Opposing activities of Notch and Wnt signaling regulate intestinal stem cells and gut homeostasis

Abstract

Proper organ homeostasis requires tight control of adult stem cells and differentiation through the integration of multiple inputs. In the mouse small intestine, Notch and Wnt signaling are required both for stem cell maintenance and for a proper balance of differentiation between secretory and absorptive cell lineages. In the absence of Notch signaling, stem cells preferentially generate secretory cells at the expense of absorptive cells. Here, we use function-blocking antibodies against Notch receptors to demonstrate that Notch blockade perturbs intestinal stem cell function by causing a derepression of the Wnt signaling pathway, leading to misexpression of prosecretory genes. Importantly, attenuation of the Wnt pathway rescued the phenotype associated with Notch blockade. These studies bring to light a negative regulatory mechanism that maintains stem cell activity and balanced differentiation, and we propose that the interaction between Wnt and Notch signaling described here represents a common theme in adult stem cell biology.

Figure 1. Distribution of Wnt and Notch signaling in crypts of the mouse small intestine

(A) Lgr5^GFP (green) and Axin2^LacZ (red) expression are co-incident in CBCs (arrows). (B) Axin2^LacZ (red) overlaps with Edu incorporation (green) in cells at the base of the crypt (arrows) and in TA cells (asterisks) adjacent to CBCs. (C) Axin2^LacZ expression (red) overlaps with secretory cell progenitors marked by Math1^GFP (green, arrowheads). (D) The active form of Notch1 (NICD, red) is localized to the nuclei of CBCs (green, arrows). (E) NICD (red) overlaps with Ki67 staining (green) in CBCs (arrows) and TA cells (asterisks). (F) Notch signaling (red) is absent from secretory progenitor cells (green).

Figure 2. Loss of Notch signaling perturbs the function of Lgr5 positive stem cells

(A) Control crypts show a normal distribution of NICD staining in CBCs and TA cells. (B) NICD immunostaining is absent from crypts treated with Notch1 and Notch2 (αN1/N2) blocking antibodies over 6 days. (C) Control crypts show a normal distribution of Lgr5^GFP expression in proliferating CBCs and a subset of TA cells (arrows). (D) Notch blockade causes an increase in Lgr5^GFP expression (green) and a decrease in proliferating cells (red) in the TA zone. (E) Lgr5-positive CBCs (green) are largely non-overlapping with lysozyme-positive Paneth cells (red). (F) Increased Lysozyme expressing cells (red) are present after 6 days αN1/N2 treatment. (G) Control crypts showing distribution of Wnt signaling (Axin2^LacZ, red) and secretory cell progenitors (Math1^GFP, green). (H) αLRP6 treatment down-regulates Axin2^LacZ (red) and Math1^GFP (green). (I) Control crypts showing Lgr5^GFP (green) and proliferating Ki67 positive cells (red). (J) Wnt attenuation with αLRP6 blocking antibody down-regulates Lgr5^GFP expression (absence of green staining) without affecting proliferating CBCs (red, arrows). (K,L) Treatment with αLRP6 does not affect the distribution of NICD. (M) Lineage tracing experiments using Lgr5^CreER/+;Rosa^RFP/+ mice show widespread labeling of crypts and villi 7 days post induction with Tamoxifen (TAM). (N) Treatment with αN1/N2 before and after induction with TAM causes decreased lineage tracing from Lgr5 positive cells. (O) Lgr5-positive stem cells were first induced to undergo recombination with TAM and then treated with αN1/N2 on days 1 and 4. (P) αLRP6 treatment causes a loss of lineage tracing from Lgr5 positive cells if provided before induction with TAM. (Q) αLRP6 treatment does not affect lineage tracing if provided after induction with TAM.

Figure 3. Notch blockade leads to Wnt signaling up-regulation

(A) Control crypts showing Axin2^LacZ staining (red). (B) Combined Axin2^LacZ (red) and Math1^GFP (green) staining. (C) Control crypts showing expression of Lgr5^GFP (green) and proliferating cells (Ki67, red). (D) Control crypts showing antibody staining of the Wnt targets SOX9 (red) and EPHB3 (green) at the base of the crypt. (E) 7 hr. time point during Notch blockade shows an increase in the Wnt reporter Axin2^LacZ. (F) 7 hr. time point shows a normal distribution of Math1^GFP relative to controls. (G) At 7 hr. time point during Notch blockade, Lgr5^GFP positive CBCs are still present but have stopped proliferating (arrows). (H) 7 hr. time point during Notch blockade shows increasing intensity of SOX9 positive nuclei at the base of the crypt (red). (I) 24 hr. time point during Notch blockade shows an increased in Axin2^LacZ staining. (J) 24 hr. time point during Notch blockade shows an increased distribution of Math1^GFP staining (green). (K) At 24 hr. time point during Notch blockade, Lgr5^GFP (green) and proliferating cells (red) are largely absent from the base of the crypt. (L) 24 hr. time point during Notch blockade shows increased staining and distribution of the Wnt targets SOX9 and EPHB3. (M,N) 24 hr. time point during LRP6 blockade shows absence of Axin2^LacZ (M, red) and loss of Math1^GFP expression (N) in treated crypts. Arrows in (N) point to residual Math1^GFP expression. (O) 24 hr. time point during LRP6 blockade shows an absence of Lgr5^GFP (green) and a normal distribution of Ki67 staining in CBCs (arrows). (P) 24 hr. time point during LRP6 blockade shows near complete down-regulation of the Wnt targets SOX9 and EPHB3. (Q) 7 hr. time point during combined Notch/LRP6 blockade shows a reduced distribution of Axin2^LacZ relative to Notch blockade alone (E). (R) 7 hr. time point during combined Notch/LRP6 blockade shows rescued of Math1^GFP expression pattern. (S) 7 hr. time point during combined Notch/LRP6 blockade shows a loss of Lgr5^GFP expression and a rescued distribution of Ki67 positive cells at the base of the crypt. (T) 7 hr. time point during combined Notch/LRP6 blockade shows the Wnt target genes Sox9 and EphB3 remain down-regulated. (U) 24 hr. time point during combined Notch/LRP6 blockade shows that Axin2^LacZ expression is reduced relative to (I). (V) 24 hr. time point during combined Notch/LRP6 blockade shows that Math1^GFP expression is reduced relative to (J). (W) 24 hr. time point during combined Notch/LRP6 blockade shows a rescued distribution of Ki67 positive cells including CBCs (arrows). (X) 24 hr. time point during combined Notch/LRP6 blockade shows the Wnt target genes Sox9 and EphB3 remain down-regulated. (Y) Fold changes in gene expression relative to controls after antibody treatments. mRNA was purified from isolated crypts. Results are mean ± SEM. (Z) Relative levels of Wnt target gene expression in Villin Cre;Math1^fl/fl mice treated with Notch blocking antibodies. (Z’) Levels of Wnt5a, Wnt9b, and Rspo4 increase in Villin Cre;Math1^fl/fl mice treated with Notch blocking antibodies.

Figure 4. Co-treatment with Notch and Wnt blocking antibodies rescues secretory cell metaplasia and ISC activity

(A) Control ileum showing proliferating cells (Ki67, brown) and Alcian Blue stained Goblet cells. (B) Notch blockade causes goblet cell metaplasia. (C) αLRP6 treatment leads to no significant changes in the distribution of Goblet cells or proliferating cells. (D) Combined αN1/N2 and αLRP6 treatment rescues the proliferation defects and Goblet cell metaplasia associated with Notch blockade alone. (E) Lineage tracing experiments using Lgr5^CreER/+;Rosa^RFP/+ mice with fully labeled crypts and villi 7 days post induction with Tamoxifen (TAM). Inset shows expression of Lgr5^GFP at crypt base. (F) Combined αN1/N2 and αLRP6 treatment rescues stem cell activity, as indicated by a recovery of lineage tracing events from Lgr5-positive stem cells. Inset shows representative fully labeled crypt with suppressed Lgr5^GFP expression. (G) Notch blockade causes goblet cell metaplasia. (H) Combined αN1/N2 and αF8CRD treatment rescues the proliferation defects and Goblet cell metaplasia.