Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium - PubMed (original) (raw)

. 2011 Mar 7;192(5):767-80.

doi: 10.1083/jcb.201010127.

Johan H van Es, Leila Makrini, Bénédicte Brulin, Georg Mellitzer, Sylvie Robine, Béatrice Romagnolo, Noah F Shroyer, Jean-François Bourgaux, Christine Pignodel, Hans Clevers, Philippe Jay

Affiliations

Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium

François Gerbe et al. J Cell Biol. 2011.

Abstract

The unique morphology of tuft cells was first revealed by electron microscopy analyses in several endoderm-derived epithelia. Here, we explore the relationship of these cells with the other cell types of the intestinal epithelium and describe the first marker signature allowing their unambiguous identification. We demonstrate that although mature tuft cells express DCLK1, a putative marker of quiescent stem cells, they are post-mitotic, short lived, derive from Lgr5-expressing epithelial stem cells, and are found in mouse and human tumors. We show that whereas the ATOH1/MATH1 transcription factor is essential for their differentiation, Neurog3, SOX9, GFI1, and SPDEF are dispensable, which distinguishes these cells from enteroendocrine, Paneth, and goblet cells, and raises from three to four the number of secretory cell types in the intestinal epithelium. Moreover, we show that tuft cells are the main source of endogenous intestinal opioids and are the only epithelial cells that express cyclooxygenase enzymes, suggesting important roles for these cells in the intestinal epithelium physiopathology.

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Figures

Figure 1.

Figure 1.

Molecular characterization of mouse intestinal tuft cells. Immunofluorescent stainings for (A) SOX9 and COX1, (B) SOX9 and COX2, (C) HPGDS and COX1, (D) villin and COX1, (E) α-tubulin and COX1, and (F) DCLK1 and COX1. Each panel contains a merged image on the left, and gray level pictures of the indicated individual markers corresponding to the yellow inset area on the right. (G) Whole-mount immunofluorescent staining for DCLK1 and F-actin on a dissociated fragment of intestinal epithelium. Panels on the right show higher magnification of the cropped area of the overlay image. Yellow arrowheads point at tuft cells. Nuclei are stained with Hoechst (blue). Bars, 10 µm.

Figure 2.

Figure 2.

DCLK1-expressing tuft cells are post-mitotic and continuously renewed. (A) Immunofluorescent staining for COX1, DCLK1, PCNA, and Hoechst. The PCNA− nucleus of a tuft cell is highlighted by a yellow dotted circle. (B) Experimental scheme of the BrdU birth dating experiment. Relative proportion and number of crypt DCLK1-expressing cells positive for BrdU are indicated. Two representative immunofluorescent stainings for DCLK1 and BrdU are shown for the indicated time point. Nuclei are stained with Hoechst (blue). Yellow dotted circles highlight tuft cell nuclei.

Figure 3.

Figure 3.

Tuft cells derive from Lgr5+ CBC stem cells. (A) Scheme explaining how chimeric Cre expression in crypts results in heterogeneous β-galactosidase staining in the adjacent villi (several crypts contribute to the generation of the cells constituting each villus). Wild-type (gray) and β-galactosidase (blue) cells coming from un-recombined (gray) and recombined (blue) crypts can migrate and colonize the same villus. The resulting cross section is shown. (B) Immunofluorescent staining for SOX9, β-galactosidase, β-catenin, and Hoechst in the Lgr5-EGFP-IRES-creERT2; Rosa26-LacZ mouse. Arrowheads point at SOX9+ tuft cells. The inset shows higher magnification of a SOX9+ tuft cells nucleus within a stretch of β-galactosidase+ cells. (C) Immunofluorescent staining for DCLK1, β-galactosidase, β-catenin, and Hoechst in intestinal sections from the Lgr5-EGFP-IRES-creERT2; Rosa26-LacZ mouse line. Arrowheads point at DCLK1+ tuft cells. The inset shows higher magnification of two DCLK1+ tuft cells within β-galactosidase+ crypts. β-galactosidase- crypts are shown by white dotted lines. Bars, 10 µm.

Figure 4.

Figure 4.

Tuft cells appear after birth. Immunofluorescent staining for DCLK1 and PCNA in the developing small intestine of E18.5, P7, and P12 mice. Arrowheads point at DCLK1-expressing tuft cells. Nuclei are stained with Hoechst (blue). Bars, 10 µm.

Figure 5.

Figure 5.

Tuft cells are found in mouse and human intestinal tumors. Immunofluorescent staining for tuft cells in K-RasV12G mouse adenoma (A and B); ApcΔ14 mouse adenoma (C–F), human adenoma (G), and human adenocarcinoma (H). Large fields (A and C) show clusters of tuft cells within the lesions. The lesion is delimited by PCNA (A) or β-catenin staining (C). DCLK1+ tuft cells coexpress the COX1 enzyme (B and D), show nuclear translocation of β-catenin (E), and are not in a proliferative state (F). Using HPGDS staining, tuft cells can also be detected in human adenomas (G) and, in rare cases, in restricted areas of human adenocarcinomas (H). For fluorescent staining, overlay (left) and individual signals of the indicated markers (right) are shown. Yellow dotted circles in E highlight tuft cell nuclei. Arrowheads point at tuft cells identified by DCLK1, COX1, and HPGDS expression. Nuclei are stained with Hoechst (blue) or hematoxylin (G and H). Bars: (A–F) 10 µm; (G and H) 100 µm.

Figure 6.

Figure 6.

Atoh1 is required for tuft cell differentiation. Immunofluorescent staining for the SOX9 transcription factor (A and C), the COX1 enzyme (A–D), and for the structural- and morphological-related tuft cells markers DCLK1 and α-tubulin (B and D) in intestines from control (A and B) and _Atoh1_-deficient mice (C and D), 3 wk after tamoxifen injection. Each panel contains the merged image on the left, and separate pictures of the indicated markers corresponding to the yellow inset on the right. Yellow arrowheads point at tuft cells revealed by SOX9 and COX1 or DCLK1, α-tubulin, and COX1 expression. Nuclei are stained with Hoechst (blue). Bars, 10 µm.

Figure 7.

Figure 7.

Neurog3 is dispensable for tuft cell differentiation. (A and B) Immunofluorescent staining for DCLK1 and ChgA expression in intestines from 6-mo-old control or _Neurog3_-deficient mice. Yellow and green arrowheads point at tuft and enteroendocrine cells, respectively. (C) Immunofluorescent staining for SOX9 and Neurog3 in wild-type mouse intestine. The arrowhead points at a Neurog3+, SOX9− cell. Nuclei are stained with Hoechst (blue). Bars, 10 µm.

Figure 8.

Figure 8.

Normal tuft cell differentiation in _Sox9_-deficient intestine. (A) Immunofluorescent staining for SOX9 and COX1 in intestines from control or _Sox9_-deficient mice, 1 mo after the first tamoxifen injection. Yellow arrowheads point at tuft cells identified by SOX9 and/or COX1 expression. The right inset shows the gray level picture of the COX1 staining, which is hardly visible in the merged image. (B) Immunofluorescent staining for SOX9, villin, and COX1 in the intestines of wild-type and _Sox9_-deficient mice. Each panel contains the merged image on the left, and individual fluorescent signals of the indicated markers corresponding to the yellow inset on the right. Arrowheads point at tuft cells identified by SOX9 and/or COX1 and villin expression. The tuft cell nucleus shown in the _Sox9_-deficient tissue is highlighted by the yellow circle. Nuclei are stained with Hoechst (blue). Bars, 10 µm.

Figure 9.

Figure 9.

Tuft cells are responsible for opioid production by the intestinal epithelium. Whole-mount immunofluorescent staining for β-endorphin, COX1, and villin in dissociated fragments of villus epithelium. Arrowheads point at tuft cells. Nuclei are stained with Hoechst (blue). Bars, 10 µm.

Figure 10.

Figure 10.

Updated model for the differentiation of the intestinal epithelial cell types. The scheme on the left represents a crypt–villus unit in the adult mouse small intestinal epithelium. The main functions, including the recently discovered function of Paneth cells in maintaining the CBC stem cell population (Sato et al., 2011), and representative molecular markers identifying each of the cell types and the intestinal stem cell are indicated. Opioid secretion is known to occur in the gut lumen (blue arrows; see Kokrashvili et al., 2009). Strong evidence suggests that tuft cells can also act as an epithelial source of prostanoids (Bezençon et al., 2008 and this paper), but the underlying secretion mechanism still has to be demonstrated. The diagram on the right summarizes the genetic hierarchy of epithelial cell lineage commitment in the intestine. Intestinal CBC stem cells proliferate and produce progenitors. Choice between absorptive or secretory cell fates is under the control of the hairy/enhancer of split 1 (Hes1) or atonal homologue 1 (Atoh1) gene. Within the cells committed to secretory types, Neurog3 is required for enteroendocrine cell differentiation. Gfi1 is required for Paneth and goblet cell differentiation, preventing the expression of Neurog3. Sox9 is essential for differentiation of Paneth cells. Spdef is required for both Paneth and goblet cell terminal maturation. M-cells are known to derive from Lgr5+ CBC stem cells (Barker and Clevers, 2010), but knowledge of the molecular pathways leading to their differentiation is still missing.

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