Structural and functional evolution of gonadotropin-releasing hormone (GnRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) in chordates
Date
2008-04-10T06:01:29Z
Authors
Adams, Bruce Alexander
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Abstract
Neuropeptide hormones arose early in evolution. Multigene families in vertebrates are proposed to have arisen initially in early vertebrates by genome duplication events. In its simplest form, the theory suggests that the copy of a duplicated, ancestral single gene diverged in sequence, and possibly function, from its original match. My goal was to understand the structural and functional evolution of two neuropeptides, gonadotropinreleasing hormone (GnRH), a member of a single gene family, and pituitary adenylate cyclase-activating polypeptide (PACAP), a member of a multigene superfamily of hormones. GnRH is the primary regulator of reproduction in vertebrates, but the evolutionary origin of GnRH is not clear. In the protochordate tunicate Ciona intestinalis, I found there are two genes that encode GnRH peptides, however each gene encodes different GnRH peptides. Furthermore, these peptides are novel structures for GnRH and quickly induce spawning in Ciona, suggesting a novel and direct action for GnRH in the control of reproduction. In studies of the novel form of GnRH in lake whitefish, wfGnRH, I provide proof wfGnRH is a gonadotropin-releasing form in whitefish by showing it to be an activator of pituitary gonadotropin and growth hormone gene expression, and is colocalized in the forebrain region. PACAP is a hormone structurally related to glucagon and has been tightly conserved in structure during evolution. PACAP is produced as either a 27 or a 38 amino acid form in vertebrates, whereas in one tunicate studied to date, PACAP is produced from each of two genes as a 27 amino acid form. PACAP regulates several endocrine systems and has direct and indirect actions on metabolism, growth, and reproduction, and is well-known for its ability to potently secrete insulin in laboratory testing. I studied a number of species to increase our understanding of PACAP gene diversity in evolution. I was unable to identify a PACAP-like gene in the tunicate species, C. intestinalis. However, I identified a number of novel PACAP peptide structures in nine fish species by molecular biological and bioinformatic approaches. I was able to identify a second copy of a PACAP gene in five of the nine species. Using these data, I constructed a phylogenetic relationship for prohormones for PACAP in chordates and propose a updated explanation for the evolution of the PACAPfglucagon superfamily of genes in vertebrates. Using morpholino-based knockdown of the PACAP peptides in zebrafish early development, I showed that each copy of these two genes is functional and important in normal development in zebrafish, suggesting that divergence in function of the two different PACAP genes coincided with divergence in sequence. I also studied mice to determine the proposed role for PACAP in themogulation. Recently, the pups born to a new model of mouse with a targeted disruption of the PACAP gene (PACAP-null) have been found to have disruption of normal lipid and carbohydrate metabolism and die early in the second postnatal week. Furthermore it has been determined this phenotype is temperature sensitive. I hypothesized that there is a disruption of the thyroid axis in these mice that contributes to their problems with thermogenesis, and because these mice have a compromised adrenergic response, they are more sensitive to obesity. I showed that there is twice as much of the active form of thyroid hormone (TH), 3,5,3'-triiodothyronine (T3), in PACAP-null mice compared to their wildtype littermates. Mice reared at different temperatures (21, 24 and 28 °C) had increased survival with increasing temperature from 14% surviving at 21°C to 79 % at 28°C, and mice held at 28°C had lower levels of THs compared to 21°C. Treatment of PACAP-null mice with methimizole decreased their level of T3 and increases their survival suggesting the levels of T3 in mice at 21°C are toxic. Mice raised at 28°C on one of two diets, regular chow (low fat) and high fat were studied for difference in appetite and in tolerance to obesity. There were no differences in either appetite or many obesity-related parameters such as mass, fed and fasted glucose levels, fat distribution or plasma levels of leptin in PACAP-null mice compared to their sex- and diet-matched wildtype comparison groups. However, there was an increase in insulin sensitivity in PACAP-null mice fed a high fat diet.