The Establishment and Characterization of H19X Loss-of-function Mouse Embryonic Stem Cells



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How the newly formed zygote develops its own identity or ‘cell fate’ is controlled by a balance of extrinsic and intrinsic signals. These signals govern gene activation and regulate the commitment of each cell towards the extraembryonic or embryonic tissues that make up the entire embryo. Initially, cell fate was thought to be mainly controlled through protein coding transcripts, which account for only 5% of the genome. More recently, non-coding RNA emerged as a key regulator of embryonic development. One genomic locus, H19X, contains such non-coding RNA. This area has been shown to have restricted expression during early heart and somite formation in the developing embryo. The aim of this dissertation was to create and characterize mouse embryonic cell lines aimed at evaluating the contributions of the non-coding locus, H19X, to the cell fate decisions in early development. Using CRISPR/CAS9 technology, we targeted specific regions within the H19X for deletion. We examined how the removal of this locus affected the stem cell state and early differentiation towards germ layer formation We found no difference between knockout lines and control lines at the pluripotent stage. Upon differentiation, we found that knockout cell lines preferentially develop into an ectoderm cell fate. This preference inhibited the ability for the knockout lines to fully specify into mesoderm, with a decrease in cTNT positive cells at Day 12 of differentiation. Additionally, we found that the KO clones displayed an atypical response to apoptosis induction using small molecules. We examined the mTORC1 pathway, known for modulation of cell growth during differentiation., and its relation to the H19X locus in development and found the H19X region may contribute to mesendoderm formation through mTOR modulation. Furthermore, we showed mTORC1 inhibition in the KO model was able to partially restore mesendoderm formation, indicating the H19X region may participate in regulating mTORC1 signaling during development. This research contributes to the theory that ncRNA are critical to specifying cell fate subtle changes in the RNA transcriptome.



mESCs, H19X, ncRNA knockout