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First published online 21 January 2009
doi: 10.1242/dev.024703
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1 Department of Molecular and Cell Biology and Center for Integrative Genomics,
University of California, Berkeley CA 94720, USA.
2 Wellcome Trust/Cancer Research UK Gurdon Institute, Tennis Court Road,
Cambridge, CB2 1QN, UK.
3 Department of Zoology, Tennis Court Road, Cambridge CB2 1QN, UK.
4 Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720,
USA.
5 California Institute for Quantitative Biosciences, Berkeley, CA 94158,
USA.
6 Department of Physiology, Development and Neuroscience, Anatomy Building,
Downing Street, Cambridge CB2 3DY, UK.
* Author for correspondence (e-mail: amacher{at}berkeley.edu)
Accepted 30 December 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Enhancer prediction, Gene regulation, No tail/brachyury, Spadetail/tbx16
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). In
addition, T-box genes play an integral role during vertebrate gastrulation,
highlighted first in the mouse by the identification of the
T/Brachyury gene
(Dobrovolskaïa-Zavadskaïa,
1927), the first T-box gene to be molecularly identified
(Herrmann et al., 1990). Since
the cloning of Brachyury, orthologs have been found in
Xenopus (Smith et al.,
1991), zebrafish [ntl
(Schulte-Merker et al., 1992)
and brachyury (Martin and
Kimelman, 2008)], Ciona intestinalis
(Corbo et al., 1997), chick
(Kispert et al., 1995) and
many others (Naiche et al.,
2005), including species as distant from vertebrates as hydra
(Technau and Bode, 1999),
suggesting that Brachyury is an ancient gene. In vertebrates,
Brachyury homologs are expressed throughout the presumptive mesoderm
during gastrulation and later in the tail bud and notochord
(Kispert et al., 1995;
Schulte-Merker et al., 1992;
Smith et al., 1991;
Wilkinson et al., 1990). Loss
of Brachyury function causes a range of deficiencies in posterior
mesoderm and notochord (Herrmann et al.,
1990; Halpern et al.,
1993; Schulte-Merker et al.,
1994; Conlon et al.,
1996; Martin and Kimelman,
2008).
Members of the Tbx6/16 subfamily of T-box factors also play important roles
in mesoderm formation. In mouse, Tbx6 is expressed in the presumptive
mesoderm during gastrulation and in the tail bud and presomitic mesoderm
during segmentation (Chapman et al.,
1996). Strikingly, the paraxial mesoderm develops as neural tissue
in Tbx6 mutants (Chapman and
Papaioannou, 1998). Zebrafish contain paralogs of mouse
Tbx6, including spadetail (spt, tbx16),
tbx6 and tbx24, that are expressed in the presumptive
non-axial mesoderm (Ruvinsky et al.,
1998; Griffin et al.,
1998; Hug et al.,
1997; Nikaido et al.,
2002). Zebrafish spt mutants have severe defects in trunk
paraxial mesoderm formation as a result of incorrect cell migration and
specification (Kimmel et al.,
1989; Ho and Kane,
1990; Amacher and Kimmel,
1998).
Although spt and ntl mutant phenotypes have been well
characterized and some Spt/Ntl targets have been identified
(Yamamoto et al., 1998;
Goering et al., 2003;
Gourronc et al., 2007),
identification of Spt- and Ntl-responsive regulatory elements is necessary to
fully understand how these genes regulate mesoderm development. First, the
identification of gene regulatory regions reveals which genes are direct
targets of the factors and how they fit into the regulatory hierarchy of
mesoderm patterning. Second, regulatory element identification may uncover
features, such as binding site number and/or orientation of co-regulator
binding sites, that are required for maximal response. The importance of T-box
factor co-regulators is highlighted by the finding that Xbra directly
interacts with the transcription factor Smad1, which is crucial for the
activation of some targets in Xenopus
(Messenger et al., 2005).
We have designed a moderate throughput method for identifying and
characterizing Spt- and Ntl-responsive regulatory elements. First, we
identified putative downstream targets by measuring genome-wide transcription
in Spt- and Ntl-deficient embryos. We performed in vitro binding selection
assays to generate binding site models to search non-coding sequence around
putative targets for binding motif clusters, and prioritized these clusters
using comparisons to other fish genomes. We demonstrate that Spt and Ntl bind
two putative regulatory regions in vivo and in vitro, and have tested both
regions for transcriptional activity using a transgenic reporter assay. Our
results place Spt and Ntl directly upstream of tbx6, a T-box gene
involved in posterior mesoderm specification
(Hug et al., 1997). In
addition, we show that T-box factors directly regulate deltaD
(dld) in the posterior tail bud. As dld has been suggested
to prime the segmentation clock (Mara et
al., 2007), we suggest that Spt and Ntl play an important role in
initiating cyclic gene expression in the presomitic mesoderm. Finally, the
T-box sites that we defined within the tbx6 regulatory region cluster
with functional Tcf/Lef transcription factor binding sites
(Szeto and Kimelman, 2004),
suggesting that Spt and Ntl are required together with Wnt signaling to
regulate some targets. Several other putative target genes we have identified
are known targets of vertebrate Wnt signaling, including the mouse
dld homolog Delta-like 1 (Hofman et al., 2004), suggesting
that an interaction between T-box genes and Wnt signaling in gastrula mesoderm
is widespread.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Morpholino injections
The morpholino (MO) combination needed to phenocopy
spt-;ntl- mutant embryos was determined by
injecting translation-blocking MOs targeting either spt or
ntl into the complementary single mutant. Injections were performed
as described by Nasevicius and Ekker
(Nasevicius and Ekker, 2000).
A combination of four MOs reliably phenocopied
spt-;ntl- mutants (see Fig. S1 in the
supplementary material).
mRNA isolation and microarray hybridization
RNA was collected at 75% epiboly from embryos injected with MOs against
spt and/or ntl or control embryos injected with Danieau
solution plus 0.25% Phenol Red. Samples were prepared and hybridized as
previously described (Ouyang et al.,
2008). Array probes consisted of the Compugen/Sigma-Genosys
zebrafish oligo library (GEO platform accession number GPL7343). Three
biological replicates were performed for each treatment and genes with
significantly different expression levels in MO-injected embryos versus
controls were identified using a Student-Newman-Keuls post hoc test.
Gene expression time course profiling
Gene expression profiles were downloaded from
http://giscompute.gis.a-star.edu.sg/~govind/zebrafish/data_download.html
(Mathavan et al., 2005) and
visualized using MapleTree
(http://rana.lbl.gov/EisenSoftware.htm).
We normalized expression levels by setting the point of maximum expression for
each gene to 1.
Recombinant Spt-GST and Ntl-GST protein preparation and in vitro DNA binding assays
spt- and ntl-GST fusion constructs were created in the
vector pGEX-5X-1. Proteins were purified basically as described by Yagi et al.
(Yagi et al., 2004). SELEX
experiments were performed as described previously
(Senger et al., 2004) and
details can be provided on request. After 3-4 rounds of selection bound
oligomers were subcloned and sequenced. Binding competition assays with
genomic sequences (Fig. 5) were
carried out as described previously (Chan
et al., 2003).
Binding site score matrix construction and genome searches
Using Spt and Ntl SELEX data and previously identified binding sites for
Tcf/Lef and Suppressor of Hairless (see Fig. S6 in the supplementary
material), the program PATSER (Hertz and
Stormo, 1999) was used to generate binding site score matrices.
Sequences matching a score matrix with a P-value of 
0.001 were
counted as binding sites. We obtained gene models from the RefSeq database
(Pruitt et al., 2005) and
genomic positions from the UCSC genome browser
(Kent et al., 2002;
Karolchik et al., 2008)
(Sanger Institute, 2004:
ftp://ftp.sanger.ac.uk/pub/sequences/zebrafish).
UCSC genome browser-annotated repetitive sequences
(Smit et al., 2007;
Jurka, 2000) were not included
in genome searches.
Chromatin Immunoprecipitation (ChIP)
Chromatin immunoprecipitation on 75-85% epiboly zebrafish embryos was
performed as previously described (Wardle
et al., 2006; Morley et al.,
2009) using anti-Ntl antibody
(Schulte-Merker et al., 1992)
or anti-Spt antibody (Amacher et al.,
2002). The microarrays used contain probes ranging from 9 kb
upstream and 3 kb downstream of transcription start sites. ChIP PCR was
carried out using standard techniques
(Morley et al., 2009).
Reporter plasmid construction
The I-SceI-based plasmids mCherry::pXex:GFP and the Tol2-based
mCherry::pXex:GFP construct were based on the previously described
GFP::pXex:BFP construct (Szeto and
Kimelman, 2004) (details can be provided on request).
Regulatory element fragments were amplified from genomic or plasmid DNA
with high fidelity polymerase and inserted into reporter constructs using
standard molecular biology techniques. The dld Tol2 construct was
made using Gateway cloning procedures and plasmids from Tol2 Gateway kits
(Villefranc et al., 2007;
Kwan et al., 2007).
Site-specific mutagenesis was carried out using PCR-based methods. Primer
sequences can be provided on request.
Generation of transgenic zebrafish
Reporter constructs were injected as previously described for
I-SceI-based transgenesis
(Thermes et al., 2002) and
Tol2-based transgenesis (Kawakami,
2004; Kwan et al.,
2007). Transiently transgenic embryos were grown to the
appropriate stage and those with GFP expression at the blastoderm margin or in
the tail bud and somites were classified as expressing mCherry in these
regions robustly, faintly or not at all. To generate stable transgenic lines,
germline transgenic founders were identified by crossing to wild-type fish.
Embryos from these crosses were raised and their offspring analyzed.
In situ hybridization, photography and mounting
Whole-mount in situ hybridization was performed as described
(Thisse et al., 1993) with
modifications (Melby et al.,
1997). Digoxigenin-labeled RNA probes were synthesized from
templates as cited in Fig. 1.
Embryos were cleared and mounted as described previously
(Griffin et al., 1998).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Halpern et al.,
1993) they also have partially redundant roles
(Amacher et al., 2002). We
measured genome-wide gene expression in spt and/or
ntl-deficient embryos to identify their putative downstream targets,
focusing on downregulated genes as Spt and Ntl homologs appear to act mainly
as transcriptional activators (Horb and
Thomsen, 1997; Conlon et al.,
1996). We depleted embryos of Spt and Ntl activity using antisense
morpholinos (MOs) targeting spt and ntl transcripts (see
Fig. S1 in the supplementary material). To identify genes most likely to be
direct targets, we measured transcript levels at mid-gastrulation [75%
epiboly, 8 hours post-fertilization (hpf)], soon after spt and
ntl expression normally begins and before mutant phenotypes become
morphologically apparent. Genes with significantly different expression levels
in depleted embryos (compared with control embryos) that were downregulated
2-fold were considered further. The list of 44 hits (representing 41
candidate target genes) is shown in Fig.
1A. Microarray results are available from the Gene Expression
Omnibus (Accession Number GSE12857).
Downregulated genes include several known Spt and/or Ntl targets, such as
protocadherin 8 [a spt target
(Yamamoto et al., 1998)],
no tail dependent gene 5 [a ntl target
(Goering et al., 2003)],
mesogenin1 [a target of both spt and ntl
(Goering et al., 2003)], and
tbx6 [which is down-regulated in spt- embryos and
not expressed in spt-;ntl- embryos
(Griffin et al., 1998)]
(Fig. 1A). Some downregulated
genes have known mutant phenotypes: for example, cdx4 mutants have
severe posterior mesoderm truncations
(Davidson et al., 2003);
mesogenin 1 (Msgn1) mutant mice fail to properly specify trunk and
tail mesoderm (Yoon and Wold,
2000); dld, her1 and her7 mutants have
mesodermal segmentation defects (Holley et
al., 2000; Henry et al.,
2002); and knypek mutants have convergence and extension
defects (Topczewski et al.,
2001). These phenotypes are reminiscent of spt and
ntl mutant phenotypes, and thus consistent with the genes being
targets of T-box factors.
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spt and ntl target genes are spatially and temporally co-expressed with spt and ntl
We presumed that direct spt and ntl target genes would be
expressed in spatial and temporal patterns overlapping with those of
spt and ntl. To examine temporal overlap, we used an
existing microarray dataset (Mathavan et
al., 2005) that reports gene expression levels for >16,000
genes at 12 early developmental time points (0-48 hpf) using the same Compugen
probe set as our experiments. We visualized the normalized expression profiles
of spt/ntl target genes from our microarray, as well as the profiles
for spt and ntl, using the program MapleTree
(Fig. 1B). Although the target
genes have a variety of expression profiles, they are generally expressed at
similar times as spt and ntl. The spatial expression
patterns of many target genes listed in
Fig. 1A have been previously
determined and, for most of them, expression partially overlaps with
spt and/or ntl (Thisse
et al., 2001) (see Fig. S2 in the supplementary material). Most
are expressed in the non-axial gastrula margin and later in the tail bud,
whereas surprisingly few are expressed in the axial midline. Typical target
gene expression patterns, including those for the genes whose regulation we
characterized further, are shown in Fig.
1C. The apparent lack of more targets (such as ntd5)
expressed in the axial midline may be due to the function of a second
ntl-like gene, brachyury, which can functionally substitute
for ntl in anterior mesoderm
(Martin and Kimelman,
2008).
Spt and Ntl bind similar sequences in vitro
Many regulatory elements have been characterized by identifying large
genomic regions that drive appropriate reporter expression, followed by
deletion and mutational analyses to determine crucial regulatory sequences. To
identify Spt- and Ntl-responsive regulatory elements, we took a more
streamlined and targeted approach by searching for Spt- and Ntl-binding motif
enrichment in genomic sequence around target genes. We first generated binding
site models using in vitro binding selection assays (SELEX assays)
(Fig. 2A,B). We performed gel
mobility shift assays with random double-stranded oligonucleotides and
GST-tagged Spt and Ntl, and amplified bound DNA using PCR
(Gold et al., 1995). The
binding selection/amplification cycle was performed four times and bound
oligomers from round four were sequenced. Limited sequencing revealed that the
Spt-bound oligomer pool from round four consisted almost entirely of near
exact matches to previously identified consensus T-box binding sites
(Kispert and Herrmann, 1993).
As Spt-responsive regulatory elements probably contain both high- and
low-affinity sites, we also sequenced oligomers from the third SELEX round and
used these to generate binding site models. More than 70 bound
oligonucleotides were sequenced for each protein and are represented by
WebLogo diagrams (Crooks et al.,
2004) (Fig. 2C,D).
The Spt model has the consensus sequence TCACACCT and the Ntl model has the
consensus sequence TCACACC(C/T), both of which are similar to sequences bound
by T-box factors Brachyury, VegT, Eomesodermin and Tbx6 from other species
(Kispert and Herrmann, 1993;
Conlon et al., 2001;
White and Chapman, 2005). This
experiment also revealed that Spt, but rarely Ntl, can bind oligomers with a C
at position 1; we confirmed this using binding assays with oligomers differing
only at the first position (see Fig. S3 in the supplementary material).
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) to construct scoring matrices based upon our SELEX data and
background base frequencies derived from zebrafish intergenic sequence (36%
G-C; 64% A-T). Using a 500 bp sliding window, we searched 5 kb upstream of
potential target genes and within intronic sequence for motifs significantly
matching the Spt- or Ntl-binding site models (P-value 0.001) and
ranked each 500 bp region based upon the number of matches. Similar binding
site cluster searches have proven effective in identifying Drosophila
regulatory elements (Berman et al.,
2002; Markstein et al.,
2002). The regions with the greatest numbers of Spt and Ntl motifs
in our dataset are shown in Table S1 in the supplementary material. In
Drosophila, conservation of binding site clusters among species is a
good indicator of regulatory activity, even when the primary sequence of the
regulatory elements is divergent (Berman et
al., 2004). To determine whether T-box site clusters were
conserved, and thus more likely to identify Spt/Ntl-responsive regulatory
elements, we aligned the genomic loci listed in Table S1 with the orthologous
loci from Fugu (Aparicio et al.,
2002), Tetraodon
(Jaillon et al., 2004),
Gasterosteus (Broad Institute, 2007:
http://www.broad.mit.edu/models/stickleback/)
and Medaka (Kasahara et al.,
2007) obtained from the ENSEMBL database
(Flicek et al., 2008), and
assessed the number of Spt/Ntl motifs in the regions aligning with the 500 bp
zebrafish T-box binding site clusters (an example from the dld locus
is shown in Fig. S4 in the supplementary material).
We chose two putative regulatory regions for detailed analysis: the
dld second intron and the upstream region of tbx6. The
dld region (+911 bp to +1316) shows conservation among all four
species for which sequence is available. In addition, previous work showed
that a dld region between +1 and +2800 bp can drive gene expression
in the gastrula margin and in the tail bud during segmentation, although
regulatory motifs have not been identified
(Hans and Campos-Ortega,
2002). Because we recognize that sequences from other fish species
will not always be available for approaches such as ours, we also chose to
examine a candidate region from the tbx6 gene that contains six
putative T-box binding sites within 500 bp of the transcription start site for
which orthologous sequence was unavailable for comparison
(Flicek et al., 2008).
Additionally, this tbx6 region is known to drive Wnt- and
BMP-dependent gene expression in the forming mesoderm
(Szeto and Kimelman, 2004).
Thus, although several regions identified in our searches are promising
candidates for regulation by T-box factors, we selected regions near
dld and tbx6 for further analysis for the above reasons and
because of the predicted high affinity of the T-box binding motifs they
contain.
Spt and Ntl bind predicted regulatory elements in vivo and in vitro
To determine whether Spt and Ntl bind tbx6 and dld
genomic regions containing conserved binding site clusters in the embryo, we
performed chromatin immunoprecipitation (ChIP) at 75-85% epiboly using Spt and
Ntl antibodies (Figs 3 and
4). Ntl-bound regions were
detected using a promoter microarray containing probes corresponding to
sequences up to 9 kb upstream and 3 kb downstream of transcription start sites
(Fig. 3) and Spt-bound regions
were detected using promoter-specific qPCR
(Fig. 4). The conserved T-box
binding site cluster upstream of tbx6 is enriched 5-fold in the
Ntl-bound sample (Fig. 3A,B).
Although our promoter microarray does not include probes corresponding to the
dld intron 2 T-box site cluster, a probe corresponding to the intron
2-exon 3 junction of dld is 2.3-fold enriched in Ntl-bound DNA
(Fig. 3C,D). Using
promoter-specific qPCR (Morley et al.,
2009), we found that the tbx6 upstream region and
dld second intron T-box clusters are enriched 7.9±1.8 fold and
4.3±0.95 fold, respectively, in Spt-bound DNA relative to untreated DNA
(Fig. 4A). Additional details
for the ChIP experiments can be provided on request. The data indicate that
Spt and Ntl bind both candidate regulatory regions in vivo.
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T-box binding sites are required for tbx6 expression
tbx6 contains six Spt/Ntl binding motifs within 500 bp of the
transcription start site (Fig.
3A; Fig. 6A),
within a region previously shown to drive Wnt- and BMP-dependent gene
expression during gastrulation and segmentation stages
(Szeto and Kimelman, 2004).
Two Tcf/Lef-binding sites were shown to be crucial for the Wnt responsiveness
of this regulatory element (Szeto and
Kimelman, 2004) and their positions are shown
(Fig. 7B). To test the ability
of the tbx6 upstream region to drive gene expression in vivo, we
built a dual fluorescence reporter construct based on one designed by Szeto
and Kimelman (Szeto and Kimelman,
2004) that allows one to assess the activity and tissue
specificity of a test regulatory region in transient transgenic assays
compared with that of a ubiquitously expressed reporter on the same plasmid.
The construct contains tbx6 upstream sequence (-1.7 kb to the start
codon) driving mCherry (Shaner et al.,
2004) expression and a ubiquitous pXex promoter driving
GFP expression to mark cells that received the plasmid
(Fig. 6A). We used
I-SceI-mediated transgenesis
(Thermes et al., 2002) to
introduce the constructs into zebrafish embryos and analyzed GFP and mCherry
expression in the injected embryos between mid-gastrulation and early
segmentation stages. We observed that mCherry expression was restricted to the
blastoderm margin during gastrulation (Fig.
6B; Fig. 7C,
compare with Fig. 1C) and later
to the tail bud and somites (Fig.
7D), as previously reported
(Szeto and Kimelman, 2004). As
previously reported (Szeto and Kimelman,
2004), we also observed that the fluorescent reporter was more
stable than endogenous tbx6 transcripts
(Fig. 1C); thus, fluorescence
perdures in presomitic cells as they incorporate into somites and become
muscle cells. Reporter expression is detected in a majority of embryos at
shield stage (6 hpf) (see Fig. S5A in the supplementary material), when
endogenous tbx6 is initially expressed
(Hug et al., 1997), suggesting
the regulatory element is involved in initiation of tbx6 expression.
Double in situ hybridization of reporter construct-injected embryos reveals
that mCherry expression overlaps extensively with endogenous tbx6
expression (see Fig. S5B,C in the supplementary material). Similar to the
endogenous tbx6 gene, mCherry expression is eliminated when
spt and ntl MOs are injected with the reporter construct
(Fig. 6B-G; see Fig. S5A broken
versus unbroken line).
|
).
Consistent with this, T-box sites a, b and c have PATSER P-values
<0.001 using the Spt matrix, but not the Ntl matrix, suggesting that Spt
has a higher affinity for these sites. Site d has a similar predicted affinity
for both factors.
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). The same study found that the region between
the dld transcription start site and +2.8 kb (including intron 2)
contains regulatory information for driving mesodermal expression, but did not
further define the sequences required. We tested activity of a dld
reporter construct containing the 1.2 kb upstream sequence, the first two
exons and introns and partial exon 3 sequence, fused in frame to mCherry
(Fig. 8C) in transient
transgenic embryos. This region drove mCherry expression in the blastoderm
margin during gastrulation and in the tailbud during segmentation
(Fig. 8E,F) and reporter
expression overlaps well with endogenous dld expression (see Fig.
S5D,E in the supplementary material). To determine whether T-box sites in the second dld intron are required for activity, we mutated binding motifs in the context of the full-length regulatory element. The dld second intron from each fish species contains an assortment of Spt/Ntl binding motifs and two of these (sites c and d) are conserved in orientation and some flanking sequence (Fig. 8B). These two sites are followed closely by a consensus T-box binding site (site e) in zebrafish, so we mutated sites c, d and e. When these sites were mutated (Fig. 8G), mCherry expression substantially decreased (compare Fig. 8H,I with Fig. 8E,F). The remaining regulatory activity could be mediated by any of the four remaining T-box sites or other regulatory sequences contained in the construct.
To further characterize the expression pattern driven by the full-length
dld construct and facilitate functional analyses, we generated stable
lines (Fig. 9). In both lines
analyzed, we observed mCherry expression in a subset of locations where
dld is normally expressed (Fig.
9B-I). Specifically, we observed mCherry transcripts in regions
where spt and ntl are strongly co-expressed: at the
blastoderm margin (Fig. 9E-G)
and later in the posterior tail bud (Fig.
9C,D,H,I). mCherry was also faintly expressed in neural tissue
(Fig. 9H), which was expected
as the construct contains an regulatory element that drives expression in the
hindbrain and other neural tissues (Hans
and Campos-Ortega, 2002). The stable lines express mCherry at the
margin by 30% epiboly (Fig.
9E), the earliest time at which dld is expressed
(Hans and Campos-Ortega,
2002), suggesting the regulatory element initiates expression of
dld.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The use of comparative genomics to identify zebrafish regulatory sequence
has been successfully implemented in the past
(Dickmeis et al., 2004;
Yang et al., 2007;
Allende et al., 2006). One
difficulty with this approach is that the positions of regulatory elements and
of transcription factor binding sites within them often change over evolution,
making them difficult to identify through alignments, particularly across
large evolutionary distances (Sanges et
al., 2006; Moses et al.,
2006). Our method avoids some of these difficulties. First, we
compared zebrafish sequence to that of closely related species, increasing the
likelihood that the regulatory elements will be conserved. Second, we
evaluated the conservation of a sequence based on the presence of
transcription factor binding sites, allowing for some flexibility in the
positions of the regulatory element regions and the binding sites they
contain.
Regulatory interactions among T-box factors in mesoderm development
Detailed characterization of spt-, ntl- and
spt-;ntl- embryos reveals that spt and
ntl function in a partially redundant manner
(Amacher et al., 2002). Other
experiments suggest spt, ntl and tbx6 interact in three
ways: (1) two factors can activate target genes that neither factor can
activate on its own (combinatorial interactions); (2) two factors can
contribute to activation additively; and (3) one factor can prevent activation
by another factor (competitive antagonism)
(Goering et al., 2003). Our
microarray-based approach indicates that additive and redundant interactions
between Spt and Ntl are the most prevalent as many target genes have
moderately decreased expression levels when one of the two T-box genes is
depleted, but a greater decrease when both are depleted
(Fig. 1A). Our data indicate
combinatorial interactions are less common, as shown by the fact that only two
targets had a greater than twofold decrease in expression level in both single
knockdowns. The distinction between additive and combinatorial interactions is
somewhat arbitrary as we define categories based on discrete cutoffs. Two
genes were downregulated in ntl-depleted embryos, but upregulated in
spt-depleted embryos. These targets may represent examples of
competitive antagonism of Ntl function by Spt. One of the two genes, sb:cb259,
encodes a transforming acidic coiled-coil-containing protein that is expressed
strongly in the axial mesoderm and very weakly in the non-axial margin during
gastrulation (Thisse et al.,
2001), suggesting that Spt may repress expression in the non-axial
margin. The other gene (transgelin3) is not spatially restricted
during gastrula stages (Thisse et al.,
2001) (see Fig. S2 in the supplementary material). Microarray
experiments with other treatments (including tbx6 and bra
depletion, and spt, ntl, bra and tbx6 overexpression), as
well as other functional experiments, will allow the interactions among T-box
genes to be more fully characterized.
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).
Consistent with this, we find several known Wnt target genes among the T-box
targets identified in our microarray screen, including tbx6
(Szeto and Kimelman, 2004),
cdx4 (Shimizu et al.,
2005), mesogenin1
(Wittler et al., 2007),
myf5 (Shi et al.,
2002) and aldh1a2
(Weidinger et al., 2005).
During gastrulation, wnt8a and ntl positively regulate one
another (Ueno et al., 2007;
Goering et al., 2003;
Martin and Kimelman, 2008),
which may explain part of the overlap in Wnt/T-box target genes. However, we
show that tbx6 is directly regulated by T-box factors, which, taken
in together with previous results demonstrating its direct regulation by
Tcf/Lef factors (Szeto and Kimelman,
2004), indicates that, in some cases, the requirement for Wnt
signaling and T-box genes is a result of direct activation by both players.
The dld regulatory element we characterized also contains
Tcf/Lef-binding motifs and further analysis will reveal if Wnt signaling
contributes to activity. Several recent studies in mouse indicate that T-box
and Tcf/Lef transcription factor binding are important for gene activation in
the forming mesoderm, including expression of Delta homologs
(Dunty et al., 2008;
Wittler et al., 2007;
Hofmann et al., 2004). This
requirement could result from a direct physical interaction between Tcf/Lef
and T-box factors, as Xenopus VegT (a close Spt homolog) can
physically interact with Tcf3 in vitro and because protein domains of VegT and
Tcf3 that are crucial for physical interaction include their well-conserved
DNA-binding domains (Cao et al.,
2007). Two T-box binding sites in the zebrafish tbx6
regulatory element are close to Tcf/Lef binding sites (8 bp and 28 bp away),
and the mouse Dll1 regulatory element also contains T-box sites
within 20 bp of Tcf/Lef binding sites (Hofman et al., 2004). This close
clustering of binding sites could result in a physical interaction between the
proteins.
Distinct T-box responsive regulatory elements drive delta expression in the tail bud of mouse and zebrafish
Previous studies have shown that Dll1 expression in mouse is
driven in the tail bud by a combination of Tbx6 and Tcf/Lef factors acting
through an upstream element (Beckers et
al., 2000; Hoffman et al., 2004;
White and Chapman, 2005). By
contrast, dld expression in the zebrafish tail bud is driven by an
element downstream the transcription start site (Figs
8 and
9)
(Hans and Campos-Ortega,
2002). We show the zebrafish dld second intron contains a
tail bud regulatory element that is bound by Spt and Ntl, and requires the
proteins for activation (Figs
3,
4,
8 and
9). Thus, Dll1 and
dld are both directly regulated by T-box factors, but through
regulatory elements in different locations. Further experiments will determine
whether Wnt signaling activates the dld tail bud regulatory element,
as it does the Dll1 regulatory element in mouse. It could be that
these two elements are controlled by the same set of transcription factors and
are functionally very similar, but located in different parts of the gene.
This type of regulatory element shuffling may be common in vertebrate
evolution (Sanges et al.,
2006). Alternatively, it could be that the two regulatory elements
are regulated by different sets of factors and the regulatory network has
changed over evolution, but the Delta expression pattern in the tail
bud has remained consistent. dld is a component of the segmentation
clock in the posterior tail bud, and has been described as a factor required
to `prime' cyclic gene expression in the presomitic mesoderm
(Mara et al., 2007). Because
T-box factors directly regulate dld expression in this posterior
zone, spt and ntl act upstream of the priming process.
Several other target genes identified in our microarray study are components
of the segmentation clock, including the cyclic genes her1, her7 and
her15.1 (Henry et al.,
2002; Gajewski et al.,
2003; Oates and Ho,
2002; Shankaran et al.,
2007). Consistent with previous studies, recent work has shown
that cyclic gene expression starts early in gastrulation
(Riedel-Kruse et al., 2007).
Our results implicate Spt and Ntl in initiating cyclic gene expression.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/5/749/DC1
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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