The ontogeny of type I antifreeze protein expression in winter flounder, Pleuronectes americanus

Murray, Harry Michael (2000) The ontogeny of type I antifreeze protein expression in winter flounder, Pleuronectes americanus. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Winter flounder (Pleuronectes americanus) produce Type I antifreeze proteins (AFPs) which are characterized as alanine rich and demonstrate an alpha-helical secondary structure. These proteins work by noncolligatively depressing the freezing point of tissues and tissue fluids. Two isotypes of Type I AFP, the liver-type and the skin-type, have been described in this species. The liver-type AFP is produced on a seasonal basis and secreted from hepatocytes into the blood and tissue fluids. The skin-type has just begun to be characterized and appears to have a wide tissue distribution and is expressed all year round. The absence of a signal sequence also suggests that this isotype has an intracellular site of activity. The aim of the present series of studies was to improve our understanding of the ontogeny of Type I AFP expression in the winter flounder, the cell types involved in skin-type AFP expression, their tissue distribution, seasonal expression characteristics, and relationship to liver AFP producing cells. -- Adult winter flounder were captured from wild populations during the summer, fall and winter seasons. Winter flounder larvae were hatched and reared in the laboratory under seasonal temperatures and photoperiod. AFP gene expression was examined in adult winter flounder seasonally. -- In situ hybridization (ISH) using AFP gene specific RNA probes and immunohistochemistry (IH) using polyclonal and monoclonal antisera to AFP were used to identify cells responsible for AFP expression in both larval, juvenile and adult tissues. ISH and IH on whole-mounted larvae stained specifically cells running laterally along the fish. These were similar or closely associated in position with superficial neuromast cells when compared to similar species. The staining declined following yolk-sac absorption suggesting that expression in these cells was only important during the early larval stages. Larval integumental mucous cells also gave a specific IH reaction but no ISH reaction suggesting a possible variation in the genes involved. IH product in these cells continued through the larval stage but was shown to disappear around the time of metamorphosis. -- ISH and IH on juvenile and adult skin sections showed a distribution of mRNA and skin-type AFP specific for the epidermis and epidermal pavement cells. Evidence of AFP mRNA and associated protein became detectable in the epidermis from early to mid fall of the fishes first year. The AFP appeared extracellular and was distributed throughout the extracellular or interstitial space usually in close association with the epidermal cells, suggesting that it may be important in slowing ice crystal formation in these interstitial regions and thus reducing cellular damage due to osmotic imbalance. -- Adult and juvenile/larval gut (stomach and intestine) did show a positive immune reaction for skin-type AFP antisera in cells throughout the stomach and intestinal mucosa. These cells also reacted with Alcian blue suggesting that they were mucous producing cells. This reaction indicated a co-secretion with mucous similar to that suggested for the integumental mucous cells of the larvae. Adult intestine did not show the IH reaction in mucous cells, but rather in-association with another population of cells. ISH with skin and liver RNA probes did not show any staining reaction in comparable regions. -- During winter, adult liver cells were positive for liver AFP mRNA and the liver type protein following ISH and IH (polyclonal and monoclonal antibodies). Neither was found present during the summer. Immunostaining reactions for liver type AFP were localized to distinct regions of the hepatocyte cytoplasm. Skin AFP RNA probes indicated that skin AFP RNA was present throughout the year in liver but interestingly the skin type protein was never detected using polyclonal antibodies. This suggested that while the skin AFP mRNA is present constitutively in liver the actual protein is undetectable in hepatocytes using IH regardless of the time of the year. Ontogenetically, expression of AFP (skin and liver types) was first detected in the livers of juvenile flounder in November of the fishes first year. Distinct ISH staining reactions were obvious in liver at this time using both liver specific and skin specific AFP probes. As with the adult liver, IH staining was only evident with liver type AFP antisera. -- Adult gill filaments showed a skin-type AFP mRNA distribution associated with cells throughout the lamellae and to some extent in the filament. The association with lamellae suggested that respiratory cells or pavement cells were likely involved. IH with antibodies for skin AFP identified specific cells corresponding to those detected using ISH. Parallel experiments with antibodies specific for chloride cells showed that these cells were not involved in AFP expression. Similarly, goblet cells did not show reactivity with the AFP antibodies. Based on these two observations the cells expressing the genes are likely gill pavement cells. Ontogenetically the gills arise from extensions of the ventral pharyngeal cartilage and continue to develop over time through increasing complexity of the filament and lamellar structures. Evidence of AFP expression in gill does not become obvious until beyond metamorphosis (October). Cellular distribution of immunopositive product was similar to that of the adult gill. Interestingly, AFP mRNA was not detected in the gill of juvenile flounders. This may have been due to the delicate nature of the tissue resulting in less stability under the conditions of in situ hybridization. -- Verification of IH and ISH results using Reverse Transcription Polymerase Chain Reaction (RT PCR) and gene specific primers showed as expected that liver AFP expression only occurred in liver tissue and only during the winter. Skin AFP expression was detectable in all adult tissues examined (skin, gill, liver, stomach and intestine) with no observed seasonal effect indicating that the constitutive nature of these genes was continuous across tissue types. Sequencing of each of the skin AFP PCR products indicated that they were all of similar nature. Isolated AFP clones from the stomach and intestine showed the most divergence from the original skin AFP clone (WFP9). Sequencing of PCR products obtained using primers specific for the pre-pro sequence of the liver AFP clone CT5 returned sequence identical to this clone, thus verifying that these primers and RT PCR can be used to examine the development of liver type AFP expression. RT PCR analysis of total RNA from larval flounder ranging in age from hatch to metamorphosis indicated that skin type AFP expression is present as early as hatch and continues through metamorphosis. Liver AFP expression however was not detectable until shortly after metamorphosis corresponding with changes in environmental conditions consistent with the onset of colder water and shortened photoperiod in the fall. This observation suggested that cold water naive juvenile fish expressed the liver AFP gene in response to environmental stimuli known to initiate expression of the same genes in adults.

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