Context-dependent differentiation of multipotential keratin 14-expressing tracheal basal cells

Abstract

Multipotential (MP) differentiation is one characteristic of a tissue-specific stem cell (TSC). Lineage tracing of tracheobronchial basal cells after naphthalene (NA) injury or in the postnatal period demonstrated that basal cells were MP progenitors for Clara-like and ciliated cells. These studies, as well as reports of spatially restricted, label-retaining basal cells, and MP differentiation by human bronchial cells support the hypothesis that a TSC maintained and repaired the tracheobronchial epithelium. However, differences in basal cell phenotype (keratin [K] 5+ versus K14+), age (postnatal versus adult), health status (normal versus injured), and injury type (acid, detergent, NA) limited comparisons among studies and thus diminished the strength of the TSC argument. The finding that K14 was up-regulated after NA injury was a caveat to our previous analysis of reparative (r)K14-expressing cells (EC). Thus, the present study lineage traced steady-state (s)K14EC and evaluated differentiation potential in the normal and repairing epithelium. We showed that sK14EC were unipotential in the normal epithelium and MP after NA, sK14EC-dervied clones were not restricted to putative TSC niches, sK14EC cells were a direct progenitor for Clara-like and ciliated cells, MP-sK14EC clones accumulated over time, and sK14EC-derived Clara-like cells were progenitors for ciliated cells.

Figure 1.

Experimental design. (A) No-exposure group. (B) Lineage tracing of steady-state keratin 14–expressing cells (sK14EC) in untreated mice (Group 2) and in naphthalene (NA)-treated mice that were recovered at 20 days (Group 3) or 40 days (Group 4). (C) Lineage tracing of sK14EC after a repeated NA exposure. Single NA exposure control (Group 5), repeated NA–exposure with a 22-day recovery (Group 6), or a 62-day recovery (Group 7). n = sample size; TAM = tamoxifen.

Figure 2.

Lineage tracing of sK14EC without and with a single NA exposure. White arrows: endogenous β-galactosidase (β-gal) activity. Black arrows: Single β-gal+ cells. Red arrows: Single-layered group of β-gal+ cells. Yellow arrows: Multilayered regions of β-gal+ cells. Green arrows: Cuboidal cells that are negative for differentiation markers. Blue arrows: Example of a clone that did not span adjacent sections. (A–D) Groups 1 through 4. Genotype is indicated in each panel. β-gal activity (blue) in tracheal tissue whole mounts at 10× magnification. (E–G) Group 4. 30× magnification. Asterisks indicate intercartilaginous (ICR) regions. (H–J) Immunohistochemical analysis (brown stain) of differentiation markers on histological sections at 200× magnification. (H) Keratin 5. (I) Acetylated tubulin. (J) Clara cell secretory protein (CCSP). 1, 2, 3, and 3′ indicate clones referred to in Results. (K) Quantification and distribution of clone types (group 4). Tracheal subregions: ICR (black), midcartilagenous (MCR, gray).

Figure 3.

-Lineage tracing of K14EC in vitro. (Top) Experimental design. Air–liquid interface cultures were generated from K14-cre:er^t/RS trachea. The 20-day culture period is divided into two phases: proliferation (Days 0–5) and differentiation (Days 6–20). Differentiation was initiated on culture Day 5. Ciliated cells were first detected on culture Days 6 and 7. Clara-like cells were detected on culture Days 9 and 10. (A–F) Analysis of K14EC differentiation as a function of time. β-gal (white pseudocolor) and differentiation markers (color code indicated at the top of each column). (A and B) Proliferation stage, Day 5. (C and D) Differentiation stage, Day 11. (E and F) Differentiation stage, Day 17. All images are en face views at 200× original magnification. A', 400× magnification of the region indicated by the arrows in A. C', 400× magnification of the region indicated by the arrows in C. E', 400S magnification of the region indicated by the green arrows in E. White arrows: β-gal+ cells. Red arrows: β-gal/ACT dual-positive cells. Green arrows: β-gal/CCSP dual-positive cells.

Figure 4.

Differentiation of sK14EC after repeated NA exposure. See Figure 1C for experimental design. (A) Distribution of clone types along the proximal to distal axis of the trachea 62 days after the second NA exposure. Cross-sections through three or four complete trachea were evaluated. (B) Quantification of the clone types detected in the steady state (black), after a single NA exposure and a 40-day recovery (NA-1X: long term, gray), after a second NA exposure and a 22-day recovery (NA-2X: short-term, stippled) or a 62-day recovery (NA-2X: long-term, hatched).

Figure 5.

Lineage tracing of CCSP-expressing cells in vivo. Bitransgenic mice harboring a CC10-cre transgene and the RS recombination substrate were evaluated in the steady state and 13 days after NA exposure. Recombined cells and their progeny were analyzed by β-gal staining and immunohistochemical detection of differentiation antigens. (A–F) Paired images showing codetection of β-gal (white, pseudocolored brightfield) and differentiation antigens (fluorescence) in sections from untreated CC10-cre/RS mice. (A, C, E) Merged brightfield and dual immunofluorescence images. (B, D, F) Dual immunofluorescence images. Antigens and the detection color are indicated within each panel. Arrows are color coded according to the antigen and serve as fiduciary points for images A and B, C and D, and E and F. Scale bar, 50 μm. (G–J) Codetection of β-gal (blue) and differentiation antigens (brown) in sections from NA-treated CC10-cre/RS bitransgenic mice. Antigens and detection color are noted in each panel. Black arrows: β-gal− basal cells. Blue arrows: β-gal+ cells that coexpress CCSP or ACT. Scale bar, 50 μm.

Figure 6.

Lineage tracing of nascent CCSP-expressing cells in vitro. ALI cultures were generated from CC10-cre:RS trachea. Differentiation kinetics were similar to those indicated in Figure 3. Cells were stained for β-gal activity and coexpression of differentiation antigens at the indicated culture time points. (A) The percentage of lineage-traced cells that coexpressed CCSP was quantified as a function of time in culture. Mean ± SEM (n = 3). *P = 0.005 with respect to Day 5. (B) Percentage of lineage-traced cells that coexpress ACT. Mean ± SEM (n = 3). *P = 0.005 with respect to Day 4.

Figure 7.

Models of multipotential differentiation. Time after NA injury is represented by the vertical axis. Black triangles: Multipotential basal cell. Gray triangles: Progeny of the multipotential basal cell. Circles: Clara-like cell. Squares: Ciliated cell. Arrows: Progenitor–progeny relationship. (A) A single differentiation program with two steps that are labeled 1a and 1b. This model is refuted by the data presented herein. (B) Three differentiation programs result in replacement of ciliated and Clara-like cells. Program 1: basal–to–ciliated cell differentiation (1). Program 2: basal–to–Clara-like cell differentiation (2). Program 3: Clara-like–to–ciliated cell differentiation (3). This model is supported by the present data set.