Spatial transcriptomics add a new dimension to our understanding of the gut.

The profound complexity of the intestinal mucosa demands a spatial approach to the study of gut transcriptomics. Although single-cell RNA sequencing has revolutionized our ability to survey the diverse cell types of the intestine, knowledge of cell type alone cannot fully describe the cells that make up the intestinal mucosa. During homeostasis and disease, dramatic gradients of oxygen, nutrients, extracellular matrix proteins, morphogens, and microbiota collectively dictate intestinal cell state, and only spatial techniques can articulate differences in cellular transcriptomes at this level. Spatial transcriptomic techniques assign transcriptomic data to precise regions in a tissue of interest. In recent years, these protocols have become increasingly accessible, and their application in the intestinal mucosa has exploded in popularity. In the gut, spatial transcriptomics typically involve the application of tissue sections to spatially barcoded RNA sequencing or laser capture microdissection followed by RNA sequencing. In combination with single-cell RNA sequencing, these spatial sequencing approaches allow for the construction of spatial transcriptional maps at pseudosingle-cell resolution. In this review, we describe the spatial transcriptomic technologies recently applied in the gut and the previously unattainable discoveries that they have produced.

IGF2BP1/IMP1 Deletion Enhances a Facultative Stem Cell State via Regulation of MAP1LC3B.

BACKGROUND & AIMS: The intestinal epithelium interfaces with a diverse milieu of luminal contents while maintaining robust digestive and barrier functions. Facultative intestinal stem cells are cells that survive tissue injury and divide to re-establish the epithelium. Prior studies have shown autophagic state as functional marker of facultative intestinal stem cells, but regulatory mechanisms are not known. The current study evaluated a post-transcriptional regulation of autophagy as an important factor for facultative stem cell state and tissue regeneration. METHODS: We evaluated stem cell composition, autophagic vesicle content, organoid formation, and in vivo regeneration in mice with intestinal epithelial deletion of the RNA binding protein IGF2 messenger RNA binding protein 1 (IMP1). The contribution of autophagy to resulting in vitro and in vivo phenotypes was evaluated via genetic inactivation of Atg7. Molecular analyses of IMP1 modulation of autophagy at the protein and transcript localization levels were performed using IMP1 mutant studies and single-molecule fluorescent in situ hybridization. RESULTS: Epithelial Imp1 deletion reduced leucine rich repeat containing G protein coupled receptor 5 cell frequency but enhanced both organoid formation efficiency and in vivo regeneration after irradiation. We confirmed prior studies showing increased autophagy with IMP1 deletion. Deletion of Atg7 reversed the enhanced regeneration observed with Imp1 deletion. IMP1 deletion or mutation of IMP1 phosphorylation sites enhanced expression of essential autophagy protein microtubule-associated protein 1 light chain 3ß. Furthermore, immunofluorescence imaging coupled with single-molecule fluorescent in situ hybridization showed IMP1 colocalization with MAP1LC3B transcripts at homeostasis. Stress induction led to decreased colocalization. CONCLUSIONS: Depletion of IMP1 enhances autophagy, which promotes intestinal regeneration via expansion of facultative intestinal stem cells.

The leptin receptor has no role in delta-cell control of beta-cell function in the mouse.

Introduction: Leptin inhibits insulin secretion from isolated islets from multiple species, but the cell type that mediates this process remains elusive. Several mouse models have been used to explore this question. Ablation of the leptin receptor (Lepr) throughout the pancreatic epithelium results in altered glucose homeostasis and ex vivo insulin secretion and Ca2+ dynamics. However, Lepr removal from neither alpha nor beta cells mimics this result. Moreover, scRNAseq data has revealed an enrichment of LEPR in human islet delta cells. Methods: We confirmed LEPR upregulation in human delta cells by performing RNAseq on fixed, sorted beta and delta cells. We then used a mouse model to test whether delta cells mediate the diminished glucose-stimulated insulin secretion in response to leptin. Results: Ablation of Lepr within mouse delta cells did not change glucose homeostasis or insulin secretion, whether mice were fed a chow or high-fat diet. We further show, using a publicly available scRNAseq dataset, that islet cells expressing Lepr lie within endothelial cell clusters. Conclusions: In mice, leptin does not influence beta-cell function through delta cells.

FOXM1 acts sexually dimorphically to regulate functional ß-cell mass.

The transcription factor FOXM1 regulates ß-cell proliferation and insulin secretion. Our previous work demonstrates that expressing an activated form of FOXM1 (FOXM1*) in ß cells increases ß-cell proliferation and mass in aged male mice. Additionally, FOXM1* enhances ß-cell function even in young mice, in which no ß-cell mass elevation occurs. Here, we demonstrate that FOXM1 acts in a sexually dimorphic manner in the ß cell. Expression of FOXM1* in female mouse ß cells does not affect ß-cell proliferation or glucose tolerance. Transduction of male but not female human islets with FOXM1* enhances insulin secretion in response to elevated glucose. Estrogen contributes to diabetes susceptibility differences between males and females, and the estrogen receptor (ER)α is the primary mediator of ß-cell estrogen signaling. We show that FOXM1* can rescue impaired glucose tolerance in female mice with a pancreas-wide ERα deletion. Further, FOXM1 and ERα binding sites overlap with each other and with other ß-cell-enriched transcription factors, including ISL1, PAX6, MAF, and GATA. These data indicate that FOMX1 and ERα cooperate to regulate ß-cell function and suggest a general mechanism contributing to the lower incidence of diabetes observed in women.

Deletion of Endogenous Neuregulin-4 Limits Adaptive Immunity During Interleukin-10 Receptor-Neutralizing Colitis.

BACKGROUND: Growth factors are essential for maintenance of intestinal health. We previously showed that exogenous neuregulin-4 (NRG4) promotes colonocyte survival during cytokine challenge and is protective against acute models of intestinal inflammation. However, the function(s) of endogenous NRG4 are not well understood. Using NRG4-/- mice, we tested the role of endogenous NRG4 in models of colitis skewed toward either adaptive (interleukin-10 receptor [IL-10R] neutralization) or innate (dextran sulfate sodium [DSS]) immune responses. METHODS: NRG4-/- and wild-type cage mate mice were subjected to chronic IL-10R neutralization colitis and acute DSS colitis. Disease was assessed by histological examination, inflammatory cytokine levels, fecal lipocalin-2 levels, and single cell mass cytometry immune cell profiling. Homeostatic gene alterations were evaluated by RNA sequencing analysis from colonic homogenates, with real-time quantitative polymerase chain reaction confirmation in both tissue and isolated epithelium. RESULTS: During IL-10R neutralization colitis, NRG4-/- mice had reduced colonic inflammatory cytokine expression, histological damage, and colonic CD8+ T cell numbers vs wild-type cage mates. Conversely, in DSS colitis, NRG4-/- mice had elevated cytokine expression, fecal lipocalin-2 levels, and impaired weight recovery. RNA sequencing showed a loss of St3gal4, a sialyltransferase involved in immune cell trafficking, in NRG4-null colons, which was verified in both tissue and isolated epithelium. The regulation of St3gal4 by NRG4 was confirmed with ex vivo epithelial colon organoid cultures from NRG4-/- mice and by induction of St3gal4 in vivo following NRG4 treatment. CONCLUSIONS: NRG4 regulates colonic epithelial ST3GAL4 and thus may allow for robust recruitment of CD8+ T cells during adaptive immune responses in colitis. On the other hand, NRG4 loss exacerbates injury driven by innate immune responses.

Neuregulin-4 (NRG4) is a growth factor that protects the epithelial cells lining the colon from injury and restrains innate (non-specific) immune responses. Here we show that NRG4's role in inflammation is context-specific, and mice that lack NRG4 have impaired adaptive immunity in a model of chronic immune-mediated colitis.

Deep Crypt Secretory Cell Differentiation in the Colonic Epithelium Is Regulated by Sprouty2 and Interleukin 13.

BACKGROUND & AIMS: Deep crypt secretory (DCS) cells are a critical component of the colonic stem cell niche. However, the regulatory mechanisms controlling DCS cell numbers and function are not well understood. Sprouty2 is an inflammation-responsive regulator of intracellular signaling that influences colonic secretory cell numbers in colitis via an epithelial-stromal interleukin (IL)33/IL13 signaling loop. Here, we tested the hypothesis that IL13, induced by epithelial Sprouty2 down-regulation, promotes DCS cell differentiation and function. METHODS: Distal colons from mice with an intestinal epithelial-specific Sprouty2 deletion (Spry2ΔIE) and littermate controls were analyzed by in situ hybridization for Reg4+ DCS cells. Single-cell RNA sequencing and immunostaining were used to identify DCS cell-derived host defense peptides (HDPs) and localization of IL13 and IL13 receptor; bulk RNA sequencing and quantitative polymerase chain reaction were used to quantify changes in expression of identified HDPs. Cytokine-treated colonoids were assessed for DCS cells. A requirement for an IL33/IL13 signaling loop in the regulation of DCS cells was assessed in vivo using IL13 null mice. RESULTS: Reg4+ DCS cell numbers were increased 2-fold in distal colons of Spry2ΔIE mice with a concomitant overall increase in DCS cell marker expression (Reg4, Spink4, and Agr2). Single-cell transcriptomics showed the HDP Retnlb/Resistin Like Beta (RELMß) is highly enriched in DCS cells. Retnlb/RELMß expression was increased in Spry2ΔIE colons. IL13, but not IL33, induced Reg4 and Retnlb expression in colonic epithelial organoids, and IL33-mediated expansion of the DCS cell population in vivo was dependent on IL13, which was expressed predominantly by type II innate lymphoid cells in the colonic mucosa. CONCLUSIONS: Sprouty2 limits colonic DCS cell differentiation through suppression of IL13 signaling. At homeostasis, DCS cells are marked by high levels of the HDP RELMß. Loss of epithelial Sprouty2 activates type II innate lymphoid cells to release IL13, promoting expansion of the DCS cell population and increased colonic RELMß levels.

Intestinal transit-amplifying cells require METTL3 for growth factor signaling and cell survival.

Intestinal epithelial transit-amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite these cells' critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit-amplifying cell function. We report that RNA methyltransferase-like 3 (METTL3) is required for survival of transit-amplifying cells in the murine small intestine. Transit-amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Sequencing of polysome-bound and methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation verified a relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit-amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine with important implications for both homeostatic tissue renewal and epithelial regeneration.

Intestinal transit amplifying cells require METTL3 for growth factor signaling, KRAS expression, and cell survival.

Intestinal epithelial transit amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite their critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit amplifying cell function. We report that the RNA methyltransferase, METTL3, is required for survival of transit amplifying cells in the murine small intestine. Transit amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Ribosome profiling and sequencing of methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of unique methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation confirmed a novel relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine, with important implications for both homeostatic tissue renewal and epithelial regeneration.

Sprouty2 limits intestinal tuft and goblet cell numbers through GSK3ß-mediated restriction of epithelial IL-33.

Dynamic regulation of intestinal cell differentiation is crucial for both homeostasis and the response to injury or inflammation. Sprouty2, an intracellular signaling regulator, controls pathways including PI3K and MAPKs that are implicated in differentiation and are dysregulated in inflammatory bowel disease. Here, we ask whether Sprouty2 controls secretory cell differentiation and the response to colitis. We report that colonic epithelial Sprouty2 deletion leads to expanded tuft and goblet cell populations. Sprouty2 loss induces PI3K/Akt signaling, leading to GSK3ß inhibition and epithelial interleukin (IL)-33 expression. In vivo, this results in increased stromal IL-13+ cells. IL-13 in turn induces tuft and goblet cell expansion in vitro and in vivo. Sprouty2 is downregulated by acute inflammation; this appears to be a protective response, as VillinCre;Sprouty2F/F mice are resistant to DSS colitis. In contrast, Sprouty2 is elevated in chronic colitis and in colons of inflammatory bowel disease patients, suggesting that this protective epithelial-stromal signaling mechanism is lost in disease.