Female mammals (XX) carry twice as much X gene content compared to males (XY). This imbalance is compensated for early during embryonic development through the broad transcriptional inactivation of one X in the female (X Chromosome Inactivation, XCI). Studies in mouse have identified the lncRNA Xist as the master regulator of XCI: strikingly, a failed X dosage compensation under Xist loss-of-function deeply impairs extraembryonic lineage development, opening exciting research grounds in the human context given its role in implantation and placenta formation. Of note, species-specific aspects of human development include X chromosome early regulatory dynamics, as XCI is seemingly preceded by the transcriptional dampening of both X-s (X Chromosome Dampening, XCD), also associated to XIST expression. Naïve human Embryonic Stem Cells (hESCs) have been capitalized to model the arbors of XCI and cell fate establishment. By employing a XIST CRISPRi system, previous work from the team has shown the importance of this lncRNA for XCI initiation and cell survival during the derivation in vitro of post-implantation human extraembryonic cell types.

We hence decided to implement a stem-cell derived 3D model, termed blastoid (KAGAWA et al., 2022), to capture more faithfully human peri-implantation development. Specifically, we aim to (1) clarify the kinetics of XCI initiation and (2) further elucidate the role of XIST expression in XCI establishment as well as lineage specification.

For this purpose, wild-type, XIST full KO as well as XIST CRISPRi hESCs are being used, describing the progression and term of blastoid formation based on morphology, lineage-specific and X-linked gene expression.

Our findings point to a scenario where XCI is at least initiated in mature wild-type blastoids. Intriguingly, our XIST full KO cells are able to segregate different and mutually exclusive fates. Even so, they repeatedly fail to cavitate and organize blastoids, a result that has been corroborated by a subset of XIST CRISPRi hESCs and implies a potentially crucial role for XCD.

With our research, we hope to enrich the current knowledge on the dynamics of human peri-implantation development, which due to ethical restrictions and paucity of samples has remained a black box. Our results will shed more light about the impact of X dosage on early development, possibly pinpointing genes and pathways whose expression level needs to be equilibrated for an increased chance of embryo survival.