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. 2014 Oct;147(4):822-834.e13.
doi: 10.1053/j.gastro.2014.07.003. Epub 2014 Jul 16.

ADAM10 regulates Notch function in intestinal stem cells of mice

Yu-Hwai Tsai  1 , Kelli L VanDussen  2 , Eric T Sawey  3 , Alex W Wade  1 , Chelsea Kasper  1 , Sabita Rakshit  1 , Riha G Bhatt  1 , Alex Stoeck  1 , Ivan Maillard  4 , Howard C Crawford  5 , Linda C Samuelson  2 , Peter J Dempsey  6
Affiliations

ADAM10 regulates Notch function in intestinal stem cells of mice

Yu-Hwai Tsai et al. Gastroenterology. 2014 Oct.

Abstract

Background & aims: A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a cell surface sheddase that regulates physiologic processes, including Notch signaling. ADAM10 is expressed in all intestinal epithelial cell types, but the requirement for ADAM10 signaling in crypt homeostasis is not well defined.

Methods: We analyzed intestinal tissues from mice with constitutive (Vil-Cre;Adam10(f/f) mice) and conditional (Vil-CreER;Adam10(f/f) and Leucine-rich repeat-containing GPCR5 [Lgr5]-CreER;Adam10(f/f) mice) deletion of ADAM10. We performed cell lineage-tracing experiments in mice that expressed a gain-of-function allele of Notch in the intestine (Rosa26(NICD)), or mice with intestine-specific disruption of Notch (Rosa26(DN-MAML)), to examine the effects of ADAM10 deletion on cell fate specification and intestinal stem cell maintenance.

Results: Loss of ADAM10 from developing and adult intestine caused lethality associated with altered intestinal morphology, reduced progenitor cell proliferation, and increased secretory cell differentiation. ADAM10 deletion led to the replacement of intestinal cell progenitors with 2 distinct, post-mitotic, secretory cell lineages: intermediate-like (Paneth/goblet) and enteroendocrine cells. Based on analysis of Rosa26(NICD) and Rosa26(DN-MAML) mice, we determined that ADAM10 controls these cell fate decisions by regulating Notch signaling. Cell lineage-tracing experiments showed that ADAM10 is required for survival of Lgr5(+) crypt-based columnar cells. Our findings indicate that Notch-activated stem cells have a competitive advantage for occupation of the stem cell niche.

Conclusions: ADAM10 acts in a cell autonomous manner within the intestinal crypt compartment to regulate Notch signaling. This process is required for progenitor cell lineage specification and crypt-based columnar cell maintenance.

Keywords: CBC; Development; Differentiation; Intestinal Epithelium.

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Figures

Figure 1
Figure 1. Reduced progenitor cell proliferation upon ADAM10 deletion
(A), ADAM10 staining in adult small intestine of wild-type and Lgr5-EGFP-ires-CreER mice. Right, Co-staining with GFP (red). (B), H&E (upper) and Ki67+ staining (lower, arrowheads) of small intestine from newborn Adam10f/f and Vil-Cre;Adam10f/f mice. Quantification of Ki67+ cells per field (n=4). (C), H&E (upper) and BrdU+ staining (lower, arrowheads) of adult small intestine from Vil-CreER;Adam10f/f mice treated with TX (100 mg/kg i.p.) or vehicle for 5 consecutive days and analyzed the next day. Quantification of BrdU+ cells in crypts (n=3). *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Scale bars (μm) = A, left, 200 and right, 35; B, upper, 100 and lower, 50; and C, 200 (inset, 65).
Figure 2
Figure 2. ADAM10 deletion converts intestinal crypt progenitors to a secretory cell fate
(A–C), Newborn small intestine from Adam10f/f and Vil-Cre;Adam10f/f mice (n=4). (D–E), Adult small intestine from Vil-CreER;Adam10f/f mice treated with TX as described in Figure 1 (n=5–7). (A,D), PAS/Alcian Blue staining (upper) and immunofluorescence analysis of goblet (MUC2), Paneth (MMP7) and enteroendocrine (CHGA) cell markers (lower). Intermediate (Paneth/goblet) and CHGA+ cells indicated by arrowheads. (B,E), Morphmometric analysis of secretory cell types. (C,F), qPCR analysis of cell lineage markers. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Scale bars (μm) = All 100 except C, upper, 200.
Figure 3
Figure 3. ADAM10 deletion leads to a loss of Notch signaling
(A–C), Perinatal small intestine from Adam10f/f and Vil-Cre;Adam10f/f mice. (D–F), Adult small intestine from Vil-CreER;Adam10f/f mice treated with TX as described in Figure 1. (A,D), qPCR analysis of Notch gene targets (upper) and cell lineage-specific transcription factors (n=3–7). (B,E), X-Gal staining of Atoh1LacZ/+ reporter mice. (C,F), Immunostaining of Neurog3YFP/+ reporter mice. Arrowheads mark Neurog3+ cells. *p<0.05, **p<0.01 and ***p<0.001. Scale bars (μm) = B, 100; C, 65; E, 200; F, 135 and (insets 65).
Figure 4
Figure 4. ADAM10 mediated Notch signaling is required for cell lineage specification and progenitor cell proliferation
(A), Immunofluorescence analysis of cell lineage markers in the newborn small intestine from Vil-Cre;Adam10f/f and in different Notch mutants. (B), Quantification of Ki67+ cells per field (n=3–4). p values of group comparisons between Adam10f/f and other genotypes are shown. *p<0.05, **p<0.01, ****p<0.0001. Scale bars (μm) = 100.
Figure 5
Figure 5. ADAM10 loss leads to crypt degeneration that can be overridden by activated Notch
(A–C), Adult mice were given a single TX (100 mg/kg, i.p.) dose and analyzed at day 9 post-treatment (n=3). (A), Body weight analysis. (B), H&E analysis. Black boxes are shown at high magnification (lower). In Vil-CreER;Adam10f/f;Rosa26YFP/+ mice, crypt degeneration (black asterisks) is observed adjacent to crypts with increased cellularity (orange asterisk). (C), Immunofluorescence analysis of ADAM10 with BrdU (upper) and YFP or NICD(nGFP) (lower). In Vil-CreER;Adam10f/f;Rosa26YFP/+ mice, degenerating crypts are ADAM10-deficient and lack cell proliferation (white asterisks) whereas adjacent regenerating crypts express ADAM10 and are highly proliferative (orange asterisk). In Vil-CreER;Adam10f/f;Rosa26NICD/+ mice, NICD(nGFP)+ ADAM10-deficient crypts (white asterisks) are filled with NICD(nGFP)+ cells (arrows) and maintain cell proliferation (arrowheads). p values from group comparisons of genotypes Vil-CreER;Adam10f/+;Rosa26YFP/+ vs Vil-CreER;Adam10f/f;Rosa26YFP/+ (red) and Vil-CreER;Adam10f/f;Rosa26YFP/+ vs Vil-CreER;Adam10f/f;Rosa26NICD/+ (blue) are shown in A. *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001.
Figure 6
Figure 6. Activated Notch restores proliferation and Olfm4 expression in AD-AM10-deficient organoids
(A), Organoid cultures from adult mice treated with 4-OH-TX (5μM) for 12 h and then analyzed at day 5 post-treatment. Whole mount analysis of morphology (upper) and EdU staining (mid-upper). Co-staining of ADAM10, EdU and YFP or NICD(nGFP) (lower panels). Arrows indicate NICD(nGFP)+ cells. Scale bars (μm) = B, 200 (inset, 65); C, 65; D, all 200. (B), qPCR analysis of ISC markers in 4-OH-TX treated organoid cultures (n=3). *p<0.05, ***p<0.001, ****p<0.0001.
Figure 7
Figure 7. ADAM10 is required for maintenance of adult Lgr5+ CBCs
(A–B), Lineage tracing of Vil-CreER;Adam10f/f;Rosa26YFP/+, Vil-CreER;Adam10f/f;Rosa26NICD/+ and genotype control mice given a single TX (50 mg/kg i.p.) dose and then chased for 28 days (n=3). Co-staining of ADAM10 and YFP or NICD(nGFP). (A), In Vil-CreER;Adam10f/f;Rosa26YFP/+ mice, no ADAM10-deficient cells were detected. Arrowheads indicate individual YFP+ Paneth cells (Supplementary Figure 8A). (B), In both genotypes, NICD(nGFP)+ cells are detected (arrowheads). Asterisks mark NICD(nGFP)+ ADAM10-deficient crypt-villus units. (C–D), Lineage tracing of Lgr5-CreER;Adam10f/f;Rosa26YFP/+, Lgr5-CreER;Adam10f/f;Rosa26NICD/+ and genotype control mice given a single TX (400 mg/kg o.g.) dose and then chased for 28 days (n=3). Immunofluorescence analysis of ADAM10 and YFP or NICD(nGFP). Note that EGFP+-Lgr5 cells can co-express YFP or NICD(nGFP). (C), In Lgr5-CreER;Adam10f/f;Rosa26YFP/+ mice, no ADAM10-deficient cells were detected. Strong YFP expression from the Rosa26YFP allele masks the weaker EGFP+- Lgr5 cells. (D), NICD(nGFP)+ cells (arrows), EGFP+-Lgr5 cells displaying stronger cytoplasmic staining (arrowheads) and NICD(nGFP)+ ADAM10-deficient crypt-villus unit (asterisk) are shown. (AD), Lower panels, Quantification of each lineage tracing event. *p<0.05; **p<0.01;***p<0.001, ****p<0.0001; na, not applicable. Scale bars (μm) = 100.
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References

    1. Noah TK, Shroyer NF. Notch in the intestine: regulation of homeostasis and pathogenesis. Annu Rev Physiol. 2013;75:263–88. - PubMed
    1. Vooijs M, Ong CT, Hadland B, et al. Mapping the consequence of Notch1 proteolysis in vivo with NIP-CRE. Development. 2007;134:535–44. - PMC - PubMed
    1. Kopinke D, Brailsford M, Shea JE, et al. Lineage tracing reveals the dynamic contribution of Hes1+ cells to the developing and adult pancreas. Development. 2011;138:431–41. - PMC - PubMed
    1. Fre S, Hannezo E, Sale S, et al. Notch lineages and activity in intestinal stem cells determined by a new set of knock-in mice. PLoS One. 2011;6:e25785. - PMC - PubMed
    1. Fre S, Huyghe M, Mourikis P, et al. Notch signals control the fate of immature progenitor cells in the intestine. Nature. 2005;435:964–8. - PubMed

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