Alternative splicing and thermosensitive expression of Dmrt1 during urogenital development in the painted turtle, Chrysemys picta

Background: The doublesex and mab-3 related transcription factor 1 (Dmrt1) is a highly conserved gene across numerous vertebrates and invertebrates in sequence and function. Small aminoacid changes in Dmrt1 are associated with turnovers in sex determination in reptiles. Dmrt1 is upregulated in males during gonadal development in many species, including the painted turtle, Chrysemys picta, a reptile with temperature-dependent sex determination (TSD). Dmrt1 is reported to play different roles during sex determination and differentiation, yet whether these functions are controlled by distinct Dmrt1 spliceoforms remains unclear. While Dmrt1 isoforms have been characterized in various vertebrates, no study has investigated their existence in any turtle.

Methods: We examine the painted turtle to identify novel Dmrt1 isoforms that may be present during urogenital development using PCR, profile their expression by RNA-seq across five embryonic stages at male- and female-producing temperatures, and validate their expression pattern via qPCR with transcript-specific fluorescent probes.

Results: A novel Dmrt1 spliceoform was discovered for the first time in chelonians, lacking exons 2 and 3 (Dmrt1 ΔEx2Ex3). Dmrt1 canonical and ΔEx2Ex3 transcripts were differentialy expressed by temperature at stages 19 and 22 in developing gonads of painted turtles, after the onset of sex determination, and displayed a significant male-biased expression pattern. This transcriptional pattern differs from studies in other turtles and vertebrates that reported Dmrt1 differential expression before or at the onset of sex determination. This study provides the first insight into Dmrt1 transcriptional diversity in turtles and opens the door for future functional studies of the alternative Dmrt1 transcript uncovered here.

Conclusions: The discovery of an isoform in turtles indicate that alternative splicing may be a common feature of Dmrt1 across vertebrates, as isoforms are also found in crocodilians, birds, mammals and fish, and this variation remains unexplained. The relatively late-onset of Dmrt1 expression observed here contrasts with other turtles, indicating that Dmrt1 is not the topmost male sex -determining factor in C. picta. When placed in a phylogenetic context, this discrepancy underscores the divergent regulation of Dmrt1, and of sexual development more generally, across vertebrates.