Loss of Myosin Vb in Intestinal Enterocytes Results in Aberrant Expression of Key Apical Transporters in vitro
Authors: Engevik Amy Christine , Weis Victoria G , Knowles Byron C , Goldenring James R 
Published: November 2023

BackgroundInactivating mutations in myosin Vb (MYO5B) result in Microvillus Inclusion Disease (MVID). MVID is a rare form of congenital diarrhea characterized by morphological intestinal enterocyte abnormalities and aberrations in the localization of brush border proteins and enzymes. Despite recent attention on MVID, the origin of the diarrheal disorder remains unclear. Our lab has generated a mouse model of MVID with a germline deletion of MYO5B (MYO5B KO). These mice rapidly develop diarrhea, have failure to thrive and demonstrate numerous microvillus inclusions in the intestinal enterocytes.HypothesisWe hypothesize that loss of MYO5B results in the improper localization of key apical membrane components away from the brush border resulting in inadequate absorption of nutrients and water.MethodsThe 3D culture of intestinal epithelial organoids, known as enteroids, allows for the in vitro propagation of all of the intestinal cell types. Enteroids were used as a simplified model to study epithelial alterations that result from loss of MYO5B. Enteroids were generated from the duodenum of neonatal mice with a germline deletion of MYO5B (MYO5B KO) and heterozygous or wildtype littermates (control). Enteroids were differentiated for 3–5 days by withdrawal of Wnt3A and analyzed for the localization of apical transporters by immunostaining. Quantitative real time PCR was used to determine the relative gene expression of transporters in MYO5B KO and control derived enteroids.ResultsF‐actin staining using phalloidin conjugates showed a mature brush border in enteroids derived from neonatal control mice. MYO5B KO derived enteroids also showed apical F‐actin staining, but F‐actin and ezrin positive inclusions were observed within enterocytes. These inclusions closely resembled inclusions seen in the intestinal tissue of MYO5B KO mice. SGLT1 and DPPIV were expressed on the apical membrane of control enteroids. In contrast, MYO5B KO derived enteroids had diffuse, subapical SGLT1 and DPPIV expression. NHE3 immunofluorescence showed apical expression of NHE3 in control enteroids and decreased expression in MYO5B KO enteroids. qRT‐PCR data showed a significant increase in SGLT1 and NHE3 mRNA expression in MYO5B KO enteroids compared to control enteroids. This could indicate that SGLT1 and NHE3 mRNA may be up‐regulated in an attempt to compensate for degradation or improper localization. Enteroids generated from MYO5B KO mice also showed a significant increase in proliferation compared to control derived enteroids, consistent with an increase in proliferation observed in the intestinal crypts of MYO5B KO mice.ConclusionsCollectively these data suggest that enteroids from MYO5B KO mice maintain the aberrant enterocyte characteristics seen in vivo and thus will be a good model for elucidating the mechanism by which loss of MYO5B results in untreatable diarrhea. The enteroid model of loss of MYO5B demonstrates intestinal epithelial changes that suggest that the improper localization of apical transporters is responsible for the diarrheal pathology seen in patients with MVID.Support or Funding InformationNIDDK Grant RO1 DK048370 and T32 Training Grant 2T32CA106183‐11.