Characterization of the vasotocin neuropeptide hormone receptor system in the sea lamprey (Petromyzon marinus)

University of Minnesota Ph.D. dissertation. November 2015. Major: Integrated Biosciences. Advisor: Benjamin Clarke. 1 computer file (PDF); xii, 242 pages.The sea lamprey (Petromyzon marinus) is a jawless vertebrate at an evolutionary nexus between invertebrates and jawed vertebrates. Lampreys are known to possess the arginine vasotocin (AVT) hormone utilized by all non-mammalian vertebrates. We postulated that the lamprey would possess AVT receptor orthologs of predecessors to the arginine vasopressin (AVP)/oxytocin (OXT) family of G protein-coupled receptors found in mammals, providing insights into the early branching into the mammalian V1a, V1b, V2 and OXT receptors. We sequenced one partial and four full-length putative lamprey AVT receptor genes that are found on separate scaffolds in the P. marinus genome. Molecular phylogeny also utilizing the Japanese lamprey (Lethenteron japonicum) genome show that the lamprey receptors cluster with the larger V1a/V1b/OXTR and V2a/b/c clades but specific orthology is unclear. Synteny analysis supports the recently proposed one-round (1R) whole-genome duplication in the vertebrate lineage as the most likely scenario, but does not refute 2R or independent 3R scenarios. The mRNA expression patterns were determined in 15 distinct tissues for these genes, showing transcription in tissues where function has been demonstrated in jawed vertebrates. The literature provides evidence of the expression of neuropeptide hormones and receptors in jawed vertebrate immune cells. For the first time, lamprey peripheral blood leukocytes were maintained in primary culture for periods of at least six days, in which mRNA transcription of a V1a/OXTR-like gene (lamprey AVT receptor Pm807) was demonstrated. These preliminary results also support the hypothesis that neuropeptide hormones may play a role in response to pathogenic challenge in all vertebrates. The possibility of AVT involvement in mediating pheromone release from glandular cells in the gills of mature male lampreys was tested. The compound petromyzonamine disulfate (PADS) was detected at higher quantities after than before injection from several AVT and OXT injected males, but this was not true for the main sex pheromone component 3-keto petromyzonol sulfate (3kPZS). The question of whether DNA methylation of cytosine-guanine (CpG) dinucleotides function to regulate lamprey gene transcription was addressed through analysis of CpG islands in the lamprey Pm807 V1a/OXT receptor gene promoter region. Using High Resolution Melt (HRM) PCR on bisulfite-converted DNA, we pinpointed a region with tissue-specific differences in DNA melt characteristics, indicating differences in methylation level. Sequencing revealed a pattern of methylation at specific CpGs at consistently higher levels in adult heart and larval liver, where Pm807 is transcribed to mRNA, than in adult liver where Pm807 is not transcribed. The methylated CpGs are associated with putative Krüppel-like factor (KLF) 4 transcription factor binding site sequences. In humans KLF4 binds to methylated DNA to initiate transcription. The results suggest that CpG methylation regulates lamprey gene transcription. Additional Pm807 putative promoter elements such as estrogen response element consensus binding sequences were found to be organized similarly to functional OXTR promoters in mammals. The results of my research support the hypothesis that, similarly to jawed vertebrates, differential mRNA expression and resultant functional pleitropy is generated through promoter region sequence and epigenetic regulatory elements in the jawless basal vertebrate lamprey