Evolutionary origin and functional divergence of totipotent cell homeobox genes in eutherian mammals

BACKGROUND: A central goal of evolutionary biology is to link genomic change to phenotypic evolution. The origin of new transcription factors is a special case of genomic evolution since it brings opportunities for novel regulatory interactions and potentially the emergence of new biological properties. RESULTS: We demonstrate that a group of four homeobox gene families (Argfx, Leutx, Dprx, Tprx), plus a gene newly described here (Pargfx), arose by tandem gene duplication from the retinal-expressed Crx gene, followed by asymmetric sequence evolution. We show these genes arose as part of repeated gene gain and loss events on a dynamic chromosomal region in the stem lineage of placental mammals, on the forerunner of human chromosome 19. The human orthologues of these genes are expressed specifically in early embryo totipotent cells, peaking from 8-cell to morula, prior to cell fate restrictions; cow orthologues have similar expression. To examine biological roles, we used ectopic gene expression in cultured human cells followed by high-throughput RNA-seq and uncovered extensive transcriptional remodelling driven by three of the genes. Comparison to transcriptional profiles of early human embryos suggest roles in activating and repressing a set of developmentally-important genes that spike at 8-cell to morula, rather than a general role in genome activation. CONCLUSIONS: We conclude that a dynamic chromosome region spawned a set of evolutionarily new homeobox genes, the ETCHbox genes, specifically in eutherian mammals. After these genes diverged from the parental Crx gene, we argue they were recruited for roles in the preimplantation embryo including activation of genes at the 8-cell stage and repression after morula. We propose these new homeobox gene roles permitted fine-tuning of cell fate decisions necessary for specification and function of embryonic and extra-embryonic tissues utilised in mammalian development and pregnancy.This work was supported primarily by the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013 ERC grant 268513) to PWHH. In addition, support was provided by the Spanish Ministry of Economy and Competitiveness (BFU2014-55076-P) to MI and a fellowship from ‘la Caixa’-Severo Ochoa to CDR

Propagation and maintenance of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) are pluripotent cells derived from preimplantation embryos that have the capacity to self-renew indefinitely in vitro. mESCs are an indispensable tool for studying cellular differentiation in vitro, generating disease in a dish models, and have been used extensively for the generation of transgenic animals. Therefore, maintaining their pluripotent state, even after extended culture, is crucial for their utility. Herein, we describe in detail a protocol for the culture of mESCs in the presence of fetal calf serum (FCS), leukemia inhibitory factor (LIF), and a layer of irradiated mouse embryonic fibroblasts (iMEFs). This culture system reliably sustains mESC pluripotency and self-renewal capacity, allowing their use in a wide range of experimental settings