Dll1- and dll4-mediated notch signaling are required for homeostasis of intestinal stem cells - PubMed (original) (raw)

Dll1- and dll4-mediated notch signaling are required for homeostasis of intestinal stem cells

Luca Pellegrinet et al. Gastroenterology. 2011 Apr.

Abstract

Background & aims: Ablation of Notch signaling within the intestinal epithelium results in loss of proliferating crypt progenitors due to their conversion into postmitotic secretory cells. We aimed to confirm that Notch was active in stem cells (SCs), investigate consequences of loss of Notch signaling within the intestinal SC compartment, and identify the physiologic ligands of Notch in mouse intestine. Furthermore, we investigated whether the induction of goblet cell differentiation that results from loss of Notch requires the transcription factor Krüppel-like factor 4 (Klf4).

Methods: Transgenic mice that carried a reporter of Notch1 activation were used for lineage tracing experiments. The in vivo functions of the Notch ligands Jagged1 (Jag1), Delta-like1 (Dll1), Delta-like4 (Dll4), and the transcription factor Klf4 were assessed in mice with inducible, gut-specific gene targeting (Vil-Cre-ER(T2)).

Results: Notch1 signaling was found to be activated in intestinal SCs. Although deletion of Jag1 or Dll4 did not perturb the intestinal epithelium, inactivation of Dll1 resulted in a moderate increase in number of goblet cells without noticeable effects of progenitor proliferation. However, simultaneous inactivation of Dll1 and Dll4 resulted in the complete conversion of proliferating progenitors into postmitotic goblet cells, concomitant with loss of SCs (Olfm4(+), Lgr5(+), and Ascl2(+)). Klf4 inactivation did not interfere with goblet cell differentiation in adult wild-type or in Notch pathway-deficient gut.

Conclusions: Notch signaling in SCs and progenitors is activated by Dll1 and Dll4 ligands and is required for maintenance of intestinal progenitor and SCs. Klf4 is dispensable for goblet cell differentiation in intestines of adult Notch-deficient mice.

Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

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Figures

Figure1

Figure1. Notch1 is activated in intestinal stem cells

(A) Schematic illustration of NIP1::CreERT2 knock-in reporter mice (see text for details). N1 ECD, Notch1 extracellular domain; TM, Notch1 trans-membrane domain. (B-C) LacZ whole mount staining and EYFP expression in different intestinal segments eight months after reporter activation. Note fully labeled crypt-villus unit from both the Rosa LacZ and EYFP reporters. (D-F) Double staining of sections derived from the small intestine of NIP1::CreERT2 Rosa EYFP reporter mice for the goblet cell marker DBA (D), Paneth cell marker lysozyme (E) and enteroendocrine marker chromogranin (F). Double positive cells are highlighted with arrowheads.

Figure2

Figure2. Increased goblet cell differentiation in Dll1 deficient intestine

(A) Alcian blue (top panel) and Ki67 (bottom panel) staining of representative sections from the proximal small intestine of control, Dll4vil-Cre-ERT2, Dll1vil-Cre-ERT2 and Jag1vil-Cre-ERT2 mice. (B) Quantification of goblet cells in control and the indicated mutant mice. The number of Alcian blue-positive goblet cells per crypt-villus unit was counted and expressed as relative values to littermate controls (1.00). Dll1 mutant mice show a 1.83 fold increase (P<.0001) in goblet cells number. (C) Relative mRNA quantification of Dll4, Dll1 and Jag1, derived from control and single mutant mice (P< .01).

Figure3

Figure3. Complete conversion of crypt progenitors into post-mitotic goblet cells in Dll1-Dll4 double mutant mice

(A) Alcian blue and (B) Ki67 antibody staining of representative sections from the small intestine of control, Dll1-Jag1vil-Cre-ERT2 and Dll1-Dll4vil-Cre-ERT2 mice. The Dll1-Jag1 double mutant mice show an increase in goblet cell numbers similar to single Dll1 mutant mice (1.88 P< .0001) while the proliferative crypt compartment of Dll1-Dll4vil-Cre-ERT2 is completely converted into post-mitotic goblet cells. Insets in panels show high magnification (400x) of the outlined crypt regions. (C) Alcian blue and Ki67 staining of colonic crypts revealed a similar phenotype. Not only the crypt but also the inter crypt epithelium is converted into goblet cells (black triangles). (D) Quantitative mRNA analysis from epithelial cells of the small intestine and colon confirmed efficient gene inactivation (P< .01).

Figure4

Figure4. Notch signaling is disrupted in the small intestine of Dll1-Dll4 and RBP-J mutant mice

(A) Active Notch signaling was visualized with anti-NICD1 antibody staining on representative sections from control, Dll1-Dll4 and RBP-J deficient intestines. Disruption of Notch signaling was confirmed by the absence of nuclear staining in the crypt compartment of Dll1-Dll4 and RBP-J deficient mice. (B) In situ hybridization of the Notch target gene Hes1 on sections from indicated mice, and (C) quantification of Math1 mRNA level derived from epithelial cells of control, Dll1-Dll4 and RBP-J mutant mice (P< .01).

Figure5

Figure5. Loss of intestinal stem cell markers in Notch signaling deficient Dll1-Dll4 and RBP-J mutant mice

(A) In situ hybridization analysis for SC markers Lgr5, Olfm4 and Ascl2 on sections of the small intestine. Simultaneous inactivation of Dll1 and Dll4 or RBP-J resulted in the loss of expression of all three SC markers described above. Insets show high magnification (400x) of outlined crypt regions. Note that for Olfm4 in situ hybridization on Dll1-Dll4 mutant mice a region with a few positive SC (presumably none deleted) was chosen on purpose to indicate successful probe labeling.

Figure6

Figure6. Klf4 is dispensable for goblet cell metaplasia induced by loss of Notch signaling

(A-B) Alcian blue (top panels) and Ki67 (bottom panels) staining on sections derived from the proximal intestine (A) and colon (B) of the indicated mice. Note the complete loss of the proliferative crypt compartment and the complete conversion into goblet cells in RBP-J and RBP-J Klf4 mutant intestine. (C) Pharmaceutical inhibition of Notch signaling using the γ-secretase inhibitor DBZ after gut specific inactivation of Klf4 results in the loss of proliferating crypt progenitors due to their conversion into postmitotic goblet cells.

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