Supplementary material from "Of eyes and embryos: subfunctionalization of the CRX homeobox gene in mammalian evolution"

ETCHbox genes are fast-evolving homeobox genes present only in eutherian (placental) mammals which originated by duplication and divergence from a conserved homeobox gene, Cone-rod homeobox (CRX). While expression and function of CRX are restricted to the retina in eutherian mammals, ETCHbox gene expression is specific to preimplantation embryos. This dramatic difference could reflect the acquisition of new functions by duplicated genes or subfunctionalization of pleiotropic roles between CRX and ETCHbox genes. To resolve between these hypotheses, we compared expression, sequence and inferred function between CRX of metatherian (marsupial) mammals and ETCHbox genes of eutherians. We find the metatherian CRX homeobox gene is expressed in early embryos and in eyes, unlike eutherian CRX, and distinct amino acid substitutions were fixed in the metatherian and eutherian evolutionary lineages consistent with altered transcription factor specificity. We find that metatherian CRX is capable of regulating embryonically expressed genes in cultured cells in a comparable way to eutherian ETCHbox. The data are consistent with CRX having a dual role in eyes and embryos of metatherians, providing an early embryonic function comparable to that of eutherian ETCHbox genes; we propose that subfunctionalization of pleiotropic functions occurred after gene duplication along the placental lineage, followed by functional elaboration.