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Dev Growth Differ
1998 Feb 01;401:97-104. doi: 10.1046/j.1440-169x.1998.t01-6-00011.x.
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Cloning and expression pattern of Xenopus prx-1 (Xprx-1) during embryonic development.
Takahashi S
,
Uochi T
,
Kawakami Y
,
Nohno T
,
Yokota C
,
Kinoshita K
,
Asashima M
.
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Homeobox genes are expressed both temporally and spatially during vertebrate development, and regulate the tissue-specific expression of other genes. A Xenopus paired-related homeobox- 1 (Xprx-1) cDNA was cloned. Xprx-1 had a paired-related homeodomain, but did not contain a paired-box. The sequence of Xprx-1 had a high level of homology with K-2(mouse) and Prx-1 (chicken), thus Xprx-1 is assumed to be the Xenopus homolog of these genes. Xprx-1 transcripts were maternally restricted, in Xenopus embryos, and a decrease in the late blastula stage was followed by an increase in zygotic transcripts after gastrulation. The transcripts were localized to the animal hemisphere of the late blastula and were concentrated in the branchial arches of the tail-bud stage embryo. In animal cap experiments, Activin A dose-dependently induced Xprx-1 gene expression. These results suggest that Xprx-1 plays a role in early Xenopus development similar to other species.
Fig. 1. Sequence of Xprx-1 and comparison with related homeodomain proteins. (a) Sequence of Xprx-1 eDNA and the corresponding
amino acid sequence. The homeodomain is boxed. Primers used for cloning and detection of expression are indicated by arrows. (b)
Phylogenetic tree of Xprx-1 and related homeodomain proteins.
Fig. 2. Embryonic expression of
Xprx-1. (a) Northern blot analysis
was performed with total RNA
(20 j..Jg) from embryos at stage 40.
The full length Xprx-1 coding region
was used as a probe. (b) RT-PCR
and Southern blot analysis showing
the temporal expression of Xprx-1.
Total RNA for RT-PCR was isolated
from five embryos at various stages.
-7.48
Xprx-1• -4.40
-2.37
Xprx-1
ODC (egg) fertilized egg; (8) stage 8,
mid-blastula; (9) stage 9, late blastula;
(10) stage 10, early gastrula;
(12) stage 12, late gastrula; (15)
stage 15, mid-neurula; (20) stage 20, late neurula; (30) stage 30, tail-bud. Subcloned PCR products were sequenced and used as
probes. The RT-PCR was performed under two conditions, 27 cycles (top) and 30 cycles (middle). ODC is a loading control that is uniformly
expressed throughout early developmental stages (bottom; Bassez et a/. 1990; Osborne et at. 1991 ) . RT- (reverse transcriptase
was not added to the total WE RNA) is a negative control, and no genomic DNA contamination was detected.
Fig. 3. Spatial expression of Xprx-
1 at stage 9. RT-PCR and Southern
blot analysis showing localized expressions
of Xprx-1. Fifty embryos
were dissected into an animal cap
(AC). vegetal cap (VC), dorsal marginal
zone (DMZ), lateral marginal
zone (LMZ). and ventral marginal
zone (VMZ), as shown in the
diagram at the right. Total RNA was
isolated and used for RT-PCR and
Southern blot analysis. EF1-a is an
VC internal loading control that is expressed
ubiquitously in embryos
(Krieg et at. 1989). WE (whole embryo, stage 9) is a positive control, and RT- (reverse transcriptase was not added to the total WE
RNA) is a negative control.
Fig. 4. Spatial expression of Xprx-
1 during tail-bud stages. Xprx-1
expression was detected by wholemount
in situ hybridization using
albino embryos. In (a)-( e), anterior
is at the left. (a) Lateral view of
stage 25 embryo. Xprx-1 is weakly
expressed in the head region
(arrowhead). (b) Lateral view of
stage 30 embryo. Xprx-1 is expressed
in head mesenchymal
areas (arrowhead) and branchial
arches (arrows, 1 ,2,3,4, are the
arch numbers). (c) Enlarged view of
(b). (d) Dorsal view of a stage 30
embryo. (e) Lateral view of a stage
35 embryo. Xprx-1 is expressed in
the same region (left-arrowhead)
and the ventral region of the tail-bud
(right-arrowhead). (f) Transverse
section of the head region in a
stage 30 embryo indicated by the
black bar in (b). Xprx-1 is expressed
in the head mesenchyme
(arrowheads).
Fig. 5. Expression of Xprx-1 in animal caps treated with peptide
growth factors. Total RNA was isolated from animal caps treated
with different concentrations of Activin A or bFGF for 6 h. RT-PCR
and Southern blot analysis showed that Activin A dose dependently
induced expression of Xprx-1 genes, but that bFGF
did not. EF1-a served as an internal loading control. WE (whole
embryo, stage 20) was a positive control, and RT- (reverse transcriptase
not added to total WE RNA) was a negative control.
Fig. 6. Temporal expression of Xprx-1 in animal caps treated
with Activin A. Animal caps were treated in Steinberg's solution
with or without 100 ng/ml Activin A for 30 min. After treatment,
the animal caps were maintained in normal Steinberg's solution
for various periods (0-24 h). RT-PCR and Southern blot analysis
showed induction of zygotic Xprx-1 expression after 6 h in
Activin A-treated animal caps, but not in non-treated animal caps.
EF1-a served as an internal loading control. WE (whole embryo,
stage 20) is a positive control, and RT- (reverse transcriptase not
added to total WE RNA) is a negative control.
prrx1 (paired related homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anteriorleft, dorsal up.
