Developmental expression and evolution of growth hormone-releasing hormone and pituitary adenylate cyclase-activating polypeptide in teleost fishes, rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio)




Krueckl, Sandra Lea

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Growth hormone-releasing hormone (GRF) and pituitary adenylate cyclase-activating polypeptide (PACAP) are members of the PACAP/Glucagon superfamily. The family is proposed to have developed from an ancestral PACAP-like molecule in invertebrates. Through successive exon, gene and genome duplications the family has grown to include seven other members. In mammals GRF and PACAP are located on different genes, but in fish, amphibians and birds they are located on the same gene. The main function of GRF is the release of growth hormone (GH) from the pituitary. Also, during development GRF influences the fetal pituitary and stimulates GH release during late gestation. In contrast, the functions of PACAP are extremely varied. PACAP is the newest member of the superfamily and there is still much work to be done before its actions are well understood. Like GRF, PACAP is a releasing hormone acting on the pituitary and in addition, the adrenal gland, pancreas and heart, as well as other organs. Also, PACAP regulates smooth muscle in the vascular system, gut, respiratory tract and reproductive tract During development PACAP affects proliferation, differentiation and apoptosis. GRF and PACAP are expressed throughout development in fish, beginning during the blastula period in rainbow trout and at the end of gastrulation in zebrafish (earliest stage examined). In rainbow trout the grf/pacap gene is expressed as two transcripts, a short and a long transcript. The short transcript is produced by alternative splicing of the gene and does not include the fourth exon which codes for GRF. The long transcript includes the coding regions for both GRF and PACAP. By this means PACAP can be regulated separately from GRF. With the extensive role PACAP appears to play in development, separate regulation of the hormone may be necessary. Expression of the grf/pacap gene in zebrafish is widespread early in development and gradually becomes localized. Of particular interest is the expression of the grf/pacap transcript in regions associated with the prechordal plate, an important organizing center in development. Although it is not yet confirmed, there is evidence to suggest GRF and PACAP are expressed in the prechordal plate and its derivatives in the gut and hatching gland. In addition, expression of the grf/pacap transcript is observed in the neuroectoderm (eye, brain and spinal cord) and the developing heart. Considering the expression pattern of GRF and PACAP, I propose that one of both of these hormones may be involved in patterning during vertebrate embryogenesis. The evolution of gene families is thought to occur through successive exon, gene and genome duplications. Duplicate exons or genes become differentiated and eventually gain new functions or become functionless. During evolution of the grf/pacap lineage, several duplication events have occurred. Analysis of rainbow trout leads me to think that this fish and other salmonids possess two copies of the grf/pacap gene. This is not unexpected considering the tetraploid nature of salmonids. Present day mammals encode GRF and PACAP on separate genes. At some point during the evolution of this lineage a duplication event has occurred, possibly in early mammals or prior to the divergence of birds. The study of multigene families is a useful way to understand evolutionary processes. To this end I examined three members of multigene families from sockeye salmon. Therefore, in addition to the evolutionary mechanisms and pathways that directed grf/pacap gene evolution, I examined the ferritin-H subunit, the alpha-tubulin subunit and the beta-globin subunit. These cDNA sequences are similar to their counterparts in other teleost. The evolution of the ferritin gene family is particularly interesting because it involves the addition or deletion of DNA sequences that affect regulation and cytosolic location.



Hormones, Gene expression, Growth hormone releasing factor, Pituitary hormone releasing factors