Mutations in the innate immune receptor NOD2 are the greatest single genetic risk factors for Crohn’s disease, yet the mechanisms by which NOD2 regulates intestinal homeostasis remain unclear. Here, we used a CRISPR-generated zebrafish model to determine the impacts of NOD2 deficiency on intestinal health. In a series of cellular, molecular, and transcriptomic studies, we uncovered substantial effects of NOD2 deficiency on epithelial and immune compartments, including deregulated expression of developmental pathways critical for establishment and maintenance of the gut epithelium, and an unexpected increase in the expression of multiple estrogen-response genes. In a series of functional assays, we uncovered a mechanistic link between estrogenic signals and NOD2-deficiency phenotypes, whereby exposure to estrogen alone replicated NOD2-deficiency phenotypes, and exposure to an estrogen receptor antagonist reverted the epithelial defects observed in NOD2 mutants. Our findings identify a critical NOD2-estrogen regulatory axis in the establishment of intestinal homeostasis and suggest that hormonal signaling may contribute to the sex-specific pathogenesis of Crohn’s disease. ### Competing Interest Statement The authors have declared no competing interest. Canadian Institutes of Health Research, https://ror.org/01gavpb45, MOP77746

Specialist intestinal epithelial cells are critical for barrier integrity and immune responses at the mucosal boundary, yet the pathways that govern their development are incompletely defined. Here, we identify an essential role for TNFRSF11A/RANK in shaping intestinal epithelial specialization in zebrafish. Using lineage trajectory analysis, we identified two tuft cell subtypes, including a subtype enriched for expression of genes required to produce pro-inflammatory leukotrienes. We showed that RANK deficiency reduced the abundance of type-2 tuft cells and BEST4 cells, increased goblet cell frequency, and promoted the accumulation of pro-inflammatory leukocytes in the gut. Functionally, we demonstrated that BEST4 cell numbers expand following infection with a pandemic strain of Vibrio cholerae , implicating this lineage in host defense against enteric pathogens. Together, our findings establish RANK as a critical determinant of epithelial diversification, linking loss of RANK to impaired epithelial immune regulation and an elevated inflammatory state. ### Competing Interest Statement The authors have declared no competing interest. Canadian Institutes of Health Research, https://ror.org/01gavpb45, MOP77746