Characterization of the neurohypophysial hormone gene loci in elephant shark and the Japanese lamprey: origin of the vertebrate neurohypophysial hormone genes

Abstract

Background: Vasopressin and oxytocin are mammalian neurohypophysial hormones with distinct functions. Vasopressin is involved mainly in osmoregulation and oxytocin is involved primarily in parturition and lactation. Jawed vertebrates contain at least one homolog each of vasopressin and oxytocin, whereas only a vasopressin-family hormone, vasotocin, has been identified in jawless vertebrates. The genes encoding vasopressin and oxytocin are closely linked tail-to-tail in eutherian mammals whereas their homologs in chicken, Xenopus and coelacanth (vasotocin and mesotocin) are linked tail-to-head. In contrast, their pufferfish homologs, vasotocin and isotocin, are located on the same strand of DNA with isotocin located upstream of vasotocin and separated by five genes. These differences in the arrangement of the two genes in different bony vertebrate lineages raise questions about their origin and ancestral arrangement. To trace the origin of these genes, we have sequenced BAC clones from the neurohypophysial gene loci in a cartilaginous fish, the elephant shark (Callorhinchus milii), and in a jawless vertebrate, the Japanese lamprey (Lethenteron japonicum). We have also analyzed the neurohypophysial hormone gene locus in an invertebrate chordate, the amphioxus (Branchiostoma floridae).

Results: The elephant shark neurohypophysial hormone genes encode vasotocin and oxytocin, and are linked tail-to-head like their homologs in coelacanth and non-eutherian tetrapods. Besides the hypothalamus, the two genes are also expressed in the ovary. In addition, the vasotocin gene is expressed in the kidney, rectal gland and intestine. These expression profiles indicate a paracrine role for the two hormones. The lamprey locus contains a single neurohypophysial hormone gene, the vasotocin. The synteny of genes in the lamprey locus is conserved in elephant shark, coelacanth and tetrapods but disrupted in teleost fishes. The amphioxus locus encodes a single neurohypophysial hormone, designated as [Ile4]vasotocin.

Conclusion: The vasopressin- and oxytocin-family of neurohypophysial hormones evolved in a common ancestor of jawed vertebrates through tandem duplication of the ancestral vasotocin gene. The duplicated genes were linked tail-to-head like their homologs in elephant shark, coelacanth and non-eutherian tetrapods. In contrast to the conserved linkage of the neurohypophysial genes in these vertebrates, the neurohypophysial hormone gene locus has experienced extensive rearrangements in the teleost lineage.

Figure 1

Organization of neurohypophysial hormone-genes in jawed vertebrates. Genes are shown as arrows. Only the neurohypophysial hormone-genes are labeled. The elephant shark genes were sequenced in this study. The arrangements of genes in other vertebrates are mainly from [8]. The neurohypophysial hormone-genes in chicken and Xenopus are arranged like their homologs in coelacanth.

Figure 2

The neurohypophysial gene locus in the elephant shark (A) and Japanese lamprey (B). The BACs used for generating the sequences are shown below. Arrows represent genes and indicate the direction of transcription. Coloured vertical lines represent repetitive sequences. VT, vasotocin gene; OT, oxytocin gene; ProSAPiP1, proline rich synapse associated protein interacting protein 1; UBOX5, U-box domain containing 5; GNRH2, gonadotropin-releasing hormone 2; PTPRA, protein tyrosine phosphatase, receptor type, A.

Figure 3

Comparison of nucleotide and deduced amino acid sequences of elephant shark vasotocin and oxytocin genes. Conserved nucleotides are indicated by an asterisk. N-glycosylation sites and Leu-rich core segment in the vasotocin precursor are boxed. Intronic sequences are shown in lower case.

Figure 4

Comparison of amino acid sequences of vasopressin family hormone precursors in vertebrates. The alignment was generated by ClustalX. Amino acid residues conserved in all vertebrates are marked with an asterisk. B. floridae, Branchiostoma floridae; X. tropicalis, Xenopus tropicalis; and T. scyllium, Triakis scyllium. VP, vasopressin and VT, vasotocin. Accession numbers of sequences used in the alignment: NP_000481.2 (human VP), BAA24026.1 (lungfish VT), O42499 (fugu VT), BAD27476.1 (T. scyllium VT) and BAA06669.1 (lamprey VT). Sequences for Xenopus tropicalis and coelacanth were generated in a previous study [8] while sequences for elephant shark (eshark) and amphioxus (B. floridae) were generated in this study.

Figure 5

Comparison of amino acid sequences of oxytocin family hormone precursors in vertebrates. The alignment was generated by ClustalX. Amino acid residues conserved in all vertebrates are marked with an asterisk. X. tropicalis, Xenopus tropicalis; and T. scyllium, Triakis scyllium. OT, oxytocin; MT, mesotocin; IT, isotocin; ASV, asvatocin and PAI, phasitocin. Accession numbers of sequences used in the alignment: NP_000906.1 (human OT), BAA24027.1 (lungfish [Phe²]MT), O42493 (fugu IT), BAD27478.1 (T. scyllium phasitocin) and BAD27477.1 (T. scyllium asvatocin). Sequences for Xenopus tropicalis and coelacanth were generated in a previous study [8] while the sequence for elephant shark (eshark) was generated in this study.

Figure 6

Expression patterns of elephant shark neurohypophysial hormone genes. Expression patterns of the elephant shark vasotocin and oxytocin genes as determined by semi-quantitative RT-PCR. Expression of actin gene was analyzed as a control for the quality of RNA and cDNA.

Figure 7

Schematic diagram of neurohypophysial hormone gene loci in chordates. Arrows represent genes and indicate the orientation of transcription. ProSAPiP1, ProSAP-interacting protein 1 gene; UBOX5, U-box domain containing 5; [Lys⁸]VP, lysipressin gene; VT, vasotocin gene; MT, mesotocin gene; OT, oxytocin gene; IT, isotocin gene, GNRH2, gonadotropin-releasing hormone 2; PTPRA, protein tyrosine phosphatase receptor type A gene; CL1, chemokine CL1 gene; UGNT, UDP glucuronic acid/N-acetylglucosamine dual transporter gene; RNR, ribonucleotide reductase M2 polypeptide; St8sia2, alpha-2,8-sialyltransferase; RPS6KA3, ribosomal protein S6 kinase 90kDa polypeptide3; Ci-VP, Ciona-vasopressin; and CELSR3, cadherin EGF LAG seven-pass G-type receptor3.