XlHbox 8: a novel Xenopus homeo protein restricted to a narrow band of endoderm

0 Department of Biological Chemistry, University of California , Los Angeles, CA 90024-1737 , USA XlHbox 8: a novel Xenopus homeo protein restricted to a narrow band of CHRISTOPHER V. E. WRIGHT, PATRICK SCHNEGELSBERG and EDDY M. DE ROBERTIS - We report the isolation of a new homeobox gene from Xenopus laevis genomic DNA. The homeodomain sequence is highly diverged from the prototype Antennapedia sequence, and contains a unique histidine residue in the helix that binds to DNA. The homeodomain is followed by a 65 amino acid carboxyterminal domain, the longest found to date in any vertebrate homeobox gene. We have raised specific antibodies against an XlHbox 8-y3-gal fusion protein to determine the spatial and temporal expression of this gene. The nuclear protein first appears in a narrow band of the endoderm at stage 33 and develops into expression within the epithelial cells of the pancreatic The majority of homeobox-containing genes in Drosophila specify or interpret positional information along the anteroposterior axis of the fly (Gehring, 1987; Akam, 1987; Ingham, 1988). Vertebrate homeobox-containing genes share a number of structural features with those of Drosophila (Cairasco et al. 1984; Fienberg et al. 1987; Dressier & Gruss, 1988; Boncinelli et al. 1988; Scott et al. 1988). One of the most compelling arguments in favour of vertebrate homeobox genes having a role similar to those of Drosophila is that many of them are expressed in restricted and precisely defined regions along the anteroposterior axis of the embryo (Awgulewitsch et al. 1986; Holland & Hogan, 1988; Oliver et al. 1988), rather than in a tissue-specific or cell type-specific manner. To date, all vertebrate homeobox genes have been found to be expressed in the neuroectoderm and sometimes also in the mesoderm (e.g. Utset et al. 1987; Dony & Gruss, 1987; Toth et al. 1987; Graham et al. 1988), but none of the genes reported are expressed in the endoderm. In Drosophila, a small amount of transient expression oifushi tarazu, engrailed, Ultrabithorax and caudal has previously been noted in small areas of the embryonic gut anlagen and duodenum. Expression within the pancreatic epithelium persists into the adult frog. This unprecedented restriction to an anteroposterior band of the endoderm suggests that vertebrate homeobox genes might be involved in specifying positional information not only in the neuroectoderm and mesoderm, but also in the endoderm. Our data suggest that XlHbox 8 may therefore represent the first member of a new class of position-dependent transcription factors affecting endodermal differentiation. (Akam & Martinez-Arias, 1985; DiNardo etal. 1985; Fjose et al. 1985; Ingham et al. 1985; Kornberg et al. 1985; Mlodzik et al. 1985; Krause et al. 1988). However, in all of these cases, the analysis did not extend to showing whether the stained cells are endodermal in origin or derived from another germ layer. Whatever the case, it is definite that no Drosophila or vertebrate gene isolated thus far is expressed exclusively in the endoderm. In this paper, we report the isolation of a new homeobox gene, called XlHbox 8, which is expressed solely in a narrow band of the endoderm in early Xenopus embryos. As development proceeds XlHbox 8 protein is restricted to the nucleus of endodermal cells of the duodenum and the developing pancreas. The homeodomain sequence and the unique pattern of expression suggest that previously undiscovered classes of homeodomain protein may be involved in the specification or interpretation of anteroposterior position within the vertebrate endoderm. In vertebrates coculturing of tissue explants has shown that the differentiation pathway followed by the embryonic endoderm (e.g. choice between lung or intestinal epithelium) is induced by the underlying mesoderm (reviewed by Gurdon, 1987; Wessels, 1977). Thus we believe that the gene isolated here promises to be a valuable marker for studying endodermal differentiation along the anteroposterior axis and its earliest induction by mesoderm. Materials and methods An XlHbox 8 genomic clone was isolated by screening a Charon 4A library (Wahli & Dawid, 1980) with Drosophila homeobox probes as described previously (Carrasco et al. 1984). The EcoRl fragments were subcloned and a 1-8 kb insert that showed the only weak hybridization with the probe was cut separately with Alul and Haelll and the EcoRl ends filled in, which allowed subcloning into M13 mp8 restricted with Smal. Two subclones were used further: an 860 bp Alul fragment and a 350 bp EcoRl/ Haelll fragment (see Fig. 1A). The Alul and EcoRl/ Haelll fragments are inserted in M13 in the opposite orientation relative to each other. Use of the M13 universal and homeobox-specific primers allowed the determination on both strands of the sequence between the homeobox EcoBJ and the Haelll site downstream of the translation stop codon. Sequencing was by the dideoxynucleotide chain-terminator method of Sanger et al. (1977). After the subcloning of all detectable EcoRl fragments, the ends of which were all double-strand sequenced in a search for the 5' portion of the homeodomain sequence, the original genomic phage stock was inadvertently lost in a transAtlantic laboratory relocation. For this reason, no reliable sequence data extending 5' to the EcoRl site in the homeobox is presently available. Completion of this sequence must await isolation of new genomic and cDNA clones. The fusion protein was constructed as follows. The Alul fragment that had been cloned into the Smal site was excised from M13 mp8 using the flanking EcoRl and BamHl restriction sites. The fragment was gel-purified and then digested further with Sau3Al (see Fig. 1A). The mixture was ligated to BamHI-cut pTRB 2 (Burglin & De Robertis, 1987), such that only the fragment with BamHIcompatible ends gave viable plasmid. Hence, the last onethird of the homeodomain and all of the carboxy-terminus of XlHbox 8 was linked in frame with the lac Z gene. The orientation of XlHbox 8 in pTRB 2 was determined using an internal Pstl site (Fig. 1A), and the maintenance of the correct frame over the junction was determined by sequencing double-stranded DNA with a primer hybridizing just 5' to the pTRB 0/pTRB l/pTRB 2 polylinker. Fusion proteins were induced in, and purified from, bacterial strain F'llrecA, essentially as described by Oliver et al. (1988). Antibody procedures Antisera were raised in NZW female rabbits using the protocol given in Oliver et al. (1988), using approximately 1-52 mg of fusion protein per inoculation. The first immune serum of reasonable titre was taken after boosting the animal twice. Subsequent boosts yielded antisera of higher titre and specificity. The anti-XlHbox 8 antibodies were purified by depletion and affinity purification as described by Oliver et al. (1988). In most cases, the XlHbox 8 antiserum was additionally depleted against a /5-gal fusion protein matrix containing an Antennapedia-\ik (...truncated)