Wnt Signaling Inhibition Induces Lateral Lumens in Enteroids

Abstract

BackgroundTo overcome the constant shedding of the intestinal epithelium, the stem cells at the base of the crypt rapidly proliferate and differentiate into epithelial cells necessary for proper intestinal function. A Wnt:Notch signaling gradient exists along the crypt‐villus axis, where distinct levels of Wnt relative to Notch signaling is necessary for the differentiation of epithelial cell populations. In our recent publication, mice lacking intestinal epithelial myosin 5B (MYO5B) have intracellular microvillus inclusions and cell differentiation deficits. The intestinal epithelium of these MYO5B knockout mice have significantly less transcript levels for genes associated with Wnt signaling relative to those associated with Notch signaling, possibly explaining these differentiation deficits. Here, we hypothesized that mice lacking germline MYO5B would have a similar lack of Wnt‐associated gene expression, and that Wnt inhibition in wildtype mouse crypt‐derived enteroids would give rise to microvillus inclusions, as observed n MYO5B knockouts.MethodsSegments of duodenum and jejunum were isolated from postnatal day (PND) 4 germline MYO5B knockout and littermate control mice. RNA was isolated from each tissue and transcript expression of Wnt ligands were analyzed by qPCR. A segment of jejunum was collected from adult wildtype C57Bl/6 mice, and the crypt was isolated to generate enteroids. After passaging in expansion media, the enteroids were cultured in differentiation‐inducing media containing Wnt inhibitors for three days. Enteroids were fixed and whole mount stained to visualize overall structure, tight junctions, and basolateral markers using confocal microscopy.ResultsLoss of germline MYO5B resulted in a decrease in transcript levels of most Wnt ligands in the duodenum and jejunum, however only the loss of Wnt4 expression was significant. Wnt secretion inhibitor (C59) enhanced microvillus inclusion formation in MYO5B deficient enteroids. Unexpectedly, C59 treatment in both MYO5B deficient and wildtype mouse enteroids induced the formation of large, intercellular lateral lumens, rather than small, intracellular microvillus inclusions (Fig. 1). Enteroids generated from adult wildtype mice similarly contained lateral lumens induced by C59, but in less numbers than that seen in neonatal mouse‐derived enteroids. These lateral lumens were not present in vehicle‐treated enteroids. Immunofluorescence studies illustrated that most of these lateral lumens expressed tight junction (ZO‐1) and basolateral (E‐cadherin) markers, again suggesting these structures are lateral lumens rather than microvillus inclusions. Similarly, treatment of wildtype enteroids with canonical Wnt signaling inhibitors, salinomycin and pyrvinium, gave rise to a similar lateral lumen phenotype.ConclusionsGermline MYO5B expression is critical for the intestinal expression of some (i.e., Wnt 4), but not all Wnt ligands. Wnt inhibition in enteroids induced the formation of lateral lumens, rather than microvillus inclusions, in wild type differentiated enteroids. This unexpected phenotype suggests that Wnt signaling promotes the establishment of proper polarity in intestinal epithelial cells.