Zebrabalığında robo2'nin pi3k/akt/tor yoluyla ilişkili olarak ın vıvo ve ın vıtro ıfade analizi

Robo2 is an axon guidance receptor, well-known for its repulsive role in thenervous system. In addition, Robo2 might regulate cell migration both duringembryogenesis or in adulthood. In this study, a novel isoform of the zebrafish robo2(robo2_tv2), which included an otherwise alternatively spliced exon (CAE), has beencharacterized. Robo2_tv2 was expressed differentially in most non-neuronal tissues ofadult zebrafish whereas Robo2_tv1 expression to a great extent was restricted to thebrain and eye. Our findings demonstrated that the amino acid sequence coded by CAEof the Robo2 gene was highly conserved between zebrafish and mammals, and alsocontained conserved motifs shared with Robo1 and Robo4 but not with Robo3.Furthermore, we provided an account of differential transcription of the CAEhomolog in various tissues of the adult rat. These results has suggested that thealternatively spliced Robo2 isoforms may exhibit tissue specificity.In addition, we investigated the transcriptional neighbors of Robo2 based oncomputational and experimental methods. Bioinformatics analysis of a publishedzebrafish microarray data by Mathavan et al. (2005) demonstrated that Robo2expression might be correlated with a number of genes involved in thePI3K/AKT/GSK3B/TOR signaling; these genes included Rheb, Gsk3alpha, PP2A,and several Wnt signaling members. Analysis of a conserved coexpression network(Stuart et al. 2003) also placed Robo2 and Tor as indirect neighbors. Accordingly,several PI3K/AKT/GS3B/TOR signaling members, namely, Pik3r2, Akt2, Gsk3bwere characterized in terms of their sequence conservation and tissue-specificexpressions for the first time in zebrafish. Phylogenetic analysis has shown that thesegenes were moderately to highly conserved among different vertebrate taxa. RT-PCRand real-time RT-PCR analyses in various adult tissues and under specific growth-regulatory conditions (e.g., TOR inhibition, and serum starvation) suggested that Tor,Pi3kr2, Akt2 and Gsk3b mRNA might be regulated at the transcriptional level.However, these preliminary findings need to be further confirmed by using multipleindependent experiments due to high variability and small fold changes thatcharacterized the expression levels.Future studies are planned to identify functional relevance for the alternativeusage of CAE and determine how different isoforms respond to conditions thatmodulate cell growth/proliferation pathways involved in cellular stress conditionsincluding cancer.