In vivo and in vitro analyses of mRNA expression of ROBO2 in zebrafish in the context of P13K/AKT/TOR pathway

Abstract

Robo2 is an axon guidance receptor, well-known for its repulsive role in the nervous system. In addition, Robo2 might regulate cell migration both during embryogenesis or in adulthood. In this study, a novel isoform of the zebrafish robo2 (robo2_tv2), which included an otherwise alternatively spliced exon (CAE), has been characterized. Robo2_tv2 was expressed differentially in most non-neuronal tissues of adult zebrafish whereas Robo2_tv1 expression to a great extent was restricted to the brain and eye. Our findings demonstrated that the amino acid sequence coded by CAE of the Robo2 gene was highly conserved between zebrafish and mammals, and also contained conserved motifs shared with Robo1 and Robo4 but not with Robo3. Furthermore, we provided an account of differential transcription of the CAE homolog in various tissues of the adult rat. These results has suggested that the alternatively spliced Robo2 isoforms may exhibit tissue specificity. In addition, we investigated the transcriptional neighbors of Robo2 based on computational and experimental methods. Bioinformatics analysis of a published zebrafish microarray data by Mathavan et al. (2005) demonstrated that Robo2 expression might be correlated with a number of genes involved in the PI3K/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, Gsk3b were characterized in terms of their sequence conservation and tissue-specific expressions for the first time in zebrafish. Phylogenetic analysis has shown that these genes were moderately to highly conserved among different vertebrate taxa. RT-PCR and real-time RT-PCR analyses in various adult tissues and under specific growthregulatory 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 multiple independent experiments due to high variability and small fold changes that characterized the expression levels. Future studies are planned to identify functional relevance for the alternative usage of CAE and determine how different isoforms respond to conditions that modulate cell growth/proliferation pathways involved in cellular stress conditions including cancer.