Gastric tuft cells express DCLK1 and are expanded in hyperplasia

Abstract

Epithelial tuft cells are named after their characteristic microtubule bundles located at the cell apex where these are exposed to the luminal environment. As such, tuft cells are found in multiple organs, including the gastrointestinal (GI) tract where the apical "tuft" is hypothesized to detect and transmit environmental signals. Thus, the goal of our study was to characterize gastric tuft cells during GI tract development, then subsequently in the normal and metaplastic adult stomach. GI tracts from mouse embryos, and newborn and postnatal mice were analyzed. Tuft cells were identified by immunohistochemistry using acetylated-α-tubulin (acTub) antibody to detect the microtubule bundle. Additional tuft cell markers, e.g., doublecortin-like kinase 1 (DCLK1), were used to co-localize with acTub. Tuft cells were quantified in human gastric tissue arrays and in mouse stomachs with or without inflammation. In the developing intestine, tuft cells in both the crypts and villi expressed all markers by E18.5. In the stomach, acTub co-localized with DCLK1 and other established tuft cell markers by E18.5 in the antrum, but not until postnatal day 7 in the corpus, with the highest density of tuft cells clustered at the forestomach ridge. Tuft cell numbers increased in hyperplastic human and mouse stomachs. In the adult GI tract, the tuft cell marker acTub co-expressed with DCKL1 and chemosensory markers, e.g.,TRPM5. In summary, tuft cells appear in the gastric antrum and intestine at E18.5, but their maximal numbers in the corpus are not achieved until after weaning. Tuft cell numbers increase with inflammation, hyperplasia, and metaplasia.

Figure 1

A) Gastric tuft cell identified by microtubule staining (yellow arrow) with anti-acetylated-α-Tubulin (acTub, red). The dotted line indicates the apical border, and the dashed line the basal membrane. B) Electromicrograph of a tuft cell in the stomach, showing the characteristic narrow apex with blunt microvilli and numerous microtubules (yellow arrow) that extend along the cell body, and lateral branches (red arrow). C) Tuft cells identified with acTub (yellow arrows) were found in high numbers at the squamous/columnar epithelium border of the stomach.

Figure 2

Gastric tuft cells identified by acTub (red) express chemosensory markers. A) The transient receptor potential cation channel, subfamily M, member 5 (TRPM5, green). B) Tuft cells in the stomach also express phospholipase Cβ2 (PLCβ2, green). C) Gastric tuft cells (white arrows) do not express TAS1R3 (yellow arrows). The epithelial basal membrane is marked with white dashed lines in all merged panels.

Figure 3

DCLK1 (green) co-localized with acTub (red)-positive tuft cells in the stomach (A and a higher power view in B), small intestine (C) and colon (D). The long basal processes of tuft cells are also evident by staining with DCLK1 (yellow arrows in A and B). The epithelial basal membrane is marked with white dashed lines.

Figure 4

A) In the embryonic stomach (E18.5), tuft cells (yellow arrows) were not positive for PLCβ2 (green). In the gastric corpus, DCLK1 mRNA abundance was increased after birth (B) and tuft cells were found in low numbers by 2 weeks (C). The high number of tuft cells in the squamous/columnar epithelium junction observed in the adult was reached only after 3 weeks of age (D). The epithelial basal membrane is marked with white dashed lines.

Figure 5

Tuft cells are expanded in hyperplasia in the stomach. A) A representative image of a hyperplastic area in the corpus of an 11 month-old gastrin knockout mouse (GKO). Tuft cells are identified with acTub (red, white arrows), and the hematoxylin-eosin stain of the same area is shown in the insert. B) Quantification of tuft cells observed in GKO mice corpus. GKO mice develop antral and corpus hyperplasia and corpus metaplasia. Areas in which expanded expression of spasmolytic peptide were observed were considered metaplastic. Tuft cells in the surrounding normal mucosa in the same sample were quantitated. P values were calculated using One-way ANOVA with Dunnet’s post-test. C) Increased expression of DCLK1 was also observed in 5 week-old Hip1rKO mice, which develop SPEM and corpus hyperplasia. Data presented are mean ±S.E.M. (N = 3; *P<0.005 with Student’s T-test). D) DCLK1 positive cells (green) were not proliferating in Hip1rKO 3 week old mouse corpus (BrdU staining in red).

Figure 6

Tuft cells were expanded in human inflammation and hyperplasia (A), intestinal metaplasia (B, gland 1), while their frequency was diminished in higher dysplastic glands (B, compare glands 2 and 3), and were absent in carcinoma samples (C).