Harry M. Murray

Ontogenetic and tissue-specific expression of preproghrelin in the Atlantic halibut, Hippoglossus hippoglossus L.

Ghrelin is a conserved vertebrate hormone that affects both GH release and appetite. We have cloned and characterized Atlantic halibut preproghrelin cDNA and examined for the first time preproghrelin expression during fish larval development using quantitative real-time PCR. In addition, cellular sites of expression in larvae and tissue-specific expression in 3-year-old halibut were studied. A full-length cDNA for preproghrelin was isolated from halibut stomach tissue. The 899 bp cDNA encodes an open reading frame of 105 amino acids that is comprised of a signal peptide and two peptides with high similarity to ghrelin and obestatin. The deduced amino acid sequence of halibut ghrelin peptide (GSSFLSPSHKPPKGKPPRA) shows significant conservation relative to other teleostean sequences and is identical to human ghrelin for the first seven amino acids of the sequence. The putative obestatin peptide is well-conserved among fishes but shares limited similarity with its human counterpart. Expression of ghrelin was localized to two different cell types in the stomach of larval halibut by in situ hybridization. However, sensitive PCR assays on tissues collected from 3-year-old fish additionally identified ghrelin transcripts in pyloric caecae, intestine, and in immature ovary and testis. Ontogenetic studies detected ghrelin expression prior to exogenous feeding during larval development (hatching and mouth-opening stages) with increased expression occurring through metamorphosis. This increase was pronounced during climax metamorphosis and coincided with stomach differentiation. Patterns of preproghrelin expression suggest that ghrelin has important roles during and after larval development in halibut, and that ghrelin is associated with digestive and gonadal tissues in this teleost.

Cellular localization of pleurocidin gene expression and synthesis in winter flounder gill using immunohistochemistry and in situ hybridization

Pleurocidin is an antimicrobial peptide isolated from winter flounder and has been previously localized to mucous cells of the skin epidermis and the intestine. The present study was designed to determine the cell type involved in pleurocidin gene expression and protein synthesis in gills from the same species. Whole-mount in situ hybridization with a pleurocidin-specific RNA probe and whole-mount immunohistochemistry with an anti-pleurocidin antibody localized the expression of this gene and the synthesis of its corresponding protein in a population of cells primarily isolated to the non-lamellar portion of the gill filament. Histological techniques allowed the presumptive identification of these cells as eosinophilic granular cells. These observations suggest that pleurocidin is expressed in not one but two distinct populations of cells within the winter flounder, one being an important group of inflammatory cells, the eosinophilic granular cells.