First published online 20 August 2003
doi: 10.1242/dev.00718
Integration of multiple signal transducing pathways on Fgf response elements of the Xenopus caudal homologue Xcad3
Tomomi Haremaki1,*,
Yasuko Tanaka1,2,
Ikuko Hongo1,
Masahiro Yuge3 and
Harumasa Okamoto1,
1 Neuroscience Research Institute, National Institute of Advanced Industrial
Science and Technology (AIST) Central 6, 1-1-1 Higashi, Tsukuba 305-8566,
Japan
2 Department of Anatomy, Institute of Basic Medical Sciences, University of
Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Japan
3 Faculty of Human Environmental Science, Fukuoka Women's University,
Kasumigaoka Higashi-ku, Fukuoka, 813-8529, Japan
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Fig. 3. Identification of multiple FREs within the intron 1. (A) Deletion and
sequence analysis of the intron 1. Reporter constructs examined contain intron
1 sequences that are deleted to various extents as depicted left of the
histogram. Reporter activities of these constructs were analyzed as in
Fig. 2, except that ectoderm
cells were cultured in the absence or presence (6 ng/ml) of bFgf. The ratio of
the luciferase activity obtained with bFgf to that obtained without bFgf is
taken as a measure of Fgf responsiveness and presented as a value of fold
induction in the histograms. Numbers on full-length intron 1 indicate
nucleotide length of respective segments. Multiple FREs appear to be
distributed within domain1 and domain 2 (light green) of intron 1. Domain 2*,
as defined in the text, is also indicated close to domain 2. Sequence analysis
of intron1 reveals that a number of Ets-binding motifs (EBM; A/CGGAA/T) and
Tcf/Lef-binding motifs (TLBM; CTTTGA/TA/T) are present throughout intron1 as
indicated by blue and red triangles, respectively. (B) Deletion analysis of a
209 bp fragment within domain 2*. Consecutive deletion of binding motifs
TLBM3, EBM4 and TLBM2, as indicated left of the histogram results in a
progressive reduction of Fgf responsiveness. Numbered red and blue triangles
point to the position of TLBMs and EBMs in the 209 bp fragment, respectively
(see Fig. 4A for precise
location).
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Fig. 4. EBMs and TLBMs as sub-elements of FREs. (A) Nucleotide sequence of the
domain 2* region of Xcad3 intron 1. EBMs and TLBMs are marked and
numbered. (B,C) Mutational analysis of EBMs (B) and TLBMs (C). Reporter
constructs examined contain the domain 2* fragments that carry various
combinations of mutations in EBMs or TLBMs, as indicated to the right of each
graph (E1, 3 means that EBM1 and EBM3 are mutated). Mutations were introduced
as follows: GAA to agA in E1, 2 and 4; TCC to ctC in E3 and 5; ATCCT to gTCga
in E6; TTTGT to Tcgag in T1; TCAAA to TagAc in T2; TCAAAGG to Tgtcgac in T3;
CTTTG to gaaTt in T4. Reporter activities of mutated constructs were analyzed
as in Fig. 3, except that
ectoderm cells were cultured in the absence or presence of increasing
concentrations of bFgf.
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Fig. 8. Direct interaction of EBM and TLBM with XTcf3, Sox2 and XEts1 proteins. An
end-labeled probe, either wild type or mutated, was incubated with XTcf3 or
Sox2, or with a combination of both proteins in A, and with Sox2 or XEts1, or
with a combination of both proteins in B, as indicated in diagrams above. All
proteins were tagged with a V5 epitope and made by in vitro translation. Probe
T2/E4/T3: an intronic DNA fragment containing TLBM2, 3 and EBM4 (overlined in
Fig. 4A). Probe E4: TLBM2 and 3
mutated. Probe T2/E4: TLBM3 mutated. Probe E4/T3: TLBM2 mutated. Mutations
were introduced as described in Fig.
4. Unlabeled probe (competitor) or antibody against V5 epitope was
added as indicated in the diagrams.
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Fig. 9. Intron 1 as an immediate early target of Fgf signaling (A) and a proposed
model of integration of multiple signaling pathways on FREs of Xcad3
intron1 (B). In A, experimental design was essentially the same as in
Fig. 2C, except for the
following changes. A reporter construct (–546/LUC/intron1) was injected
into animal blastomeres of eight-cell stage embryos. Ectodermal cells at stage
10 were cultured for 30 minutes with or without 10 µg/ml cycloheximide and
then for an additional 1.5 hours with or without bFgf (10 ng/ml). The
transcriptional levels of the reporter gene (luciferase-coding portion) were
analyzed by RT-PCR.
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© The Company of Biologists Ltd 2003
XFgf-4a or d59 (a control inactive version of 
transcript, an internal standard (upper panels). Each RT-PCR product was
quantified by a laser image analyzer and values for En2 (
) and
Xcad3 (
). (E,F) Presence of FRE
in the intron 1. Chimeric constructs injected are indicated below each graph:
they contained either Xcad3 intron1 (E) or SV40 enhancer sequence
(F). Reporter activities of these constructs were analyzed as in D and
presented in arbitrary units. Note that inclusion of intron1 sequence in
reporter constructs is essential for dose-dependent response to Fgf.
),
dnXER81 (
) and EnR (
