Lysophosphatidic acid enhances intestinal tuft cell differentiation in Myo5b KO mice independent of Pou2f3

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We have reported that adult tamoxifen‐induced, intestine‐specific, Myosin Vb knockout (Myo5b KO) mice recapitulate malabsorption syndrome in a congenital diarrheal disease, microvillus inclusion disease, and that daily treatment with lysophosphatidic acid (LPA) stimulated brush border maturation and sodium/glucose absorption in the Myo5b KO mice. Influence of Myo5b loss and LPA in sensory cell differentiation had not been studied. The present study evaluated the global changes in gene expression following Myo5b loss and LPA treatment, focusing on the differentiation of a nutrient sensor cell lineage, tuft cell. VillinCreERT2;Myo5bflox/flox mice were treated with LPA or vehicle for 4 days after a single tamoxifen injection. Immunostaining on small intestinal sections and whole‐mounted enteroids was used to quantify tuft cell density. RNA sequencing was performed on isolated jejunal epithelial cells. The sections of entire small intestine were analyzed by whole‐slide scanning and digital analysis with Cell Profiler. The frequency of DCLK1+ tuft cell was decreased by 80% with Myo5b loss, whereas enteroendocrine cells showed no difference between Myo5b KO mice and littermate controls. Paneth cell numbers were significantly increased in Myo5b KO, correlating with hyperproliferation in crypts. LPA did not alter the increased Paneth cells or Ki67+ cells. Intraperitoneal LPA treatment re‐established the frequency of tuft cells in Myo5b KO mice, indicating that a tuft cell‐specific differentiation mechanism was activated by LPA. In contrast, enteroids that were generated from VillinCreERT2;Myo5Bflox/flox jejunum had no difference in tuft cell number between hydroxytamoxifen‐treated (iKO) and control enteroids. The disrupted tuft cell differentiation by Myo5b loss was not represented in in vitro system, although microvillus formation was immature in iKO enteroids. RNA‐seq revealed that 844 genes in jejunal epithelial cells were significantly down‐regulated, and 657 genes were up‐regulated by tamoxifen‐induced loss of Myo5b compared to control. Three stem cell transcription factors were significantly increased in KO, whereas two tuft cell‐specific transcription factors (Spib and Pou2f3) were decreased. Epithelial Myo5b loss demonstrated a broad impact on epithelial cell differentiation and maturation. Treatment of Myo5b KO mice with ip LPA up‐regulated only 9 genes and had no effect on the expression of transcription factors, suggesting that LPA may modulate transporter trafficking to stimulate nutrient absorption rather than change the transcription. Myo5b loss abolished the differentiation of a sensory tuft cell, and in vivo treatment with LPA re‐established the tuft cell population independent of Pou2f3 expression. These findings suggest unexplored functions for Myo5b and LPA signaling in cell lineage choice in the intestinal epithelial cells, interacting with non‐epithelial cells.Support or Funding InformationR01 DK48370 and R01 DK70856 and a gift from the Christine Volpe Fund to J.R.G. This work was supported by core resources of the Vanderbilt Digestive Disease Center (P30 DK058404), the Vanderbilt‐Ingram Cancer Center (P30 CA68485), VUMC Digital Histology Shared Resource (supported by a VA Shared Equipment Grant 1IS1BX003097).