Sato SM and Sargent TD (1991), Localized and inducible expression of Xenopus-p...

XB-ART-24722

Development 1991 Jul 01;1123:747-53.

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Localized and inducible expression of Xenopus-posterior (Xpo), a novel gene active in early frog embryos, encoding a protein with a 'CCHC' finger domain.

Sato SM , Sargent TD .

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Xenopus-posterior (Xpo) is a gene that is activated at or shortly after the midblastula transition (MBT). The RNA accumulates to a relatively low level, which remains constant until gastrulation, then rapidly and transiently increases in posterior ectoderm and mesoderm. A single copy of a putative finger motif, of the 'CCHC' type, is located near the carboxyl terminus. One or two copies of similar sequence motifs are found in the nucleocapsid protein of retroviruses where they are involved in protein-RNA interactions, and in cellular nucleic acid binding protein (CNBP), a protein that binds to the sterol regulatory element. Xpo expression is induced in ectodermal explants by treatment with basic fibroblast growth factor (bFGF) and with polypeptide growth factors found in medium conditioned by the Xenopus XTC cell line (XTC-CM). Taken together, these properties suggest a possible role for Xpo in the organization of the anteroposterior axis during development.

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Species referenced: Xenopus
Genes referenced: acta4 actc1 actl6a cnbp fgf2 post tbx2

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	Fig. 1. Localization of Xpo transcripts in tailbud embryos. Stage 22-25 embryos were dissected into head, trunk, and tail regions. Total RNA (2 fig per lane) was analyzed by RNA gel blotting, using as a probe a P-labeled DNA fragment derived from the Xpo cDNA clone. RNA samples were prepared in parallel and were equally intact as judged from the ethidium bromide staining pattern (not shown). The blot was exposed to X-ray film with an intensifier screen for 3 days at -70°C.
	Fig. 2. Developmental expression of Xpo. Total RNA (2 fig per lane) from embryos of the indicated stages (st) was analyzed by RNA gel blotting as described in Fig. 1. A faint band is visible in the stage 8-9 lane in the original autoradiogram. Exposure to X-ray film was as for Fig. 1.
	Fig. 3. Localization of Xpo transcript during mid- to late gastrulation by in situ hybridization. Cross sections of embryos at stage 11 (A) or stage 12.5-13 (B) were hybridized to 35S-labeled antisense Xpo RNA probe. A schematic of the plane of section and hybridization pattern (cross-hatching) is shown to the right of the dark field image. In panel A the dorsal side of the embryo is oriented to the right. Note that in gastrula endoderm, air bubbles create an apparent background in the dark field image; however, these bubbles can be distinguished clearly from silver grains under the microscope.
	Fig. 4. Partial DNA sequence and deduced protein sequence of Xpo. The DNA sequence of the Xpo cDNA clone, A72A is shown only for the existing 5' untranslated leader and the protein coding region. The remaining 2080 nt of 3' untranslated sequence has been included in the GenBank submission (Accession Number X58487). Stop codons in the 5' untranslated region are underlined, as is the 'CCHC motif. Nucleotide and amino acid (parentheses) residue numbers are given at right.
	Fig. 5. Comparison of CCHC motifs from Xpo and other proteins. Identical residues are shaded. References for sequences are as follows: HIV (Human Immunodeficiency Virus), Ratner et al. 1985; Copia, Mount and Rubin, 1985; CNBP con (consensus of 7 copies in Cellular Nucleic Acid Binding Protein), Rajavashisth et al. 1989.
	Fig. 6. Induction of Xpo by treatment of ectodermal explants with XTC-CM or bFGF. Explants dissected from stage 8 blastulae were incubated in various conditions until sibling controls reached stages 11, 13 or 28 as indicated. Incubation conditions were as follows: lane b, no treatment; lane c, bFGF at SOngml"1; lane d, XTC-CM (1:10 dilution). Total RNA prepared from whole embryo siblings (lane a, 1 embryo equivalent) or explants (lanes b-d, 10 explants) was subjected to gel blot analysis. The blot was hybridized to a mixture of nick-translated -"P-labelled Xpo and a'-actin probes. Arrows indicate the position of the RNA for Xpo, cytoskeletal actin (CA), a'-actin (muscle actin, MA), and the 28S and 18S ribosomal RNAs.