Intestinal epithelial stem cells do not protect their genome by asymmetric chromosome segregation - PubMed (original) (raw)

Intestinal epithelial stem cells do not protect their genome by asymmetric chromosome segregation

Marion Escobar et al. Nat Commun. 2011.

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Abstract

The idea that stem cells of adult tissues with high turnover are protected from DNA replication-induced mutations by maintaining the same 'immortal' template DNA strands together through successive divisions has been tested in several tissues. In the epithelium of the small intestine, the provided evidence was based on the assumption that stem cells are located above Paneth cells. The results of genetic lineage-tracing experiments point instead to crypt base columnar cells intercalated between Paneth cells as bona fide stem cells. Here we show that these cells segregate most, if not all, of their chromosomes randomly, both in the intact and in the regenerating epithelium. Therefore, the 'immortal' template DNA strand hypothesis does not apply to intestinal epithelial stem cells, which must rely on other strategies to avoid accumulating mutations.

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Figures

Figure 1

Figure 1. Chromosome fates and labelling in the stem-cell lineages depending on the mode of chromosome segregation (CS).

Solid lines represent parental template DNA strands and dotted lines represent newly synthesized strands. (a) Nonrandom chromosome segregation (CS) implies an asymmetric mode of cell division generating stem and non-stem daughter cells. The 'immortal' DNA template (*) inherited by a stem cell at generation _g_−1, is again inherited by the stem progeny at generations g and g+1. (b) In the case of nonrandom CS, and no matter how long the labelling period (1 to n generations), only one strand of each chromosome can be labelled (red) in stem cells as they systematically inherit the chromatid containing the initially unlabelled 'immortal' strand (blue*). (c) In the case of nonrandom CS, stem cells replicating their DNA in the absence of label are unlabelled from the first generation. (d) On the contrary, because random CS implies that both templates of generation _g_−1 can be transmitted to generation g+1 with equal probability, the proportion of labelled chromosomes steadily increases with each generation of stem cells produced in the presence of label (e), and steadily decreases with each generation of stem cells produced during the chase period (f).

Figure 2

Figure 2. Cytokinetic parameters of CBC cells.

(a) Schematic representation of the protocol of cumulative CldU labelling. The solid black line represents the length of the S phase relative to the other phases of the cycle. The solid red line represents the distribution of cells within the cycle, which have been labelled with CldU. (b) Kinetics of cumulative CldU incorporation in CBC stem cells. We performed CldU immunohistochemistry on thin paraffin sections of rolled intestine, using Ki67 as a marker of cycling cells and phospho (ser10)-histone H3 to evaluate the percentage of cells in the G2/M transition. Analysis of the relationship between the ratio of CldU+ Ki67+ CBC cells to the total number of Ki67+ CBC cells and time revealed a linear increase in labelled CBC cells until CldU labelling became saturated. Data points represent mean percentages of Ki67+ CldU+ CBC cell nuclei from two mice (A-F and I-M), except for the 21-h and 25-h time points (mice G,H). The average length of the cell cycle (_T_c=28.5 h) is given by the slope of the regression line (1/0.0351), and that of the DNA synthetic phase (_T_s=9.4 h) is derived from the y-intercept _T_s/_T_c=0.3301. _T_c–_T_s is the time needed to reach the maximum labelling value. The length of the G2/M phase was calculated using the following equation: TG2/M=(% Histone H3–P+ CBC cells×_T_c).

Figure 3

Figure 3. Analysis of the CBC and cp4 cell-modes of chromosome segregation during a labelling-dilution experiment.

(a,b) Simulation of the CldU content fluctuations in ten individual CBC cell lineages (one colour per lineage) during a pulse-chase experiment. A CldU content beyond 2 cannot be reached before the beginning of the S phase, when ploidy increases. (a) In the case of nonrandom CS, the number of labelled DNA strands per chromosome (CldU content) should substantially increase after 2 days of labelling (−2 to 0) but should decrease abruptly within 2–3 days of chase. (b) In the case of random CS, the CldU content should decay at a slower rate and labelled DNA should still be present after 5 days of chase. (c,d) Mathematical modelling of the DNA label dilution kinetics. The proportion of CldU+ cells depends on the minimal amount of CldU-labelled DNA that can be detected by immunofluorescence. The parameter δ corresponds to this threshold in our simple detection model. Predictions are represented for the nonrandom and random CS scenarios and six thresholds of detection ranging from _δ_=1 (CldU content >one labelled strand per chromosome) to the limit when δ approaches 0 (black line perfect detection). (c) In the case of nonrandom CS, CldU should be lost from the stem cell compartment after 3 days of chase, whereas in the case of random CS (d), CldU should still be present and detected provided that δ<0.5. (**e**) Kinetics of CldU dilution in the fully labelled CBC (dark blue) and cp4 (turquoise blue) cell populations. Mean±s.d. (_n_=3). One initial injection of CldU was followed by CldU in drinking water for 60 h (>2Tc). Mice were killed at the end of the labelling period and every day for 5 consecutive days. The differences between day 4 and day 3 and between day 5 and day 4 are significant (P< 0.05, Fisher's exact test). (f) Model fit for CBC cells. The observed kinetics (data represented by open circles) is compared with the nonrandom CS scenario (grey hatched area) and the random CS scenario after estimating the label-detection threshold (orange line) and a more sophisticated two-parameter model of label detection (blue line). The fraction of variance (_r_2) explained by this second model reaches 0.96 compared with 0.74 with the simple model. (g) Modelling the probability of label detection as a function of the CldU content. The orange line represents the probability of detection, using the simple model estimated on the experimental data (_δ_=0.088). The second model represented by the blue line accounts for randomness in the detection process. It involves two parameters, δ and ɛ, estimated from the experimental data (_δ_=0.055 and _ɛ_=0.085).

Figure 4

Figure 4. CldU and Ki67 immunostaining of the CBC and cp4 cell populations.

Mice were killed at the beginning of the chase period (T0) and every day for 5 consecutive days. (a–f) Merged lower power views of ileal crypt sections stained for CldU (green) and Ki67 (red), and counterstained with 4′-6-diamidino-2-phenylindole (DAPI) on (a) day 0, (b) day 1, (c) day 2, (d) day 3, (e) day 4 and (f) day 5. (g–r) Enlarged single-channel views of the crypt sections present in the insets. (g–l) CldU staining on (g) day 0, (h) day 1, (i) day 2, (j) day 3, (k) day 4 and (l) day 5. Note that the number but not the intensity of the spots steadily decreased from T0 (g) to day 5 (l), attesting the progressive loss of labelled chromosomes through successive divisions. (m–r) Ki67 staining on (m) day 0, (n) day 1, (o) day 2, (p) day 3, (q) day 4 and (r) day 5. Closed and open arrowheads point to CBC cell and cp4 cell nuclei, respectively. P, unlabelled (white) and CldU-labelled (green) Paneth cell nuclei. Scale bars, 10 μm.

Figure 5

Figure 5. Quantification of CBC cell depletion, followed by recovery in the ileum of irradiated mice.

Lgr5-EGFP mice were irradiated (or not) with a dose of 6 Gy of Cs137 to the whole body. They were then administered BrdU in drinking water for 5 days and killed 2, 5, 14 and 23 days post irradiation. (a) Temporary decrease followed by recovery in GFP expression in the crypt epithelium of irradiated mice. (b) Transient irradiation-induced depletion in CBC cells (dark blue) but not cp4 cells (turquoise blue) 2 days post irradiation. (c) Similar rates of BrdU incorporation (day 5) and BrdU dilution (day 14) in CBC cells (red) and cp4 cells (purple) in irradiated versus control mice. Note that 2 days post irradiation, 100% of surviving CBC cells were Ki67+ BrdU+. Data are presented as mean with error bars. Error bars with * (_n_=3) correspond to standard deviations and bars without * (_n_=2) indicate the range. The values obtained from distinct mice for the same time point (see Supplementary Tables S2-S5) were compared using Fisher's exact test.

Figure 6

Figure 6. Immunofluorescence CBC cell BrdU-labelling at 2 and 5 days post irradiation.

Control (a–e,k–o) and irradiated (f–j,p–t) Lgr5-EGFP mice were continuously exposed to BrdU administered in drinking water and killed after 2 and 5 days. Ileum sections were simultaneously stained for nuclear BrdU (green), nuclear Ki67 (red), cytoplasmic lysozyme (red) and cytoplasmic green fluorescent protein (GFP, white). Nuclei were counterstained with DAPI (blue). Closed and open arrowheads point at CBC and to cp4 cells, respectively. (a) Merge picture of neighbouring GFP+ and GFP− crypt sections. (b) Merge picture of a crypt section with four CBC cells staining positive for Ki67 (c, green arrowheads) BrdU (d, red arrowheads) and GFP (e, yellow arrowheads). (f) Merge picture of crypt sections devoid of CBC cells. (g) Merge picture of a single crypt section. The cp4 cell of this crypt (open arrowhead) is Ki67+ (h), BrdU+ (i) and GFP− (j). (k) Merge picture of GFP+ crypt sections. (l) Merge picture of a single crypt section with four CBC cells and one cp4 cell, all of which are Ki67+ (m), BrdU+ (n) and GFP+ (o). (p) Merge picture of a succession of GFP+ and GFP−crypt sections. (q) Merge picture of a single GFP+ crypt section with four CBC cells, all of which are Ki67+ (r), BrdU+ (s) and GFP+ (t). Note the presence of a Ki67+ BrdU+ GFP− cp4 cell and the BrdU+Ki67− nucleus (*) of a post-mitotic cell sandwiched between Ki67+ BrdU+ progenitor nuclei. P, Paneth cell nuclei. Scale bar, 10 μm. DAPI, 4′-6-diamidino-2-phenylindole.

Figure 7

Figure 7. CBC and cp4 cells of irradiated Lgr5-EGFP mice are not label-retaining cells.

Control and irradiated Lgr5-EGFP mice were continuously exposed to BrdU for 5 days and killed 14 days (9 days of chase) and 23 days (18 days of chase) after the beginning of the treatment. Ileum sections were either simultaneously stained for BrdU, GFP and lysozyme (a–l) or BrdU, GFP, lysozyme, chromogranin A, Dclk1 and Ki67 (m–x). (a–d) Single crypt section from a control mouse. (a) Merge. (b) BrdU+ CBC cell nuclei (green arrowheads) sandwiched between BrdU+ and BrdU− Paneth cell nuclei (p). The CBC cells of this crypt section are all GFP+ (arrowheads in c) and the Paneth cells are lysozyme+ (d). (e–h) Single crypt section from an irradiated mouse killed 14 days post irradiation. (e) Merge. The two nuclei that stain positive for BrdU (f) and negative for GFP (g) belong to Paneth cells with cytoplasmic lysozyme granules (h). (i–l) Single crypt section from an irradiated mouse killed 23 days post irradiation. (i) Merge. The BrdU+ nucleus (j) belongs to a GFP− (k) lysozyme+ (l) Paneth cell. (m–p) Single crypt from a control mouse. (m) Merge. (n) The small arrow points at an entero-endocrine cell (E) with nuclear BrdU and cytoplasmic chromogranin A staining. Arrowheads point at BrdU+ nuclei that belong to GFP+ (o), KI67+ (p) CBC cells. (q–t) Single crypt section from an irradiated mouse killed 14 days post irradiation. (q) Merge. (r) Two BrdU+ nuclei, one of which belongs to a GFP+ cell (arrowhead in s) and the other to a lysozyme+ Paneth cell (t). (u–x) Single crypt section from an irradiated mouse killed 23 days post irradiation. (u) Merge. (v) The BrdU+ nucleus belongs to a GFP− (w) lysozyme+ (x) Paneth cell. White arrows in (x) point at BrdU−Dclk1+ tuft cells. Scale bar, 10 μm. DAPI, 4′-6-diamidino-2-phenylindole.

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