Paedomorphic patterns of gene expression in the eyes of Brook Salamanders

Abstract Shifts in the timing of developmental processes (heterochrony) can lead to phenotypic diversification, yet the underlying molecular mechanisms explaining variation remain largely unexplored outside a few model systems. Extensive remodeling of the visual system occurs during amphibian metamorphosis making this a valuable process for studying the evolution of developmental mechanisms. Here we investigated how life cycle mode and ecology can influence heterochronic patterns of expression for eye development and maintenance genes across Brook Salamanders (genus Eurycea). This clade exhibits repeated evolution of larval-form paedomorphosis and independent instances of cave-dwelling (troglomorphy), which allows for testing how gene expression evolves across different life cycle modes and ecologies. We used RNA-Seq to quantify the expression of 34 eye development and maintenance genes for larvae and adults across 13 lineages of Brook Salamanders (eight biphasic and five paedomorphic), including two troglomorphic lineages. We found numerous lineage-specific differences between larvae and adults. Across biphasic specimens, but not considering phylogeny, we found the transcription factors visual system homeobox 1 (vsx1) had higher expression in larvae versus adults, while Krüppel-like factor 9 (klf9) and mineralocorticoid receptor (mr) had higher expression in adults than larvae. In contrast, we found no significant differences between larvae and adults across paedomorphic specimens. When considering phylogeny, the expression of klf9 and meis homeobox 2 (meis2) were significantly higher in biphasic adults compared to paedomorphic adults. There was a significant increase in klf9 expression after metamorphosis, but meis2 expression was maintained in biphasic salamanders across development. Conversely, the expression of meis homeobox 1 (meis1) and ISL LIM homeobox 1 (isl1) maintained higher levels of gene expression in paedomorphic adults compared to biphasic adults. Some of these transcription factors are known to be highly expressed in vertebrate embryos and are involved in retinal cell specification and maintenance. The sustained high expression in paedomorphic adults might reflect juvenilization of eye development with embryonic expression patterns continuing into adulthood. In the troglomorphic Grotto Salamander (E. spelaea) that degenerates its eyes during metamorphosis, we found striking downregulation of orthodenticle homeobox 2 (otx2) in adults compared to their larvae, and to conspecific larvae and adult stages of surface-dwelling relatives. Overall, our results highlight the importance of utilizing phylogenetic comparative approaches for inferring generalizable expression patterns. We also demonstrated that the loss of metamorphosis in larval-form paedomorphs and derived metamorphic patterns in some troglomorphs are associated with heterochronic shifts in expression of eye development and maintenance genes.