OBSERVING CELL LONGEVITY AND DYNAMICS IN THE TASTE BUD OVER TIME - PROJECT SUMMARY OUR ABILITY TO TASTE IS FACILITATED BY MAINTENANCE OF DIFFERENT TYPES OF CELLS WITHIN TASTE BUDS THAT ARE SPECIALIZED TO DETECT DIVERSE TASTE MODALITIES. TASTE BUD CELLS UNDERGO RAPID TURNOVER, WHICH LIKELY REFLECTS THE HARSH ENVIRONMENT IN WHICH THEY EXIST, YET WE RETAIN CONSTANCY IN OUR TASTE PERCEPTION. PREVIOUS STUDIES OF POSTMORTEM TISSUE USING TRITIATED THYMIDINE OR NUCLEAR ANALOGS HAVE REPORTED A PROPOSED AVERAGE HALF-LIFE OF 8 DAYS FOR TYPE II TASTE BUD CELLS, AND 22 DAYS FOR TYPE III CIRCUMVALLATE TASTE BUD CELLS BY LABELING THE PROLIFERATING STEM CELL POPULATION. THIS METHOD ALLOWS REPORTING OF POPULATION-LEVEL LIFESPAN AVERAGES FOR SPECIFIC CELL TYPES BUT NOT INDIVIDUAL CELL VARIABILITY FOR SPECIFIC CELL TYPES. LIFESPAN VARIABILITY WITHIN A SPECIFIC CELL TYPE IS LIKELY INFLUENCED BY BOTH INTRINSIC FACTORS, SUCH AS GENETICS, AND EXTRINSIC FACTORS, SUCH AS THE ENVIRONMENT. THESE FACTORS MAY NOT ONLY SHAPE DIFFERENCES IN CELL LONGEVITY BUT ALSO INFLUENCE HOW TASTE BUD CELLS ARE REPLENISHED OVER TIME IN TASTE BUDS. THE OVERALL HYPOTHESIS FOR THIS PROJECT IS THAT TYPE II AND TYPE III TASTE BUD CELLS HAVE DISTINCT LIFESPANS, EACH WITH SHORT-LIVED AND LONG-LIVED SUBPOPULATIONS, AND THAT CELL LOSS DRIVES DIFFERENTIATION BOTH UNDER NORMAL CONDITIONS AND AFTER TASTE BUD INJURY. TO TEST THIS, LONGITUDINAL IN VIVO TWO-PHOTON LASER SCANNING MICROSCOPY WILL BE USED TO REPEATEDLY IMAGE THE SAME TASTE BUDS OVER TIME, TRACKING INDIVIDUAL CELL POPULATIONS AS CELLS ENTER, MATURE, AND DISAPPEAR FROM THE TASTE BUD. THIS APPROACH WILL ALLOW TRACKING OF THE FULL LIFE CYCLE OF INDIVIDUAL TYPE II AND TYPE III TASTE BUD CELLS, REVEALING HOW THEIR LIFESPANS VARY AND HOW ENVIRONMENTAL FACTORS INFLUENCE THEIR TURNOVER. THE GOALS OF THIS PROJECT ARE: 1) TO DETERMINE THE TIME IT TAKES FOR K14+ PROGENITOR CELLS AND SHH+ POSTMITOTIC PRECURSOR CELLS TO DIFFERENTIATE INTO TYPE II AND TYPE III TASTE BUD CELLS; 2) TO DETERMINE THE LIFESPAN OF MATURE TYPE II AND TYPE III TASTE BUD CELLS; AND 3) TO DETERMINE THE TIMING OF SHH+ POSTMITOTIC PRECURSOR CELL ENTRY AND DIFFERENTIATION INTO TASTE BUDS AFTER TASTE BUD INJURY. TO ACHIEVE THIS, I WILL INTEGRATE SPARSE CELL GENETICS, TIME- LAPSE IN VIVO IMAGING, AND TWO-PHOTON LASER SCANNING MICROSCOPY, UTILIZING INNOVATIVE AUTOMATED QUANTIFICATION METHODS FOR ANALYSIS. IF OUR HYPOTHESES ARE CORRECT, THEN THIS WOULD INDICATE THAT DIFFERENCES IN THE TIME K14+ PROGENITOR CELLS AND SHH+ POSTMITOTIC PRECURSOR CELLS SPEND IN EARLY STAGES MAY CONTRIBUTE TO VARIABILITY IN MATURE TYPE II AND III TASTE BUD CELL LIFESPANS; THAT SUBPOPULATIONS OF TASTE BUD CELLS EXIST FOR SPECIFIC CELL TYPES; AND THAT HOMEOSTATIC DRIVE REGULATES CELL NUMBER DIFFERENTIATION BOTH UNDER NORMAL CONDITIONS AND AFTER TASTE BUD INJURY. THIS IS THE FIRST STEP IN INVESTIGATING THE REGULATORY MECHANISMS THAT GOVERN TASTE BUD CELL TURNOVER AND MAINTAIN TASTE PERCEPTION.