Over the few days that follow fertilisation, the mammalian embryo must specify the cell types it needs to implant in the uterus and begin somatic differentiation: first the Trophectoderm (TE), which will give rise to the placenta, segregates from the inner cell mass (ICM). Then, cells in the ICM are allocated to the primitive endoderm (PrE), forming the yolk sack, or to the epiblast (Epi), which is able to give rise to all embryonic tissues. How this key trait of epiblast cells, defined as pluripotency, is established remains incompletely understood. Our previous work suggests that two transcription factors (TFs) belonging to the class of orphan nuclear receptors - NR5A2 and ESRRB - may play important roles during these cell fate choices. We showed that embryos lacking NR5A2 fail to activate expression of early regulators of the Epi, TE and PrE. These mutants are thus unable to begin the process of lineage specification, and arrest at the morula stage. Moreover, we found that the concomitant KO of NR5A2 and ESRRB in mouse embryonic stem cells leads to the collapse of the regulatory network sustaining pluripotency: this suggests that the two TF might later play a crucial role also in the establishment of pluripotency in the epiblast.

We have thus developed mice that allow triggering NR5A2 and ESRRB loss of function after a morula has formed, and bypass the early lethality of NR5A2 mutants. Single embryo RNA-seq, ATAC-seq, and immunostaining at the blastocyst stage shows mild effects on TE markers and highlights how the two orphan receptors exert distinct roles in controlling PrE fate. In contrast, we describe an additive effect of NR5A2 and ESRRB on epiblast specification: the KO of both TFs entirely ablates transcription of key pluripotency TFs, including Nanog and Sox2. However, Oct4 expression, despite being diminished, persists. We are thus analysing development after implantation, to understand if mutant epiblast cells remain pluripotent, and are indeed competent to begin somatic differentiation. Moreover, we are exploring if these epiblast cells are competent to initiate germline specification, and whether primordial germ cells, which reactivate the pre-implantation pluripotency gene regulatory network, can be maintained in the absence of NR5A2 and ESRRB. Taken together, our studies might define orphan nuclear receptors as key family of regulators of cell fate that act throughout the development cycle.