Chemosensory brush cells of the trachea. A stable population in a dynamic epithelium

Abstract

Tracheal brush cells (BCs) are specialized epithelial chemosensors that use the canonical taste transduction cascade to detect irritants. To test whether BCs are replaced at the same rate as other cells in the surrounding epithelium of adult mice, we used 5-bromo-2'-deoxyuridine (BrdU) to label dividing cells. Although scattered BrdU-labeled epithelial cells are present 5-20 days after BrdU, no BCs are labeled. These data indicate that BCs comprise a relatively static population. To determine how BCs are generated during development, we injected 5-day-old mice with BrdU and found labeled BCs and non-BC epithelial cells 5 days after BrdU. During the next 60 days, the percentage of labeled BCs increased, whereas the percentage of other labeled cell types decreased. These data suggest that BCs are generated from non-BC progenitor cells during postnatal tracheal growth. To test whether the adult epithelium retains the capacity to generate BCs, tracheal epithelial cells were recovered from adult mice and grown in an air-liquid interface (ALI) culture. After transition to differentiation conditions, BCs are detected, and comprise 1% of the total cell population by Day 14. BrdU added to cultures before the differentiation of BCs was chased into BCs, indicating that the increase in BC density is attributable to the proliferation of a non-BC progenitor. We conclude that: (1) BCs are normally a static population in adult mice; (2) BC progenitors proliferate and differentiate during neonatal development; and (3) BCs can be regenerated from a proliferative population resident in adult epithelium.

Figure 1.

Brush cells (BCs) comprise a distinct cell type in the tracheal epithelium. (A) Triple-labeled, whole-mount tracheal epithelium shows transient receptor potential melastatin 5 (TRPM5)–green fluorescent protein (GFP; green), protein gene product 9.5 (PGP9.5; blue), and acetylated tubulin (ACT; red). BCs appear green. Nerve fibers (magenta) are immunoreactive for both PGP9.5 and acetylated tubulin. Neuroendocrine cells are immunoreactive for PGP9.5 (blue), whereas cilia appear red, and are immunoreactive for ACT. (B) BCs coexpress Gα-gustducin (Gust; red) and choline acetyltransferase (ChAT; green), and occur within the tracheal epithelium (arrowheads) and in submucosal glands (arrow). (C) Most BCs are immunoreactive for both Gα-gustducin (red) and TRPM5 (green), and have multiple processes. (D) Gα-gustducin (green) immunoreactive BCs are not immunoreactive for ACT (magenta), a marker for ciliated cells. (E) Gα-gustducin (green) immunoreactive BCs are not immunoreactive for club (Clara)–cell secretory protein (CCSP) (red), a marker for club (Clara)–like cells. (F and G) TRPM5 (green)–expressing BCs appear morphologically similar to neuroendocrine cells, but are not immunoreactive for the known neuroendocrine cell markers (E) calcitonin gene related peptide (CGRP; red) and (F) 5-hydroxytryptamine (5HT; red). (B–G) Counterstains with DRAQ5 are shown in cyan. Photographs are of tissues from mice that ranged in age from 90–180 days. Scale bars = 10 μm.

Figure 2.

BCs comprise a static population in the adult trachea, but are generated by a population of proliferative progenitor cells during perinatal development. (A) Double-labeled image with Nomarski overlay shows 5-bromo-2′-deoxyuridine (BrdU; red)–labeled epithelial cell nuclei near an unlabeled Gα-gustducin (green) immunoreactive BC in an adult mouse, 20 days after BrdU treatment. (B) Percentages of tracheal epithelial cells and BCs labeled with BrdU at 5–20 days after BrdU treatment of adult mice. Bars depict the mean ± SEM (n = 4) for each point. (C) Double-labeled image shows Ki67 (red)–labeled epithelial cell nuclei near a Gα-gustducin (green) immunoreactive BC in a trachea from a 35-day-old mouse. (D) Percentages of perinatal tracheal epithelial cells and BCs immunoreactive for the mitotic marker Ki67. Bars depict the mean ± SEM (n = 3) for each time. (E) Double-labeled image with Nomarski overlay shows a BrdU (red)–labeled, Gα-gustducin (green) immunoreactive BC in a 35-day-old mouse, 30 days after BrdU treatment. (F) Percentages of perinatal tracheal epithelial cells and BCs labeled with BrdU at different times after the injection of 5-day-old mice. Bars depict the mean ± SEM (n = 4) for each time point. **P < 0.01 and ***P < 0.001, according to χ² test. Scale bars = 10 μm.

Figure 3.

New BCs can be generated from adult tracheal epithelium in vitro. (A) Gα-gustducin (red) immunoreactive BCs in tracheal epithelial cell culture on air–liquid interface (ALI) Day 7. Cultured BCs have multiple processes like their in vivo counterparts. (B) BCs in cultures produced from TRPM5-GFP (green) transgenic mice were immunoreactive for Gα-gustducin (red) and expressed GFP on ALI Day 7. (C) Gα-gustducin (green) immunoreactive BCs were negative for the ciliated cell marker acetylated tubulin (ACT, magenta) on ALI Day 7. (D) A double-labeled cell with a BrdU (green) and Gα-gustducin (red) double-immunoreactive BC (arrow). BrdU also labeled other cells in culture (ALI Day 14). (E) Several doublet Gα-gustducin (red) immunoreactive BCs, suggestive of daughter cells, were observed culture on ALI Day 1. (F) Double-labeled image shows a Gα-gustducin (red) immunoreactive doublet, where both nuclei are labeled with BrdU (green), on ALI Day 14. Counterstains with 4′6-diamidino-2-phenylindole are shown in blue. Scale bars = 10 μm. (G) Tracheal epithelial cell density (red line) and the fraction of BCs (green line) over time in ALI cultures. Points depict the mean ± SEM (n = 3) for each time. (H) Fraction of cultured BCs labeled when dosed with BrdU, either during the proliferation period (Pro 3) or on ALI Day 0. *P < 0.001, according to t test. Bars depict the mean ± SEM (n = 3) for each point.