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Using a subtracted Xenopus cDNA library based on the differential sensitivity of anterior and posterior genes to retinoic acid, we isolated a novel Xenopus nuclear GTP-binding protein (XGB). XGB is expressed prominently in the optic primordia at the tailbud stage. The N-terminal region of XGB contains a set of GTP-binding protein motifs, and the C-terminal region contains two putative nuclear localization signals and two coiled regions. A GFP-XGB fusion protein was expressed in the nucleus of NIH3T3 cells where it bound to subnuclear structures. Truncated C-terminal constructs of XGB containing both nuclear localization signal(s) and coiled region(s) suppressed eye formation, whereas neither the N-terminal construct nor constructs with a mutated GTP-binding protein motif affected eye formation. Expression of Pax6 and Rx1 genes, which are crucial for eye development, was reduced in embryos overexpressing the C-terminal constructs of XGB. Suppression of Pax6 and Rx1 at earlier developmental stages as well as perturbation of eye formation at later stages was counteracted by co-expression of wild-type XGB. We conclude that XGB plays a role in the formation of optic primordia through activation of at least two eye field transcription factors.
Fig. 1. (A) Comparison of amino acid sequence of Xenopus
nuclear GTP-binding protein (XGB) and human chronic renal
failure gene (CRFG). Dots in the CRFG sequence indicate
residues identical to XGB. Double lines indicate the set of GTPbinding
protein motifs. Dotted underlines indicate putative
nuclear localization signals, and plain underlines indicate
putative coiled regions. (B) Schematic drawings of the four
truncated constructs (N-terminal, C-terminal, C1, and C2) of
XGB used in subsequent studies.
Fig. 2. Spatiotemporal expression pattern of Xenopus nuclear
GTP-binding protein (XGB). (A) Ribonuclease protection
analysis of XGB transcripts in developing Xenopus embryos.
Total RNA from embryos of stages 0–27 was hybridized with
an antisense RNA probe for XGB and then subjected to
ribonuclease protection analysis (lanes 0–27). XGB mRNA was
first detected at stage 13 and increased towards stage 27. In
lane Y, the RNA probe was hybridized with yeast RNA, and in
lane C, the RNA probe was not digested with ribonuclease. (B)
Whole-mount in situ hybridization showing expression of XGB in
developing Xenopus embryos. B1, Dorsal view of a stage 13
embryo showing expression of XGB in the anterior neural plate.
B2, Dorsal view of a stage 16 embryo showing expression of
XGB in a broader area of the neural plate. B3, Dorsal view of a
stage 20 embryo showing expression of XGB in the prospective
eye region. B4, Lateral view of a stage 23 embryo showing
expression of XGB in the optic primordium and branchial arch
region. Anterior and posterior sides are indicated by A and P,
respectively.
Fig. 3. Malformation of the optic structure in the embryo
injected with plasmid DNA encoding the C-terminal domain of
Xenopus nuclear GTP-binding protein (XGB). Forty pg of the
plasmid DNA encoding either the N-terminal or C-terminal
domain of XGB was injected into two animal blastomeres on the
dorsal side of an 8-cell stage embryo, and the embryo was
reared until the tailbud stage. The embryo overexpressing the
C-terminal domain (A) but not the N-terminal domain (B) of XGB
lacked an eye structure. (C) Non-injected embryo.
Fig. 4. Malformation of eye in embryos overexpressing
truncated forms of Xenopus nuclear GTP-binding protein
(XGB). Electroporation was performed after injection of 1.5 ng
of mRNA into the prospective eye regions on both sides of
stage 12 embryos. Embryos were photographed at stage 35 (A,
C, E, G, I and K) or stage 41 (B, D, F, H, J and L). (A,B) Embryos
overexpressing the C-terminal domain of XGB. (C,D) Noninjected
embryos. (E,F) Embryos overexpressing the N-terminal
domain of XGB. (G,H) Embryos overexpressing both the Cterminal
domain and wild-type XGB. (I,J) Embryos overexpressing
the C1 domain of XGB. (K,L) Embryos overexpressing the C2
domain of XGB. Scale bar, 1 mm.
Fig. 5. Internal structure of an eye-defected embryo. Shown are
a series of histological sections of stage 35 embryos overexpressing
either an N-terminal construct (A-F) or a C-terminal construct (G-L).
The truncated constructs were overexpressed by electroporation
of the respective mRNAs. Scale bar, 500 μm.
Fig. 6. Localization of Xenopus nuclear GTP-binding protein
(XGB) fusion proteins in subnuclear structures. Mouse NIH3T3
cells and HEK293 cells were transfected with plasmid DNA
encoding a fusion protein, and the fluorescence was observed
by laser scanning confocal microscopy. (A) Fluorescent micrograph
image of a mouse NIH3T3 cell transfected with plasmid DNA
encoding green fluorescent protein (GFP)-XGB fusion protein.
(B) Phase contrast image of the same cell as in (A). (C)
Superimposed image of (A) and (B). (D) Fluorescent micrograph
image of a HEK293 cell transfected with plasmid DNA encoding
the GFP-N-terminal XGB fusion protein. (E) Fluorescent micrograph
image of a HEK293 cell transfected with plasmid DNA encoding
DsRed2-C-terminal XGB fusion protein. (F) Phase contrast
image of the same cell as in (D). (G) Phase contrast image of
the same cell as in (E). (H) Superimposed image of (D) and (F).
(I) Superimposed image of (E) and (G).
Fig. 7. Suppression of Pax6 and Rx1 by overexpression of the
C-terminal domain of Xenopus nuclear GTP-binding protein
(XGB). XGB C-terminal domain and green fluorescent protein
(GFP) mRNAs were electroporated into the two prospective eye
regions of stage 12 embryos. At stage 20, the embryo was fixed
and subjected to whole-mount in situ hybridization. Shown is the
expression of GFP in the anterior neural tissue at stage 18 (A,
dorsal view) and stage 23 (B, lateral view). Overexpression of
the C-terminal domain leads to a drastic reduction of expression
of both Pax6 (C) and Rx1 (D) in the optic primordial, as
compared with the expression of Pax6 (E) and Rx1 (F) in the
embryo electroporated without mRNA. Suppression of Pax6 (G)
and Rx1 (H) by the C-terminal construct was rescued by coelectroporation
of mRNA encoding wild-type XGB.
Fig. 8. Specific mode of action of the C-terminal construct of
Xenopus nuclear GTP-binding protein (XGB). Plasmid DNA of
the C-terminal construct was injected into two animal blastomeres
on the dorsal side of 8-cell stage embryos in combination with
lineage-tracing GFP mRNA. Embryos were reared until stage
20. Double in situ hybridization was performed to visualize GFP
mRNA (red signal), Pax6 transcript (C and D, blue signal,
indicated by white arrowhead), Rx1 (E and F, blue signal,
indicated by white arrowhead), and En2 (E and F, blue signal,
indicated by white arrow). (A) Fluorescent view of a stage 18
embryo. (B) Bright-field view of the same embryo. (C, E) Embryos
overexpressing the C-terminal domain of XGB. (D, F) Embryos
overexpressing both the C-terminal domain and wild-type XGB.
gtpbp4 (GTP binding protein 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, dorsal view, anteriorleft.
gtpbp4 (GTP binding protein 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, dorsal up, anteriorleft.
