Multiple alternative splicing of Dmrt1 during gonadogenesis in Indian mugger, a species exhibiting temperature-dependent sex determination.

Dmrt1 is an evolutionarily conserved gene having important role in the sex determination from lower vertebrates to mammals. Recent studies show transcriptional diversity for this important gene during gonadal differentiation in a few vertebrate species having genetic sex determination (GSD). In this study, we show for the first time that the transcriptional diversity of Dmrt1 is also found in the Indian mugger that exhibits temperature-dependent sex determination (TSD). We report here isolation and characterization of eight novel isoforms of Dmrt1 from Crocodylus palustris, along with its genomic locus that is referred as, cpDmrt1. Further, by sequence comparisons of cpDmrt1 and its expressed isoforms, we demonstrate that all the isoforms are generated by alternative splicing, exonization of intronic sequences and alternative polyA sites from the same locus. The eight transcripts range from 494 to 2060 bp and encode six predicted proteins having the characteristic DM domain of Dmrt1. The major heterogeneity in the isoforms and their predicted proteins is seen only in their C-termini and 3'-UTRs, which do not match with any similar sequences reported for other vertebrates. The cpDmrt1 expression was seen mainly in developing GAM (genital ridge-adrenal-mesonephros complex) with significant upregulation only in male embryos from the start of the temperature sensitive period (TSP). More significantly, approximately 70% of this expression was contributed by only one isoform (cpDmrt1e) that also has a unique 15 amino acid domain towards its C-terminal. cpDmrt1 expression was also detected at a lower level in brain and developing kidney. The study thus provides the first account of Dmrt1 locus, its transcriptional diversity and sex-specific expression in Indian mugger, a TSD species.