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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Isolation and developmental expression of growth hormone-releasing hormone (GRF), pituitary adenylate cyclase-activating polypeptide (PACAP) and their receptors in the zebrafish, Danio rerio

Fradinger, Erica Aileen 16 August 2018 (has links)
The growth and development of an organism requires the coordinated actions of many factors. During development individual cells undergo proliferation, migration and differentiation to form the adult organism. Two structurally related members of the glucagon superfamily, growth hormone releasing hormone (GRF) and pituitary adenylate cyclase-activating polypeptide (PACAP), are thought to modulate vertebrate development. In mammals, GRF modulates the development of pituitary somatotrophs and the release of fetal growth hormone. In contrast, PACAP appears to have a more general role during development. PACAP may be involved in the patterning of the embryonic axis and in the development of the neural tube. The objectives of my study were to isolate GRF, PACAP and their receptors from the zebrafish, characterize their expression in the developing embryo and adult embryo and examine the role of PACAP during brain development. To study the role of GRF and PACAP, I isolated a genomic clone encoding the GRF and PACAP peptides from the zebrafish genomic library and characterized its gene copy number and adult tissue expression pattern. The GRF-PACAP gene isolated from the zebrafish was comprised of five exons with the GRF peptide encoded on the fourth exon and the PACAP peptide encoded on the fifth exon. This gene structure is similar to that found in other non-mammalian vertebrates and supports the hypothesis that the gene duplication leading to the encoding of the GRF and PACAP peptides on separate genes occurred later in evolution. In addition, the zebrafish genome was found to contain only one copy of the GRF-PACAP gene. The GRF-PACAP gene was widely expressed in the adult zebrafish in tissues developmentally derived from all three germ layers, suggesting that the gene may be widely expressed in the embryo as well. To examine the functional significance of the co-expression of GRF and PACAP in zebrafish, I isolated the GRF and PACAP receptors and characterized their expression pattern. I isolated three distinct cDNAs from zebrafish encoding the GRF receptor, the PACAP specific PAC1 receptor and the shared vasoactive intestinal peptide/PACAP receptor VPAC1. In addition, four isoforms of the PAC1 receptor were isolated from zebrafish including a novel isoform found in the gill. All three receptors were widely expressed in adult zebrafish and receptors for both GRF and PACAP were found in most tissues. This indicates that GRF and PACAP may modulate each other’s function. To determine the developmental role of GRF and PACAP, I characterized the expression pattern of the GRF-PACAP gene and the GRF, PAC1 and VPAC1 receptors in the zebrafish embryo. The GRF and PAC1 receptors are the earliest to be expressed in development starting at the cleavage stage. Later, the GRF-PACAP gene and the VPAC1 receptor are first expressed at the late blastula/early gastrula stage in the zebrafish and are expressed throughout the developmental period. Strong expression of the GRF, PACAP and their receptors during mid gastrulation indicates that these peptides may be involved in modulating the formation of the embryonic axis. During the segmentation period the GRF-PACAP gene is widely expressed in the zebrafish embryo and the PAC1 receptor short and hop isoforms are differentially expressed. Therefore, PACAP may regulate cell cycle exit or cell proliferation through activation of different PAC1 receptor isoforms during the segmentation stage. In the subsequent pharyngula period, the GRF-PACAP transcript is localized mainly to the hatching gland. However, expression is seen also in tissues that undergo differentiation during this stage. Therefore, the timing of the expression of the GRF-PACAP gene indicates that it may be involved in early patteming events and promoting cell cycle exit prior to differentiation. To investigate the role of GRF and PACAP in the developing brain, I localized the expression of GRF, PACAP and the PAC1 receptor in neuroblasts derived from an embryonic day 3.5 chick. PACAP was found to stimulate the cAMP pathway in these cells, indicating that PACAP may modulate brain development. This work indicates that GRF and PACAP play an important role in vertebrate development. / Graduate

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