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1.
Figure 5

Figure 5. Type III cells become EdU labeled only after 3 days and they persist beyond 40 days.. From: Functional Cell Types in Taste Buds Have Distinct Longevities.

A, B, C, a representative taste bud from a mouse 10 days after i.p. injection, stained for EdU (red), KCNQ1 (grey) and 5HT (green). D, Aggregate data on the incidence of EdU+5HT+ cells in circumvallate taste buds from 35 mice. The solid line is a non-linear decay curve that yields a half-life of 22 days (R2 = 0.54) for Type III cells. The dashed line is a smoothed curve through the mean values at each time point. Scale bar, 20 µm.

Isabel Perea-Martinez, et al. PLoS One. 2013;8(1):e53399.
2.
Figure 4

Figure 4. EdU-labeled nuclei are present in Type II cells 2 days post-injection and are mostly eliminated by 25 days.. From: Functional Cell Types in Taste Buds Have Distinct Longevities.

A, a representative taste bud from a mouse analyzed 7 days post-injection, viewed for EdU and KCNQ1 immunofluorescence as above. One EdU-labeled nucleus (arrowhead) is completely embedded in the KCNQ1-stained taste bud. B,C, the same nucleus (arrowhead) is seen to be contained in a PLCβ2+ cell. D, Aggregate data from 35 mice for EdU+ nuclei located in PLCβ2+ cells. As in Figures 2,3, each symbol represents data from one mouse. The solid line is a 1-phase exponential decay curve. The average half-life of EdU+ PLCβ2+ cells is calculated to be 8 days (R2 = 0.84). The dashed line is a smoothed line through averages at each time point. Scale bar, 20 µm.

Isabel Perea-Martinez, et al. PLoS One. 2013;8(1):e53399.
3.
Figure 1

Figure 1. Estimating the efficacy of EdU detection through successive mitoses.. From: Functional Cell Types in Taste Buds Have Distinct Longevities.

CHO cells plated at clonal density were exposed to a 4 h pulse of EdU (10 µM) followed by 24 h or 48 h chase in medium lacking EdU. Cultures were fixed and stained for EdU and well-isolated clones were imaged. A, B, EdU-labeled cells (arrowhead) are brightly fluorescent and distinguished from unlabeled cells (arrow). C, D, Fluorescence in 2- and 4-cell clones is well above background. E, In this clone, asynchronous cell divisions produced bright fluorescence in top two cells after two mitoses, and less bright fluorescence in the lower four cells after three mitoses. F, After three mitoses, fluorescence is barely above background. Scale, 20 µm.

Isabel Perea-Martinez, et al. PLoS One. 2013;8(1):e53399.
4.
Figure 7

Figure 7. Paired “sister” cells are seen in and around taste buds. A–C,. From: Functional Cell Types in Taste Buds Have Distinct Longevities.

a pair of cells inside and near the base of a circumvallate taste bud, 1 day after EdU injection. Note that in this single plane confocal micrograph, KCNQ1 staining surrounds each of the two labeled nuclei. D–F, A pair of sister cells with one cell (arrow) still on the outside of the taste bud; the other cell (arrowhead) appears to be transitioning into the KCNQ1-positive area of the taste bud. These images are from a single optical plane micrograph from a mouse 1 day after EdU injection. G-I, An example of an EdU-labeled cell located centrally and away from the basement membrane region. The three panels are consecutive optical sections, each 4–5 µm thick, of a single taste bud.from a mouse 4 h after EdU injection. The appearance of the same taste bud at planes above and below the plane of the labeled cell demonstrates this cell was mid-way through the taste bud in the Z-dimension. Scale bar, 20 µm.

Isabel Perea-Martinez, et al. PLoS One. 2013;8(1):e53399.
5.
Figure 3

Figure 3. EdU-labeled nuclei appear in taste buds within 1 day of injection.. From: Functional Cell Types in Taste Buds Have Distinct Longevities.

Cryosections of circumvallate papillae were stained for EdU (red) and KCNQ1 (grey) as in Figure 2. All micrographs are confocal single (≈1.5 µm) optical sections. A, EdU+ nuclei are closely apposed to the base of the taste bud 1 day post-injection. B, The same field as in A, viewed for KCNQ1 alone reveals “dimples” where EdU+ nuclei (arrowheads) appear to push in without penetrating into the taste bud. C, Example of an EdU+ nucleus (arrowhead) located inside a taste bud 4 h after EdU injection. Optical section was selected passing through the widest, most brightly stained part of the nucleus. D, E, Representative high magnification views of taste buds from mice killed 3 days or 7 days after EdU injection. F, Aggregate data for EdU+ nuclei located within taste buds. Each symbol represents data from one mouse. Solid line is a 1-phase exponential decay curve that yields a half-life of 11 days for EdU+ taste cell nuclei (R2 = 0.92). A smoothed (dashed) line through averages at time points is also included. Scale bars, 20 µm.

Isabel Perea-Martinez, et al. PLoS One. 2013;8(1):e53399.
6.
Figure 6

Figure 6. EdU-labeled non-Type II, non-Type III cells enter the taste bud rapidly and constitute the largest fraction of EdU+ cells.. From: Functional Cell Types in Taste Buds Have Distinct Longevities.

This heterogeneous group of cells is defined here simply by the absence of both PLCβ2 and 5HT. A, B, C, a taste bud from a mouse 3 days post-injection displays several EdU-labeled cells (arrowheads) of this class. EdU, KCNQ1, PLCβ2 and 5HT were visualized as in previous figures. The indicated EdU+ nuclei do not map onto immunoreactivity for either cell-type marker; but they are clearly within the taste bud perimeter. D, Aggregate data from 30 mice for EdU+, PLCβ2, 5HT cells, fit with a two-phase exponential decay curve. The faster component comprises 80% of the population and exhibits a half-life of 8 days while the slower component contains 20% of the EdU+,PLCβ2,5HT cells and disappears with a half-life of 24 days (R2 = 0.97). The exponential fit was ambiguous insofar as the relative fraction of the two sub-populations could not be discerned precisely. Equivalent goodness-of-fit was obtained for the fast component constituting 60–80% of the cellular population. The EdU+,PLCβ2,5HT cells are obviously a heterogeneous group as discussed in the text. Different constitutent cell types (e.g. mature Type I cells, immature taste cells, quiescent undifferentiated cells, and perhaps some progenitor cells) may all have dramatically different lifetimes. E, Incidence of these EdU+ PLCβ2 5HT cells among all EdU+ cells within the taste buds. As with other graphs, each symbol represents data from a separate mouse. Scale bar, 20 µm.

Isabel Perea-Martinez, et al. PLoS One. 2013;8(1):e53399.
7.
Figure 2

Figure 2. EdU-labeled nuclei in non-taste lingual epithelium surrounding taste buds turn over rapidly. A,. From: Functional Cell Types in Taste Buds Have Distinct Longevities.

wide-field fluorescence micrograph of circumvallate papilla, 1 day after a single pulse of EdU. Many EdU-labeled nuclei (red) are visible along the base of the epithelium. Taste buds are immunostained (grey) for KCNQ1, a marker for all taste cells. The location of EdU+ nuclei, whether inside or outside of taste buds cannot be judged from a wide-field micrograph such as this. B–D, Higher power single plane confocal micrographs of circumvallate taste buds, 1, 3, or 5 days after injecting EdU. The large majority of EdU-labeled epithelial cells are outside of taste buds. Basal and apical limits of the epithelium are indicated with white solid and dotted lines respectively to highlight the rapid migration of newly-born non-taste cells from the stratum basale to the superficial epithelial strata. E, Cartoon depicting progression of EdU-labeled non-taste nuclei vertically through the epithelium and their simulataneous change of shape from ovoid at the base to horizontally flattened at the apical surface. F, Plot of EdU labeled non-taste nuclei per unit area versus days post-injection. Data were obtained from micrographs of circumvallate trench such as those in B–D. Each symbol represents data from a separate mouse. The solid line is a best-fit 1-phase exponential decay curve that yields a half-life of 2 days (R2 = 0.98) for non-taste epithelial cells surrounding taste buds. The dashed line is a smoothed line through the average values at each time point in the time course. Scale bars, 20 µm.

Isabel Perea-Martinez, et al. PLoS One. 2013;8(1):e53399.

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