Heterogeneity and stochastic growth regulation of biliary epithelial cells dictate dynamic epithelial tissue remodeling
- Institute of Molecular and Cellular Biosciences, The University of Tokyo, Japan
Figures
Figure 1 with 3 supplements
Visualization and lineage labeling of BECs using Prominin1 (Prom1) expression.
(a) Immunofluorescent (IF) staining for CK19 and Prom1 in the adult mouse liver (scale bars, 100 μm). (b) Representative expression pattern of Prom1 and EpCAM by fluorescence-activated cell sorting …
Figure 1—figure supplement 1
Quantification of lineage-labeled cells in Prom1-CreERT2; R26R-tdTomato mice.
(a) BEC labeling ratio in the Prom1-CreERT2;R26R-tdTomato mice was evaluated by FACS analysis. Left panel shows the experimental design. Two weeks after tamoxifen administration, the mice were …
Figure 1—figure supplement 2
EdU uptake assay in lineage-labeled cells in Prom1-CreERT2;R26R-tdTomato mice.
EdU uptake assay was performed to examine the proliferative characteristics of lineage-labeled and non-labeled BECs. After tamoxifen administration (10 mg/20 g body weight), the Prom1-CreERT2;R26R-td…
Figure 1—figure supplement 3
No labeled cells were detected in the absence of tamoxifen in Prom1-CreERT2;R26R-tdTomato mice.
(a and b) Leaky labeling in the Prom1-CreERT2;R26R-tdTomato mice was evaluated by FACS analysis. Left panel shows the experimental design. The mice were analyzed in the presence (a) or absence (b) …
Figure 2 with 2 supplements
Pre-existing BECs contribute to the nascent biliary epithelial tissue structure upon injury.
(a) Schematic illustration of the 3D imaging method used to observe biliary tree structures. (b) 3D imaging view of the normal biliary tree structure revealed by anti-CK19 immunostaining. Z-stacked …
Figure 2—figure supplement 1
The level and distribution pattern of the ductular reaction in a microscopic view is highly diversified within a liver.
(a) Progression of the ductular reaction is not uniform microscopically. Sections were prepared from different regions of the same liver sample derived from a mouse treated with TAA for 6 weeks, and …
Figure 2—figure supplement 2
Macroscopic view of the ductular reaction upon TAA injury over time.
The entire tissue structure of the biliary tree was visualized by whole-mount X-gal staining of the liver from the Prom1-CreERT2 mice harboring the nLacZ gene knocked-in to the Prom1 locus. …
Figure 3 with 2 supplements
Lineage tracing of hepatocytes.
(a) R26R-tdTomato mice were used in combination with rAAV2/8-iCre for the labeling of hepatocytes. rAAV2/8-iCre is designed to transduce only hepatocytes. (b) Representative image of FACS analysis …
Figure 3—figure supplement 1
FACS analysis of hepatocyte-derived cells in the TAA and DDC models.
The experimental design is described on the left side of the figure. Hepatocytes of R26R-tdTomato mice were labeled with rAAV2/8-iCre. Dissociated liver cells were stained with anti-MIC1-1C3 …
Figure 3—figure supplement 2
FACS quantification of labeled ratio in the Prom1-CreERT2; R26R-tdTomato mice before/after DDC injury.
(a and b) Labeling ratio in EpCAM+or MIC1-1C3+ cell populations in the Prom1-CreERT2;R26R-tdTomato mice was evaluated by FACS. The panels on the left show the experimental design. The mice were …
Figure 4 with 1 supplement
BECs exhibit heterogeneity in terms of proliferative capacity in vivo.
(a) Schematic diagram showing the rationale for quantitative in vivo single-BEC tracing. (b) Experimental design. (c) Upon administration of a very low dosage of tamoxifen (0.25 mg/kg body weight), …
Figure 4—figure supplement 1
Labeling ratio in the Prom1-CreERT2; R26R-tdTomato mice was not changed after 8 weeks of TAA injury.
Left panel shows the experimental design. Mice were subjected to TAA injury after the low-dosage tamoxifen injection (0.25 mg/kg body weight). Before injury, the labeling ratio of BECs was less than …
Figure 5 with 1 supplement
Proliferating BECs are scattered in the peripheral branching architectures in the biliary tree.
(a) 3D images of the biliary tree (CK19 immunostaining; green) and the cell cycle marker Ki67 (magenta) in TAA-injured liver samples. Middle and right panels show magnified views of the region of …
Figure 5—figure supplement 1
Distribution of Ki67+ BECs in the duct unit.
3D images corresponding to the ROI 2 area in Figure 5a represent the duct compartment of a mouse liver at TAA 6 weeks. Signals of 3D immunofluorescence for CK19 and Ki67 (magenta) were converted …
Figure 6
BECs do not proliferate uniformly and can be subdivided into those in the proliferative state and those in the quiescent state.
(a) Schematic diagram for the experimental design. At week 8 of the TAA injury model, mice were given BrdU via drinking water (0.8 mg/ml) for 8 days (1st label). After an interval of a further 8 …
Figure 7 with 2 supplements
Mathematical modeling predicts the stochastic nature of the BEC proliferation.
(a) Schematic diagram of the proposed growth model. We set five parameters. The parameter p represents the initial ratio of the cells in the proliferative state compared to total cells; m represents …
Figure 7—figure supplement 1
Discordance observed during the model-fitting process before taking account of the timing of the start of BEC proliferation upon injury.
Simulation data obtained from a model that is not adjusted for the proliferation start timing (gray bars) did not fit the experimental data for BEC colony formation in vivo (black bars). Early in …
Figure 7—figure supplement 2
TAA causes death of hepatocytes around CV, but not death of BECs.
Cell death of BECs was analyzed using a cell-death detection method by in vivo administration of EthD-3. After staining with EthD-3 in vivo, liver tissue sections were prepared and immunostaining …
Author response image 1
Tables
Table 1
List of antibodies used in this study.
| Antibody | Company/Source | Host animal | Method | Dilution |
| Prominin1/CD133 (APC-conjugated) | Biolegend (San Diego, CA) | Rat | FACS | 1:100 |
| Prominin1/CD133 (purified) | eBioscience (Santa Clara, CA) | Rat | IF | 1:100 |
| EpCAM (FITC-conjugated) | (Okabe et al., 2009) | Rat | FACS | 1:200 |
| EpCAM (purified) | BD Pharmingen (Franklin Lakes, NJ) | Rat | IF | 1:200 |
| CD45 (APC-conjugated or APC-Cy7-conjugated) | Biolegend | Rat | FACS | 1:200 |
| MIC1-1C3 | STEMGENT (Lexington, MA) | Rat | FACS | 1:200 |
| Spp1 | R&D systems (Minneapolis, MN) | Goat | IF | 1:200 |
| Cytokeratin 19 | (Tanimizu et al., 2003) | Rabbit | IF | 1:200 |
| Ki67 | eBioscience | Rat | IF | 1:200 |
| LacZ | Abcam (United Kingdom) | Chicken | IF | 1:200 |
| BrdU | Abcam | Rat | IF | 1:200 |
| Hnf4a | Santa Cruz (Dallas, TX) | Goat | IF | 1:200 |
