An unbiased approach to identify genes involved in development in a turtle with temperature-dependent sex determination

Abstract

Background: Many reptiles exhibit temperature-dependent sex determination (TSD). The initial cue in TSD is incubation temperature, unlike genotypic sex determination (GSD) where it is determined by the presence of specific alleles (or genetic loci). We used patterns of gene expression to identify candidates for genes with a role in TSD and other developmental processes without making a priori assumptions about the identity of these genes (ortholog-based approach). We identified genes with sexually dimorphic mRNA accumulation during the temperature sensitive period of development in the Red-eared slider turtle (Trachemys scripta), a turtle with TSD. Genes with differential mRNA accumulation in response to estrogen (estradiol-17β; E(2)) exposure and developmental stages were also identified.

Results: Sequencing 767 clones from three suppression-subtractive hybridization libraries yielded a total of 581 unique sequences. Screening a macroarray with a subset of those sequences revealed a total of 26 genes that exhibited differential mRNA accumulation: 16 female biased and 10 male biased. Additional analyses revealed that C16ORF62 (an unknown gene) and MALAT1 (a long noncoding RNA) exhibited increased mRNA accumulation at the male producing temperature relative to the female producing temperature during embryonic sexual development. Finally, we identified four genes (C16ORF62, CCT3, MMP2, and NFIB) that exhibited a stage effect and five genes (C16ORF62, CCT3, MMP2, NFIB and NOTCH2) showed a response to E(2) exposure.

Conclusions: Here we report a survey of genes identified using patterns of mRNA accumulation during embryonic development in a turtle with TSD. Many previous studies have focused on examining the turtle orthologs of genes involved in mammalian development. Although valuable, the limitations of this approach are exemplified by our identification of two genes (MALAT1 and C16ORF62) that are sexually dimorphic during embryonic development. MALAT1 is a noncoding RNA that has not been implicated in sexual differentiation in other vertebrates and C16ORF62 has an unknown function. Our results revealed genes that are candidates for having roles in turtle embryonic development, including TSD, and highlight the need to expand our search parameters beyond protein-coding genes.

Figure 1

Development Cluster from DAVID functional annotation clustering with high stringency. Functional cluster of developmental genes and GO terms from the resulting known genes from the SSH libraries with high stringency.

Figure 2

Macroarray results showing sexually dimorphic expression patterns. Log view of the fold change between female and male expression patterns determined from a macroarray.

Figure 3

Semi-quantitative PCR Results. A. An example of a semi-quantitative gel image. B. Semi-quantitative results for sexual dimorphism (M = male, F = female), stage effect between males (green bars), and an E₂ time trial (orange bars).

Figure 4

Quantitative RT-PCR showing sexual dimorphic expression at stage 17 and 19. The expression of MALAT1 (n = 5) consists of sex and stage (e.g. M17 = male, stage 17) and is relative to the control gene (PP1). The relative expression for M17 was set as 1 to allow easier comparison between groups. Gene expression was analysed using two-tailed t-test to examine differences between sexes at the same stage (stage 17, P = 0.04 and stage 19, P = 0.01) and between stages of the same sex (male, P = 0.18 and female, P = 0.24).