The importance of MYO5B in intestinal epithelial progenitor cell identity and function

Microvillus inclusion disease (MVID) is a congenital disorder characterized by severe diarrhea, caused by inactivating mutations in myosin Vb (MYO5B). Mice lacking MYO5B in their intestinal epithelium ( VilCre ERT2 ; MYO5B fl/fl ) demonstrate the importance of MYO5B in enterocyte brush border development and crypt function, including differentiation and proliferation. However, previous MYO5B knockout models lacked specificity to the crypt, therefore, the observed crypt deficits could be secondary effects of nutrient malabsorption. This study aims to better understand the crypt-specific effects caused by MYO5B loss. Tamoxifen-inducible Lrig1-Cre ERT2 ;R26R-YFP;MYO5B fl/fl (Lrig1DMYO5B) mice were generated by crossbreeding of established strains. Adult Lrig1DMYO5B mice and control littermates received tamoxifen at Day 0, and GI tissues were collected up to Day 5. Single cell RNA-sequencing (scRNA-seq) was conducted on the jejunal epithelium and lamina propria of Day 3 and 5. To validate the small intestinal stem cell-specific effects of MYO5B loss, we generated Olfm4-Cre ERT2 ;R26R-TdTOM;MYO5B fl/fl (Olfm4DMYO5B) mice. Lrig1DMYO5B mice began exhibiting weight loss at Day 4 and lost 20% of their starting body weight on Day 5. The proliferative crypt region was expanded in Lrig1DMYO5B mice beginning at Day 3. Correspondingly, MYO5B expression was diminished in the crypts at Day 3 and nearly absent from the entire epithelium by Day 5. Differentiation of epithelial cells derived from MYO5B-lacking progenitors was altered: decreased tuft cells, abnormal goblet cell shape, and mislocalized Paneth cells. Electron Microscopy of the Day 3 jejunum revealed a disruption in progenitor cell mitochondrial morphology. Day 3 Lrig1DMYO5B progenitor cells, but not villus cells, demonstrated significantly less Fatty Acid Oxidation (FAO) activity compared to control. scRNA-seq data revealed a lack of stem cell identity in progenitor cells of the Day 3 and 5 Lrig1DMYO5B jejunum, marked by a decrease in stem cell gene expression and numbers. TA, absorptive and secretory progenitor cell populations were expanded at both timepoints. Lrig1DMYO5B fibroblasts had decreased expression of Wnt s, Bmp s, and R-spondin s, which are critical for epithelial progenitor cell function. Mitochondrial oxidation gene expression was decreased in Day 3 Lrig1DMYO5B progenitor cells. In the Olfm4DMYO5B mouse small intestine, MYO5B – /TdTOM+ cells were present across the crypt-villus axis at Day 7. Mirroring the observations in Lrig1DMYO5B, the Olfm4DMYO5B small intestine demonstrated a progressive crypt expansion and cell differentiation deficits. Lrig1DMYO5B and Olfm4DMYO5B strains revealed the negative impact of MYO5B loss on intestinal epithelial cell differentiation and interaction between progenitor cell and mesenchymal niche. Funding: NIH R01DK128190 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.