An annual cycle of gene regulation in the red-legged salamander mental gland: from hypertrophy to expression of rapidly evolving pheromones

Abstract

Cell differentiation is mediated by synchronized waves of coordinated expression for hundreds to thousands of genes, and must be an exquisitely regulated process to produce complex tissues and phenotypes. For many animal species, sexual selection has driven the development of elaborate male ornaments, requiring sex-specific differentiation pathways. One such male ornament is the pheromone-producing mental gland of the red-legged salamander (Plethodon shermani). Mental gland development follows an annual cycle of extreme hypertrophy, production of pheromones for the ~2 month mating season, and then complete resorption before repeating the process in the following year. At the peak of the mating season, the transcriptional and translational machinery of the mental gland are almost exclusively redirected to synthesis of many rapidly evolving pheromones. Of these pheromones, Plethodontid Modulating Factor (PMF) has experienced an unusual history of disjunctive evolution: following gene duplication, positive sexual selection has diversified the protein coding region while the untranslated regions have been conserved by purifying selection. However, the molecular underpinnings that bridge the processes of gland hypertrophy, pheromone synthesis, and disjunctive evolution remain to be determined and are the focus of the present investigation. Using Illumina sequencing, we prepared a de novo transcriptome of the mental gland at six stages of development. Differential expression analysis and immunohistochemistry revealed that the mental gland initially adopts a highly proliferative, almost tumor-like phenotype, followed by a rapid increase in pheromone mRNA and protein levels. One likely player in this transition is Cold Inducible RNA Binding Protein (CIRBP), which selectively and cooperatively binds the highly conserved PMF 3' UTR. CIRBP, along with other stress response proteins, have seemingly been co-opted to aid in mental gland development by helping to regulate pheromone synthesis. The P. shermani mental gland utilizes a complex system of transcriptional and post-transcriptional gene regulation to facilitate its hypertrophication and pheromone synthesis. The data support the evolutionary interplay of both coding and noncoding segments in rapid gene evolution, and necessitate study of the co-evolution of pheromone gene products along with their transcriptional and translational regulators. Additionally, the mental gland could be a powerful emerging model of regulated proliferation and subsequent resorption of a tissue, within the dermis, thus having potential links to skin cancer biology.