The expanding roles of Nr6a1 in development and evolution

Abstract

The Nuclear Receptor (NR) family of transcriptional regulators possess the ability to sense signalling molecules and directly couple that to a transcriptional response. While this large class of proteins are united by sequence and structural homology, individual NR functional output varies greatly depending on their expression, ligand selectivity and DNA binding sequence specificity. Many NRs have remained somewhat enigmatic, with the absence of a defined ligand categorising them as orphan nuclear receptors. One example is Nuclear Receptor subfamily 6 group A member 1 (Nr6a1), an orphan nuclear receptor that has no close evolutionary homologs and thus is alone in subfamily 6. Nonetheless, Nr6a1 has emerged as an important player in the regulation of key pluripotency and developmental genes, as functionally critical for mid-gestational developmental progression and as a possible molecular target for driving evolutionary change in animal body plan. Here, we review the current knowledge on this enigmatic nuclear receptor and how it impacts development and evolution.

Keywords: GCNF; Hox genes; Nr6a1; Oct4; axial elongation; orphan nuclear receptor; retinoic acid.

FIGURE 1

Nr6a1 Structural insight (A,B) Schematised structure of a canonical Nuclear Receptor. Nuclear receptors have a less conserved N-terminal domain (NTD) which harbours an activator function-1 (AF-1) region, a highly conserved DNA binding domain (DBD), a ligand-binding domain (LBD) that harbours an activator function-2 (AF2), and a hinge domain linking the DBD and LBD. (C) Predicted 3-Dimensional structure of human Nr6a1, generated by AlphaFold (Jumper et al., 2021; Varadi et al., 2022), model confidence indicated. (D) A timeline of key milestones in the identification and functional assessment of Nr6a1.

FIGURE 2

Nr6a1 genomic structure and transcript regulation (A) The Nr6a1 genomic locus of Mus musculus. Exons marked in red, not to scale. Multiple Nr6a1 transcripts with coding potential have been identified on the sense strand, while both long non-coding antisense and micro-RNA encoding transcripts are produced from the opposite strand. (B) Nr6a1 expression is defined by key developmental signals/regulators known to control axial elongation. Nr6a1 expression increases in response to Wnt signalling, while the synergistic actions of Gdf11 and miR-196, and potentially let-7 expression, terminate Nr6a1 expression at the trunk-to-tail transition.

FIGURE 3

The dynamic expression of Nr6a1 controls key developmental genes and transitions. The expression of Nr6a1 over time in vivo and during in vitro differentiation (A). In vivo, Nr6a1 expression is detected within the inner cell mass (ICM) at very early stage of mouse development, broadly within the epiblast (epi) at embryonic day (E)6.5, across most tissues and germ layers at E8.5, with a gradual clearing of expression beginning with the posterior tail bud from E9.5 and expression largely cleared from the embryo by E12.5. A similar expression dynamic is seen during in vitro ESC-to-NMP differentiation: Nr6a1 expression within ESCs increases following exposure to Fgf2, and further increases following activation of Wnt signalling (CHIR). The transition in vitro from a trunk NMP to a tail NMP following exposure to Gdf11 downregulates Nr6a1 to low/basal levels. The dynamic in vivo expression of Nr6a1 overlaid with key target genes Oct4 and posterior Hox genes (B,C). The rise of Nr6a1 within the epiblast directly repress Oct4 levels, leading to broadly complementary patterns of expression between early and mid-gestation (B). Conversely, the rise of Nr6a1 prevents precocious expression of posterior Hox genes, leading to broadly complementary patterns of expression between mid and late-gestation (C). Whether posterior Hox repression by Nr6a1 is via direct mechanisms is currently unclear. Images created in Biorender.