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
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/9924 |
| Date | 16 August 2018 |
| Creators | Fradinger, Erica Aileen |
| Contributors | Sherwood, Nancy |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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