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The p8 gene encodes a transcription factor with a basic helix-loop-helix motif and has been cloned in rat, mouse and human. It is upregulated in acute pancreatitis. In the present study, the Xenopus laevis homolog of p8 (Xp8) was isolated by PCR. The full-length Xp8 cDNA consists of 677 bp and encodes 82 amino acids. The basic helix-loop-helix region is well conserved between X. laevis and mammals. Overexpression of an Xp8-EGFP fusion protein indicated that Xp8 was localized to nucleus, as in mammals. Analysis by RT-PCR showed that expression of Xp8 was increased after gastrulation and maintained into later developmental stages. Expression was detected in the prospective neural region at the neurula stage by whole-mount in situ hybridization. At the larval stage, Xp8 was expressed in central nervous regions, such as ventrolateral brain, trigeminal nerve, vestibulocochlear nerve and dorsal neural tube. Expression was also detected in the basal cement gland, olfactory placode, ear vesicle, notochord and anus. This specific pattern of expression suggests that Xp8 may play a role in the development of the central nervous system in X. laevis.
Fig. 1. Sequence of Xenopus
laevis p8 cDNA and the deduced
peptide sequence. The polyadenylation
site AATAAA is underlined.
A full-length Xp8 clone
was obtained by a modified
PCR method. The Xp8 cDNA is
677 bp in length and encodes
82 amino acids, including a
basic helix-loop-helix motif. It has
246 bp in the ORF, 114 bp in the
3 UTR and 317 bp in the 5 UTR.
Fig. 2. Comparisons of p8 peptide
sequences among Xenopus
laevis, human, mouse and rat.
(a) Alignments of Xp8 and the
three mammalian p8 peptides.
Conserved amino acids are
highlighted. A putative basic
helix-loop-helix (bHLH) region is
illustrated under the sequence.
The region defined by arrows
indicates a nuclear targeting
sequence (Mallo et al. 1997). (b)
Full-length amino acid sequence
identity between the four species.
(c) Sequence identity within the p8
bHLH regions of the four species.
Comparison of the full-length and bHLH regions indicates that only the bHLH region is well conserved. Arrowheads show conserved
serine, threonine and tyrosine residues, which may be targets for phosphorylation.
Fig. 3. Intracellular localization
of the Xp8. (a) Xp8-EGFP construct.
An Xp8-EGFP or EGFP-only
sequence was ligated into the
pCS2 plasmid vector. Enhanced
green fluorescent protein (EGFP)
or Xp8-EGFP transcripts were
injected into the animal pole at the
2-cell stage. The enclosed regions
in (b) and (e) are enlarged in (c)
and (f), respectively. Animal cap
cells injected with 2 ng EGFP
had fluorescence throughout the
cytoplasm (b,c), whereas Xp8-
EGFP was specifically localized to
the nucleus (e,f). Arrows show
the nucleus. Injected embryos
were cultured to stage 38 and
observed. Larvae injected with
0.2 ng EGFP mRNA or 2 ng
Xp8-EGFP mRNA are shown in
(d) and (g), respectively. (d) The
fluorescence remained strong
in whole embryo. (g) Only low
level fluorescence was observed
on the abdominal surface.
Fig. 4. Reverse transcription–polymerase chain reaction analysis
of Xp8 expression. Expression of Xp8 mRNA was increased
from stages 12 to 15 and was maintained until stage 40. Maternal
Xp8 mRNA was detected at very low levels.
Fig. 5. Whole-mount in situ
hybridization. Expression pattern
of Xp8 at stages (a, b) 14, (c, d)
18, (e, f) 22 and (g, h) 30. (a,c,e)
Dorsal (D) view, (d,f–h) lateral
view, (b) ventroposterior view.
The anterior (A) is situated on
the right side in all parts except
(b). Arrowheads show the central
nervous system. V, ventral; P,
posterior; AN, anus; NC, notochord;
CG, cement gland; VN,
vestibulocochlear nerve; TN,
trigeminal nerve; OP, olfactory
placode.
Fig. 6. After whole-mount in situ
hybridization, embryos were
embedded in paraffin and sectioned
at 12 μm. The larva shown
(stage 30) was sectioned at lines
A–D and sections are shown in
(A–D). CG, cement gland; Re,
retina; Ev, ear vesicle; Nt, neural
tube.
nupr1 (nuclear protein, transcriptional regulator, 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anteriorright.
nupr1 (nuclear protein, transcriptional regulator, 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anteriorright, dorsal up.
nupr1 (nuclear protein, transcriptional regulator, 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anteriorright, dorsal up.
