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Fig. 1 Amino acid sequences
of msx-2 in human, mouse and
chick are compared with the
two versions of the Xenopus
msx-2 genes, Xmsx-2 and
Xhox-7.1’. Comparisons are
based on a clustal multiplesequence
alignment of predicted
amino acid sequences published
in the EMBL database.
The boxed areas indicate homology
of all the members,
whereas the stippled area indicates
conservative changes.
Where Xmsx-2 and Xhox-7.1’
differ the most, around 64
to112 a.a., Xmsx-2 has higher
conservation with the other
members. The homeodomain is
underlined. There is one amino
acid residue difference in the
homeodomain of the human
gene compared to the rest of
the members. Surrounding the
homeodomain region are extensive
areas of homology, as
indicated by the numerous
boxed areas. The last row of
sequence indicates the shared
homologous sequence of all
the members of the genes with
conserved changes indicated
by a period (.)
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Fig. 2A,B Temporal expression profile of Xmsx-2. A Exposed
film of a Northern blot analysis of Xmsx-2 and EF1-α. Total
RNAs extracted from Xenopus oocytes (O) and embryos at stages
9, 13, 20, 27 and 41 were electrophoresed on a 1% agarose formaldehye
gel and transferred to a membrane that was hybridized
to probe 7.3. It was rehybridized with radiolabelled EF1-α (bottom
lane) to check for loading. The migration of the 28S and 18S ribosomal
RNAs are indicated on the left. Signal is first detected at
stage 13; two transcripts are apparent in some lanes while the larger
transcript is evident first at stage 13 and gains intensity by stage
41. The slighter smaller transcript is first seen at stage 27 but
seems to decrease by stage 41. B Reverse transcriptase polymerase
chain reaction (RT-PCR) analysis of Xmsx-2 expression at the embryonic
stages indicated (E, egg). Elongation factor-1 alpha (EF1-
α) is used as the internal control. These experiments were performed
by reverse transcribing RNAs extracted from egg and different
staged embryos as indicated. Polymerse chain reactions
were performed on the cDNAs with primers specific to Xmsx-2
and EF1-α at 59°C for 27 cycles in the same reaction tube in a
50-μl volume. The reaction products were loaded and electrophoresed
on a 1% agarose gel. Four hundred nanograms of φΧHaeIII
were also loaded as a marker (M). Expression of the Xmsx-2 is
first detected at stage 11 and continues to be present until stage 41
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Fig. 3A, B Spatial expression profile of Xmsx-2 in developing Xenopus
embryos. A In situ hybridization with a digoxigenin-labeled
antisense Xmsx-2 gene-specific probe was performed on tissue
sections prepared from embryos at different stages. Xmsx-2 transcripts
were present in the posterior part of the stage-13 embryo (a
transverse section through the dorsal blastoporal lip) around the
blastoporal lip region (arrow). At stage 15 (b), expression was obvious
in both the ventral (v) and dorsal (d) parts of the blastoporal
collar. Sections through the sagittal (c) and transverse (d) planes of
stage-17 embryos revealed expression of the Xmsx-2 gene in the
anterior and posterior part of the embryo (arrows in c) and dorsal
expression specifically in the lateral neural crest cells (d, lnc). At a
later stage of development, transcripts were observed anteriorly in
areas behind the cement gland (cg), heart primordium, and posteriorly
in the tail fin area (arrow). The expression of the transcripts
at stage 34 (f) seemed to be localized to the branchial arches (br)
and in the area behind the cement gland; expression was also present
in the tail fin area (not shown). B RT-PCR analysis of the spatial
pattern of Xmsx-2 expression. Embryos were dissected as
shown on the top row of a stage-23 embryo from the dorsal view
(left) and lateral view (R). A cut is made dividing the embryo into
anterior (A) and posterior (P) halves. To obtain dorsal (D) and
ventral (V) tissues, cuts were made along the lines as indicated.
Dorsal tissues were further dissected into anterior (DA) and posterior
(DP). RNAs were extracted from the different embryo parts as
well as whole (W) embryos and RT-PCR performed as described
in Methods. The PCR products were then electrophoresed in 1%
agarose gels as shown above. In all stages examined, there was no
expression in the ventral region. There appears to be a shift in the
expression from the posterior to the anterior part of the embryo at
different stages of development. Initially (stages13–17), expression
seems more abundant in the posterior region, but by stages
18–23, posterior expression has declined, eventually increasing by
stage 30; M marker, W whole, A anterior, P posterior, D dorsal, V
ventral, DA dorsal anterior, DP dorsal posterior
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Fig. 4a, b Xmsx-2 gain-of-function phenotype of Xenopus embryos.
a One nanogram of in vitro transcribed msx-2 mRNA was microinjected
randomly into the marginal zone of newly fertilized
Xenopus embryos. The embryos were allowed to grow and their
phenotype noted. The control uninjected embryo is depicted at the
top. Note the anterior truncation of the bottom embryo, although
the overall morphology of the posterior part seems to be intact.
b There is a range in the extent to which anterior structures are
missing. The control, uninjected embryo is on the far right. Note
the progressive decrease in the amount of anterior structures, ranging
from reduced eyes and cement glands to missing head structures.
The tail structure still appears intact in all the embryos
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Fig. 5a, b Lineage tracing with green fluorescent protein (GFP).
About 600 ρg synthetic GFP mRNA was coinjected with about
1 ηg msx-2 mRNA into the marginal zone of one-cell Xenopus
embryos that were then allowed to grow. a The embryo depicted
here is approximately stage 41 and shows some phenotypic effect
of the overexpression of the msx-2 gene; the cement gland and
eyes are reduced, and there is some slight bending of the body axis.
b The same embryo under fluorescent light shows the presence
of the green protein in the head and somitic region
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Fig. 6 Analysis of marker genes in Xmsx-2 gain-of-function embryos.
Graphs showing levels of genes normalized against those of
EF1-α. RNAs were extracted from pools of about 10 stage-12 and
-18 embryos that had been microinjected with the msx-2 mRNA at
the one-cell stage. The cDNAs that were reverse transcribed from
these RNAs were subject to PCR reactions with primers specific to
the Xhox-3, Xwnt-8, Xvent-1, and Xbra genes in combination with
primers specific to the EF1-α. The PCR reactions were electrophoresed
on a 1% gel and blotted onto membranes that were then hybridized
to radiolabeled probes of the specific genes being tested.
After hybridization, the membranes were stripped and probed with
EF1-α. Levels of the genes of interest and of EF1-α, corresponding
to the intensities in the graphs, were analyzed and measured with a
phospho-imager. These graphs represent levels of the genes normalized
against those of EF1-α. The stippled boxes indicate normal
uninjected embryos, while the clear boxes indicate embryos that
had been injected with the exogenous mRNA. Markers of a ventral-
posterior nature, e.g. Xhox-3, Xvent-1, and Xwnt-8 are upregulated,
more in the anterior region than posteriorly
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