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We characterized Xenopus SIP1 (XSIP1), Smad interacting protein, from activin-treated animal caps by differential screening. The XSIP1 is very similar to mouse SIP1 in the protein coding region including the zinc finger domain and homeodomain. The expression pattern was analyzed by RT-PCR and whole mount in situ hybridization. XSIP1 expression was initially restricted to the dorsal marginal zone in the late gastrula and was subsequently expressed at the lateral edge of neural plate and, in the tailbud stage, in the forebrain, neural tube, and eye. Overexpression of XSIP1 at the animal caps resulted in activation of anterior neural markers without mesodermal markers. Ectopic expression of XSIP1 induced enlargement of neural cells and disordered eye formation. In addition to abnormal head phenotypes, many embryos were short-tailed. Our findings suggest that XSIP1 is a transcriptional repressor, which may be involved in the activin-dependent signal pathway.
FIG. 2. RT-PCR analysis was performed at various stages.
Stages according to Niewcoop and Faber (1956) are shown over the
lanes. ODC (lower panel) is indicated as a loading control.
FIG. 2. RT-PCR analysis was performed at various stages.
Stages according to Niewcoop and Faber (1956) are shown over the
lanes. ODC (lower panel) is indicated as a loading control.
FIG. 3. Whole mount in situ hybridization with an XSIP antisense probe. (A) Stage 9 embryo, vegetal view. XSIP was not detected. (B
and C) Stage 10.5 embryo, dorsovegetal view and vegetal view. XSIP1 is detected in the dorsal ectoderm. (D) Stage 12 embryo, dorsal view.
(E) Stage 17 embryo, dorsal view. (F) Stage 26 embryo. Large white arrowhead and small white arrowhead indicate the telencephalon and
mesencephalon. d, dorsal; v, ventral; b, blastopore.
FIG. 4. XSIP1 overexpressing embryos. (A) Stage 38 embryo, uninjected control side. (B) Stage 38 embryo, XSIP1 mRNA (500 pg) injected
side. (C) Stage 38 embryo, XSIP1 mRNA (1 ng) injected side. (D) Stage 38 embryo, uninjected side. (E and F) Transverse section of the embryo
shown in B. The section levels are indicated in B. (G) Transverse section of the embryo shown in C. The section levels are indicated in C. l,
lens; r, retina; nt, neural tube.
FIG. 5. XSIP1 induced neural marker genes without mesoderm
induction in animal cap explants. Embryos were injected with 1 ng of
XSIP1 or control mRNA into the 2-cell stage animal pole. Animal
caps were explanted at stage 9 and cultured for 2 days. N-CAM,
general neural marker; XAG1, cement gland marker; F-spondin and
X1Hbox6, trunk/tail neural marker; ODC, internal positive control.
Vol. 271, No. 1, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
156
