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. 2012 Jun 5;109(23):8965-70.
doi: 10.1073/pnas.1201652109. Epub 2012 May 14.

Functional intestinal stem cells after Paneth cell ablation induced by the loss of transcription factor Math1 (Atoh1)

Aurélie Durand  1 , Bridgitte Donahue, Grégory Peignon, Franck Letourneur, Nicolas Cagnard, Christian Slomianny, Christine Perret, Noah F Shroyer, Béatrice Romagnolo
Affiliations

Functional intestinal stem cells after Paneth cell ablation induced by the loss of transcription factor Math1 (Atoh1)

Aurélie Durand et al. Proc Natl Acad Sci U S A. .

Abstract

Intestinal epithelium has the capacity to self-renew and generate differentiated cells through the existence of two types of epithelial stem cells: active crypt base columnar cells (CBCs) and quiescent +4 cells. The behaviors of these cells are regulated both by intrinsic programs and by extrinsic signals sent by neighboring cells, which define the niche. It is clear that the β-catenin pathway acts as an essential intrinsic signal for the maintenance and proliferation of CBC, and it was recently proposed that Paneth cells provide a crucial niche by secreting Wingless/Int (Wnt) ligands. Here, we examined the effect of disrupting the intestinal stem cell niche by inducible deletion of the transcription factor Math1 (Atoh1), an essential driver of secretory cell differentiation. We found that complete loss of Paneth cells attributable to Math1 deficiency did not perturb the crypt architecture and allowed the maintenance and proliferation of CBCs. Indeed, Math1-deficient crypt cells tolerated in vivo Paneth cell loss and maintained active β-catenin signaling but could not grow ex vivo without exogenous Wnt, implying that, in vivo, underlying mucosal cells act as potential niche. Upon irradiation, Math1-deficient crypt cells regenerated and CBCs continued cycling. Finally, CBC stem cells deficient in adenomatous polyposis coli (Apc) and Math1 were able to promote intestinal tumorigenesis. We conclude that in vivo, Math1-deficient crypts counteract the absence of Paneth cell-derived Wnts and prevent CBC stem cell exhaustion.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Paneth cell ablation does not alter CBCs in Math1Δint mice. (A–C) Hematoxylin and eosin (H.E.) staining in control and Math1-deficient mice 7 and 22 d after Tam injections. (D–I) In situ hybridization of lysozyme and Olfm4 were carried out on serial sections from control and Math1-deficient mice 7 and 22 d after Tam injections. (J and K) Double-staining of lysozyme (brown) and Olfm4 (blue) in representative crypts of control and Math1-deficient mice. (L and M) Ascl2 immunostaining of representative crypts of control and Math1-deficient mice. (N and O) Transmission electron microscopy showing ablation of the Paneth cell lineage and the presence of normal CBCs with numerous supranuclear mitochondria and prominent supranuclear Golgi apparatus in Math1-deficient mice (O) compared with control mice (N). Dashed lines delimit the CBCs and P indicates the Paneth cells.
Fig. 2.
Fig. 2.
β-Catenin signaling and cell proliferation of progenitors and CBC in Math1-deficient mice. (A–F) In situ hybridization of Wnt3 (A and B), Wnt6 (C and D), and Wnt9 (E and F) in control and Math1-deficient mice 7 d after Tam injection. (G–P) β-Catenin (G and H), Ki67 (I and J), c-Myc (K and L), and Sox9 (O and P) immunostaining and in situ hybridization of Axin2 (M and N) in control and Math1-deficient mice 7 d after Tam injection. (Q) Real-time quantitative RT-PCR analyses of β-catenin/Tcf target genes in control and Math1Δint mice 7 and 22 d after Tam injections. Significant differences between the expression levels of control and Math1-deficient mice are marked by asterisks: *P < 0.05; **P < 0.01.
Fig. 3.
Fig. 3.
Growth of Math1 mutant intestinal organoids required exogenous Wnt. (A) Crypt organoids from adult control mice form efficiently during the first 3 d of culture. Note the presence of Paneth cells (indicated by asterisks) in the crypts from control mice. Progressive degeneration of Math1-deficient crypts occurred between days 2 and 3 in culture. Addition of Wnt3a supported the growth of Math1 mutant crypts into organoids. (B and C) Quantification of the total number of organoids (B) and the organoid structural complexity (C) of crypts from each mouse genotype during the first 3 d of culture in presence or absence of exogenous Wnt3a.
Fig. 4.
Fig. 4.
Math1 is dispensable for intestinal regeneration. (A–D) H.E. (A and B) and BrdU (C and D) staining of regenerative crypts from control and Math1-deficient mice 72 h following 10 Gy irradiation. (E and F) Lysozyme staining showing the efficient recombination of Math1-deficient crypts after Tam injection. (G and H) Costaining of Olfm4 (blue) and BrdU (brown) showing actively cycling CBCs in control and in Math1-deficient mice 72 h after irradiation.
Fig. 5.
Fig. 5.
Paneth cells are not required to initiate the intestinal tumorigenesis driven by Apc loss. (A–H) β-catenin (A and B), lysozyme (C and D), H.E. (E and F), and Ki67 (G and H) staining of small intestine from ApcΔInt and ApcΔIntMathΔInt mice 5 d after Tam injections. (I–L) β-Catenin (I and J) and lysozyme(K and L) staining of adenomas from ApcΔInt/+ and ApcΔInt/+MathΔInt mice, respectively, 70 and 22 d after Tam injections. (M) Real-time quantitative RT-PCR analysis of stem cell markers in the small intestine of ApcΔInt and ApcΔIntMathΔInt mice 5 d after Tam injections.
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