Australian lungfish neurohypophysial hormone genes encode vasotocin and [Phe2]mesotocin precursors homologous to tetrapod-type precursors (molecular cloningyprecursor organizationymolecular evolutionyphylogenetic tree)

In view of the well-established role of neurohypophysial hormones in osmoregulation of terrestrial vertebrates, lungfishes are a key group for study of the molecular and functional evolution of the hypothalamo-neurohypophysial system. Here we report on the primary structure of the precursors encoding vasotocin (VT) and [Phe2]mesotocin ([Phe2]MT) of the Australian lungfish, Neoceratodus forsteri. Genomic sequence analysis and Northern blot analysis confirmed that [Phe2]MT is a native oxytocin family peptide in the Australian lungfish, although it has been reported that the lungfish neurohypophysis contains MT. The VT precursor consists of a signal peptide, VT, that is connected to a neurophysin by a Gly-Lys-Arg sequence, and a copeptin moiety that includes a Leu-rich core segment and a glycosylation site. In contrast, the [Phe2]MT precursor does not contain a copeptin moiety. These structural features of the lungfish precursors are consistent with those in tetrapods, but different from those in teleosts where both VT and isotocin precursors contain a copeptin-like moiety without a glycosylation site at the carboxyl terminals of their neurophysins. Comparison of the exonyintron organization also supports homology of the lungfish [Phe2]MT gene with tetrapod oxytocinyMT genes, rather than with teleost isotocin genes. Moreover, molecular phylogenetic analysis shows that neurohypophysial hormone genes of the lungfish are closely related to those of the toad. The present results along with previous morphological findings indicate that the hypothalamoneurohypophysial system of the lungfish has evolved along the tetrapod lineage, whereas the teleosts form a separate lineage, both within the class Osteichthyes. Neurohypophysial hormones are nonapeptides regulating various physiological events related especially to water and salt metabolism and reproduction. Twelve distinct nonapeptide principles have been chemically characterized in a wide variety of vertebrates, and are classified into two groups: the vasopressin (VP) and the oxytocin (OT) families. They are believed to have developed from a common ancestral molecule by gene duplication (1). All vertebrate species, except for the cyclostomes, contain at least one VP family peptide and one OT family peptide. Several schemes have been proposed as pathways of hormonal molecular evolution based on amino acid sequences of nonapeptides and their phyletic distribution (1). Complementary DNA and genomic analyses have shown that neurohypophysial nonapeptides are synthesized as large precursor molecules (2). Analysis of the precursor molecules, which are much larger than the nonapeptides, gives more precise and reliable information for estimating the molecular evolutionary relationships than does analysis based on substitutions only in the nine amino acid residues. Using statistical comparison of gene structures and the predicted amino acid sequences of precursors, Urano and colleagues (3–5) have proposed that teleost neurohypophysial hormone genes have their own evolutionary history separate from that of the tetrapod genes. Their hypothesis is further supported by the structural characteristics of neurohypophysial hormone precursors, including composition and the presence or absence of posttranslational modification sites (2–5). Lungfishes are the closest surviving freshwater relatives of the Devonian fish group from which early amphibians are assumed to have evolved. Acher and his colleagues (6, 7) reported that lungfishes have mesotocin (MT), the nonmammalian tetrapod type hormone, as their OT family hormone, whereas all teleosts examined have isotocin (IT). They have proposed an evolutionary lineage of IT (teleosts)-MT (non-mammalian tetrapods)-OT (mammals) and suggest a close relationship between lungfishes and amphibians. However, the data on amino acid substitutions in nonapeptides are insufficient to estimate the molecular phylogeny of the whole neurohypophysial hormone genes. For their VP family peptide, lungfishes have vasotocin (VT), which is common to all non-mammalian vertebrate species. These previous reports led us to study lungfish neurohypophysial hormone precursors and their genes to further clarify the molecular evolution of neurohypophysial hormone genes. As material, we used the Australian lungfish, Neoceratodus forsteri, which is considered to be more closely related to other freshwater fish than other lungfish species because of the well-developed gills on all gill arches. We isolated cDNAs encoding the neurohypophysial hormone precursors of this species. We report here characteristics of the nucleotide and deduced amino acid sequences of the isolated cDNAs and the structural organization of the precursors. These characteristics, together with the molecular phylogenetic analysis, clearly show that neurohypophysial hormone genes of the Australian lungfish have evolved along the tetrapod lineage, whereas the teleosts appear to form a separate lineage within the class Osteichthyes. Furthermore, we present evidence for a peptide, [Phe2]MT, as a native neurohypophysial hormone in addition to VT in the Australian lungfish. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9413339-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: IT, isotocin; MT, mesotocin; OT, oxytocin; VP, vasopressin; VT, vasotocin. Data deposition: The sequences reported in this paper have been deposited in the GenBank database {accession nos. AB001977 (VT cDNA) and AB001978 ([Phe2]mesotocin cDNA)}. †To whom reprint requests should be addressed. e-mail: cshyodo@ komaba.ecc.u-tokyo.ac.jp.