Overexpression of a Novel XenopusRel mRNA Gene Induces Tumors in Early Embryos

Abstract

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 novelXenopus Rel protein, called Xrel3. Xrel3 is a member of the cRel subfamily and is most closely related to but distinct from otherXenopus Rel members. The expression of Xrel3 mRNA was investigated using Northern analysis, RNase protection assay, reverse transcriptase-linked polymerase chain reaction and in situhybridization. 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. 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 novelXenopus Rel protein, called Xrel3. Xrel3 is a member of the cRel subfamily and is most closely related to but distinct from otherXenopus Rel members. The expression of Xrel3 mRNA was investigated using Northern analysis, RNase protection assay, reverse transcriptase-linked polymerase chain reaction and in situhybridization. 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. The Rel family of transcriptional activators is a group of higher eukaryotic proteins that participate in cell differentiation (for reviews, see Refs. 1Verma I.M. Stevenson J.K. Schwarz E.M. Antwerp D.V. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1662) Google Scholar, 2Baeurele P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2932) Google Scholar, 3Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5581) Google Scholar, 4Huguet C. Crepieux P. Laudet V. Oncogene. 1997; 15: 2965-2974Crossref PubMed Scopus (49) Google Scholar, 5Ip Y.T. Kraut R. Levine M. Rushlow C.A. Cell. 1991; 64: 439-446Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 6Wasserman S.A. Mol. Biol. Cell. 1993; 4: 767-771Crossref PubMed Scopus (77) Google Scholar, 7Barinaga M. Science. 1996; 273: 735-736Crossref PubMed Scopus (73) Google Scholar). The family includes the c-Rel oncoprotein, RelB, NF-κB (both p65 or RelA, and p50 subunits), and the DORSAL patterning morphogen of Drosophila. They are expressed in a wide variety of tissues and transactivate genes that are involved in the immune response, apoptosis, cancer, and development. Rel transcription complexes exist as homo- or heterodimers, but only specific dimer pairs have been demonstrated in vivo. Although there are different levels of rel gene regulation in different cells, the activity of all of the proteins is determined by their subcellular localization. Thus, Rel proteins are active in the nucleus, but are retained in the cytoplasm in an inactive state. Depending on the cell type, Rel complexes activate transcription from target genes in either a constitutive or inducible manner. Inducible complexes are retained in the cytoplasm by a regulatory subunit (1Verma I.M. Stevenson J.K. Schwarz E.M. Antwerp D.V. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1662) Google Scholar, 2Baeurele P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2932) Google Scholar, 3Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5581) Google Scholar). Upon induction, the regulatory subunit releases the Rel complex and allows it to translocate to the nucleus where it binds to target DNA sequences. Rel proteins share motifs required for their similar functions. The amino terminus contains the highly conserved Rel Homology Domain (RHD) 1The abbreviations used are: RHD, Rel Homology Domain; RT-PCR, reverse transcriptase-linked polymerase chain reaction. of about 300 amino acids, which is required for DNA binding, dimerization with other Rel proteins, and a nuclear localization signal. The remainder of the protein is variable and is necessary for transcriptional activation (8Schmitz M.L. Baeuerle P.A. EMBO J. 1991; 10: 3805-3817Crossref PubMed Scopus (666) Google Scholar). In some cases, such as the p50 subunit of NF-κB, the activation domain is absent so that the protein is essentially an RHD. The DORSAL protein of Drosophila is localized to the nuclei of ventral cells that form the embryonic body axis (9Stein D. Roth S. Vogelsang E. Nüsslein-Volhard C. Cell. 1991; 65: 725-735Abstract Full Text PDF PubMed Scopus (198) Google Scholar). Genetic and biochemical studies indicate that DORSAL activates body patterning genes such as twist and snail (6Wasserman S.A. Mol. Biol. Cell. 1993; 4: 767-771Crossref PubMed Scopus (77) Google Scholar). In mice, Rel/NF-κB activity is detected at the fetal stage and in the developing brain (10Schmidt-Ullrich R. Memet S. Lilienbaum A. Feuillard J. Raphael M. Israel A. Development. 1996; 122: 2117-2128PubMed Google Scholar). Mice deficient for c-rel andrelB develop defects in their immune systems while NF-κB(p50)-defective mice die of liver failure before birth (for review, see Ref. 11Attar R.M. Caamano J. Carrasco D. Iotsova V. Ishikawa H. Ryseck R.P. Weih F. Bravo R. Semin. Cancer Biol. 1997; 8: 93-101Crossref PubMed Scopus (74) Google Scholar). These fetuses develop apparently normal bodies suggesting that rel genes are not required for embryogenesis. It is possible, however, that redundant pathways exist to compensate for the loss of specific rel gene function. Furthermore, there are likely other undiscovered vertebrate embryonic patterning rel-related genes since homologues totwist and snail are expressed in axis-forming tissues of the postimplantation mouse embryo (12Smith D.E. Del Amo F.F. Gridley T. Development. 1992; 116: 1033-1039PubMed Google Scholar, 13Stoetzel C. Weber B. Bourgeois R. Bolcato-Bellemin A.L. Perrin-Schmidt F. Mech. Dev. 1995; 51: 251-263Crossref PubMed Scopus (74) Google Scholar) and since humantwist is associated with congenital defects of the mesoderm (14Dixon M. Nat. Genet. 1997; 15: 3-4Crossref PubMed Scopus (7) Google Scholar). Xenopus homologues of twist (15Hopwood N.D. Pluck A. Gurdon J.B. Cell. 1989; 59: 893-903Abstract Full Text PDF PubMed Scopus (267) Google Scholar) andsnail (16Sargent M.G. Bennett M Development. 1990; 109: 967-973PubMed Google Scholar) are expressed in tissues that form the body axis. None of the Xenopus genes, Xrel2 (17Tannahill D. Wardle F. Int. J. Dev. Biol. 1995; 39: 549-558PubMed Google Scholar),XrelA (18Bearer E. Eur. J. Cell Biol. 1994; 63: 255-268PubMed Google Scholar, 19Kao K. Hopwood N. Proc. Natl. Acad. Sci. 1991; 88: 2697-2701Crossref PubMed Scopus (42) Google Scholar, 20Richardson J.C. Garcia-Estrabot A.M. Woodland H.R. Mech. Dev. 1994; 45: 173-189Crossref PubMed Scopus (25) Google Scholar), and XrelB (21Suzuki K. Yamamoto T. Inoue J. Nucleic Acids Res. 1995; 23: 4664-4674Crossref PubMed Scopus (15) Google Scholar), have significant spatiotemporal restriction of mRNA expression in embryos. The lack of an embryonic patterning phenotype in Rel-defective mice is paradoxical since both twist and snail homologues are expressed and/or required for correct body patterning in frogs, mice, and humans. In this paper, we identify a new Xenopus rel family member,Xrel3, that is a version of c-rel, but unlike any other rel gene, messenger RNA encoding Xrel3 protein is expressed in two phases of early development. Xrel3 messenger RNA is present in embryos up to the late blastula stage followed by a dramatic decline to undetectable levels at gastrulation. Messages then accumulate in the forebrain and otic placode of neurula-stage embryos and in the dorsal part of the mid-hindbrain, the forebrain, the otocysts, and finally the notochord of tailbud and later stage embryos. This novel expression pattern indicates a new role for relgenes in vertebrate embryonic development. Interestingly, overexpression of Xrel3 mRNA in the animal hemisphere of 2-cell embryos results in the formation of tumors. The size of the tumors is proportional to the dosage of RNA injected, and their position on the embryo depends on the location of injection: epidermal tumors develop from animal pole injection (prospective ectoderm) while tumors in the endoderm (digestive system) develop from vegetal pole injection. Recent evidence indicates that Rel protein overexpression directly causes oncogenesis in mammalian cells. Our results support this observation and suggest that Xrel3 might function in the decision for cells to differentiate or proliferate in the embryo. Hybridization and washing of filters at high stringency were performed as described previously (18Bearer E. Eur. J. Cell Biol. 1994; 63: 255-268PubMed Google Scholar). cDNAs retrieved from library screening were subcloned into pBluescript SK+ (Stratagene) and sequenced on both strands at least twice by the dideoxynucleotide method as described (22Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. (U. S. A.). 1977; 74: 5463-5467Crossref PubMed Scopus (52688) Google Scholar) using Sequenase (Amersham Pharmacia Biotech) and oligonucleotide primers corresponding to overlapping regions of sequence. Wild-type and albino Xenopus embryos were obtained by in vitro fertilization as described (23Kao K.R. Elinson R.P. Dev. Biol. 1988; 127: 64-77Crossref PubMed Scopus (423) Google Scholar). Embryos were raised at room temperature and dissected in normal amphibian medium (24Kao K. Lockwood A. Mech. Dev. 1996; 58: 129-139Crossref PubMed Scopus (17) Google Scholar). RNA from embryos was prepared as described (15Hopwood N.D. Pluck A. Gurdon J.B. Cell. 1989; 59: 893-903Abstract Full Text PDF PubMed Scopus (267) Google Scholar). Northern blots and RNase protection assays were performed as described (19Kao K. Hopwood N. Proc. Natl. Acad. Sci. 1991; 88: 2697-2701Crossref PubMed Scopus (42) Google Scholar, 25Krieg P. Melton D. Nucleic Acids. Res. 1984; 12: 7057-7070Crossref PubMed Scopus (1082) Google Scholar). For RNase protection assays, anEcoRI-DraI fragment from the entire 5′-nontranslated region of d10 that protects a 210-base pair region inXrel3 was subcloned into pBluescript. As a control, anXrelA probe was added to the hybridization mix, which protected a 229-base pair region (19Kao K. Hopwood N. Proc. Natl. Acad. Sci. 1991; 88: 2697-2701Crossref PubMed Scopus (42) Google Scholar). Reverse transcriptase-linked polymerase chain reaction (RT-PCR) was performed as described with 32P-ATP trace labeling (24Kao K. Lockwood A. Mech. Dev. 1996; 58: 129-139Crossref PubMed Scopus (17) Google Scholar, 26Hemmati-Brivanlou A. Melton D Cell. 1994; 77: 273-281Abstract Full Text PDF PubMed Scopus (409) Google Scholar) and gene-specific primers for Xrel3 (forward primer, 5′-TCCTTGGAGATATTTGTGGGG-3′; reverse primer, 5′-TTTAAACCGGCCATGTTGATG-3′), Vg1 (Ref. 27Rebagliati M.R. Weeks D.L. Harvey R.P. Melton D.A. Cell. 1985; 42: 769-777Abstract Full Text PDF PubMed Scopus (320) Google Scholar; forward primer, 5′-GAGGATATTCAACCAAAGG-3′; reverse primer, 5′-CGTCTCTATCGCACATTAC-3′), and histone (Ref. 28Niehrs C. Steinbesser H. DeRobertis E.M. Science. 1994; 263: 817-820Crossref PubMed Scopus (144) Google Scholar). The cycling parameters were as follows: 94 °C for 5 min, followed by 26 cycles for Xrel3and Vg1 (19 for histone) with the program: 30 min at 94 °C, 30 min at 62 °C, and 30 min at 72 °C. Reactions were then extended at 72 °C for 5 min. Whole mount in situ hybridization with digoxigenin-UTP (Boehringer Mannheim) incorporated probes was carried out as described (29Harland, R. (1991) in Methods in Cell Biology, Vol. 36, pp. 685–695Google Scholar). In situ hybridization was detected by alkaline phosphatase-linked anti-digoxigenin antibodies using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as a chromogenic reagent. Microinjection of embryos was done as described (24Kao K. Lockwood A. Mech. Dev. 1996; 58: 129-139Crossref PubMed Scopus (17) Google Scholar) using capped, synthetic Xrel3 mRNA made using the Ribomax kit (Promega). The Xrel3 coding domain was subcloned into the CS2+ expression vector, linearized with EcoRI, and used as a template. Control RNA was made using the D10 cDNA subcloned into CS2+ and linearized with NotI. D10 lacks a significant region (~30 amino acids) of the carboxyl terminus which represents the transcriptional activation domain. We searched for rel genes in Xenopus by screening a neurula stage λ cDNA library (a gift from Dr. H. Sive, Massachusetts Institute of Technology) with XrelA cDNA sequences at high stringency. Several of the largest cross-hybridizing clones were used as templates for RNA probes for whole mount in situhybridization on neurula stage embryos. One of the probes, clone d10, hybridized to embryos in a pattern of interest, localizing to anterior neural structures (see below). When completely sequenced, d10 revealed an open reading frame truncated at the carboxyl terminus. By screening a Xenopus stage 8 λ ZAP library (30Gillespie L.L. Chen G. Paterno G. J. Biol. Chem. 1995; 270: 22758-22763Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) at very high stringency with d10, we isolated a larger clone with identical overlapping sequence to d10 (Fig.1). The putative protein from of this cDNA was most similar to that of Xrel2 and mammalian cRel Refs. E. N. N. Oncogene. 1989; 4: Google and M.L. Israel A. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). The of not appear to be the of a but it a protein size in with in vitro protein made from this clone putative of sequences in all reading and it is in the position as that of other related Rel family of the protein sequence of that of other cRel the Xrel3 sequence was with that of Xrel2 (17Tannahill D. Wardle F. Int. J. Dev. Biol. 1995; 39: 549-558PubMed Google sequences indicate Xrel3 and position at at of identical amino with Xrel3. amino sequence of Xrel3 with other of the and indicates most to this of the The sequence of Xrel3 is most closely related to Xrel2 at identical amino the RHD, the two proteins share their coding there are significant of coding sequence The region in the carboxyl terminus of Xrel3 contains a high of and with the role of this region as a domain (8Schmitz M.L. Baeuerle P.A. EMBO J. 1991; 10: 3805-3817Crossref PubMed Scopus (666) Google Scholar). At the the sequences Xrel2 are completely different their coding determine the pattern of Xrel3 accumulation early embryonic development, we Northern blots of RNA from embryos at made from d10 cDNA hybridized to a of about pairs in The was present in RNA from embryos but at embryonic transcription is to at the stage Ref. J. M. Cell. Full Text PDF PubMed Scopus Google Scholar, J.C. D. Woodland H.R. Mech. Dev. 1995; PubMed Scopus (15) Google Scholar), we that the early accumulation of Xrel3 mRNA be in hybridization was in later but the was on Northern to DNA levels in that the to The RNase protection using an RNA probe made from a sequence of Xrel3 later The probe protected a fragment of the size of with RNase and RNase These assays the of in embryos. The and to but then increases late neurula to the early stage made XrelA (19Kao K. Hopwood N. Proc. Natl. Acad. Sci. 1991; 88: 2697-2701Crossref PubMed Scopus (42) Google Scholar), RNA is present development, were added to the hybridization to determine the levels of Xrel3 mRNA to this of the mRNA levels two genes indicates that the of Xrel3 mRNA is about that of XrelA In studies with XrelA (19Kao K. Hopwood N. Proc. Natl. Acad. Sci. 1991; 88: 2697-2701Crossref PubMed Scopus (42) Google Scholar) and Xrel2 (17Tannahill D. Wardle F. Int. J. Dev. Biol. 1995; 39: 549-558PubMed Google situ hybridization of mRNA was not performed dissected embryo the embryo. Our for Xrel3 was on screening to spatial restriction of mRNA In this Xrel3 represents a different gene from other Xenopus rel The whole mount was extended to a of to determine the of mRNA localization development. embryos in all cells 5 As late stage embryos not for the or of at embryos stage not using probes RNase protection assays of at a to this This be to in the two RNA to embryos was used in the protection assays, the of in the with in situ At about stage accumulation in the forebrain and in a region to the We this to to the ear or otocyst, on and in later the is with the forebrain, the dorsal part of the and and the notochord 5 of messages in the notochord with absent in stage 30 embryos but is in embryos a 5 made from templates not the of probe 5 Whole mount RNA of embryos is to significant of that the hybridization and expression in we using oligonucleotide primers specific to Xrel3 mRNA on RNA from dissected of RNA not a that the we were not to Our the in and RNase protection assays, of Xrel3 cDNA in and with a decline in expression and equatorial all Xrel3 mRNA in stage 8 that it is present all the axis is a in Xrel3 mRNA accumulation in the equatorial as with the vegetal and animal on using histone mRNA Microinjection of up to of Xrel3 synthetic mRNA into the animal or vegetal pole not embryos from after gastrulation, the embryos develop or tumors location and size on the of injection. develop in the from animal pole injection They appear at the neurula stage as a of on the ventral and the region into a and is associated with animal pole as might be since structures develop from animal cells not In some cases, the tumors are so normal for anterior into the vegetal pole or endoderm (prospective system) but tumors were the of the embryo after been by Control embryos with of a truncated Xrel3 develop The size of tumors with the of injection. The that any of formation was At this only a of embryos tumors and appeared only in the of the of the embryo in the ventral At or 300 the tumors were about of the embryo and appeared in embryo tumors appeared in the anterior ventral two and the remainder appeared on the or ventral At the tumors appeared in embryo and most of the ventral The tumors that form are very large and with the of normal tissues We have a novel rel gene, RNA is expressed in a manner. The Xrel3 protein is most similar to the previously Xrel2 The in sequences their coding regions suggest that Xrel3and two genes with different regulatory At the protein in the most highly conserved RHD, Xrel2 a very to the of at to of Xrel3 there is a of that are only in Xrel3 and not by any other Rel These two regions the putative DNA and dimerization domain. These suggest that Xrel3 and Xrel2 might with different to form a transcription and/or to different DNA of the RHD, Xrel2 and Xrel3 in amino sequence. are with a large of to of Xrel3 corresponding to the transcriptional activation domain of Rel proteins (9Stein D. Roth S. Vogelsang E. Nüsslein-Volhard C. Cell. 1991; 65: 725-735Abstract Full Text PDF PubMed Scopus (198) Google Scholar). Thus, the of and amino indicates and Xrel2 are distinct The mRNA expression indicates that Xrel3 is distinct from other Xenopus Xrel3 mRNA is expressed in all cells maternally but is to anterior neural structures after gastrulation. The DORSAL morphogen expression development. Although RNA and protein are present the entire DORSAL protein is localized only to ventral nuclei of early Drosophila embryos (8Schmitz M.L. Baeuerle P.A. EMBO J. 1991; 10: 3805-3817Crossref PubMed Scopus (666) Google Scholar). XrelA protein be localized to nuclei of blastula and neurula embryos but not stage embryos (18Bearer E. Eur. J. Cell Biol. 1994; 63: 255-268PubMed Google Scholar). This observation with the expression pattern of Xrel3 and it be to determine the XrelA and Xrel3. We are antibodies Xrel3 to determine and it is localized to nuclei of specific structures of the embryo. The formation of tumors by Xrel3 overexpression a to of tumors be induced by overexpression of N. J. P. P. A. 1997; PubMed Scopus Google Scholar), a transcription that is involved in neural differentiation J. P. A. Development. 1997; PubMed Google Scholar) and is in cell N. J. P. P. A. 1997; PubMed Scopus Google Scholar). We are Xrel3 but there is gene The of by Xrel3 however, is that of since of Xrel3 as with mRNA Ref. N. J. P. P. A. 1997; PubMed Scopus Google Scholar), larger tumors. Xrel3 mRNA induced tumors it was into the vegetal pole (prospective suggesting that it have a generalized role in Rel protein overexpression been to be associated with or directly mammalian tumors. For the is involved in and activates the p65 subunit of NF-κB in cells M. J.C. Oncogene. 1997; 15: PubMed Scopus Google Scholar). active form of NF-κB causes tumors to form in P. A. C. F. M. Oncogene. 1997; 14: PubMed Scopus Google Scholar) and expression is and required for the phenotype of cell R. J. S. M. F. K. F. F. M. A. Oncogene. 1997; 15: PubMed Scopus Google Scholar). Our results are not with formation by Xrel3 overexpression in embryos is likely similar to the formation of which from of In this results indicate that Xrel3 be involved in regulation of the decision for embryonic cells to we that Xrel3 is involved in then role in tissues in which it is expressed is to of tissues before This is with Xrel3 mRNA expression in the embryo it is In any the formation of tumors by Xrel3 overexpression in embryos a to the in We and for for the CS2+ and for the histone