Resumen de Establishing an in vitro model for x chromosome reactivation in the germline
The X-Chromosome dosage compensation is a prime model for the study of epigenetic regulation. In female mammals, one of the X chromosomes is inactivated through X chromosome Inactivation (XCI) during the epiblast differentiation for dosage compensation between sexes. However, XCI is later specifically reversed in female germ cells by reactivation of the inactive X chromosome (XCR) via diffusible signaling molecules produced by female gonadal cells.
The XCR observed in female germ cells is an attractive epigenetic phenomenon, but its mechanistic study is impeded by the low cell numbers present in vivo and the need for scalable readouts of X chromosome activity at allelic and single-cell resolution.
To implement a model for the study of germ cell XCR and its signaling effectors, we characterized the XCI and XCR kinetics of a new cell differentiation protocol that differentiates embryonic stem cells (ESCs) into X-inactive epiblast-like cells (Epi-LCs), from which in vitro germ cell (PGC-LCs) differentiation is stimulated through a set of cytokines. The goal was to obtain a source of X-inactive PGC-LCs that could be characterized while exposed to XCR-inducing cues.
We discovered that, contrarily to what was believed in the field, PGC-LCs could undergo XCR mediated by the cytokines present in the PGC-LC differentiation media without exposure to female gonadal cells, providing a working hypothesis for ulterior testing of the role of those cytokines in germ cell XCR.
In the search for scalable readouts of X chromosome activity, we tested several oligo- FISH methodologies based on oligonucleotide labeling and hairpin chain reaction.
Of all methodologies tested, we discovered that the signal intensity of hairpin chain reaction coupled with the split-paired probe design of V3.0 smHCR provided a scalable platform to monitor the X activity at allelic resolution.
In summary, this thesis allowed the implementation of an in vitro model for XCR characterization in the germline and provided the conceptual foundation for a scalable readout to monitor the X activity at allelic and single-cell resolution.
