ABSTRACT
A key aspect of nutrient absorption is the exquisite division of labour across the length of the small intestine, with individual nutrients taken up at different proximal:distal positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum and ileum. By examining the fine-scale longitudinal transcriptional patterns that span the mouse and human small intestine, we instead identified five domains of nutrient absorption that mount distinct responses to dietary changes, and three regional stem cell populations. Molecular domain identity can be detected with machine learning, which provides a systematic method to computationally identify intestinal domains in mice. We generated a predictive model of transcriptional control of domain identity and validated the roles of Ppar-δ and Cdx1 in patterning lipid metabolism-associated genes. These findings represent a foundational framework for the zonation of absorption across the mammalian small intestine.
Subject(s)
Duodenum , Intestine, Small , Humans , Mice , Animals , Intestine, Small/metabolism , Duodenum/metabolism , Intestines , Jejunum/metabolism , Ileum/metabolism , MammalsABSTRACT
A key aspect of nutrient absorption is the exquisite division of labor across the length of the small intestine, with individual classes of micronutrients taken up at different positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum, and ileum. By examining fine-scale longitudinal segmentation of the mouse and human small intestines, we identified transcriptional signatures and upstream regulatory factors that define five domains of nutrient absorption, distinct from the three traditional sections. Spatially restricted expression programs were most prominent in nutrient-absorbing enterocytes but initially arose in intestinal stem cells residing in three regional populations. While a core signature was maintained across mice and humans with different diets and environments, domain properties were influenced by dietary changes. We established the functions of Ppar-Ạand Cdx1 in patterning lipid metabolism in distal domains and generated a predictive model of additional transcription factors that direct domain identity. Molecular domain identity can be detected with machine learning, representing the first systematic method to computationally identify specific intestinal regions in mice. These findings provide a foundational framework for the identity and control of longitudinal zonation of absorption along the proximal:distal small intestinal axis.
ABSTRACT
The gastrointestinal tract relies on the production, maturation, and transit of mucin to protect against pathogens and to lubricate the epithelial lining. Although the molecular and cellular mechanisms that regulate mucin production and movement are beginning to be understood, the upstream epithelial signals that contribute to mucin regulation remain unclear. Here, we report that the inflammatory cytokine tumor necrosis factor (TNF), generated by the epithelium, contributes to mucin homeostasis by regulating both cell differentiation and cystic fibrosis transmembrane conductance regulator (CFTR) activity. We used genetic mouse models and noninflamed samples from patients with inflammatory bowel disease (IBD) undergoing anti-TNF therapy to assess the effect of in vivo perturbation of TNF. We found that inhibition of epithelial TNF promotes the differentiation of secretory progenitor cells into mucus-producing goblet cells. Furthermore, TNF treatment and CFTR inhibition in intestinal organoids demonstrated that TNF promotes ion transport and luminal flow via CFTR. The absence of TNF led to slower gut transit times, which we propose results from increased mucus accumulation coupled with decreased luminal fluid pumping. These findings point to a TNF/CFTR signaling axis in the adult intestine and identify epithelial cell-derived TNF as an upstream regulator of mucin homeostasis.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator , Mucins , Humans , Animals , Mice , Mucins/genetics , Mucins/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Tumor Necrosis Factor Inhibitors , Epithelial Cells/metabolism , Cell Differentiation , Tumor Necrosis Factors , HomeostasisABSTRACT
Epithelial organoids derived from intestinal tissue, called enteroids, recapitulate many aspects of the organ in vitro and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identified an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells and feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown enteroids, and EREG-grown enteroids showed that EGF enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.
Subject(s)
Epidermal Growth Factor , Intestines , Humans , Epiregulin , Intestinal Mucosa , Cell DifferentiationABSTRACT
The intestine is a site of direct encounter with the external environment and must consequently balance barrier defense with nutrient uptake. To investigate how nutrient uptake is regulated in the small intestine, we tested the effect of diets with different macronutrient compositions on epithelial gene expression. We found that enzymes and transporters required for carbohydrate digestion and absorption were regulated by carbohydrate availability. The "on-demand" induction of this machinery required γδ T cells, which regulated this program through the suppression of interleukin-22 production by type 3 innate lymphoid cells. Nutrient availability altered the tissue localization and transcriptome of γδ T cells. Additionally, transcriptional responses to diet involved cellular remodeling of the epithelial compartment. Thus, this work identifies a role for γδ T cells in nutrient sensing.
Subject(s)
Dietary Carbohydrates/administration & dosage , Dietary Carbohydrates/metabolism , Enterocytes/physiology , Interleukins/metabolism , Intestinal Mucosa/physiology , Receptors, Antigen, T-Cell, gamma-delta , T-Lymphocyte Subsets/physiology , Adaptation, Physiological , Animals , Cell Communication , Dietary Proteins/administration & dosage , Digestion , Gene Expression Regulation , Interleukins/genetics , Intestinal Absorption , Intestinal Mucosa/cytology , Intestine, Small/cytology , Intestine, Small/metabolism , Mice, Inbred C57BL , Nutrients/administration & dosage , Nutrients/metabolism , T-Lymphocyte Subsets/immunology , Transcription, Genetic , Transcriptome , Interleukin-22ABSTRACT
The intestinal epithelium is a structured organ composed of crypts harboring Lgr5+ stem cells, and villi harboring differentiated cells. Spatial transcriptomics have demonstrated profound zonation of epithelial gene expression along the villus axis, but the mechanisms shaping this spatial variability are unknown. Here, we combine laser capture micro-dissection and single cell RNA sequencing to uncover spatially zonated populations of mesenchymal cells along the crypt-villus axis. These include villus tip telocytes (VTTs) that express Lgr5, a gene previously considered a specific crypt epithelial stem cell marker. VTTs are elongated cells that line the villus tip epithelium and signal through Bmp morphogens and the non-canonical Wnt5a ligand. Their ablation is associated with perturbed zonation of enterocyte genes induced at the villus tip. Our study provides a spatially-resolved cell atlas of the small intestinal stroma and exposes Lgr5+ villus tip telocytes as regulators of the epithelial spatial expression programs along the villus axis.
Subject(s)
Enterocytes/metabolism , Intestinal Mucosa/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Enterocytes/cytology , Intestinal Mucosa/cytology , Intestine, Small/cytology , Intestine, Small/metabolism , Male , Mice , Mice, Inbred C57BL , Receptors, G-Protein-Coupled/genetics , Stromal Cells/metabolism , Wnt-5a Protein/metabolismABSTRACT
The gastrointestinal (GI) tract renews frequently to sustain nutrient digestion and absorption in the face of consistent tissue stress. In many species, proliferative intestinal stem cells (ISCs) are responsible for the repair of the damage arising from chemical and mechanical aspects of food breakdown and exposure to pathogens. As the cellular source of all mature cell types of the intestinal epithelium throughout adulthood, ISCs hold tremendous therapeutic potential for understanding and treating GI disease in humans. This review focuses on recent advances in our understanding of ISC identity, behavior, and regulation during homeostasis and injury-induced repair, as revealed by two major animal models used to study regeneration of the small intestine: Drosophila melanogaster and Mus musculus. We emphasize recent findings from Drosophila that are likely to translate to the mammalian GI system, as well as challenging topics in mouse ISC biology that may be ideally suited for investigation in flies. For context, we begin by reviewing major physiological similarities and distinctions between the Drosophila midgut and mouse small intestine.
Subject(s)
Cell Proliferation/physiology , Intestines/physiology , Regeneration/physiology , Stem Cells/cytology , Animals , Drosophila , Homeostasis/physiology , Humans , Intestines/cytology , Mice , Stem Cells/metabolismABSTRACT
During tissue repair, myofibroblasts produce extracellular matrix (ECM) molecules for tissue resilience and strength. Altered ECM deposition can lead to tissue dysfunction and disease. Identification of distinct myofibroblast subsets is necessary to develop treatments for these disorders. We analyzed profibrotic cells during mouse skin wound healing, fibrosis, and aging and identified distinct subpopulations of myofibroblasts, including adipocyte precursors (APs). Multiple mouse models and transplantation assays demonstrate that proliferation of APs but not other myofibroblasts is activated by CD301b-expressing macrophages through insulin-like growth factor 1 and platelet-derived growth factor C. With age, wound bed APs and differential gene expression between myofibroblast subsets are reduced. Our findings identify multiple fibrotic cell populations and suggest that the environment dictates functional myofibroblast heterogeneity, which is driven by fibroblast-immune interactions after wounding.
Subject(s)
Macrophages/physiology , Myofibroblasts/physiology , Re-Epithelialization/physiology , Skin/injuries , Wound Healing/physiology , Adipocytes/physiology , Animals , Cell Proliferation , Extracellular Matrix/metabolism , Fibrosis , Integrin beta1/genetics , Keloid/pathology , Lectins, C-Type/analysis , Lectins, C-Type/metabolism , Lymphokines/metabolism , Mesenchymal Stem Cells/physiology , Mice , Mice, Inbred C57BL , Platelet-Derived Growth Factor/metabolism , Re-Epithelialization/genetics , Skin/immunology , Skin/pathology , Skin Aging/physiology , Transcriptome , Wound Healing/geneticsABSTRACT
Adipocytes undergo pronounced changes in size and behavior to support diverse tissue functions, but the mechanisms that control these changes are not well understood. Mammary gland-associated white adipose tissue (mgWAT) regresses in support of milk fat production during lactation and expands during the subsequent involution of milk-producing epithelial cells, providing one of the most marked physiological examples of adipose growth. We examined cellular mechanisms and functional implications of adipocyte and lipid dynamics in the mouse mammary gland (MG). Using in vivo analysis of adipocyte precursors and genetic tracing of mature adipocytes, we find mature adipocyte hypertrophy to be a primary mechanism of mgWAT expansion during involution. Lipid tracking and lipidomics demonstrate that adipocytes fill with epithelial-derived milk lipid. Furthermore, ablation of mgWAT during involution reveals an essential role for adipocytes in milk trafficking from, and proper restructuring of, the mammary epithelium. This work advances our understanding of MG remodeling and tissue-specific roles for adipocytes.
Subject(s)
Adipocytes/cytology , Lipid Metabolism , Mammary Glands, Animal/cytology , Mammary Glands, Human/cytology , Adipocytes/metabolism , Adipocytes, White/cytology , Adipocytes, White/physiology , Animals , Breast Feeding , Cell Size , Epithelial Cells/cytology , Epithelial Cells/physiology , Fatty Acids/metabolism , Female , Humans , Lactation/physiology , Mammary Glands, Animal/physiology , Mammary Glands, Human/physiology , Mice, Inbred C57BL , Mice, Transgenic , PregnancyABSTRACT
Adipose tissue depots can exist in close association with other organs, where they assume diverse, often non-traditional functions. In stem cell-rich skin, bone marrow, and mammary glands, adipocytes signal to and modulate organ regeneration and remodeling. Skin adipocytes and their progenitors signal to hair follicles, promoting epithelial stem cell quiescence and activation, respectively. Hair follicles signal back to adipocyte progenitors, inducing their expansion and regeneration, as in skin scars. In mammary glands and heart, adipocytes supply lipids to neighboring cells for nutritional and metabolic functions, respectively. Adipose depots adjacent to skeletal structures function to absorb mechanical shock. Adipose tissue near the surface of skin and intestine senses and responds to bacterial invasion, contributing to the body's innate immune barrier. As the recognition of diverse adipose depot functions increases, novel therapeutic approaches centered on tissue-specific adipocytes are likely to emerge for a range of cancers and regenerative, infectious, and autoimmune disorders.
Subject(s)
Adipose Tissue/anatomy & histology , Adipose Tissue/physiology , Animals , Humans , Models, Biological , Organ SpecificityABSTRACT
Activation of the human embryonic stem cell (hESC) signature genes has been observed in various epithelial cancers. In this study, we found that the hESC signature is selectively induced in the airway basal stem/progenitor cell population of healthy smokers (BC-S), with a pattern similar to that activated in all major types of human lung cancer. We further identified a subset of 6 BC-S hESC genes, whose coherent overexpression in lung adenocarcinoma (AdCa) was associated with reduced lung function, poorer differentiation grade, more advanced tumor stage, remarkably shorter survival, and higher frequency of TP53 mutations. BC-S shared with hESC and a considerable subset of lung carcinomas a common TP53 inactivation molecular pattern which strongly correlated with the BC-S hESC gene expression. These data provide transcriptome-based evidence that smoking-induced reprogramming of airway BC toward the hESC-like phenotype might represent a common early molecular event in the development of aggressive lung carcinomas in humans.
Subject(s)
Embryonic Stem Cells/metabolism , Gene Expression Profiling , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung/pathology , Smoking/genetics , Smoking/pathology , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adenocarcinoma of Lung , Animals , Cell Line, Tumor , Epithelium/metabolism , Epithelium/pathology , Gene Expression Regulation, Neoplastic , Humans , Lung/metabolism , Mice , Multivariate Analysis , Phenotype , Proportional Hazards Models , Survival Analysis , Tumor Suppressor Protein p53/metabolismABSTRACT
BACKGROUND: Ciliated cells play a central role in cleansing the airways of inhaled contaminants. They are derived from basal cells that include the airway stem/progenitor cells. In animal models, the transcription factor FOXJ1 has been shown to induce differentiation to the ciliated cell lineage, and the RFX transcription factor-family has been shown to be necessary for, but not sufficient to induce, correct cilia development. METHODS: To test the hypothesis that FOXJ1 and RFX3 cooperatively induce expression of ciliated genes in the differentiation process of basal progenitor cells toward a ciliated cell linage in the human airway epithelium, primary human airway basal cells were assessed under conditions of in vitro differentiation induced by plasmid-mediated gene transfer of FOXJ1 and/or RFX3. TaqMan PCR was used to quantify mRNA levels of basal, secretory, and cilia-associated genes. RESULTS: Basal cells, when cultured in air-liquid interface, differentiated into a ciliated epithelium, expressing FOXJ1 and RFX3. Transfection of FOXJ1 into resting basal cells activated promoters and induced expression of ciliated cell genes as well as both FOXJ1 and RFX3, but not basal cell genes. Transfection of RFX3 induced expression of RFX3 but not FOXJ1, nor the expression of cilia-related genes. The combination of FOXJ1 + RFX3 enhanced ciliated gene promoter activity and mRNA expression beyond that due to FOXJ1 alone. Corroborating immunoprecipitation studies demonstrated an interaction between FOXJ1 and RFX3. CONCLUSION: FOXJ1 is an important regulator of cilia gene expression during ciliated cell differentiation, with RFX3 as a transcriptional co-activator to FOXJ1, helping to induce the expression of cilia genes in the process of ciliated cell differentiation of basal/progenitor cells.
Subject(s)
Cilia/metabolism , DNA-Binding Proteins/metabolism , Forkhead Transcription Factors/metabolism , Respiratory Mucosa/metabolism , Respiratory Mucosa/ultrastructure , Stem Cells/cytology , Stem Cells/metabolism , Transcription Factors/metabolism , Cell Differentiation , Cells, Cultured , Cilia/ultrastructure , Gene Expression Regulation/physiology , Humans , Regulatory Factor X Transcription FactorsABSTRACT
BACKGROUND: The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. METHODOLOGY/PRINCIPAL FINDINGS: Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the "human airway basal cell signature" as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. CONCLUSION/SIGNIFICANCE: The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem/progenitor cells of the human airway epithelium.