Yang S et al. (1998), Overexpression of a novel Xenopus rel mRNA gene...

XB-ART-14922

J Biol Chem 1998 May 29;27322:13746-52. doi: 10.1074/jbc.273.22.13746.

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Overexpression of a novel Xenopus rel mRNA gene induces tumors in early embryos.

Yang S , Lockwood A , Hollett P , Ford R , Kao K .

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The Rel family of transcriptional activators form a large diverse group of proteins that are involved in the activation of genes involved in immunity, development, apoptosis and cancer. So far, none of the rel genes cloned in mammals appear to be required for embryonic development. We have cloned and characterized a cDNA from an embryonic cDNA library that encodes a novel Xenopus Rel protein, called Xrel3. Xrel3 is a member of the cRel subfamily and is most closely related to but distinct from other Xenopus Rel members. The expression of Xrel3 mRNA was investigated using Northern analysis, RNase protection assay, reverse transcriptase-linked polymerase chain reaction and in situ hybridization. Messages are present maternally and are slightly enriched in the equatorial region of the blastula stage embryo. At gastrulation, the accumulation of Xrel3 messages declines to undetectable levels but then increases after neurulation. In situ RNA hybridization was used to determine the spatial location of Xrel3 messenger RNA in embryos. Messages are localized to the developing forebrain, dorsal mid-hindbrain region, the inner ear primordium, or otocyst, and in the notochord. Overexpression by microinjection of Xrel3 RNA induced tumors in the developing embryo that appeared after gastrulation. The location of the tumors depended on the location of the injection site. These results suggest that Xrel3 might have a generalized role in regulation of cell differentiation in the embryo.

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Species referenced: Xenopus
Genes referenced: gdf1 mt-tr rel rela tp53 trna

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	FIG. 1. Nucleotide and predicted protein sequence of Xrel3 cDNA. The rel homology domain is underlined.
	FIG. 2. Comparison of the predicted protein sequence of Xrel3 with that of other cRel proteins. A, the Xrel3 sequence was aligned with that of Xrel2 (17). Underlined bold-faced sequences indicate differences between Xrel3 and Xrel2. Pos., position number starting at 1 at start of translation; %ID, percent identical amino acids compared with Xrel3. B, amino acid sequence analysis of Xrel3 with other members of the cREL and RELA members indicates its most probable relationship to this branch of the family.
	FIG. 3. Northern analysis of developmental expression of Xrel3. RNA from four embryos was loaded per lane. Histone (H4) was used as a loading control. Numbers at bottom represent stage of development (Nieuwkoop and Faber), and arrow indicates ;3 kilobase Xrel3 mRNA size.
	FIG. 4. Distribution of Xrel3 transcripts during embryogenesis. RNA from two embryos was analyzed for Xrel3 and XrelA messages by RNase protection assay. Embryo stages are shown below. tRNA is used as a hybridization control, whereas XrelA is used as an RNA loading control since it is relatively ubiquitously expressed throughout development.
	FIG. 5. Spatial distribution of Xrel3 mRNA during development. Albino embryos were subjected to whole mount in situ hybridization analysis using Xrel3- specific probes. The dark blue staining indicates localization of message. A, blastula; B, gastrula; C, late neurula; D and E, larva; F, control; op, otic placode; fb, forebrain; mhb, mid-hindbrain; oto, otocyst; No, notochord; sc, spinal cord; E, eye; Oc, oral cavity. Scale bar 5 0.5 mm.
	FIG. 6. Distribution of Xrel3 mRNA along the animal-vegetal axis of blastula-stage embryos. A, quantitative RT-PCR of staged embryo RNA indicates down-regulation of expression of Xrel3 in the late gastrula/early neurula stage. B, Xrel3 mRNA is unevenly distributed in the stage 8 blastula. Six animal caps, four equatorial regions, and twelve vegetal pieces were dissected from stage 8 embryos, pooled, and processed for RT-PCR using 1/50 the total mass of the extracted RNA. Vg1 and histone are used as controls. Note the accumulation of Vg1 messages in the vegetal region, whereas histone messages are present relatively evenly in all the samples to control for loading.
	FIG. 7. Tumor formation by overexpression of Xrel3 in Xenopus embryos. Embryos were injected with 500 pg (A-C, and E) or 200 pg (D) of Xrel3 mRNA into the animal (A-D) or vegetal (E) pole at the 2-cell stage. F, control embryo injected with a truncated Xrel3 mRNA. Tumors are indicated by arrowheads. Embryos on the left of panels A and B were injected with control RNA.
	rel (v-rel avian reticuloendotheliosis viral oncogene homolog) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up.