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Li DH
,
Chan T
,
Satow R
,
Komazaki S
,
Hashizume K
,
Asashima M
.
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We isolated and characterized the Xenopus translocon-associated protein XTRAP-gamma, one of four subunits of the translocon-associated protein complex. TRAP has been proposed to aid the translocation of nascent polypeptides into the lumen of the endoplasmic reticulum, but this has not been demonstrated until now. XTRAP-gamma was specifically expressed in the pronephros tubules of Xenopus laevis from stage 25 during kidney development. Antisense morpholino oligonucleotide-mediated knockdown of XTRAP-gamma suppressed tubulogenesis and decreased expression of the pronephros marker genes Pax-2 and Wnt-4. XTRAP-gamma morpholinos also inhibited differentiation of the pronephros in activin/retinoic acid-treated animal caps. We conclude that XTRAP-gamma plays an important role in the process of pronephros differentiation.
Fig. 2. Whole-mount in situ hybridization analyses
demonstrate the localization of XTRAP-γ
transcripts. The expression of XTRAP-γ was
weakly ubiquitous during all stages of development.
(A-F) Localization of XTRAP-γ was observed
from stage 12 onward. (D) Condensed
XTRAP-γ signals were detected in the otic vesicles
(arrowhead) and pronephric tubules (arrow) from
stage 25 onward. (E) In the early tadpole stages,
XTRAP-γ transcripts were localized in the eyes
(arrow). (F) By stage 33/34, XTRAP-γ was expressed
in liver (arrow). (G-I) Histology section of
the Xenopus embryo from Fig. 2F. e, eye; ov, otic
vesicle; p, pronephros; l, liver.
Fig. 3. XTRAP-γ MO affects pronephric tubulogenesis. (A) XTRAP-γ MO
specificity in vitro. Western-blotting analysis showed significant inhibition of
XTRAP-γ- HA (100 pg/embryo) protein synthesis by XTRAP-γ MO (40 ng/embryo).
XTRAP-γ- HA (100 pg/embryo) protein synthesis was not affected with 5-mis-
XTRAP-γ MO (40 ng/embryo). (B-G) Whole-mount immunohistochemistry analysis
on pronephros. (B) XTRAP-γ MO (40 ng), (D) 5-mis-XTRAP-γ MO (40 ng) and
(E) XTRAP-γ MO (40 ng) plus 5-mis-XTRAP-γ mRNA (1 ng) were injected into one
side of the lateral marginal zone of 16-cell-stage embryos. Embryos were fixed at
stage 40. Development of the pronephros was analyzed by staining with the
monoclonal antibody 3G8. (B) Pronephric tubule formation was inhibited in XTRAP-
γ MO-injected embryos (arrow in B) compared with the non-injected-side (arrow
in C) and the embryos injected 5-mis-XTRAP-γ MO (arrow in D). The phenotype of
the pronephric tubules was rescued by co-injection of XTRAP-γ MO with 5-mis-
XTRAP-γ mRNA (E). (F,G) Histological analysis of XTRAP-γ MO injected embryo
and 5-mis-XTRAP-γ MO injected embryos. Pronephros tubules in XTRAP-γ MOinjected
embryos showed severe developmental defects (arrow in F) compared
with that of the 5-mis-XTRAP-γ MO-injected embryos (arrow in G).
Fig. 4. Induction experiments in
XTRAP-γ MO-injected animal caps.
Two-cell stage embryos were injected
with 40 ng/embryo XTRAP-γ MO. All
animal caps were treated with activin/
retinoic acid for 3 h. (A,B) For light
microscopy, semi-thin sections were
stained with 0.1% toluidine blue solution.
(A) Pronephros differentiation
was markedly inhibited by XTRAP-γ
MO (arrow indicates muscle tissues)
compared with (B) animal caps of the
control (arrowhead indicates pronephros
tissues). (C,D,E) For electron microscopy,
ultra-thin sections were
double stained with uranyl acetate
and lead citrate. Only samples injected
with XTRAP-γ MO are shown (C,D).
The results show the condensation of
mesodermal cells and a radial arrange-
Fig. 5. Whole-mount in situ hybridization
shows the effect on
expression of pronephros marker
genes XTRAP-γ MO injection.
Embryos were injected with XTRAP-
γ MO (40 ng) or 5-mis-XTRAP-γ MO
(40 ng) and synthetic of β-galactosidase
mRNA (250 ng), then fixed at
stage 30/31 (A-P) and stage 22/23
(Q-T). Expression of Lim-1 was
increased in the side of embryos
injected with XTRAP-γ MO (A),
compared the non-injected side (B).
Expression of Pax-2 and Wnt-4
was inhibited by injection of XTRAP-
γ MO (E,I), compared with noninjected
side (F,J). For those embryos
fixed at stage 22/23, expression
of Pax-2 (Q) and Wnt-4 (S) in
embryos fixed at stage 22/23 was
not affected by injection of XTRAP-
γ MO or the control 5-mis-XTRAP-
γ MO (R,T). Expression of Pax-8
was not affected by XTRAP-γ MO
(M,N). The expression of Lim-1
(C,D), Pax-2 (G,H), Wnt-4 (K,L)
and Pax-8 (O,P) was unchanged in
embryos injected with 5-mis-
XTRAP-γ MO.The cells with the basal membrane (arrows), however the unique pronephric cells with microvillus was invisible. (E) Muscle differentiation was
normal. Ld, lipid droplet; Yg, yolk granule. Scale bar of (A,B) is 100 μm; scale bar of (C,D) is 1 μm; scale bar of (E) is 5 μm.
Fig. 5. Whole-mount in situ hybridization
shows the effect on
expression of pronephros marker
genes XTRAP-γ MO injection.
Embryos were injected with XTRAP-
γ MO (40 ng) or 5-mis-XTRAP-γ MO
(40 ng) and synthetic of β-galactosidase
mRNA (250 ng), then fixed at
stage 30/31 (A-P) and stage 22/23
(Q-T). Expression of Lim-1 was
increased in the side of embryos
injected with XTRAP-γ MO (A),
compared the non-injected side (B).
Expression of Pax-2 and Wnt-4
was inhibited by injection of XTRAP-
γ MO (E,I), compared with noninjected
side (F,J). For those embryos
fixed at stage 22/23, expression
of Pax-2 (Q) and Wnt-4 (S) in
embryos fixed at stage 22/23 was
not affected by injection of XTRAP-
γ MO or the control 5-mis-XTRAP-
γ MO (R,T). Expression of Pax-8
was not affected by XTRAP-γ MO
(M,N). The expression of Lim-1
(C,D), Pax-2 (G,H), Wnt-4 (K,L)
and Pax-8 (O,P) was unchanged in
embryos injected with 5-mis-
XTRAP-γ MO.
Fig. 6. The phenotype of pronephric tubules was rescued by coinjection
with Pax-2, Wnt-4, or Pax-2 and Wnt-4. (A) XTRAP-γ MO (40
ng), (C) XTRAP-γ MO (40 ng) plus Pax- 2 (100 pg), (E) XTRAP-γ MO (40 ng)
plus Wnt-4 (100 pg), (G) XTRAP-γ MO (40 ng) plus Pax-2 (100 pg) and Wnt-
4 (100 pg) were injected into one side of the lateral marginal zone of 16-
cell-stage embryos. Embryos were fixed at stage 40. Development of the
pronephros was analyzed by staining with the monoclonal antibody 3G8.
(A) Pronephric tubule formation was inhibited in the side of embryos
injected with XTRAP-γ MO (arrow in A) compared to non-injected-side
(arrow in B). (C) Pronephric tubule formation was significantly rescued in
embryos injected with XTRAP-γ MO (40 ng) plus Pax-2 (100 pg) (arrow in
C) compare to the XTRAP-γ MO-injected embryos (arrow in A). Although
the dorsal branches of the pronephric tubules were shorter in the injected
side than in the non-injected side (arrow in D), three dorsal branches of the
pronephric tubules were still visible. Injection of (E) XTRAP-γ MO (40 ng)
plus Wnt-4 (100 pg), or (G) XTRAP-γ MO (40 ng) + Pax-2 (100 pg) and Wnt-
4 (100 pg) almost completely rescued pronephric tubule formation, with
relatively normal pronephric tubules being observed.
ssr3 (signal sequence receptor, gamma (translocon-associated protein gamma)) gene expression in a Xenopus laevis embryo, NF stage 33. Lateral view: anteriorleft, dorsal up
