Program in Cancer Biology

Research project: 
My pilot experiments of models of TSC using CreER driven by the promoters of transcription factors that have spatially restricted expression in the mouse V-SVZ with a Tsc2 floxed line have been completed. Large-scale tumor formation has not been observed at any time points in Gli1CreER; Tsc2 fl/fl; Ai14fl/fl  (ventral) nor in Emx1CreER; Tsc2 fl/fl; Ai14fl/fl (dorsal) however, mice have displayed varying phenotypes including hydrocephalus (enlarged ventricles), fewer stem cell progeny in the olfactory bulb, ventricular tortuosity and enlarged cell size starting at 3 months, and more severe phenotypes have been observed at 6 months. These experiments have suggested that the timing of Tsc2 removal is a key factor as to whether or not tumors will form. To move forward with this hypothesis, we have obtained mice from our Vanderbilt collaborators, Cary Fu and Kevin Ess, that have Cre-driven by the promoters of transcription factors that have spatially restricted expression in the mouse V-SVZ with a Tsc2-floxed line. In these mice, the Cre is not temporally controlled and leads to neonatal lethality thus precluding long-term analysis. Despite this, analysis of brains collected at postnatal day 7 mice from Nkx2.1Cre; Tsc2 fl/fl; Ai14fl/fl (ventral) and Emx1Cre; Tsc2 fl/fl; Ai14fl/fl (dorsal) is promising. Thus far, I have seen structures in the ventricles resembling either smaller nodules or larger tumors in 5/5 of Nkx2.1Cre; Tsc2 fl/fl; Ai14fl/fl (ventral) mice and no nodules/tumors in Emx1Cre; Tsc2 fl/fl; Ai14fl/fl (dorsal) mice (n=9). Conversely, I have observed that 9/9 Emx1Cre; Tsc2 fl/fl; Ai14fl/fl (dorsal) mice display severe hydrocephaly (enlarged ventricles) as compared to Nkx2.1Cre; Tsc2 fl/fl; Ai14fl/fl(ventral) mice. This suggests that separate progenitor pools contribute to the variety of symptoms observed in patients with TSC- it is possible that Emx1+ progenitors are responsible for the epilepsy phenotype while Nkx2.1+ progenitors form tumors, as suggested by these experiments. This would explain the wide genotype-phenotype presentations currently observed in the clinic.

I have continued to develop and optimize assays to study neural stem/progenitor cell signaling at the single cell level using fluorescent flow cytometry for both cultured and acutely dissected cells from the mouse V-SVZ. My continued experiments have supported my preliminary finding that stem cells within different regions of the V-SVZ have different basal signaling profiles in the pathways affected in tumor development in TSC patients. I have now incorporated additional signaling markers into my panel including some downstream of mTORc1 (p-STAT3 S727) and some not directly associated with mTORc1 (p-ERK T202/Y204). My continued analysis suggests that the signaling differences I have observed are specific to mTORc1 signaling. I have now optimized an additional fluorescent panel for use on acutely dissociated mouse V-SVZ regions to determine if cells of all stages of the neural stem cell lineage (i.e. quiescent neural stem cell, activated neural stem cell, transit amplifying cell, migrating neuroblasts) display the dorsal vs. ventral differences in p-S6 signal or if this is restricted to a specific part of the lineage. Preliminary results suggest that the differences between sub-regions are still observed and that this difference is more abundant in cells positive for the neural stem cell marker, glial fibrillary acidic protein (GFAP). Additionally, I have utilized assays to measure cell proliferation including measuring acute analog uptake (EdU) and the cell cycle marker Ki67 in cultured cells via immunofluorescence and flow cytometry. Both of these measures indicate no differences in proliferation between V-SVZ sub-regions.

Publications and manuscripts

1. Rushing, G.V. and Ihrie, R.A. (2016). Neural Stem Cell Heterogeneity through Time and Space in the Ventricular-Subventricular Zone. Frontiers in Biology, 11(4): 261-284. PMCID: PMC5371406.

2. Rushing, G.V., Brockman, A.B., Bollig, M.K., Leelatian, N., Mobley, B.C., Irish, J.M., Fu, C., Ess, K.C., and Ihrie, R.A. Subgroups of Neural Stem Cells have Differential mTORC1 Activity and Distinct Responses to Tsc2 Loss. In preparation for submission.