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Fig. 1. Structural analysis of the Xenopus Krox-20 gene and comparison with human and mouse Krox-20 genes. (A) Schematic illustration of the XKrox-20
genomic clone isolated (an uppermost trace). A part of the clone that includes two exons and an intron is zoomed and compared with a corresponding part of
human Krox-20, egr-2 (lower traces). The two exons are represented by boxes, in which the consensus C2H2 type Zinc finger regions are dotted. Numbers
represent those from the transcription initiation site. The translation initiation (0) and termination (A) sites are also indicated. The 3Vend of exon 2 in XKrox-20
is not determined. (B) Alignment of the nucleotide sequences around the transcription initiation site of Krox-20 genes from three different species. The
respective initiation sites are indicated (!). Gaps in the sequence are positioned with dashes and numbers are indicated for the Xenopus sequence. The positions
of sequence identity are also indicated (*). The previously known sequence elements are boxed. These include the Ets-binding site (EBS), the CArG-box, the
cyclic AMP response element (CRE), and the TATA-box.
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Fig. 2. Analysis of promoter activity within the genomic XKrox-20 sequence. (A) Schematic illustration of subcloning strategy. In the upper trace, the coding
sequence of XKrox-20 is boxed. The 5Vflanking sequence is subcloned into a site immediately upstream of the firefly-luciferase-coding sequence (Luc) in the
reporter plasmid pGL3, while the intron and 3Vflanking sequences are subcloned into a site immediately downstream of the luciferase-coding sequence (the
lower trace). (B) Schematic illustration of injection strategy. The luciferase reporter constructs were injected into eight-cell stage embryos together with an
internal standard plasmid pRL-CMV that contained Renilla luciferase-coding sequence. Arrows indicate injection sites. AD; animal-dorsal. VV; vegetalventral.
(C) The promoter activity of the genomic XKrox-20 fragments. Injected reporter constructs are shown schematically on the left of histogram. Luciferase
activities were measured at stage 20 and the firefly luciferase reporter activity normalized to the pRL-CMV internal standard activity was represented in the
histogram as percentages of the maximum value.
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Fig. 3. Identification of a minimal promoter element within the 5Vflanking sequence of XKrox-20. A series of deletion constructs was injected into two AD
blastomeres at the eight-cell stage and relative luciferase activities are presented as described for Fig. 2C. (A and B) A series of 5Vdeletion constructs from
3987 to 56 that were indicated on the left of each histogram was assayed. (C) A series of 3Vdeletion constructs from 1 to 55, which had a fixed 5V
boundary at 72, was assayed. These constructs had a heterologous TATA-box-containing sequence in the immediate upstream of the luciferase-coding
sequence. Injected reporter constructs are shown schematically on the left of histogram.
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Fig. 4. Analysis of the effects of mutation of CArG-box and CRE on the
minimal promoter activity. The original construct had the minimal promoter
element ( 72 to 51) and the heterologous TATA-box-containing
sequence as described for Fig. 3C. The CArG-box and CRE were mutated
as indicated on the left of histogram ( ). Mutated promoter fragments were
double-stranded synthetic oligonucleotides. The original and mutated
constructs were injected into two AD blastomeres at the eight-cell stage
and assayed. Relative luciferase activities are presented as percentages of
the maximum value.
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Fig. 5. A ternary complex formation on the minimal promoter element by SRF and CREB proteins. Binding reactions were carried out with end-labeled probe,
either wild type or mutated, in the absence or presence of whole cell extracts from stage 20 embryos. Further additives are indicated below. Reaction mixtures
were electrophoresed on 3.5% native polyacrylamide gel. In A and B, end-labeled wild-type probes ( 73 to 43) were used. (A) Lane 1, probe alone; 2, probe
plus extracts and no competition; 3, competition with unlabeled probe at 125-fold excess; 4, components in lane 2 plus anti-SRF; 5, components in lane 2 plus
anti-pCREB; 6, components in lane 2 plus anti-SRF and anti-pCREB. (B) Lane 1: probe plus extracts and no competition; 2, competition with unlabeled CArGbox-
mutated fragment at 125-fold excess; 3, competition with unlabeled CRE-mutated fragment at 125-fold excess. (C) Lane 1: end-labeled wild-type probe
plus extracts; 2, end-labeled CRE-mutated probe plus extracts; 3, end-labeled CArG-box-mutated probe plus extracts; 4, components in lane 2 plus anti-SRF; 5,
components in lane 3 plus anti-pCREB; 6, components in lane 1 plus anti-pCREB and anti-SRF.
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Fig. 6. Suppression of the minimal promoter activity in the reporter
construct by inhibition of SRF. (A) Structural features of wild-type XSRF
and its dominant-negative version employed (XSRFDC). Black and gray
boxes represent the C-terminal transactivation and the N-terminal DNA
binding domains, respectively. (B) Suppression of the minimal promoter
activity by XSRFDC and its rescue by wild-type XSRF. Synthetic mRNAs
encoding XSRFDC or XSRF were coinjected with the minimal reporter
construct described in Fig. 4 and pRL-CMV into two animal blastomeres of
eight-cell stage embryos. The injected amounts of XSRFDC and XSRF
mRNAs were 1.5 and 4.5 pg/blastomere, respectively. The total amount of
injected mRNA was adjusted to 6.0 pg/blastomere by adding control GFP
mRNA. Reporter activities were analyzed and presented as in Fig. 2B.
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Fig. 7. Involvement of SRF in the expression of endogenous XKrox-20. In
each panel, mRNA was injected into two animal blastomeres on one side
(*) at the eight-cell stage embryo and in situ hybridization was performed at
the stage 20 (C to G and I) or stage 11.5 (H). (A) XSRFDC mRNA and
lineage-tracing GFP mRNA were coinjected at 60 and 6 pg/blastomere,
respectively. Florescent view of a stage 20 embryo. (B) Bright-field view of
A. The arrow points to the cement gland. (C) Control GFP mRNA was
injected at 60 pg/blastomere. (D) XSRFDC and lineage-tracing GFP
mRNAs were coinjected at 60 and 6 pg/blastomere, respectively. (E)
XSRFDC and XSRF mRNAs were coinjected at 60 and 80 pg/blastomere,
respectively. In C, D, and E, arrows point to the expression of XKrox-20,
whereas arrowheads point to that of BF-1. (F) XSRFDC and lineage-tracing
GFP mRNAs were coinjected as in D and double in situ hybridization was
performed to visualize GFP mRNA (red signal), BF-1 transcript (green
signal, indicated by arrowhead), and XKrox-20 transcript (green signal,
indicated by arrow). (G) The same injection scheme as in F. Double in situ
hybridization was performed to visualize GFP mRNA (red signal), BF-1
transcript (green signal, indicated by arrowhead), and XmafB transcript
(green signal, indicated by arrow). (H) The same injection scheme as in F.
Double in situ hybridization was performed to visualize GFP mRNA (red
signal) and chordin transcript (green signal, indicated by arrow). (I) The
same injection scheme as in F. Double in situ hybridization was performed
to visualize GFP mRNA (red signal) and actin transcript (green signal,
indicated by arrow).
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Fig. 8. Involvement of SRF in FGF-induced expression of XKrox-20 in ectoderm cells. (A) Experimental design for ectodermal cell culture assay used in B.
Control GFP or XSRFDC mRNA was injected at 60 pg/blastomere into four animal blastomeres of eight-cell stage embryos. When they reached stage 10,
ectodermal tissues were isolated. The dissociated ectodermal cells were then inoculated into microculture wells at 200 cells/well. After completion of
reaggregation by brief centrifugation, cells were cultured in the presence of increasing concentrations of bFGF until control embryos reached stage 23. The
transcriptional levels of XKrox-20 and BF-1 were analyzed by RT-PCR (Kengaku and Okamoto, 1995; Hongo et al., 1999). (B) Suppression of FGF-induced
XKrox-20 expression in ectoderm cells by XSRFDC. Autoradiographs are shown of RT-PCR products of the transcripts from XKrox-20 and BF-1, both of
which were coamplified with EF1a transcript, an internal standard (upper panels). Each RT-PCR product was quantified by a laser image analyzer and values
for XKrox-20 and BF-1 transcripts with (.) or without (o) overexpression of XSRFDC, which are normalized to EF1a transcript, are presented as percentages
of the respective maximum value and plotted against bFGF dose (lower graphs).
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