Two groups of neuropeptides (secretin and gonadotropin-releasing hormone groups, GnRH) and their receptors are of considerable interest because they are highly conserved in structure during evolution and have important physiological effects. To study the role of these neuropeptides during development, the zebrafish was selected initially as a model because the embryo is transparent and accessible for genetic manipulation. Later, a mouse model was added to evaluate the effects of permanent gene loss. The purpose of this thesis was to 1) characterize the structure and function of several peptides and receptors within the secretin group that had not been previously identified for zebrafish, 2) examine brain development in zebrafish after gene knock down of an important peptide in each of the secretin and GnRH groups, 3) identify the pattern and location of expression of the GnRH receptors in zebrafish embryos, and 4) study the functional importance of the GnRH receptor in a mouse line deficient in the receptor. The secretin superfamily of hormones includes 10 structure-related polypeptides in mammals, but fewer in zebrafish. In this thesis it was discovered that within the secretin group, zebrafish have two peptides, peptide histidine-isoleucine (PHI) and vasoactive intestinal polypeptide (VIP), in addition to two receptors, PHI receptor and growth hormone-releasing hormone-like peptide (GHRH-LP) receptor that were not previously identified. After isolation of the cDNA for each, the signaling pathways were characterized in vitro by transfection of the receptors into COS cells. The PHI-R was activated by PHI but not by VIP or pituitary adenylate cyclase-activating polypeptide (PACAP). The GHRH-LP receptor was activated by GHRH-LP 1 and GHRH but not by GHRH-LP2. A novel observation was that the PAC, receptor is activated by both PACAP and VIP, whereas the PAC, receptor is usually specific to PACAP. Also novel was evidence that a VPAC2 receptor in zebrafish was structurally and functionally a PHI receptor. In examining the role of PACAP in early brain development in zebrafish, morpholinos were used to knockdown PACAP1 and PACAP2 in the zygote. The study revealed that loss of PACAP leads to change in the expression of brain markers.
GnRH and its receptors (GnRH-R) are key regulators of reproduction and sexual behaviour. In zebrafish, morpholino-induced knock down of GnRH revealed that GnRH is critical in eye and brain development by affecting transcription factors and/or secreted factors: pax2.1, fgf8, pax6.1 and mab. Further, the GnRH receptor was expressed at 24h post fertilization showing that GnRH and its receptor could influence early development. To determine whether the GnRH receptor is critical at all stages of reproduction, a mouse line was created in which the GnRH receptor was disrupted using the gene trap method. This model has a similar phenotype to the clinical syndrome of hypogonadotropic hypogonadism. In mice, GnRH receptor disruption results in small sexual organs, low gonadotropin and steroid hormone levels, failure of sexual maturation, and lack of reproduction. Also, the GnRH receptor may be important in the fetal testis. This thesis provides evidence that 1) in evolution the PAC, receptor may have responded to both PACAP and VIP in fish, 2) zebrafish have a PHI receptor rather than a VPAC2 receptor, 3) PACAP and GnRH peptides have a fundamental role in early brain development, 4) the GnRH receptor is not only important in reproduction but unexpectedly, in zebrafish embryos is localized in hindbrain motor neurons and fibers, which are involved normally in movement and escape, and 5) GnRH receptor-disrupted mice should be a valuable model for understanding reproduction and clinical approaches to hypogonadotropic hypogonadism.
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/2484 |
| Date | 07 April 2010 |
| Creators | Wu, Sheng |
| Contributors | Sherwood, Nancy |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
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