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Goblet cell diversity An adult human has a gut surface area averaging 30 square meters that is bombarded daily by xenobiotics and microorganisms. Mucus synthesized by goblet cells supplies a protective barrier coating. Nyström et al. discovered that goblet cells are not all the same along the length of the gut; rather, they form different functional populations depending on location. Small-intestine mucus is laced with antimicrobial peptides and is permeable to small molecules; downstream, thicker mucus is generated that excludes bacteria and xenobiotics. Mucus oozes in thick plumes from goblet cells within the crypts to shield the stem cell niche. Between the crypts lie highly differentiated goblet cells producing permeable mucus. Together, both types of mucus form a network that shelters the gut epithelium. If, however, the intercrypt goblet cells become dysfunctional, the exposed epithelium is exposed to bacteria and vulnerable to developing colitis. Science, this issue p. eabb1590 Mucus lining the mammalian gut is produced by anatomically distinct subpopulations of goblet cells, which produce mucus with different functions. INTRODUCTION An intricate balance with our intestinal microbes is pivotal to human health. A key interface of host-microbial interactions occurs in the mucus that covers the intestinal epithelial surface. In the colon, the mucus layer serves as a barrier that inhibits direct epithelial contact with the dense population of microbes. Defects in this system are a hallmark of colitis. The mucus layer is structurally dependent on the polymeric mucin MUC2, which is synthesized by goblet cells (GCs)—specialized secretory cells classically viewed as a homogeneous cell type. Studies identifying divergent functional features in GC subpopulations, including differential mucus biosynthesis rates and responses to bacteria, suggest that GC populations may in fact be heterogeneous. RATIONALE In the present study, we characterized intestinal GC expression diversity and defined how a specific GC subtype, localized in the intercrypt surface epithelium, functionally contributes to the formation of the mucus barrier. RESULTS Using mCherry-MUC2 transgenic mice to sort and isolate GCs, we generated transcriptomic and proteomic profiles to characterize the GC expression landscape in both the small intestine and the colon. Single-cell transcriptomic analysis revealed several distinct GC clusters in each tissue region, which segregated into two separate trajectories. One trajectory had enriched expression of known GC-specific genes (e.g., Clca1 and Fcgbp) and was designated as canonical GCs. Conversely, the other trajectory was enriched for the expression of genes typically associated with enterocytes (e.g., Dmbt1 and Gsdmc4), so we designated this trajectory as noncanonical. In the colon, the most differentiated GCs are the high–mucus turnover cells localized to the surface epithelium between crypts, which were designated as intercrypt GCs (icGCs). These cells had expression profiles that were distinct from those of crypt-resident GCs, so we investigated their role in forming the mucus barrier. We exploited the lectin-binding features of mucus to resolve the three-dimensional organization of mucus in live tissue explants. Results demonstrated that icGCs secreted distinct mucus that filled the spatial regions between mucus plumes secreted from crypt openings. The intercrypt mucus was impenetrable to bacteria-sized beads; however, it was more penetrable to smaller molecules compared with crypt plume mucus. Penetrable surface mucus may be important for the absorption of ions and other compounds, whereas denser mucus within the crypt compartment contributes to the shielding of the stem cell niche. Both mucus subtypes appear to be important for the overall protective function of mucus, because a barrier impenetrable to bacteria was formed by the mixed, net-like organization of intercrypt and crypt plume mucus. A mouse model with dysfunctional icGCs lacking normal intercrypt mucus exhibited an inadequate mucus barrier and was more susceptible to both chemically induced and spontaneous (age-dependent) colitis, which demonstrates the indispensable role of icGCs in maintaining a functional mucus barrier. Furthermore, biopsies from patients with ulcerative colitis—including those in remission—exhibited increased GC shedding and reduced icGC numbers. These characteristics were associated with structural defects in the mucus barrier, including gaps in the intercrypt mucus that exposed areas of the surface epithelium. CONCLUSION The current study identifies GCs as a heterogeneous population of cells with diverse functional features that indicate a dynamic cellular system. GCs at different locations along the crypt-surface axis contribute to a functional mucus barrier that protects the epithelium from microorganisms. The icGCs possess a specific role in mucus organization, where their malfunction is associated with colitis in both mice and humans. Diversity of GC expression and function. Intestinal GCs can be divided into several distinct clusters that form canonical and noncanonical trajectories, and gene expression profiles outline their topographical position. The specialized surface GC type—the icGCs—secrete mucus crucial for forming a protective barrier between bacteria and the epithelium. Depletion of icGCs impairs mucus function, a phenomenon observed in ulcerative colitis (UC) patients. The intestinal mucus layer, an important element of epithelial protection, is produced by goblet cells. Intestinal goblet cells are assumed to be a homogeneous cell type. In this study, however, we delineated their specific gene and protein expression profiles and identified several distinct goblet cell populations that form two differentiation trajectories. One distinct subtype, the intercrypt goblet cells (icGCs), located at the colonic luminal surface, produced mucus with properties that differed from the mucus secreted by crypt-residing goblet cells. Mice with defective icGCs had increased sensitivity to chemically induced colitis and manifested spontaneous colitis with age. Furthermore, alterations in mucus and reduced numbers of icGCs were observed in patients with both active and remissive ulcerative colitis, which highlights the importance of icGCs in maintaining functional protection of the epithelium.