Multivariate expression analysis of the gene network underlying sexual development in turtle embryos with temperature-dependent and genotypic sex determination.

Sexual development has long been the target of study and despite great advances in our understanding of the composition and regulation of the gene network underlying gonadogenesis, our knowledge remains incomplete. Of particular interest is the relative role that the environment and the genome play in directing gonadal formation, especially the effect of environmental temperature in directing this process in vertebrates. Comparative analyses in closely related taxa with contrasting sex-determining mechanisms should help fill this gap. Here I present a multivariate study of the regulation of the gene network underlying sexual development in turtles with temperature-dependent (TSD; Chrysemys picta) and genotypic sex determination (GSD; Apalone mutica). I combine novel data on SOX9 and DMRT1 from these species with contrasting sex-determining mechanisms for the first time with previously reported data on DAX1, SF-1 (NR5A1), WT1, and aromatase (CYP19A1) from these same taxa. Comparative expression analyses of SOX9 and DMRT1 from these and other species indicate additional elements whose expression has diverged among TSD taxa, further supporting the notion that significant evolutionary changes have accrued in the regulation of the TSD gene network in reptiles. A non-parametric MANOVA revealed that temperature had a significant effect in multivariate gene expression in C. picta that varied during embryonic development, whereas the covariation of gene expression in A. mutica was insensitive to temperature. A phenotypic trajectory analysis (PTA) of gene expression comparing both species directly indicated that the relative covariation in gene expression varied between temperatures in C. picta. Furthermore, the 25 degrees C trajectory of C. picta differed from that of A. mutica in the magnitude of gene expression change. Additional analyses revealed a stronger covariation in gene expression and a more interconnected regulatory network in A. mutica, consistent with the hypothesis that sexual development is a more canalized process in A. mutica, as would be expected if GSD evolved in this lineage through directional selection from its TSD ancestor.