Complete physiologic renewal of intestinal epithelium occurs about every week and is driven by actively proliferating ISCs (aISCs) located in the epithelial-crypt base. A rare subset cells in the crypt, still not completely defined but that are collectively known as `reserve' ISCs (rISCs), are `quiescent' or slowly dividing, and can convert into aISCs when certain health conditions or radio- or chemotherapeutic exposures damage and deplete the native aISC pool. The rISC to aISC conversion process is traditionally studied in mice where irradiation (IR) is used to deplete aISCs and induce rISCs. Here, rISCs are defined by their resistance to IR-induced death, and then by their `plastic' ability to generate actively dividing ISC progeny, which replenish the aISC pool and drive subsequent epithelial regeneration. The mechanisms conferring rISC radio-resistance and plasticity are unknown. In prior work we demonstrated that the transcription factor Sox9 is required for the generation and function of rISCs in mice. Lineage tracing with a Sox9CreERT2 driver showed that after IR, all regenerating epithelium is derived from cells that expressing Sox9, and epithelium-specific genetic ablation of Sox9 profoundly impeded epithelial regeneration and cell survival post-IR. These findings indicate that Sox9-dependent mechanisms govern rISC function. Growing evidence in the stem cell and radiation fields suggest that slowing the cell-cycle rate in dividing cells can enhance radioresistance after IR-exposure, and can modulate cell fate commitment versus self-renewal decisions. We have found that elevated Sox9 levels are associated with slowly dividing cells in the crypt, and that Sox9-overexpression in rapidly dividing aISCs can slow or halt their proliferation. We hypothesize that Sox9-expression levels modulate cell-cycle progression to determine and diversify rISC (Sox9HI) and aISC (Sox9LO) functions in the intestinal crypt. If supported by the results of our experiments, this study will uncover the underlying pathways governing rISC radioresistance and plasticity, and could provide a unifying mechanism describing whether and how rISC properties exist among a broad range of cell types in the crypt.
Aim 1 : Assess the effects of increasing Sox9 levels on cell-cycle progression in ISCs.
Aim 2 : Determine if Sox9-mediated G1-elongation confers radio-resistance.
Aim 3 : Determine if Sox9- mediated G1-elongation confers a `secretory precursor' rISC phenotype.
Mechanisms underlying cellular plasticity are poorly understood. Intestinal stem cells (ISCs) exist in actively proliferating (aISCs) or non-proliferative `reserve' states (rISCs). Because of their `quiescent' proliferative state, rISCs are also resistant to genotoxic stress and are important for the epithelial regenerative response after IR- and chemotherapeutic damage. Here we test a defined Sox9-dependent mechanism, where different Sox9 levels regulate the rISC properties of radioresistance and plasticity through elongation of the G1 phase of the cell-cycle. This study stands to uncover the underlying pathways governing rISC radioresistance and plasticity, and could provide a unifying mechanism describing whether and how rISC properties exist among a broad range of cell types in the crypt.
