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Show CELL CYCLE ALTERATIONS IN CHX10 NULL AND CHX10, P27KIPI DOUBLE NULL RETINAL PROGENITOR CELLS REVEAL THE IMPORTANCE OF PROLIFERATION RATE IN PROMOTING RETINAL DEVELOPMENT T. Raleigh, E.S. Green, B.W. Jones, R. Marc, (E.M. Levine) John A. Moran Eye Center, School of Medicine Purpose. To quantify the changes in cell cycle dynamics (i.e. duration of the overall cell cycle time (Tc) and the times for S-phase (Ts), Gl-phase (TG1), and G2/M-phase (TG2/M)) in the cell-deficient ChxlO null retina and partially rescued ChxlO, p27KiP] double null retina. Methods. Flow Cytometry: Cell cycle phase ratios of dissociated retinal progenitor cells (RPCs) were measured from wildtype (wt), ChxlO null, and ChxlO, p27Kjp] double null mice at postnatal day 0 (PO) on the basis of fluorescent quantification of DNA content. Growth Fraction Analysis: The growth fraction is the ratio of (1) proliferating RPCs determined with anti-Proliferating Cell Nuclear Antigen (PCNA) to (2) the total cell population determined with DAPI staining in cryosections. Cell Cycle Kinetics: Tc, Ts and the percentage of cells in S-phase were calculated in each strain by a high-resolution double-label version of the 3H-Thymidine/BrdU window labeling technique. TG1 and TG2/M were derived from the parameters obtained in these three methods. Results. In comparison to wt mice, the PO ChxlO null RPC population has a slower cell cycle due to a large increase in TG1, even though Ts is shortened. The ChxlO, p27KiP] double null RPC population has a faster cell cycle than the ChxlO null RPC population and, surprisingly, the wt RPC population as well. This was due to unexpected decreases in all phase times. Conclusion. The cell number deficit of the ChxlO null retina is strongly correlated with a slowing of the progenitor cell cycle. While the changes in cell cycle dynamics confirm the importance of ChxlO in regulating RPC proliferation rate, the unexpectedly fast cell cycle and phase times in the ChxlO, p27K'P] double null RPC population suggests that ChxlO and p27K'P1 control proliferation rate by both common and distinct mechanisms. CR: None Support: NIH Grants EY013760 (EMU, EY002576 (RM), EYO15128 (RAA); RPB Career Development Award and FFB Award (EMU; RPB Senior Investigator Award (RM) Todd Raleigh Biology / Bioengineering Faculty Sponsor Edward Levine |
