Tissue Distribution, Developmental Expression And Functions Of Galanin Receptor1a Gene In Zebrafish

Galanin, a29amino acid neuropeptide in mammals (30in humans), was firstisolated in1983from pig intestine. In mammals, galanin is involved in themodulation of hormone and neurotransmitter release, cognitive functions, feedingbehavior, intestinal secretion and motility, and neuronal development. Furthermore,recent studies revealed both mitogenic and anti-proliferative effects of galanin onvarious carcinoma cells, suggesting its potential roles in cancer pathology. Thediverse physiological effects of galanin are mediated by three galanin receptors,GalR1, GalR2and GalR3, and all of which are G protein-coupled receptors (GPCR).Galanin receptors are highly conserved among species, and share a low percentage ofhomology within the same species. The three receptors have substantial difference intheir tissue distribution and functions. It has been known that human GalR1ispredominantly located in the brain and spinal cord. A potential role for GalR1ingrowth and development in human, as well as in the regulation of insulin, glucose andfat homeostasis was suggested. GalR2is widely distributed in almost all tissues,numerous physiological functions of GalR2such as feeding, emotion, memory,nociception, cellular growth, nerve regeneration and growth hormone release werereported. The third known galanin receptor GalR3is relatively abundant in peripheraltissues, but expression in the CNS is more limited, being largely confined to thehypothalamus and the midbrain and hindbrain. And GalR3might be involved inemotion, feeding, pituitary hormone release, nociception and metabolism.In contrast to the extensive studies in mammals, relatively little information withregard to fish galanin receptors is reported. Galanin receptor sequences from somefishes are known, but there are few reports about its distribution and functions to date. Recently, we have found that no GalR3gene was present in fish through investigatingthe evolution of GalRs, but two GalR1genes (GalR1a and GalR1b) were found in fishincluding Takifugu rubripes, Tetraodon nigroviridis and Danio rerio. In additionfurther research indicates that fish GalR1a may represent the primitive gene formduring fish evolution. To uncover the functions of GalR1a in fish, here the tissuedistribution in adult fish, developmental expression and the role in nutrition regulationof GalR1a were investigated in zebrafish (Danio rerio). We found that the expressionof GalR1a mRNA was restricted to the intestine and brain in adult zebrafish, whileanother subtype of GalR in zebrafish GalR1b mRNA was present in all tissues tested,these results indicate that GalR1a resembles its homologous GalR1in mammal.During embryogenesis, the expression of GalR1a mRNA was first decreased and thenincreased,which presents the typical characteristics of maternal expression. In situhybridization of embryos and larvae, the GalR1a gene was widely expressed at beforegastrula stage while at after segmentation period the expression site was changed intovery fairly specific, and the expression signals were concentrated in the area of headand belly. In addition, larva after hybridization in situ were sectioned and resultsshowed that the expression of GalR1a in gut were notable which resembles theexpression pattern in adult zebrafish. Whether in vitro or in vivo, a significantincrease in gut GalR1a mRNA levels was observed after fasting in zebrafish, also,theexpression of gut GalR1a mRNA was decreased to the normal level when the fastedzebrafish were refed with a standard laboratory chow. These results indicate thatstimulation of fasting could induce the levels of GalR1a, which consisted the researchresults in mammalian.Gut GalR1a mRNA levels are up-regulated after high fat overfed compared to thelow fat feed. Also in the cell transfection experiment,GalR1a expression in FG cellswas up-regulated after fatty acid (linoleic acid, LA) treatment, and which wereconsisted with the functions of GalR1in rat. Our data further suggest that zebrafishGalR1a may resemble mammalian GalR1, but not GalR2or GalR3. In addition, inthe FG cells transfected with zebrafish GalR1a gene had much more lipid dropletsthan the control groups (cells without GalR1a gene transfection), which suggesting that the overexpression of GalR1a in FG cells results in the accumulation of lipiddroplets. While the underlying mechanism needs further research. Our study lays afoundation for further investigation of GalR1a function and evolution in fish.In addition, the successful construction of a pair of GalR1a-pCS2Talen vectorsprovides basis for knockout of targeted gene, and this will be a good contribution tofuture research of GalR1a function and mechanism in zebrafish using the geneticmutation technique.