Sex-biased expression of sex-differentiating genes FOXL2 and FGF9 in American alligators, alligator Mississippiensis

Abstract

Across amniotes, sex-determining mechanisms exhibit great variation, yet the genes that govern sexual differentiation are largely conserved. Studies of evolution of sex-determining and sex-differentiating genes require an exhaustive characterization of functions of those genes such as FOXL2 and FGF9. FOXL2 is associated with ovarian development, and FGF9 is known to play a role in testicular organogenesis in mammals and other amniotes. As a step toward characterization of the evolutionary history of sexual development, we measured expression of FOXL2 and FGF9 across 3 developmental stages and 8 juvenile tissue types in male and female American alligators, Alligator mississippiensis. We report surprisingly high expression of FOXL2 before the stage of embryonic development when sex is determined in response to temperature, and sustained and variable expression of FGF9 in juvenile male, but not female tissue types. Novel characterization of gene expression in reptiles with temperature-dependent sex determination such as American alligators may inform the evolution of sex-determining and sex-differentiating gene networks, as they suggest alternative functions from which the genes may have been exapted. Future functional profiling of sex-differentiating genes should similarly follow other genes and other species to enable a broad comparison across sex-determining mechanisms.

Fig. 1

Relative expression in male and female alligators of FGF9 at both early and late stages of embryogenesis and at a juvenile stage. (a) Analysis of FGF9 expression in all queried samples indicates significant variation across developmental stages. (b) Comparison of FGF9 expression before (termed early) and after (termed late) the thermally sensitive period of sex determination indicates a significant increase in FGF9 expression after sex determination among male but not female embryos. Expression increased significantly from late embryos to juvenile tissues in both males and females. (c) Comparing male and female tissue types from juvenile alligators indicates significant variation among male but not female tissues. Means were log₂-transformed for ease of visual interpretation. Lines on bars represent standard error. Numbers above bars indicate the number of biological replicates. The black diamond in figures a and b indicates the developmental stage in which sex is determined. Asterisks represent significance (*: p < 0.05; ***: p < 0.001).

Fig. 2

Relative expression in male and female alligators of FOXL2 at both early and late stages of embryogenesis and at a juvenile stage. (a) Analysis of FOXL2 expression in all queried samples does not indicate significant variation across developmental stages. (b) Comparison of FOXL2 expression before (termed early) and after (termed late) the thermally sensitive period of sex determination indicates a significant decrease in FOXL2 expression after sex determination among both male and female embryos. Expression increased significantly from late embryos to juvenile tissues in both males and females. (c) Comparing male and female tissue types from juvenile alligators indicates significant variation among both male and female tissues. Means were log₂-transformed for ease of visual interpretation. Lines on bars represent standard error. Numbers above bars indicate the number of biological replicates. The black diamond in figures a and b indicates the developmental stage in which sex is determined. Asterisks represent significance (*: p < 0.05; ***: p < 0.001).

Fig. 3

The housekeeping gene LDHA amplified from each queried tissue. (a) LDHA amplification varies by stage, suggestive of ontogenetic differences in cell proliferation (Merchant et al., 2009). (b) Subdivided by sex, the dataset shows stage-differential but not sex-differential expression of LDHA, suggesting the utility of the marker as a normalizer of expression data for genes of interest. Means were log₂-transformed for ease of visual interpretation. Lines on bars represent standard error. Numbers above bars indicate the number of biological replicates. The black diamond in each figure indicates the developmental stage in which sex is determined. Asterisks represent significance (***: p < 0.001).