Pluripotent embryonic stem (ES) cells are heterogeneous mixtures of
naïve and lineage-primed states defined by distinct transcription factor
expression profiles. However, the events that prime pluripotent cells for
differentiation are not well understood. Id proteins, which are inhibitors of
basic helix-loop-helix (bHLH) transcription factors, contribute to
pluripotency by blocking differentiation. Using Yeast-Two-Hybrid screening,
our lab identified Tcf15 as an Id-regulated transcription factor.
In this study, I first examined the expression of Tcf15 during
differentiation in vitro and during early development in vivo in the mouse.
Tcf15 expression is higher in primed pluripotent embryonic stem (ES) cells
than in naïve ES cells or epiblast stem cells (EpiSCs). In addition, Tcf15 is
expressed heterogeneously in ES cells and is also detected in the inner cell
mass (ICM) of E4.5 mouse embryos. Expression of Tcf15 was upregulated
during early stages of differentiation and downregulated before cells
committed to any specific lineage. Using Tcf15-Venus reporter cells, I found
that expression of Tcf15 is specifically associated with a novel subpopulation
of ES cells primed for somatic lineages.
Gain of function and loss of function studies were then performed to
perturb Tcf15 expression in ES cells in order to assess the function of Tcf15 in
self-renewal and during differentiation. An inducible Id-resistant form of
Tcf15 accelerates somatic lineage commitment by maturating naïve
pluripotent ES cells transit toward primed epiblast and later on epiblastderived
somatic lineages whilst suppressing differentiation towards
extraembryonic endoderm. Preliminary loss of function studies also suggest
that down-regulation of Tcf15 may promote a naïve state within pluripotent
cells.
I investigated the mechanism by which Tcf15 expression becomes
associated with the epiblast-primed state by identifying the upstream
regulators and downstream targets of Tcf15. Tcf15 expression is dependent
on FGF signalling. Microarray analysis identified that Tcf15 downregulates
the naïve pluripotency determinant Nanog and upregulates the epiblast
determinant Otx2. Taken together, our results suggest that Tcf15 acts in
opposition to the pluripotency network to prime pluripotent cells towards
differentiation