Research Projects

During mammalian development, the totipotent blastomeres of the morula make the first cell fate decision to segregate into either the extraembryonic trophectoderm, which will contribute to the placenta, or the inner cell mass which will give rise to the embryo proper. The identity of these cells is safeguarded by certain molecular barriers which are encoded in the chromatin structure, preventing them from interconverting into other type of cells in the embryo. Perturbations in this process can hamper embryo implantation or cause aberrant placentation. The embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) represent the invitro counterparts of the Inner cell mass and the trophoblast layer of embryo respectively.The pluripotent stem cells and trophoblast linage are considered to adhere to strict lineage restriction and do not cross contribute/differentiate to other linage cells. However recent reports of derivation of human TSC from ESC have started to question this dogma of strict linage adherence. The inability to derive TSCs from Mouse ESC have led to conclusion that the molecular/chromatin barriers between Pluripotent cells and trophoblast may be of different strength is different mammalian species and the mouse pluripotent cells may have stronger molecular barriers to convert to trophoblast lineage. In contrast to current understanding, data from our lab (preprint on bioRxiv) show that mouse pluripotent ESCs and ICM can readily differentiate to trophoblast linage by a two-step protocol under appropriate culture conditions. We also identify at least 3 different intermediate stages during this process. Here we propose to understand which molecular/ chromatin barriers are important and which barriers are the first one to be overcome for ESC to TSC conversion. An understanding of the barriers between TSCs and pluripotent stem cells can be gained from in vitro models of ESC conversion to TSCs. In our lab, we have found that mouse ES cells can be pushed to the TS fate without any genetic manipulation by modulating the Wnt signaling pathway. The TS cell lines derived through this method robustly express TS-specific genes, contribute exclusively to the trophectoderm in chimera experiments, and can differentiate into the different placental cell types. Using this trans-differentiation model, our approach will be to generate reporter cell lines for core TS-specific transcription factors to track ES-TS conversion and perform ATAC-seq for active chromatin landscape, ChIP-seqs for differentially regulated histone methylation and acetylation marks in ES and TS cells, whole genome bisulphite sequencing for changes in global methylation landscape, and RNA-seq at different stages in this process. This should give us an understanding of the crosstalk between signaling pathways and chromatin dynamics that underlies the first lineage segregation.