Cell differentiation is disrupted by MYO5B loss through Wnt/Notch imbalance

Abstract

Functional loss of myosin Vb (MYO5B) induces a variety of deficits in intestinal epithelial cell function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). The impact of MYO5B loss on differentiated cell lineage choice has not been investigated. We quantified the populations of differentiated epithelial cells in tamoxifen-induced, epithelial cell-specific MYO5B-knockout (VilCreERT2 Myo5bfl/fl) mice utilizing digital image analysis. Consistent with our RNA-sequencing data, MYO5B loss induced a reduction in tuft cells in vivo and in organoid cultures. Paneth cells were significantly increased by MYO5B deficiency along with expansion of the progenitor cell zone. We further investigated the effect of lysophosphatidic acid (LPA) signaling on epithelial cell differentiation. Intraperitoneal LPA significantly increased tuft cell populations in both control and MYO5B-knockout mice. Transcripts for Wnt ligands were significantly downregulated by MYO5B loss in intestinal epithelial cells, whereas Notch signaling molecules were unchanged. Additionally, treatment with the Notch inhibitor dibenzazepine (DBZ) restored the populations of secretory cells, suggesting that the Notch pathway is maintained in MYO5B-deficient intestine. MYO5B loss likely impairs progenitor cell differentiation in the small intestine in vivo and in vitro, partially mediated by Wnt/Notch imbalance. Notch inhibition and/or LPA treatment may represent an effective therapeutic approach for treatment of MVID.

Keywords: Epithelial transport of ions and water; Gastroenterology; Genetic diseases; Protein traffic.

Figure 1. Changes in frequency of intestinal tuft cells by MYO5B loss and ip administration of LPA.

(A) Representative overlaid images of immunostaining and digitally identified cells. Whole-slide images of small intestine were analyzed. All nuclei were identified by Hoechst signals (shown in blue) and circled by light blue, and DCLK1⁺ tuft cells (shown in red) were defined with yellow circles. Original magnification is 20×. (B) Quantification of tuft cell frequency. Total cell number was counted by nuclei. Mean ± SD. Each data point represents a value from individual mouse. *P < 0.05, **P < 0.01 analyzed by 2-way ANOVA with Tukey’s multiple comparison. n = 3–6 mice per group. (C) Z-stack confocal images of DCLK1 (red) and ACTG1 (green) in tuft cells in control and induced MYO5B-deficient mouse jejunum. Vertical sections of tuft cell show dense ACTG1⁺ microtubule structure in both villi and crypts of control intestine. Villus tuft cells were present in MYO5B-deficient intestine with similar morphologies to those in controls. Arrows: microvillus inclusions in MYO5B-deficient enterocytes. Scale bars: 10 μm. ACTG1, actin gamma 1.

Figure 2. Lack of changes in the frequency of enteroendocrine cells in MYO5B-deficient intestine.

(A) Representative immunostaining for chromogranin A (ChgA) in jejunal sections from control and induced MYO5B-knockout mice 4 days after tamoxifen injection. ChgA⁺ cells were scattered throughout the crypt-to-villus axis in all treatment groups. Scale bar: 100 μm. (B) Representative high-magnification image of ChgA immunoreactivity. ChgA⁺ secretory granules were distributed in the cytosol. Scale bar: 10 μm. (C) Quantification of enteroendocrine cells in whole small intestine. Mean ± SD. Each data point represents a value from an individual mouse. No significant difference was detected by 1-way ANOVA.

Figure 3. Decrease in frequency of goblet cells in MYO5B-deficient mouse small intestine.

(A) Colocalization of TFF3-immunoreactivity with PAS staining in goblet cells of control mouse intestine. TFF3 staining was detected in luminal mucus and in the cytosol of goblet cells similar to the intense red staining by PAS. UEA-1 staining was found in a subset of mucus granules. Scale bars: 10 μm. (B) Representative staining for goblet cells in jejunum from control and tamoxifen-induced MYO5B-knockout mice after 4-day treatment with vehicle, og LPA, or ip LPA. Scale bars: 100 μm. (C) Quantification of UEA-1– and/or TFF3-immunoreactive goblet cells in whole small intestine using digital image analysis. n = 4 mice per group. Mean ± SD. *P < 0.05 in total goblet cell numbers analyzed by 1-way ANOVA with Dunnett’s multiple comparison.

Figure 4. Increased Paneth cells in MYO5B-deficient small intestine.

(A) Colocalization of lysozyme and autofluorescence in apical granules of Paneth cells. Scale bar: 10 μm. (B) Representative immunostaining for lysozyme in jejunum from control and induced MYO5B-knockout mice 4 days after tamoxifen injection. Some Paneth cells in MYO5B-knockout mice migrated toward villi in addition to the crypt bottom. Scale bars: 100 μm. (C) Quantification of Paneth cell frequency using digital image analysis. Mean ± SD. Each data point represents a value from individual mouse. n = 3–4 mice per group. *P < 0.05; ***P < 0.001 by 1-way ANOVA with Dunnett’s multiple comparison.

Figure 5. Changes in immunostaining pattern for OLFM4 and MCT1 induced by MYO5B loss.

(A) OLFM4⁺ stem/progenitor cells were limited to the bottom half of crypts in control intestine. Tamoxifen-induced MYO5B-deficient intestines with or without LPA supplementation demonstrated an extended OLFM4⁺ cell zone along with expanded crypt length. MCT1 was localized on the basolateral membranes of epithelial cells. Control intestine had ubiquitous expression of MCT1 along the crypt-to-villus axis, whereas MYO5B-deficient intestine showed clear MCT1 expression only in the crypt cells. Scale bars: 100 μm in merged images in the left column, 20 μm in the right column. (B) OLFM4⁺ crypt length was compared. Each data point represents an average value of 10 measured sites in an individual mouse. n = 3 mice per group. ****P < 0.0001 by 1-way ANOVA with Dunnett’s multiple comparison.

Figure 6. Alteration in the balance of Wnt/Notch signaling induced by MYO5B loss.

(A) mRNA expressions of Wnt ligands and Notch signaling molecules in isolated epithelial cells were compared between control and tamoxifen-induced MYO5B-deficient mouse jejunum. Mean ± SD. Each data point represents a value from individual mouse. n = 3–5 mice per group. *P < 0.05, by 2-way ANOVA with Bonferroni’s post hoc test. (B) Immunostaining for a Notch signaling effector, HES1 (red), and a proliferating cell marker, proliferating cell nuclear antigen (PCNA) (green), in mouse jejunum. (C) Immunostaining for the apoptosis marker cleaved caspase-3 (CCP3). CCP3⁺ cells were infrequently identified in upper part of villi similarly in control and MYO5B-deficient intestine. Scale bars: 50 μm. Dll, delta like canonical Notch ligand; Hes1, hes family bHLH transcription factor 1.

Figure 7. Tuft cell differentiation in VilCre^ERT2 Myo5B^fl/fl intestinal organoids.

(A) Immunostaining for DCLK1 (magenta) and F-actin (green) in whole-mount organoids. MYO5B loss was induced by 4-OH-tamoxifen in vitro (iKO) and control organoids were incubated with ethanol as vehicle. (Inset) Confocal images of F-actin show brush borders and the long microvillus structure in tuft cells. Scale bars: 20 μm. Insets: original magnification is 60×. (B) Tuft cell numbers were determined per organoid. ND, nondifferentiated organoids that were cultured for same period in IntestiCult Organoid Growth Medium as differentiated organoids. (C) Wnt signaling inhibitor C59 (10 μM) significantly reduced tuft cell differentiation in control organoids to a similar level as in iKO organoids. Each data point represents a value of individual organoid. n = 15–36 (B) and n = 98–133 (C) samples from 3 mice per group. ****P < 0.0001 by 1-way ANOVA with Dunnett’s multiple comparison.

Figure 8. DBZ treatment improved enterocyte differentiation in tamoxifen-induced MYO5B-knockout mice.

(A) PAS-stained jejunum of vehicle- or DBZ-treated VilCre^ERT2 Myo5B^fl/fl mice. Paneth and goblet cells and brush borders were stained in dark purple. Scale bars: 100 and 10 μm. (B) Villus and crypt lengths were determined in 10 regions of jejunum of each mouse. Data point indicates the average value of individual mouse. n = 4 mice per group. **P < 0.01 by unpaired 2-tailed t test. (C) Brush border localization of NHE3 and SGLT1 in DBZ-treated MYO5B-deficient intestine. NHE3 was colocalized with the microvillus marker ACTG1 in a subset of enterocytes in villi. Some mice received EdU (5 mg/kg) 24 hours before euthanasia that was identified in some SGLT1⁺ villus cells, indicating that newly differentiated cells express SGLT1 with proper localization.

Figure 9. Cell lineage differentiation 3 days after acute Notch inhibition.

(A) PAS-stained jejunum of DBZ-treated control and VilCre^ERT2 Myo5B^fl/fl mice. Arrows indicate luminal mucus covering the villi. (B–E) Immunostaining for tuft cell marker DCLK1 (B), goblet cell marker TFF3 (C), Paneth cell marker lysozyme (D), and proliferating cell marker PCNA (E). Nuclei were counterstained with Hoechst in cyan or blue. Scale bars: 100 μm.