Live imaging of alveologenesis in precision-cut lung slices reveals dynamic epithelial cell behaviour

Abstract

Damage to alveoli, the gas-exchanging region of the lungs, is a component of many chronic and acute lung diseases. In addition, insufficient generation of alveoli results in bronchopulmonary dysplasia, a disease of prematurity. Therefore visualising the process of alveolar development (alveologenesis) is critical for our understanding of lung homeostasis and for the development of treatments to repair and regenerate lung tissue. Here we show live alveologenesis, using long-term, time-lapse imaging of precision-cut lung slices. We reveal that during this process, epithelial cells are highly mobile and we identify specific cell behaviours that contribute to alveologenesis: cell clustering, hollowing and cell extension. Using the cytoskeleton inhibitors blebbistatin and cytochalasin D, we show that cell migration is a key driver of alveologenesis. This study reveals important novel information about lung biology and provides a new system in which to manipulate alveologenesis genetically and pharmacologically.

Fig. 1

Experimental protocol for time-lapse imaging of precision cut lung slices. a Lung harvesting, inflation, PCLS generation and imaging. b Comparative durations of ex vivo PCLS culture experiments. c–e Deconvolved widefield, single plane z-stack image of P3 PCLS. Epithelial cells were labelled with EpCAM-FITC antibody (green) and cell nuclei were labelled with SiR-DNA (red). Boxed areas show EpCAM-FITC +ve cells in alveolar (d) and airway (e) epithelial cells. Scale bar = 50 µm. f MTT cell viability assay comparing control and EpCAM-FITC/SiR-DNA labelled adult PCLS at 1 and 48 h. n = 3 independent experiments, with duplicate slices per group, per experiment. g Confocal images of P3 PCLS labelled with EpCAM-FITC, Sp-C (alveolar type 2 cell marker) and SiR-DNA. EpCAM-FITC (green)/SP-C (red) +ve epithelial cells (arrows) EpCAM-FITC (green)/ Sp-C (red) −ve cells (arrowheads). Nuclei were stained with SiR-DNA (blue). n = 2 independent experiments, with duplicate slices from one mouse per experiment. Scale bar = 50 µm. h Deconvolved widefield, single plane, z-stack image of P3 PCLS labelled with EpCAM-FITC (green)/CD11c-PE (red, macrophage marker)/SiR-DNA (blue). n = 2 independent experiments, with duplicate slices from one mouse per experiment. Scale bar = 50 µm. i Raw and deconvolved, widefield, single plane, z-stack images labelled with EpCAM-FITC (green)/SiR-DNA(magenta). Scale bar = 50 µm. j MTT cell viability assay on PCLS pre and post imaging and in normal culture conditions, n = 3 independent experiments using three separate mice, with duplicate slices per group per experiment. Yellow ‘a’ indicates alveolar airspaces. Error bars are defined as s.e.m

Fig. 2

Quantification of alveologenesis and alveolar epithelial cell migration in postnatal mice. H&E staining of left lung sections from P3, P7, P14 and adult mice (a–d); n = 3 mice from each age group. Scale bar = 100 µm. Mean linear intercept (Lm) from P3, P7, P14 and adult mice (e); n = 3 separate mice from each age group, 3 H&E lung sections were quantified per mouse, ***p < 0.001; ns = not significant, one-way ANOVA with Tukey’s post hoc test. Mean linear intercept in PCLS from P3 mice (f), n = 6 separate mice, 3 H&E lung sections were quantified per mouse; ns = not significant, one-way ANOVA with Tukey’s post hoc test. Airspace count from P3, P7, P14 and adult lung sections (g); n = 3 separate mice from each age group, 3 H&E lung sections were quantified per mouse, ***p < 0.001; ns = not significant, one-way ANOVA with Tukey’s post hoc test. Individual cell tracking over 8 h in P3, P7, P14 and adult PCLS, 70–160 cells were tracked per field (h). Mean net epithelial cell migration over 8 h in P3, P7, P14 and adult lungs (i); **p < 0.01, ***p < 0.001; ns = not significant, one-way ANOVA with Tukey’s post hoc test, n = 3 mice for each age group, 2–3 fields were quantified from one slice per mouse, per experiment. Individual net cell migration in P3, P7, P14 and adult lungs over 8 h (each dot represents a single cell). P3, 607 cells, P7, 944 cells, P12, 950 cells and adult, 613 cells were tracked (j), n = 3 mice from each age group, 2–3 fields were quantified from one slice per mouse, per experiment, **p < 0.01, ***p < 0.001, ns = not significant. Error bars are defined as s.e.m

Fig. 3

Septation, cell clustering and cell migration in PCLSi. Deconvolved widefield images of P3 PCLS from 13 h time-lapse video (Supplementary movie 5A and higher magnification 5B, 5Ci) show two examples of septation, i.e., extension of secondary septum into an airspace (dashed red line demarcates airspaces a1/a2 and a3/a4, red boxed arrows point to secondary septa (a, b). Epithelial cell clustering, a common feature noted during postnatal alveologenesis, is indicated by blue circles (a, b) (Supplementary movie 5A and higher magnification 5D). The images also show EpCAM-FITC (green) labelled alveolar epithelial cell migration (yellow arrows) towards existing airspaces (a5 and a6) (a–c). Enlarged still images from 13 h time-lapse video (Supplementary movie 5A higher magnification 5E at 0, 3, 6, 9 and 13 h, show epithelial cell migration (yellow arrows) towards airspace a5 (c). Nuclei were labelled with SiR-DNA (magenta). Scale bar = 50 µm (a, b), 25 µm (c). Deconvolved EpCAM-FITC (red)/SiR-DNA (cyan) labelled still images from 14 h time-lapse video (Supplementary movie 6) showing EpCAM positive epithelial cell migration (green arrows) towards alveolar airspace (white dashed lines outline airspaces) in P3 PCLS (d). Cells 1 and 2 migrate towards a small airspace (white dashed outline) within the interstitium and meet cell 3 (d, upper panel). Corresponding images labelled with SiR-DNA only are displayed in d, lower panel. Yellow ‘a’ indicates alveolar airspaces. Scale bar = 50 µm

Fig. 4

Cell hollowing in PCLSi. a Brightfield images from 18 h time-lapse video of P3 PCLS (Supplementary movie 7) shows ‘hollowing’ within the parenchyma at 0, 9 and 18 h (red arrow, and red arrowheads in white boxed area, dotted yellow lines outline surrounding airspaces). Inserts in a enlarged views of the white boxed area. Pink horizontal lines in insert depict width of ‘hole’ expanding (compare a, middle panel to a, right panel). Scale bar = 50 µm. b EpCAM-FITC (red)/SiR-DNA (cyan) labelled raw images of P3 PCLS from 14 h time-lapse video (Supplementary movie 8) show ‘hollowing’ at 0, 7 and 14 h (white dashed areas, A and B, yellow arrows). Scale bar = 50 µm. c Deconvolved still images from 14 h time-lapse video (Supplementary movie 9) show EpCAM +ve epithelial cell migration and rearrangement around the newly formed hollows/airspaces (white circles, A and B yellow arrows,). White ‘a’ indicates alveolar airspaces, n = 3 independent experiments using 3 separate mice, with duplicate slices from each mouse per experiment. Scale bar = 50 µm

Fig. 5

Cell extension during postnatal alveologenesis. Brightfield still images from P4 PCLS time-lapse video (Supplementary movie 10) at 0 (a, d), 5 (e), 10 (f) and 16 (b, g) hours showing cell extension along an alveolar wall (dashed yellow line in a, b, d–g); yellow ‘a’ indicates airspaces, yellow arrow indicates cell position at 0 h (a, d), red arrow indicates the final cell position (b). Measurement of cell extension during 16 h of time-lapse imaging (c). Enlargement of white boxed areas in a and b show the extending cell (white dashed arrow) during a 16 h video (e–g). Scale bar = 50 µm

Fig. 6

Blebbistatin inhibits epithelial cell migration in PCLS. Deconvolved widefield, single plane z-stack images from 14 h time-lapse videos (Supplementary movies 14A, B) of control (a–g) and blebbistatin-treated (h, i) P3 PCLS labelled with EpCAM-FITC (green) and SiR-DNA (magenta). Still images are taken from supplementary video 14A at 0 (c), 4 (d), 8 (e) 12 (f) and 14 (g) hours represent the white boxed regions in a and b. Scale bar = 50 µm. MTT cell viability assay comparing control and blebbistatin-treated P3 PCLS at the end of time-lapse, (j), n = 3 independent experiments using three separate mice, with duplicate slices per condition, per experiment, ns = not significant; paired Student t-test. Individual cell tracking over 14 h in a single field from P3 PCLS treated with control DMSO (k) or blebbistatin (l) containing media. Mean net epithelial cell migration in P3 control and blebbistatin-treated PCLS movies over 14 h (m). n = 3 independent experiments using three separate mice, with duplicate slices per condition, per experiment. Two fields were quantified per slice. Each dot represents mean net epithelial cell migration per field. Individual net cell migration in blebbistatin treated vs. DMSO control P3 PCLS after 14 h of time-lapse imaging (each dot represents a single cell) (n). A total of 1243 cells for DMSO control and 1340 cells for blebbistatin-treated PCLS were tracked. n = 3 independent experiments using three separate mice, with duplicate slices per condition, per experiment. Two fields were quantified per slice. ***p < 0.0001, **p = 0.001; Mann-Whitney U-test,. Yellow ‘a’ indicates alveolar airspaces. Error bars are defined as s.e.m

Fig. 7

Blebbistatin inhibits ex vivo alveologenesis. H&E stained sections from P3 PCLS at 0 and 72 h, cultured with DMSO control (top panels) or 50 μM blebbistatin (bottom panels) (a). Scale bar = 100 µm. Mean linear intercept (Lm) (b) and airspace count (c) obtained from H&E sections of P3 PCLS treated with DMSO control or 50 μM blebbistatin at 0 and 72 h, n = 3 independent experiments using 3 separate mice, 3 H&E sections from each PCLS from each mouse were quantified per group, per experiment, each dot represents per field count (b, c); ***p < 0.001, ns = not significant, one-way ANOVA with Tukey’s post hoc test. Confocal single plane z-stack images of DMSO control (top panels) and 50 µM blebbistatin (bottom panels) P3 PCLS at 0 and 72 h culture, immunostained with Ki67 (red), Sp-C (green) and DAPI (blue) (d). Scale bar = 50 µm. Quantification of Ki67 and Sp-C +ve cells in control and blebbistatin-treated P3 PCLS at 0 and 72 h culture (e), n = 3 independent experiments using 3 separate mice, with duplicate slices per group per experiment. Two fields were quantified per slice. Each dot represents mean value of per field counts per experiment; **p < 0.01; ***p < 0,001; ns = not significant; one-way ANOVA with Tukey’s post hoc test. Quantification of Ki67 +ve cells in DMSO control and blebbistatin-treated P3 PCLS at 0 and 24 h in culture (f), n = 3 independent experiments using 3 separate mice, with duplicate slices per group, per experiment. Two fields were quantified per slice. Each dot represents mean value of per field counts, per experiment; one-way ANOVA with Tukey’s post hoc test. Confocal single plane z-stack images of DMSO control (top panels) and 50 µM blebbistatin-treated (bottom panels) P3 PCLS at 0 and 24 h culture, immunostained with Ki67 (red) and DAPI (blue) (g). Scale bar = 50 µm. Error bars are defined as s.e.m

Fig. 8

Cytochalasin D inhibits ex vivo alveologenesis. Deconvolved, widefield single plane z-stack images from 14 h time-lapse videos (Supplementary movies 15A, B) of control (a, top panels) and cyto-D treated (a, bottom panels) P3 PCLS labelled with EpCAM-FITC (green) and SiR-DNA (magenta) at 0 and 14 h. Individual cell tracking over 14 h in a single field from P3 PCLS treated with DMSO, control (b) or 100 ng/ml cyto-D containing media (c). Mean net epithelial cell migration over 14 h in DMSO control or 100 ng/ml cyto-D containing media (d), n = 3 independent experiments using three separate mice; two fields were quantified from each PCLS from each mouse per condition, per experiment. Each dot represents mean net epithelial cell migration per field. Mann-Whitney U-test, p = 0.0022. Individual net cell migration in cyto-D treated vs. DMSO control P3 PCLS after 14 h of time-lapse imaging (each dot represents a single cell) (e). A total of 778 cells in DMSO control and 718 cells in cyto-D treated P3 PCLS were tracked. Student t-test; n = 3 independent experiments using three separate mice, 2 fields were quantified from each PCLS from each mouse per condition, per experiment, p = 0.0024. MTT cell viability assay comparing P3 PCLS after 15 h under normal culture conditions or after 15 h time-lapse imaging in DMSO control or cyto-D containing media (f), n = 3 independent experiments using 3 separate mice, quantification was from a single PCLS per condition, per experiment, one-way ANOVA with Tukey’s post hoc test. Mean linear intercept (Lm) (g) and airspace count (h) from H&E sections of P3 PCLS at 0 and 72 h of ex vivo culture; n = 3 independent experiments using 3 separate mice; 3 H&E sections from each PCLS from each mouse were quantified per group, per experiment, each dot represents per field count (g, h); ns = not significant, ***p < 0.001; one-way ANOVA with Tukey’s post hoc test. MTT cell viability assay on P3 PCLS at 0 and 72 h of culture in DMSO control or cyto-D containing media (i), n = 3 independent experiments using three separate mice, quantification was from a single PCLS per condition, per experiment; ns = not significant; paired Students t-test. Error bars are defined as s.e.m

Fig. 9

Diagram depicting cell behaviours identified in time-lapse videos. Schematic to show migratory epithelial behaviours that contribute to different stages of alveolar development. a Epithelial cells migrate and form clusters; b a small hole forms within a cell cluster, this widens and deepens to form a hollow following which additional epithelial cells migrate and integrate into the alveolus (g); c a cell extends around the wall of a mature alveolus