of 1-crystallin by Delta2Tr was rescued by
co-injection of mRNA encoding wild-type Delta2 (75%, n=67,
Fig. 5E,I), but not Delta1
(n=38, Fig. 5I),
indicating the specific activity of Delta2 for lens induction. These results
show that Delta2 activity in the optic vesicle is necessary for FoxE3
expression in the PLE and for subsequent lens placode formation.
To examine how the responses to Notch signaling might directly impinge on
FoxE3 expression in lens cells, we used a construct encoding an
inducible dominant-negative form of Su(H), GR-Su(H)DBM
(Rones et al., 2000). This
construct was generated by fusing the human glucocorticoid receptor
ligand-binding domain (GR) to a modified version of Xenopus Su(H),
which contains a point mutation in its DNA-binding domain. This GR-Su(H)DBM
protein, which inhibits Notch signaling in response to dexamethasone (Dex) by
sequestering NICD from endogenous Su(H), allowed us to circumvent possible
head defects that could be caused by constitutive inhibition of Notch
signaling. mRNA encoding GR-Su(H)DBM was injected along with nlacZ
mRNA into one dorsal blastomere of four-cell stage X. laevis embryos.
The injected embryos were cultured in the absence of Dex until stages 15-16,
and then maintained with Dex (10 µM) either present (induced) or absent
(uninduced) until fixation at early tailbud stages (stages 22-24). This time
period was chosen to yield functional GR-Su(H)DBM protein at the time when
endogenous FoxE3 expression is upregulated in the PLE following
neural tube closure. The fixed embryos were subjected to lacZ
staining to select embryos in which expression was targeted to the anterior
ectoderm including the PLE. As observed in embryos injected with
Delta2Tr mRNA, FoxE3 expression was lost or severely
reduced on injected sides of the Dex-treated embryos (55%, n=33,
Fig. 5J). Downregulation of
FoxE3 was not observed on uninjected sides of any of these embryos
(Fig. 5K) or on injected sides
of any sibling embryos untreated with Dex (n=65,
Fig. 5L), indicating that Dex
itself had no effect on FoxE3 expression and the FoxE3
downregulation in the Dex-treated embryos depended on the activation of
GR-Su(H)DBM by hormone treatment.
|
). Similar
to the experiments with GR-Su(H)DBM, GR-Su(H)VP16 was expressed by injecting
its mRNA, and the resulting embryos were cultured with or without Dex until
fixation at stages 22-24. Ninety-five percent of the injected embryos treated
with Dex exhibited ectopic FoxE3 expression in the anterior region of
the injected sides stained with lacZ (n=55,
Fig. 5M). In contrast to the
extended lacZ staining, the ectopic FoxE3 expression was
spotty and localized in a domain of the ectoderm overlying the anterior brain
and that surrounding the cement gland. Ectopic FoxE3 expression was
not observed on the uninjected sides of any of these embryos
(Fig. 5N) or on the injected
sides of any sibling embryo untreated with Dex (n=69,
Fig. 5O). The activation and inhibition of Notch signaling using the active and dominant-negative forms of Su(H), respectively, induced upregulation and downregulation of FoxE3. These results demonstrate the essential role of Notch signaling in PLE-specific FoxE3 expression, and suggest that Notch signaling in the PLE is likely to be activated by Delta2 expressed in the adjacent optic vesicle. Interestingly, the ectopic FoxE3 expression induced by the active form of Su(H) was regionally restricted to part of the anterior ectoderm, which suggests pre-localization there of a factor enabling responsiveness to Notch signaling. In addition, this restricted ectopic expression is consistent with a role for Notch signaling as a cue to turn on FoxE3 at the right place within this competent domain.
Otx2 confers the ability to activate FoxE3 in response to Notch signaling
We examined Otx2, as a candidate factor responsible for the regional
responsiveness to Notch signaling, for two reasons. First, we identified a
putative Otx motif as among the most essential of the transcription
factor-binding motifs in the FoxE3 enhancer
(Fig. 3D, mt5). Second, during
the time window chosen for the activation of GR-Su(H)VP16 (from the neural
plate to early tailbud stages), Otx2 shows diffuse expression in the
head ectoderm, including not only the PLE, but also the surrounding region
where the ectopic FoxE3 expression was observed
(Fig. 6A-C'', compare
Fig. 5M and
Fig. 6C)
(Zygar et al., 1998).
|
). This construct allows us to circumvent severe
gastrulation defects and spina bifida caused by misexpression of wild-type
Otx2 in Xenopus embryos (Blitz and
Cho, 1995). mRNAs encoding GR-Su(H)VP16 and Otx2-GR were injected
separately or together into a ventral blastomere of four-cell stage embryos to
target expression in the trunk region instead of the head ectoderm expressing
endogenous Otx2. All injected embryos were treated with Dex at stages
15-16, fixed at stages 22-24, and subjected to lacZ staining and in
situ hybridization with the FoxE3 probe. None of embryos misexpressing either GR-Su(H)VP16 or Otx2-GR exhibited ectopic FoxE3 expression in the trunk region, where the lacZ staining indicated broad distribution of the injected mRNAs (n=66 and n=52, respectively; Fig. 6D,E). This indicates that neither Notch signaling nor Otx2 activity is sufficient for FoxE3 activation. However, when embryos were co-injected with GR-Su(H)VP16 and Otx2-GR mRNAs, 91% (n=54) exhibited striking FoxE3 expression in the trunk region throughout the lacZ-stained region (Fig. 6F, black arrowheads). This trunk expression was restricted to the ectoderm layer, and was absent from the underlying neural and mesodermal tissues (Fig. 6F, inset). Ectopic FoxE3 expression was also detected in embryos that were induced with Dex in the presence of a protein synthesis inhibitor, cycloheximide, verifying a direct effect of GR-Su(H)VP16 and Otx2-GR on the FoxE3 promoter (see Fig. S6 in the supplementary material).
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The Otx2-dependent activation of FoxE3 was also observed when NICD was misexpressed instead of GR-Su(H)VP16 to activate Notch signaling: misexpression of NICD alone in the trunk ectoderm did not induce ectopic FoxE3 expression in any injected embryos (n=33, Fig. 6G), but the combination of NICD and Otx2-GR did (49%, n=45; Fig. 6H, black arrowheads). This showed that endogenous Su(H) activates FoxE3 as effectively as the artificial construct GR-Su(H)VP16, if Notch is activated.
In addition to these gain-of-function experiments, we designed
loss-of-function experiments for Otx2 to verify that its activity is required
for FoxE3 expression in the PLE. Since constitutive loss of Otx2
activity impairs anterior neural fate determination
(Gammill and Sive, 2001), we
chose to inject mRNA encoding an inducible dominant-negative form of Otx2,
GR-Otx2-En. We generated this construct by fusing a coding sequence of GR to a
dominant-negative form of Otx2, Otx2-En (Xenopus Otx2 fused to the
minimal repressor domain of Engrailed), which has been shown previously to
specifically block Otx2 function (Gammill
and Sive, 2001). The injected embryos induced with Dex from stage
18 onward did not exhibit any detectable head abnormalities
(Fig. 6I,J), suggesting that
anterior neural defects were circumvented by the use of this inducible
construct. Loss, or significant reduction, of FoxE3 expression in the
PLE was detected on their injected sides by in situ hybridization (58%,
n=33, Fig. 6I), but
not in their uninjected sides (Fig.
6J) nor in the injected sides of any sibling embryos untreated
with Dex (n=25, not shown).
These experiments demonstrate that Otx2 enables FoxE3 activation in response to Notch signaling. To test whether Otx2 and Notch inputs are sufficient to direct PLE-specific expression, we generated a reporter construct carrying four copies of a pair of consensus Otx- and Su(H)-binding motifs in front of the β-actin promoter-GFP cassette [Otx-Su(H)-βGFP]. Transgenic embryos injected with this Otx-Su(H) reporter exhibited GFP expression not only in the PLE, but also in the optic vesicle and spinal cord (Fig. 6K-L'), although the expression in the PLE did not appear so strong as that driven by the FoxE3 enhancer. This eye-specific expression was not detected when transgenic embryos were generated with reporter constructs in which either the Otx or Su(H) motifs were mutated [mtOtx-Su(H)-βGFP and Otx-mtSu(H)-βGFP, respectively; Fig. 6K], or when a reporter construct that carried eight copies of the Su(H) motifs and no copy of the Otx motif was used (not shown). These reporter assays suggest that both Otx2 and Notch inputs are necessary and sufficient to drive expression in PLE, but additional inputs are required for boosting the expression level in the PLE and for repressing expression in neural tissues, thereby directing the more defined expression of the FoxE3 enhancer.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Yoshimoto et al., 2005). Because the dominant-negative form of Delta1 induced head defects in embryos, we could not examine possible roles for Delta1 in FoxE3 regulation and subsequent lens formation. However, neural tube formation is accompanied by a dynamic change in Delta1 expression from the anterior neural ridge to the optic vesicle (Fig. 4C-E'), which might be responsible for the shift of FoxE3 expression from the pre-placodal ectoderm to the PLE (Fig. 4I-K'). FoxE3 expression in the PLE of embryos expressing the dominant-negative form of Delta2 was severely reduced, but remained in some cases, which might be due to some contribution of Delta1 to FoxE3 regulation.
As in Xenopus, Otx2 is expressed in the PLE of mouse embryos, and
the lens placode of Otx2 heterozygous mutant mice fail to form a
normal lens vesicle on an Otx1 homozygous mutant background
(Martinez-Morales et al.,
2001). deltaC, a zebrafish Notch-ligand gene that has the
highest sequence similarity to Xenopus Delta2, is expressed in the
developing optic vesicle in the same manner as Xenopus Delta2
(Smithers et al., 2000),
whereas a mammalian homolog of Delta2/deltaC has not been identified
yet. Interestingly, Jag1, a mammalian homolog of Xenopus
Serrate1, is expressed in the optic vesicle of rat embryos
(Bao and Cepko, 1997), and is
deleted in the mouse mutant coloboma (Cm), whose lens fails to detach
from the ectoderm as in the Foxe3 mutant mouse, dysgenetic lens
(dyl) (Blixt et al.,
2000; Brownell et al.,
2000; Theiler and Varnum,
1981; Xue et al.,
1999). Hence the role of Delta2/deltaC in the lens
induction of lower vertebrates might be taken over by Jag1 in
mammals. Regarding Notch receptors, mammalian Notch2 and
Notch3 are expressed in the developing lens
(Lindsell et al., 1996), but
their expression at earlier stages has not been characterized in detail. In
mammalian embryos, cells in the optic vesicle and lens ectoderm are physically
separated by a space, but are connected by cytoplasmic extensions
(McAvoy, 1980), which may
permit direct contact for Notch signaling. It is also possible that Notch
ligands might have secreted forms that are involved in Notch signaling
(Qi et al., 1999).
Otx2-Notch interactions in lens determination programs, and their analogy with selector-signaling system in Drosophila
The data presented here have significant implications for molecular
mechanisms underlying the stepwise determination of the lens. Otx2
expression in head ectoderm might constitute a part of the lens-forming bias
suggested by embryological studies, and Notch signaling is likely to be one of
the inducing signals provided from the optic vesicle to turn on the
lens-specification programs in this competent/biased ectoderm. Unlike
FoxE3, expression of a lens differentiation marker,
1-crystallin, was not induced in the trunk ectoderm by
misexpression of Otx2-GR and GR-Su(H)VP16 (not shown), suggesting that the
Otx2-Notch combination is not sufficient to activate the whole
lens-differentiation program. However, severe reduction or loss of
1-crystallin-positive lens placode cells in embryos expressing
the dominant-negative Delta2 suggests a crucial role for Notch signaling in
lens specification. The lens differentiation programs are presumably turned on
when a set of all terminal regulators, such as FoxE3 and
L-maf, is activated in the PLE by different, but possibly
overlapping, mechanisms.
Genetic studies in mouse have shown that Pax6 lies upstream of
Mab21l1, and that Mab21l1 lies upstream of Foxe3
(Yamada et al., 2003). We
found that the combination of Otx2 and Notch signaling induced ectopic
FoxE3 expression in the trunk ectoderm without activating
Pax6 (not shown). These findings imply that the Pax6-Mab21l1
pathway controls FoxE3 expression indirectly through regulation of
Otx2 and/or Notch signaling. Notch2 might be a downstream
target of Pax6, as the broad expression of Notch2 in the
head ectoderm is, as development proceeds, gradually localized to the lens and
olfactory fields expressing Pax6
(Fig. 4A-B' and see Fig.
S5 in the supplementary material).
Interestingly, the combinatorial mechanism of FoxE3 regulation is
similar to the selector-signaling system in Drosophila, in which
selective gene activation by signals for cell fate specification is achieved
by obligate integration of both inputs of field-specific transcription factors
(selectors) and signal-activated transcription factors at the level of their
target cis-regulatory elements (Guss et
al., 2001). Although this system has not been previously examined
in vertebrate development, our study suggests that the same mechanism
underlies the `multiple-step induction' of the lens. As classic embryological
studies demonstrate similar stages in many vertebrate organ systems
(Gurdon, 1992), a
selector-signaling system might be broadly used in vertebrates for specifying
a variety of organ and tissue identities by reiteratively using a limited
number of signaling pathways. While the events we are studying in this paper
occur during the bias and specification phases of lens induction, the same
principle could apply earlier, during the competence period, when a broadly
expressed selector gene may contribute to the period of competence and
converge with an early lens-inducing signal.
Xenomics (Xenopus genomics) for analysis of genomic regulatory networks for development
The results of our classic-style deletion analysis are in close agreement
with those from the comparative analysis of human and Xenopus
genomes, which demonstrates the effectiveness of using the Xenopus
genome for in silico prediction of conserved regulatory elements in
vertebrates. The conserved enhancer of mouse Foxe3 identified in our
study (-3529 to -3107) is included in the lens element that was independently
identified by Kondoh's group by deletion analysis in transgenic mice (-4.40 to
-2.63 kb) (Yoshimoto et al.,
2005), showing that Xenopus and mouse assays give
consistent results.
An important challenge in the post-genomic era is to untangle the complex
wiring of gene regulatory networks controlling development, growth and
differentiation. As shown in the pioneering study of the gene regulatory
network for sea urchin endomesoderm specification
(Davidson et al., 2002), this
type of study requires a high-throughput assay system for comprehensive
analysis of cis-regulatory elements. The mammalian-Xenopus genome
comparison and an approach developed in the course of this study -
co-transgenesis - which rapidly tests enhancer activities by co-injection of
PCR products along with the basal promoter-GFP cassette, will allow
Xenopus to serve as a vertebrate model system that fulfils this
requirement.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/2/249/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bao, Z. Z. and Cepko, C. L. (1997). The
expression and function of Notch pathway genes in the developing rat eye.
J. Neurosci. 17,1425
-1434.
Blitz, I. L. and Cho, K. W. (1995). Anterior neurectoderm is progressively induced during gastrulation: the role of the Xenopus homeobox gene orthodenticle. Development 121,993 -1004.[Abstract]
Blixt, A., Mahlapuu, M., Aitola, M., Pelto-Huikko, M., Enerback,
S. and Carlsson, P. (2000). A forkhead gene,
FoxE3, is essential for lens epithelial proliferation and closure of
the lens vesicle. Genes Dev.
14,245
-254.
Bourguignon, C., Li, J. and Papalopulu, N. (1998). XBF-1, a winged helix transcription factor with dual activity, has a role in positioning neurogenesis in Xenopus competent ectoderm. Development 125,4889 -4900.[Abstract]
Brownell, I., Dirksen, M. and Jamrich, M. (2000). Forkhead Foxe3 maps to the dysgenetic lens locus and is critical in lens development and differentiation. Genesis 27,81 -93.[CrossRef][Medline]
Chitnis, A., Henrique, D., Lewis, J., Ish-Horowicz, D. and Kintner, C. (1995). Primary neurogenesis in Xenopus embryos regulated by a homologue of the Drosophila neurogenic gene Delta. Nature 375,761 -766.[CrossRef][Medline]
Davidson, E. H., Rast, J. P., Oliveri, P., Ransick, A.,
Calestani, C., Yuh, C. H., Minokawa, T., Amore, G., Hinman, V., Arenas-Mena,
C. et al. (2002). A genomic regulatory network for
development. Science
295,1669
-1678.
Eastman, Q. and Grosschedl, R. (1999). Regulation of LEF-1/TCF transcription factors by Wnt and other signals. Curr. Opin. Cell Biol. 11,233 -240.[CrossRef][Medline]
Faber, S. C., Dimanlig, P., Makarenkova, H. P., Shirke, S., Ko, K. and Lang, R. A. (2001). Fgf receptor signaling plays a role in lens induction. Development 128,4425 -4438.[Medline]
Gammill, L. S. and Sive, H. (1997). Identification of otx2 target genes and restrictions in ectodermal competence during Xenopus cement gland formation. Development 124,471 -481.[Abstract]
Gammill, L. S. and Sive, H. (2001). otx2 expression in the ectoderm activates anterior neural determination and is required for Xenopus cement gland formation. Dev. Biol. 240,223 -236.[CrossRef][Medline]
Gan, L., Mao, C. A., Wikramanayake, A., Angerer, L. M., Angerer, R. C. and Klein, W. H. (1995). An orthodenticle-related protein from Strongylocentrotus purpuratus. Dev. Biol. 167,517 -528.
Grainger, R. M. (1992). Embryonic lens induction: shedding light on vertebrate tissue determination. Trends Genet. 8,349 -355.[Medline]
Gurdon, J. B. (1992). The generation of diversity and pattern in animal development. Cell 68,185 -199.[CrossRef][Medline]
Guss, K. A., Nelson, C. E., Hudson, A., Kraus, M. E. and
Carroll, S. B. (2001). Control of a genetic regulatory
network by a selector gene. Science
292,1164
-1167.
Ishibashi, S. and Yasuda, K. (2001). Distinct roles of maf genes during Xenopus lens development. Mech. Dev. 101,155 -166.[CrossRef][Medline]
Jen, W. C., Wettstein, D., Turner, D., Chitnis, A. and Kintner, C. (1997). The Notch ligand, X-Delta-2, mediates segmentation of the paraxial mesoderm in Xenopus embryos. Development 124,1169 -1178.[Abstract]
Kaufmann, E., Muller, D. and Knochel, W. (1995). DNA recognition site analysis of Xenopus winged helix proteins. J. Mol. Biol. 248,239 -254.[Medline]
Kenyon, K. L., Moody, S. A. and Jamrich, M. (1999). A novel fork head gene mediates early steps during Xenopus lens formation. Development 126,5107 -5116.[Abstract]
Kiyota, T., Jono, H., Kuriyama, S., Hasegawa, K., Miyatani, S. and Kinoshita, T. (2001). X-Serrate-1 is involved in primary neurogenesis in Xenopus laevis in a complementary manner with X-Delta-1. Dev. Genes Evol. 211,367 -376.[CrossRef][Medline]
Ko, L. J. and Engel, J. D. (1993). DNA-binding
specificities of the GATA transcription factor family. Mol. Cell.
Biol. 13,4011
-4022.
Kroll, K. L. and Amaya, E. (1996). Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122,3173 -3183.[Abstract]
Kusanagi, K., Inoue, H., Ishidou, Y., Mishima, H. K., Kawabata,
M. and Miyazono, K. (2000). Characterization of a bone
morphogenetic protein-responsive Smad-binding element. Mol. Biol.
Cell 11,555
-565.
Lai, E. C. (2004). Notch signaling: control of
cell communication and cell fate. Development
131,965
-973.
Lang, R. A. (2004). Pathways regulating lens induction in the mouse. Int. J. Dev. Biol. 48,783 -791.[CrossRef][Medline]
Lindsell, C. E., Boulter, J., diSibio, G., Gossler, A. and Weinmaster, G. (1996). Expression patterns of Jagged, Delta1, Notch1, Notch2, and Notch3 genes identify ligand-receptor pairs that may function in neural development. Mol. Cell. Neurosci. 8,14 -27.[CrossRef][Medline]
Martinez-Morales, J. R., Signore, M., Acampora, D., Simeone, A.
and Bovolenta, P. (2001). Otx genes are required for
tissue specification in the developing eye.
Development 128,2019
-2030.
Mathers, P. H., Grinberg, A., Mahon, K. A. and Jamrich, M. (1997). The Rx homeobox gene is essential for vertebrate eye development. Nature 387,603 -607.[CrossRef][Medline]
McAvoy, J. W. (1980). Cytoplasmic processes interconnect lens placode and optic vesicle during eye morphogenesis. Exp. Eye Res. 31,527 -534.[CrossRef][Medline]
Muller, F., Blader, P. and Strahle, U. (2002). Search for enhancers: teleost models in comparative genomic and transgenic analysis of cis regulatory elements. BioEssays 24,564 -572.[CrossRef][Medline]
Nieuwkoop, P. D. and Faber, J. (1967). Normal XTable of Xenopus laevis. Amsterdam: North-Holland.
Offield, M. F., Hirsch, N. and Grainger, R. M. (2000). The development of Xenopus tropicalis transgenic lines and their use in studying lens developmental timing in living embryos. Development 127,1789 -1797.[Abstract]
Ogino, H. and Yasuda, K. (1998). Induction of
lens differentiation by activation of a bZIP transcription factor, L-Maf.
Science 280,115
-118.
Ogino, H. and Yasuda, K. (2000). Sequential activation of transcription factors in lens induction. Dev. Growth Differ. 42,437 -448.[CrossRef][Medline]
Pommereit, D., Pieler, T. and Hollemann, T. (2001). Xpitx3: a member of the Rieg/Pitx gene family expressed during pituitary and lens formation in Xenopus laevis. Mech. Dev. 102,255 -257.[CrossRef][Medline]
Qi, H., Rand, M. D., Wu, X., Sestan, N., Wang, W., Rakic, P.,
Xu, T. and Artavanis-Tsakonas, S. (1999). Processing of the
notch ligand delta by the metalloprotease Kuzbanian.
Science 283,91
-94.
Rones, M. S., McLaughlin, K. A., Raffin, M. and Mercola, M. (2000). Serrate and Notch specify cell fates in the heart field by suppressing cardiomyogenesis. Development 127,3865 -3876.[Abstract]
Sive, H., Grainger, R. and Harland, R. (2000). Early Development of Xenopus laevis - A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Smith, J. C. (2005). Xenopus genetics and genomics. Mech. Dev. 122,259 -262.[CrossRef][Medline]
Smithers, L., Haddon, C., Jiang, Y. J. and Lewis, J. (2000). Sequence and embryonic expression of deltaC in the zebrafish. Mech. Dev. 90,119 -123.[CrossRef][Medline]
Theiler, K. and Varnum, D. S. (1981). Development of coloboma (Cm/+), a mutation with anterior lens adhesion. Anat. Embryol. 162,121 -126.[CrossRef][Medline]
Tun, T., Hamaguchi, Y., Matsunami, N., Furukawa, T., Honjo, T.
and Kawaichi, M. (1994). Recognition sequence of a highly
conserved DNA binding protein RBP-J. Nucleic Acids
Res. 22,965
-971.
Wasserman, W. W. and Sandelin, A. (2004). Applied bioinformatics for the identification of regulatory elements. Nat. Rev. Genet. 5,276 -287.[CrossRef][Medline]
Woolfe, A., Goodson, M., Goode, D. K., Snell, P., McEwen, G. K., Vavouri, T., Smith, S. F., North, P., Callaway, H., Kelly, K. et al. (2005). Highly conserved non-coding sequences are associated with vertebrate development. PLoS Biol. 3, e7.[CrossRef][Medline]
Xue, Y., Gao, X., Lindsell, C. E., Norton, C. R., Chang, B.,
Hicks, C., Gendron-Maguire, M., Rand, E. B., Weinmaster, G. and Gridley,
T. (1999). Embryonic lethality and vascular defects in mice
lacking the Notch ligand Jagged1. Hum. Mol. Genet.
8, 723-730.
Yamada, R., Mizutani-Koseki, Y., Hasegawa, T., Osumi, N.,
Koseki, H. and Takahashi, N. (2003). Cell-autonomous
involvement of Mab21l1 is essential for lens placode development.
Development 130,1759
-1770.
Yoshimoto, A., Saigou, Y., Higashi, Y. and Kondoh, H.
(2005). Regulation of ocular lens development by Smad-interacting
protein 1 involving Foxe3 activation.
Development 132,4437
-4448.
Zhou, X., Hollemann, T., Pieler, T. and Gruss, P. (2000). Cloning and expression of xSix3, the Xenopus homologue of murine Six3. Mech. Dev. 91,327 -330.[CrossRef][Medline]
Zygar, C. A., Cook, T. L. and Grainger, R. M., Jr (1998). Gene activation during early stages of lens induction in Xenopus. Development 125,3509 -3519.[Abstract]
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