Notch2 depletion ameliorates MYO5B loss-induced differentiation defects in enteroids

Loss-of-function variants of Myosin Vb (MYO5B) cause microvillus inclusion disease (MVID), which is a devastating congenital diarrheal and malabsorption disorder. Utilizing MVID patient-derived enteroids and MYO5B deficient mice, we previously reported that MYO5B loss disrupts cell lineage differentiation. Since our transcriptomic datasets of mouse tissues and enteroids indicate the upregulation of Notch signaling molecules, including Notch2, we hypothesize that suppression of Notch2, a minor subtype in the intestine, may reverse the epithelial phenotype induced by MYO5B loss. Vil1-CreER T2 ;Myo5b fl/fl (MYO5B∆IEC) mice on the C57Bl6 background and Notch1 fl/fl ;Notch2 fl/fl on the CD-1 background were crossbred to obtain the double knockout (KO) mice for MYO5B and either Notch receptor subtype (MYO5B:Notch1∆IEC and MYO5B:Notch2∆IEC), MYO5B∆IEC with heterozygous for Notch, and controls in the same litters. Adult mice received a single dose of tamoxifen, and intestinal tissues were investigated on day 4 through 6 post tamoxifen. Enteroids were generated from jejunal crypts of KO mice without tamoxifen treatment, and Cre-recombinase was induced by 4-OH-tamoxifen in vitro prior to the differentiation of enteroids. Tamoxifen-treated MYO5B∆IEC, MYO5B:Notch1∆IEC, and MYO5B:Notch2∆IEC mice similarly lost an average of 12% of body weight within 4 days. As compared to the previous MYO5B∆IEC mice on the Bl6 background, KO mice on CD-1 background showed a wider range of diarrheal symptoms and intestinal tissue histology, such as villus blunting, crypt expansion, and mislocalized apical proteins. This heterogeneity might be caused by differential tamoxifen kinetics among mouse strains. In enteroid cultures, MYO5B deletion was induced more uniformly than in vivo. Tuft cell differentiation was significantly decreased in MYO5B∆IEC enteroids compared to control enteroids, which were untreated with 4-OH-tamoxifen. MYO5B:Notch1∆IEC enteroids reversed the tuft cell deficit and MYO5B:Notch2∆IEC enteroids further increased tuft cell numbers. Immunostaining on enteroid sections distinguished proliferative cells (Ki67 + /CFTR + ) vs. mature enterocytes (Ki67 – /SGLT1 + ) that well recapitulate crypt vs. villus cell types in jejunum. Control enteroids showed brush border localization of CFTR, SGLT1, NHE3, and MUC13, whereas MYO5B∆IEC enteroids demonstrated cytoplasmic signals for SGLT1, NHE3, and MUC13, but normal CFTR distribution. SGLT1 localization was normalized in MYO5B:Notch2∆IEC enteroids, suggesting that double KO for Notch2 and MYO5B can rescue epithelial cell differentiation towards mature enterocytes and tuft cells. TFF3 and LYZ were co-immunostaining for goblet and Paneth cells, respectively. TFF3+ and LYZ+ cell numbers were comparable in enteroids among genotypes, however, the majority of positive cells expressed both markers. The mixed characteristics of secretory cell lineages may represent a stressed condition in enteroids. Our observations suggest that enteroid is a useful model for enterocyte and tuft cell differentiation defects that are seen in MVID patients. Notch2-targeted therapeutics might be an effective treatment for MVID. NIH R01DK128190, RC2DK118640, Volpe Foundation This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.