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Mist1 Expression Is Required for Paneth Cell Maturation - PubMed

Mist1 Expression Is Required for Paneth Cell Maturation

Christopher M Dekaney et al. Cell Mol Gastroenterol Hepatol. 2019.

Abstract

Background: Paneth cells are professional secretory cells found within the small intestinal crypt epithelium. Although their role as part of the innate immune complex providing antimicrobial secretory products is well-known, the mechanisms that control secretory capacity are not well-understood. MIST1 is a scaling factor that is thought to control secretory capacity of exocrine cells.

Methods: Mist1+/+ and Mist1-/- mice were used to evaluate the function of MIST1 in small intestinal Paneth cells. We used histologic and immunofluorescence staining to evaluate small intestinal tissue for proliferation and lineage allocation. Total RNA was isolated to evaluate gene expression. Enteroid culture was used to evaluate the impact of the absence of MIST1 expression on intestinal stem cell function.

Results: Absence of MIST1 resulted in increased numbers of Paneth cells exhibiting an intermediate cell phenotype but otherwise did not alter overall epithelial cell lineage allocation. Muc2 and lysozyme staining confirmed the presence of intermediate cells at the crypt base of Mist1-/- mice. These changes were not associated with changes in mRNA expression of transcription factors associated with lineage allocation, and they were not abrogated by inhibition of Notch signaling. However, the absence of MIST1 expression was associated with alterations in Paneth cell morphology including decreased granule size and distended rough endoplasmic reticulum. Absence of MIST1 was associated with increased budding of enteroid cultures; however, there was no evidence of increased intestinal stem cell numbers in vivo.

Conclusions: MIST1 plays an important role in organization of the Paneth cell secretory apparatus and managing endoplasmic reticulum stress. This role occurs downstream of Paneth cell lineage allocation.

Keywords: Intermediate Cells; MIST1; Paneth Cells.

Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

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Figures

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Graphical abstract
Figure 1
Figure 1

MIST1 protein is expressed in Paneth cells within the intestinal epithelium. (Left) Immunofluorescence staining of lysozyme (green), MIST1 (red), and DNA (DAPI) in wild-type (Mist1+/+) and Mist1 knockout (Mist1–/–) mice. Scale bar equals 100 μm. (Right) Quantitative reverse transcriptase polymerase chain reaction to show relative expression of Mist1 mRNA in wild-type and Mist1–/– mice. n = 6 mice per group. **P <.01.

Figure 2
Figure 2

Loss of MIST1 expression does not affect epithelial proliferation or migration. (A) Top panel: IdU was given 90 minutes before death to monitor proliferation by labeling cells in S phase. Scale bar equals 100 μm. Bottom panel: CldU was given 24 hours before death to evaluate migration of cells. Scale bar equals 100 μm. (B) Quantification of percentage of IdU+ cells per crypt. n = 3 mice per group. (C) Quantification of average cell position of highest CldU+ cells along the crypt/villus axis. n = 3 mice per group.

Figure 3
Figure 3

Loss of Mist1–/–in small intestine does not alter secretory allocation. (A) Staining for sucrase-isomaltase (enterocytes), alcian blue (goblet cells), and lysozyme (Paneth cells). Scale bars equal 100 μm, 50 μm, and 50 μm, respectively. Black arrows point out alcian blue+ Paneth cells. (B) Quantification of chromogranin (Cg) A+ cells in crypt and villus compartments. n = 3 mice per group. Quantification of alcian blue+ cells in crypt and villus compartments. n = 3 mice per group. **P <.01. Quantification of lysozyme+ cells per crypt. n = 3 mice per group. (C) mRNA expression of transcription factors associated with secretory cell lineage allocation in isolated jejunal crypts from Mist1+/+ and Mist1–/– mice. n = 3–6 mice per group.

Figure 4
Figure 4

Mist1 expression prevents Paneth cells from becoming intermediate cells. (A) Immunofluorescence staining of jejunal tissue from Mist1+/+ and Mist1–/– mice for muc2 (green) and lysozyme (red). Dual positive cells (yellow) are intermediate cells. Scale bar equals 50 μm. (B) Representative PTAB staining of jejunal tissue from Mist1+/+ and Mist1–/– mice. Phloxine tartrizine stains protein-dense areas, Paneth cell–like granules brown, and alcian blue stains mucins blue. Paneth cell granules from Mist1+/+ Paneth cells stain brown (white arrows), and granules from Mist1–/– Paneth cells, which contain mucins, stain purple, suggesting an intermediate cell phenotype (black arrows). Scale bar equals 10 μm. (C) Quantification of PTAB staining of jejunal tissue from Mist1+/+ and Mist1–/– mice from (B) showing alcian blue+ (AB), phloxine/tartrizine+ (PT), dual positive (PTAB), and total stained cells (PT + PTAB) per crypt. n = 3 mice per group. *P <.05. **P <.01. ****P <.0001.

Figure 5
Figure 5

MIST1 expression plays a role in Paneth cell granule size and maturation. Transmission electron micrographs of crypts from jejunal tissue of (A) and (a) Mist1+/+ and (B) and (b) Mist1–/– mice showing ultrastructural characteristics of Paneth cells. (C) Quantification of Paneth cell granule size. n = 3 mice per group. **P <.01.

Figure 6
Figure 6

Intermediate cell phenotype of Mist1–/–mice is not due to impaired Notch signaling. (A) Mist1+/+ and Mist1–/– mice were treated with DBZ to inhibit Notch signaling, and jejunal tissues were stained with alcian blue. Scale bar equals 125 μm. (B) Jejunal tissues from (A) were stained with PTAB to evaluate Paneth cells (yellow arrows) and intermediate cells (black arrows). Paneth cell granules from Mist1+/+ Paneth cells stain brown, and granules from Mist1–/– Paneth cells that contain mucins stain purple, suggesting an intermediate cell phenotype. Scale bar equals 50 μm. (C) Quantification of Paneth cell and intermediate cell numbers. n = 4 mice per group. ****P <.0001. **P <.01.

Figure 7
Figure 7

Loss of MIST1 expression improves active ISC function in culture. (A) Representative in situ hybridization of Olfm4 in jejunal tissue from Mist1+/+ and Mist1–/– mice. Scale bar equals 20 μm. (B) Quantification of Olfm4 in situ hybridization staining using ACD scoring system. NS, not significant. (C) Micrographs of enteroid cultures derived from jejunal crypts from Mist1+/+, Mist1–/– and Atoh1–/– mice 1, 3, and 6 days after culture. (D) Quantification of budding percentage of Mist1+/+, Mist1–/– and Atoh1–/– enteroids over time. n = 5 Mist1+/+ and Mist1–/– mice were used for 4 independent culture experiments. n = 2 Atoh1–/– mice for 2 independent culture experiments to verify the inability of Atoh–/– crypts to grow in minimal culture medium. For each experiment n = 6 separate wells from each mouse were used for quantifying budding. **P <.01. ***P <.001.

Comment in

  • DeMISTifying Paneth Cell Maturation.

    Spatz LB, Mills JC. Spatz LB, et al. Cell Mol Gastroenterol Hepatol. 2019;8(4):643-644. doi: 10.1016/j.jcmgh.2019.08.005. Epub 2019 Oct 5. Cell Mol Gastroenterol Hepatol. 2019. PMID: 31593647 Free PMC article. No abstract available.

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