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Dev Growth Differ
2015 Dec 01;579:591-600. doi: 10.1111/dgd.12246.
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Pou5f3.2-induced proliferative state of embryonic cells during gastrulation of Xenopus laevis embryo.
Nishitani E
,
Li C
,
Lee J
,
Hotta H
,
Katayama Y
,
Yamaguchi M
,
Kinoshita T
.
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POU class V (POU-V) transcription factors play the important role in maintenance of pluripotency and cell differentiation. Pou5f3.2 (Oct25), one of Xenopus POU-V transcription factors, shows the zygotic expression prior to gastrulation. In order to know the molecular mechanism of pou5f3.2 expression at gastrula stage, we examined a responsiveness of pou5f3.2 to Nodal signaling. Animal cap assay demonstrated that Xnr2 activates the gene expression of pou5f3.2. In comparative analysis of the 5'-flanking region of pou5f3.2 between Xenopus laevis and X. tropicalis, two conserved regions were detected within the flanking region. Reporter analyses showed that one of the conserved regions contained an enhancer region, which had several Smad2/3 and FoxH1 binding motifs. ChIP assay demonstrated that Smad2 binds to the enhancer region. These results suggest that Nodal signaling induces zygotic expression of pou5f3.2 at gastrula stage. To understand a role of pou5f3.2 in gastrula embryos, morpholino oligo DNA of pou5f3.2 was injected into the lateral side of one blastomere at the 2-cell stage. The morphant embryos showed diminution of Xbra1 expression and gastrulation defect in the injection side, suggesting the essential role of pou5f3.2 at the gastrula stage. Xbra1 expression and gastrulation were also inhibited by injecting with the synthesized RNAs of pou5f3.2. Furthermore, in the pou5f3.2-injected embryo, gene expression of p27Xic1 was drastically suppressed, and the number of dividing cells increased in the injection side. These results suggest that one role of pou5f3.2 is to keep the embryonic cells in undifferentiated and proliferative state during gastrulation.
Fig. 1. Localized expression of pou5f3.2 during gastrulation. (A)
Expression profile of pou5f3.2 in the early development of Xenopus
embryos. Gene expression was quantitatively examined by
RT-PCR according to the Nieuwkoop and Faber’s stages. egg:
unfertilized egg. Histone H4 is an internal control. -RT: without
reverse transcriptase reaction. (B) Distribution of pou5f3.2
expression in whole-mount in situ hybridization. Localized
expression of pou5f3.2 (arrow) was observed in the marginal
zone. Lateral view with the animal pole upside and the dorsal
side right. (C) RT-PCR analysis showing pou5f3.2 expression in
the isolated ectoderm (ect), mesoderm (mes) and endoderm
(end) at St.10.5. WE: whole embryo. XK81, Xbra1 and Sox17a
are marker genes for ectoderm, mesoderm and endoderm,
respectively. Histone H4 is an internal control. -RT: without
reverse transcriptase reaction.
Fig. 2. Effect of Nodal signal on gene expression of pou5f3.2.
Synthesized RNA of Xnr2 was injected into both blastomeres of
2-cell stage embryos. Animal caps were isolated from the Xnr2-
injected embryos at St.8, and were reared until St.10.5 with or
without cycloheximide (CHX). Total RNA was extracted from the
animal caps and gene expression of pou5f3.2 was examined by
RT-PCR using reduced number of PCR cycles to detect an effect
of Xnr2. Gene expression of Xbra1 was examined as a positive
control for Nodal signaling. Histone H4 is an internal control. -RT:
without reverse transcriptase reaction.
Fig. 3. Enhancer in the 50-flanking region of pou5f3.2 gene. (A)
Reporter construct, 5056 pou5f3.2/luc, which consists of
5056 bp upstream region of pou5f3.2 gene and the firefly luciferase
(upper panel). CR1 and 2: the DNA regions conserved
between Xenopus laevis pou5f3.2 and X. tropicalis pou5f3.2.
Luciferase activity was examined using St.10.5 embryos injected
with various deletion constructs of pou5f3.2 50-flanking region
(lower panel). Luciferase activity is relatively shown on the base of
the value of 2842 pou5f3.2/luc. (B) DNA sequence of the
pou5f3.2 50-flanking region showing the enhancer activity. This
region has two putative Smad binding motifs (red box), two putative
FoxH1 binding motifs (green box) and three FoxH1 binding
motif-like sequences (light green box). (C) ChIP assay of Smad2
binding to the enhancer region of pou5f3.2 promoter. Myc-tagged
Smad2 (MT-Smad2) RNA was injected into the 2-cell stage
embryo, and immunoprecipitation with anti-myc antibody was performed
at St.10.5. PCR analysis of the enhancer region was performed
using DNA extract before precipitation (input) or after
precipitation with antibody (anti-myc) or without antibody (no Ab).
Fig. 4. Effect of pou5f3.2 and pou5f3.2MO on the early development.
(A) Gene expression of Xbra1 in pou5f3.2 or pou5f3.2MOinjected
embryos. Synthesized RNA of GFP (a, d), pou5f3.2 (b, e)
or pou5f3.2MO (c, f) was injected into lateral side of one blastomere
at 2-cell stage together with b-galactosidase as a tracer,
and gene expression of Xbra1 was examined at St.10.5 by
whole-mount in situ hybridization. Arrowheads indicate the injection
side, which is indicated by red reaction product of b-galactosidase.
a–c, lateral view with the animal pole upside and the
dorsal side left. d–f, vegetal view with the dorsal side up. (B)
Gastrulation of pou5f3.2 or pou5f3.2MO-injected embryo. Synthesized
RNA of GFP (a, e), pou5f3.2 (b, f), pou5f3.2MO (c, g) or
pou5f3.2MO and pou5f3.2 (d, h) was injected into lateral side of
one blastomere at 2-cell stage, together with GFP RNA as a tracer,
and morphological changes were examined at St.11. a–d,
vegetal view with the dorsal side up. Arrowheads indicate the
injection side. e–h, GFP fluorescence showing the injection side
in a, b, c and d, respectively. (C) Gastrulation defect of
pou5f3.2MO-injected embryo. Effect of pou5f3.2MO on the gastrulation
was evaluated as a percentage of the embryos showing
gastrulation defect (black) or normal gastrulation (white) at St. 11.
Bar indicates the standard deviation calculated from more than
70 samples. Gastrulation defect was caused in a dose-dependent
manner, and rescued by the co-injection with synthesized
RNAs of 4-base mismatch pou5f3.2.
Fig. 5. Cell proliferation in pou5f3.2-injected embryos. (A) Cell
division in the marginal zone of St.10.5 embryo injected with
800 pg GFP RNA, 800 pg pou5f3.2 RNA or 4.2 ng pou5f3.2MO.
M-phase cells were detected as red fluorescence by immunostaining
with anti-PH3 antibody. Nuclei were stained with Hoechst.
(B) Mitotic index in the marginal zone of St.10.5 embryo injected
with GFP RNA, pou5f3.2 RNA or pou5f3.2MO. Bar indicates the
standard deviation calculated from more than seven samples.
Fig. 6. Gene expression of p27Xic1 in pou5f3.2-injected
embryos. (A) Whole-mount in situ hybridization showing p27Xic1
expression at St. 10.5. The embryo was injected with 800 pg
GFP RNA or pou5f3.2 RNA into the animal pole at 1-cell stage.
Lateral view with the animal pole upside. (B) RT-PCR analysis of
p27Xic1 expression in the pou5f3.2-injected embryo at St.10.5.
Histone H4 is an internal control. -RT: without reverse transcriptase
reaction. (C) RT-PCR analysis of p27Xic1 expression in the
pou5f3.2MO-injected embryo at St. 10.5. Histone H4 is an internal
control. -RT: without reverse transcriptase reaction.
Fig. 7. Schematic diagram showing a feedback loop of pou5f3.2
and Nodal. Nodal signaling activates the gene expression of
pou5f3.2, which in turn inhibits the Nodal signaling. Such a feedback
loop might play a role in maintaining the moderate expression
of pou5f3.2. Consequently, pou5f3.2 is able to keep cells in
proliferative state instead of cell differentiation through the downregulation
of Nodal and p27Xic1.
