Mammalian embryo comparison identifies novel pluripotency genes associated with the naïve or primed state

Abstract

During early mammalian development, transient pools of pluripotent cells emerge that can be immortalised upon stem cell derivation. The pluripotent state, ‘naïve’ or ‘primed’, depends on the embryonic stage and derivation conditions used. Here we analyse the temporal gene expression patterns of mouse, cattle and porcine embryos at stages that harbour different types of pluripotent cells. We document conserved and divergent traits in gene expression, and identify predictor genes shared across the species that are associated with pluripotent states in vivo and in vitro. Amongst these are the pluripotency-linked genes Klf4 and Lin28b. The novel genes discovered include naïve- (Spic, Scpep1 and Gjb5) and primed-associated (Sema6a and Jakmip2) genes as well as naïve to primed transition genes (Dusp6 and Trip6). Both Gjb5 and Dusp6 play a role in pluripotency since their knockdown results in differentiation and downregulation of key pluripotency genes. Our interspecies comparison revealed new insights of pluripotency, pluripotent stem cell identity and a new molecular criterion for distinguishing between pluripotent states in various species, including human.

Multivariate Meta-Analysis of Differential Principal Components underlying Human Primed and Naive-like Pluripotent States

“…Moreover, GJB5, identified from C13, encodes the Gap junction beta-5 protein that mediates cell-cell contacts and direct intracellular communication. Knockdown of Gjb5 in mESCs results in downregulation of critical pluripotency genes, thereby leading to cellular differentiation (Bernardo et al, 2018). Thus, several gene markers identified in this analysis have already shed some light on the biochemical and epigenetic aspects of naive-like pluripotency.…”

Allele-specific RNA-seq expression profiling of imprinted genes in mouse isogenic pluripotent states

“…To gain insight into the expression of imprinted genes in the developing B6D2F1 embryo in vivo, we used RNAseq that we generated previously [28] on three replicate tissues of (1) Expanded Blastocyst: Inner cell mass (E3.5; "EBI"); (2) Epithelial cells of the pre-gastrulation Radially Symmetrical Epiblast (E6.25; "ERSE"); and (3) midgastrulation Anterior-Posterior Epiblast (E7.25; "APE"). Given the importance of imprinting in the placenta [38], Expanded Blastocyst: mural TrophEctoderm tissue (E3.5; "EB-TE"; two replicates), we performed RNA-seq profiling to provide insight into imprinted gene expression in the very early extraembryonic trophoblast lineage ( Fig.…”

“…a Mouse strains and crosses used to derive the B6D2F1 samples as profiled by RNA-seq. b Embryos were collected at different stages of development (top) and microdissected for RNA-seq as shown in the illustration (bottom) [28]. The epiblast is depicted in shades of pink, trophectoderm is in gray, extraembryonic endoderm is light brown, and extraembryonic mesoderm is dark brown.…”

“…The embryo sample preparation and RNA-seq has been described previously [28]. In short, for preimplantation B6D2F1 mouse embryonic tissues, samples were collected by flushing embryos out of the uterus.…”

“…To study the degree to which imprinted gene expression is preserved in pluripotent cell lines as compared to their founder mouse embryonic cells in vivo, we performed high-resolution allele-specific RNA-seq on embryonic tissues as well as on ESCs and EpiSCs derived after normal fertilization, NT and PGA. For the embryonic tissues, we focused on the very early embryonic stages (between E3.5 and E7.25 [28]) from which ESCs and EpiSCs are derived. Although two recent studies performed in-depth analysis of allelic gene expression during embryonic development, these studies focused on much later stages (from E9.5 onward) [11,20].…”