Sox4 drives Atoh1-independent intestinal secretory differentiation toward tuft and enteroendocrine fates

Background & Aims The intestinal epithelium is maintained by intestinal stem cells (ISCs), which produce post-mitotic absorptive and secretory epithelial cells. Initial fate specification toward enteroendocrine, goblet, and Paneth cell lineages is dependent on Atoh1 , a master regulator of secretory differentiation. However, the origin of tuft cells, which participate in Type II immune responses to parasitic infection, is less clear and appears to occur in an Atoh1 -independent manner. Here we examine the role of Sox4 in ISC proliferation and differentiation. Methods We used mice with intestinal epithelial-specific conditional knockout of Sox4 (Sox4fl/fl:vilCre; Sox4cKO) to study the role of Sox4 in the small intestine. Crypt- and single cell-derived organoids were used to assay proliferation and ISC potency between control and Sox4cKO mice. Lineage allocation and genetic consequences of Sox4 ablation were studied by immunofluorescence, RT-qPCR, and RNA-seq. In vivo infection with helminths and in vitro cytokine treatment in primary intestinal organoids were used to assess tuft cell hyperplasia in control and Sox4cKO samples. Atoh1 GFP reporter mice and single cell RNA-seq (scRNA-seq) were used to determine co-localization of SOX4 and Atoh1 . Wild-type and inducible Atoh1 knockout (Atoh1fl/fl:vilCreER; Atoh1iKO) organoids carrying an inducible Sox4 overexpression vector (Sox4OE) were used to determine the role of Atoh1 in Sox4 driven secretory differentiation. Results Loss of Sox4 impairs ISC function and secretory differentiation, resulting in decreased numbers of enteroendocrine and tuft cells. In wild-type mice, SOX4+ cells are significantly upregulated following helminth infection coincident with tuft cell hyperplasia. Sox4 is activated by IL13 in vitro and Sox4cKO knockout mice demonstrate impaired tuft cell hyperplasia and parasite clearance following infection with helminths. A subset of Sox4 -expressing cells colocalize with Atoh1 and enteroendocrine markers by scRNA-seq, while Sox4+ / Atoh1 -cells correlate strongly with tuft cell populations. Gain-of-function studies in primary organoids demonstrate that Sox4 is sufficient to drive both enteroendocrine and tuft cell differentiation, and can do so in the absence of Atoh1 . Conclusion Our data demonstrate that Sox4 promotes enteroendocrine and tuft cell lineage allocation independently of Atoh1 . These results challenge long-standing views of Atoh1 as the sole regulator of secretory differentiation in the intestine and are relevant for understanding host epithelial responses to parasitic infection.