Complement in breast milk modifies offspring gut microbiota to promote infant health.

Breastfeeding offers demonstrable benefits to newborns and infants by providing nourishment and immune protection and by shaping the gut commensal microbiota. Although it has been appreciated for decades that breast milk contains complement components, the physiological relevance of complement in breast milk remains undefined. Here, we demonstrate that weanling mice fostered by complement-deficient dams rapidly succumb when exposed to murine pathogen Citrobacter rodentium (CR), whereas pups fostered on complement-containing milk from wild-type dams can tolerate CR challenge. The complement components in breast milk were shown to directly lyse specific members of gram-positive gut commensal microbiota via a C1-dependent, antibody-independent mechanism, resulting in the deposition of the membrane attack complex and subsequent bacterial lysis. By selectively eliminating members of the commensal gut community, complement components from breast milk shape neonate and infant gut microbial composition to be protective against environmental pathogens such as CR.

Osr2 functions as a biomechanical checkpoint to aggravate CD8+ T cell exhaustion in tumor.

Alterations in extracellular matrix (ECM) architecture and stiffness represent hallmarks of cancer. Whether the biomechanical property of ECM impacts the functionality of tumor-reactive CD8+ T cells remains largely unknown. Here, we reveal that the transcription factor (TF) Osr2 integrates biomechanical signaling and facilitates the terminal exhaustion of tumor-reactive CD8+ T cells. Osr2 expression is selectively induced in the terminally exhausted tumor-specific CD8+ T cell subset by coupled T cell receptor (TCR) signaling and biomechanical stress mediated by the Piezo1/calcium/CREB axis. Consistently, depletion of Osr2 alleviates the exhaustion of tumor-specific CD8+ T cells or CAR-T cells, whereas forced Osr2 expression aggravates their exhaustion in solid tumor models. Mechanistically, Osr2 recruits HDAC3 to rewire the epigenetic program for suppressing cytotoxic gene expression and promoting CD8+ T cell exhaustion. Thus, our results unravel Osr2 functions as a biomechanical checkpoint to exacerbate CD8+ T cell exhaustion and could be targeted to potentiate cancer immunotherapy.

SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2.

Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.

A patterned human neural tube model using microfluidic gradients.

The human nervous system is a highly complex but organized organ. The foundation of its complexity and organization is laid down during regional patterning of the neural tube, the embryonic precursor to the human nervous system. Historically, studies of neural tube patterning have relied on animal models to uncover underlying principles. Recently, models of neurodevelopment based on human pluripotent stem cells, including neural organoids1-5 and bioengineered neural tube development models6-10, have emerged. However, such models fail to recapitulate neural patterning along both rostral-caudal and dorsal-ventral axes in a three-dimensional tubular geometry, a hallmark of neural tube development. Here we report a human pluripotent stem cell-based, microfluidic neural tube-like structure, the development of which recapitulates several crucial aspects of neural patterning in brain and spinal cord regions and along rostral-caudal and dorsal-ventral axes. This structure was utilized for studying neuronal lineage development, which revealed pre-patterning of axial identities of neural crest progenitors and functional roles of neuromesodermal progenitors and the caudal gene CDX2 in spinal cord and trunk neural crest development. We further developed dorsal-ventral patterned microfluidic forebrain-like structures with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic development of the human forebrain pallium and subpallium, respectively. Together, these microfluidics-based neurodevelopment models provide three-dimensional lumenal tissue architectures with in vivo-like spatiotemporal cell differentiation and organization, which will facilitate the study of human neurodevelopment and disease.

Paneth Cell-Derived Lysozyme Defines the Composition of Mucolytic Microbiota and the Inflammatory Tone of the Intestine.

Paneth cells are the primary source of C-type lysozyme, a ß-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn's disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1-/- hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti- and pro-inflammatory responses, with implications for IBD.

Stress Can Induce Transcription of Toxin-Antitoxin Systems without Activating Toxin.

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in bacterial genomes, but their functions are controversial. Although they are frequently postulated to regulate cell growth following stress, few null phenotypes for TA systems have been reported. Here, we show that TA transcript levels can increase substantially in response to stress, but toxin is not liberated. We find that the growth of an Escherichia coli strain lacking ten TA systems encoding endoribonuclease toxins is not affected following exposure to six stresses that each trigger TA transcription. Additionally, using RNA sequencing, we find no evidence of mRNA cleavage following stress. Stress-induced transcription arises from antitoxin degradation and relief of transcriptional autoregulation. Importantly, although free antitoxin is readily degraded in vivo, antitoxin bound to toxin is protected from proteolysis, preventing release of active toxin. Thus, transcription is not a reliable marker of TA activity, and TA systems do not strongly promote survival following individual stresses.

Infection and inflammation stimulate expansion of a CD74+ Paneth cell subset to regulate disease progression.

Paneth cells (PCs), a specialized secretory cell type in the small intestine, are increasingly recognized as having an essential role in host responses to microbiome and environmental stresses. Whether and how commensal and pathogenic microbes modify PC composition to modulate inflammation remain unclear. Using newly developed PC-reporter mice under conventional and gnotobiotic conditions, we determined PC transcriptomic heterogeneity in response to commensal and invasive microbes at single cell level. Infection expands the pool of CD74+ PCs, whose number correlates with auto or allogeneic inflammatory disease progressions in mice. Similar correlation was found in human inflammatory disease tissues. Infection-stimulated cytokines increase production of reactive oxygen species (ROS) and expression of a PC-specific mucosal pentraxin (Mptx2) in activated PCs. A PC-specific ablation of MyD88 reduced CD74+ PC population, thus ameliorating pathogen-induced systemic disease. A similar phenotype was also observed in mice lacking Mptx2. Thus, infection stimulates expansion of a PC subset that influences disease progression.

High-throughput and genome-scale targeted mutagenesis using CRISPR in a nonmodel multicellular organism, Bombyx mori.

Large-scale genetic mutant libraries are powerful approaches to interrogating genotype-phenotype correlations and identifying genes responsible for certain environmental stimuli, both of which are the central goal of life science study. We produced the first large-scale CRISPR-Cas9-induced library in a nonmodel multicellular organism, Bombyx mori We developed a piggyBac-delivered binary genome editing strategy, which can simultaneously meet the requirements of mixed microinjection, efficient multipurpose genetic operation, and preservation of growth-defect lines. We constructed a single-guide RNA (sgRNA) plasmid library containing 92,917 sgRNAs targeting promoters and exons of 14,645 protein-coding genes, established 1726 transgenic sgRNA lines following microinjection of 66,650 embryos, and generated 300 mutant lines with diverse phenotypic changes. Phenomic characterization of mutant lines identified a large set of genes responsible for visual phenotypic or economically valuable trait changes. Next, we performed pooled context-specific positive screens for tolerance to environmental pollutant cadmium exposure, and identified KWMTBOMO12902 as a strong candidate gene for breeding applications in sericulture industry. Collectively, our results provide a novel and versatile approach for functional B. mori genomics, as well as a powerful resource for identifying the potential of key candidate genes for improving various economic traits. This study also shows the effectiveness, practicality, and convenience of large-scale mutant libraries in other nonmodel organisms.

The H1/H5 domain contributes to OsTRBF2 phase separation and gene repression during rice development.

Transcription factors (TFs) tightly control plant development by regulating gene expression. The phase separation of TFs plays a vital role in gene regulation. Many plant TFs have the potential to form phase-separated protein condensates; however, little is known about which TFs are regulated by phase separation and how it affects their roles in plant development. Here, we report that the rice (Oryza sativa) single Myb TF TELOMERE REPEAT-BINDING FACTOR 2 (TRBF2) is highly expressed in fast-growing tissues at the seedling stage. TRBF2 is a transcriptional repressor that binds to the transcriptional start site of thousands of genes. Mutation of TRBF2 leads to pleiotropic developmental defects and misexpression of many genes. TRBF2 displays characteristics consistent with phase separation in vivo and forms phase-separated condensates in vitro. The H1/H5 domain of TRBF2 plays a crucial role in phase separation, chromatin targeting and gene repression. Replacing the H1/H5 domain by a phase-separated intrinsically disordered region from Arabidopsis (Arabidopsis thaliana) AtSERRATE partially recovers the function of TRBF2 in gene repression in vitro and in transgenic plants. We also found that TRBF2 is required for trimethylation of histone H3 Lys27 (H3K27me3) deposition at specific genes and genome-wide. Our findings reveal that phase separation of TRBF2 facilitates gene repression in rice development.

Filament structure and subcellular organization of the bacterial intermediate filament-like protein crescentin.

The protein crescentin is required for the crescent shape of the freshwater bacterium Caulobacter crescentus (vibrioides). Crescentin forms a filamentous structure on the inner, concave side of the curved cells. It shares features with eukaryotic intermediate filament (IF) proteins, including the formation of static filaments based on long and parallel coiled coils, the protein's length, structural roles in cell and organelle shape determination and the presence of a coiled coil discontinuity called the "stutter." Here, we have used electron cryomicroscopy (cryo-EM) to determine the structure of the full-length protein and its filament, exploiting a crescentin-specific nanobody. The filament is formed by two strands, related by twofold symmetry, that each consist of two dimers, resulting in an octameric assembly. Crescentin subunits form longitudinal contacts head-to-head and tail-to-tail, making the entire filament non-polar. Using in vivo site-directed cysteine cross-linking, we demonstrated that contacts observed in the in vitro filament structure exist in cells. Electron cryotomography (cryo-ET) of cells expressing crescentin showed filaments on the concave side of the curved cells, close to the inner membrane, where they form a band. When comparing with current models of IF proteins and their filaments, which are also built from parallel coiled coil dimers and lack overall polarity, it emerges that IF proteins form head-to-tail longitudinal contacts in contrast to crescentin and hence several inter-dimer contacts in IFs have no equivalents in crescentin filaments. Our work supports the idea that intermediate filament-like proteins achieve their shared polymerization and mechanical properties through a variety of filament architectures.

Dysregulated inter-mitochondrial crosstalk in glioblastoma cells revealed by in situ cryo-electron tomography.

Glioblastomas (GBMs) are the most lethal primary brain tumors with limited survival, even under aggressive treatments. The current therapeutics for GBMs are flawed due to the failure to accurately discriminate between normal proliferating cells and distinctive tumor cells. Mitochondria are essential to GBMs and serve as potential therapeutical targets. Here, we utilize cryo-electron tomography to quantitatively investigate nanoscale details of randomly sampled mitochondria in their native cellular context of GBM cells. Our results show that compared with cancer-free brain cells, GBM cells own more inter-mitochondrial junctions of several types for communications. Furthermore, our tomograms unveil microtubule-dependent mitochondrial nanotunnel-like bridges in the GBM cells as another inter-mitochondrial structure. These quantified inter-mitochondrial features, together with other mitochondria-organelle and intra-mitochondrial ones, are sufficient to distinguish GBM cells from cancer-free brain cells under scrutiny with predictive modeling. Our findings decipher high-resolution inter-mitochondrial structural signatures and provide clues for diagnosis and therapeutic interventions for GBM and other mitochondria-related diseases.

TET3-mediated DNA oxidation is essential for intestinal epithelial cell response to stressors.

DNA methylation functions as a repressive epigenetic mark that can be reversed by the Ten-eleven translocation (TET) family of DNA dioxygenases that sequentially oxidize 5-methylcytosine into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be excised by DNA base-excision repair factors leading to unmodified cytosines. TET enzymes were recently implicated as potential risk factors for inflammatory bowel disease (IBD), but the contribution of TET-mediated DNA oxidation to intestinal homeostasis and response to environmental stressors are unknown. Here, we show prominent roles of TET3 in regulating mouse intestinal epithelial differentiation and response to luminal stressors. Compared with wild-type littermates, mice with intestinal epithelial cell-specific ablation of Tet3 (Tet3ΔIEC) demonstrated a decreased transcriptome involved in innate immune response, Paneth cell differentiation, and epithelial regeneration. Tet3IEC mice exhibited an elevated susceptibility to enteric pathogen infection that is correlated with a decreased epithelial 5hmC abundance. Infection of human enterocytes or mice with the pathogenic bacteria acutely increased 5hmC abundance. Genome-wide 5hmC profiling revealed a shift of genomic enrichment of 5hmC toward genes involved in activating Notch, Wnt, and autophagy pathways. Furthermore, chemical stressor dextran sulfate sodium (DSS) represses epithelial 5hmC abundance in a temporal fashion, and Tet3IEC mice exhibited increased susceptibility to DSS experimental colitis with reduced regenerative capacity. TET3 is a critical regulator of gut epithelial DNA methylome and transcriptome, especially in response to luminal stressors, for the maintenance of tissue homeostasis.

CRISPR metabolic screen identifies ATM and KEAP1 as targetable genetic vulnerabilities in solid tumors.

Cancer treatments targeting DNA repair deficiencies often encounter drug resistance, possibly due to alternative metabolic pathways that counteract the most damaging effects. To identify such alternative pathways, we screened for metabolic pathways exhibiting synthetic lethality with inhibition of the DNA damage response kinase Ataxia-telangiectasia-mutated (ATM) using a metabolism-centered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 library. Our data revealed Kelch-like ECH-associated protein 1 (KEAP1) as a key factor involved in desensitizing cancer cells to ATM inhibition both in vitro and in vivo. Cells depleted of KEAP1 exhibited an aberrant overexpression of the cystine transporter SLC7A11, robustly accumulated cystine inducing disulfide stress, and became hypersensitive to ATM inhibition. These hallmarks were reversed in a reducing cellular environment indicating that disulfide stress was a crucial factor. In The Cancer Genome Atlas (TCGA) pan-cancer datasets, we found that ATM levels negatively correlated with KEAP1 levels across multiple solid malignancies. Together, our results unveil ATM and KEAP1 as new targetable vulnerabilities in solid tumors.

AP2S1 regulates APP degradation through late endosome-lysosome fusion in cells and APP/PS1 mice.

AP2S1 is the sigma 2 subunit of adaptor protein 2 (AP2) that is essential for endocytosis. In this study, we investigated the potential role of AP2S1 in intracellular processing of amyloid precursor protein (APP), which contributes to the pathogenesis of Alzheimer disease (AD) by generating the toxic ß-amyloid peptide (Aß). We found that knockdown or overexpression of AP2S1 decreased or increased the protein levels of APP and Aß in cells stably expressing human full-length APP695, respectively. This effect was unrelated to endocytosis but involved lysosomal degradation. Morphological studies revealed that silencing of AP2S1 promoted the translocalization of APP from RAB9-positive late endosomes (LE) to LAMP1-positive lysosomes, which was paralleled by the enhanced LE-lysosome fusion. In support, silencing of vacuolar protein sorting-associated protein 41 (VPS41) that is implicated in LE-lyso fusion prevented AP2S1-mediated regulation of APP degradation and translocalization. In APP/PS1 mice, an animal model of AD, AAV-mediated delivery of AP2S1 shRNA in the hippocampus significantly reduced the protein levels of APP and Aß, with the concomitant APP translocalization, LE-lyso fusion and the improved cognitive functions. Taken together, these data uncover a LE-lyso fusion mechanism in APP degradation and suggest a novel role for AP2S1 in the pathophysiology of AD.

A Comparison of Rapid Rule-Learning Strategies in Humans and Monkeys.

Interspecies comparisons are key to deriving an understanding of the behavioral and neural correlates of human cognition from animal models. We perform a detailed comparison of the strategies of female macaque monkeys to male and female humans on a variant of the Wisconsin Card Sorting Test (WCST), a widely studied and applied task that provides a multiattribute measure of cognitive function and depends on the frontal lobe. WCST performance requires the inference of a rule change given ambiguous feedback. We found that well-trained monkeys infer new rules three times more slowly than minimally instructed humans. Input-dependent hidden Markov model-generalized linear models were fit to their choices, revealing hidden states akin to feature-based attention in both species. Decision processes resembled a win-stay, lose-shift strategy with interspecies similarities as well as key differences. Monkeys and humans both test multiple rule hypotheses over a series of rule-search trials and perform inference-like computations to exclude candidate choice options. We quantitatively show that perseveration, random exploration, and poor sensitivity to negative feedback account for the slower task-switching performance in monkeys.

O-GlcNAcylation stimulates the deubiquitination activity of USP16 and regulates cell cycle progression.

Histone 2A monoubiquitination (uH2A) underscores a key epigenetic regulation of gene expression. In this report, we show that the deubiquitinase for uH2A, ubiquitin-specific peptidase 16 (USP16), is modified by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation involves the installation of the O-GlcNAc moiety to Ser/Thr residues. It crosstalks with Ser/Thr phosphorylation, affects protein-protein interaction, alters enzyme activity or protein folding, and changes protein subcellular localization. In our study, we first confirmed that USP16 is glycosylated on Thr203 and Ser214, as reported in a previous chemoenzymatic screen. We then discovered that mutation of the O-GlcNAcylation site Thr203, which is adjacent to deubiquitination-required Cys204, reduces the deubiquitination activity toward H2AK119ub in vitro and in cells, while mutation on Ser214 had the opposite effects. Using USP16 Ser552 phosphorylation-specific antibodies, we demonstrated that O-GlcNAcylation antagonizes cyclin-dependent kinase 1-mediated phosphorylation and promotes USP16 nuclear export. O-GlcNAcylation of USP16 is also required for deubiquitination of Polo-like kinase 1, a mitotic master kinase, and the subsequent chromosome segregation and cytokinesis. In summary, our study revealed that O-GlcNAcylation of USP16 at Thr203 and Ser214 coordinates deubiquitination of uH2A and Polo-like kinase 1, thus ensuring proper cell cycle progression.

Intestinal lysozyme engagement of Salmonella Typhimurium stimulates the release of barrier-impairing InvE and Lpp1.

Lysozyme is a ß-1,4-glycosidase that hydrolyzes the polysaccharide backbone of bacterial cell walls. With an additional bactericidal function mediated by a separate protein domain, lysozyme is considered a uniquely important antimicrobial molecule contributing to the host's innate immune response to infection. Elevated lysozyme production is found in various inflammatory conditions while patients with genetic risks for inflammatory bowel diseases demonstrate abnormal lysozyme expression, granule packaging, and secretion in Paneth cells. However, it remains unclear how a gain- or loss-of-function in host lysozyme may impact the host inflammatory responses to pathogenic infection. We challenged Lyz1-/- and ectopic Lyz1-expressing (Villin-Lyz1TG) mice with S. Typhimurium and then comprehensively assessed the inflammatory disease progression. We conducted proteomics analysis to identify molecules derived from human lysozyme-mediated processing of live Salmonella. We examined the barrier-impairing effects of these identified molecules in human intestinal epithelial cell monolayer and enteroids. Lyz1-/- mice are protected from infection in terms of morbidity, mortality, and barrier integrity, whereas Villin-Lyz1TG mice demonstrate exacerbated infection and inflammation. The growth and invasion of Salmonella in vitro are not affected by human or chicken lysozyme, whereas lysozyme encountering of live Salmonella stimulates the release of barrier-disrupting factors, InvE-sipC and Lpp1, which directly or indirectly impair the tight junctions. The direct engagement of host intestinal lysozyme with an enteric pathogen such as Salmonella promotes the release of virulence factors that are barrier-impairing and pro-inflammatory. Controlling lysozyme function may help alleviate the inflammatory progression.

Autotaxin in encephalitogenic CD4 T cells as a therapeutic target for multiple sclerosis.

Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the CNS. A defining characteristic of MS is the ability of autoreactive T lymphocytes to cross the blood-brain barrier and mediate inflammation within the CNS. Previous work from our lab found the gene Enpp2 to be highly upregulated in murine encephalitogenic T cells. Enpp2 encodes for the protein autotaxin, a secreted glycoprotein that catalyzes the production of lysophosphatidic acid and promotes transendothelial migration of T cells from the bloodstream into the lymphatic system. The present study sought to characterize autotaxin expression in T cells during CNS autoimmune disease and determine its potential therapeutic value. Myelin-activated CD4 T cells upregulated expression of autotaxin in vitro, and ex vivo analysis of CNS-infiltrating CD4 T cells showed significantly higher autotaxin expression compared with cells from healthy mice. In addition, inhibiting autotaxin in myelin-specific T cells reduced their encephalitogenicity in adoptive transfer studies and decreased in vitro cell motility. Importantly, using two mouse models of MS, treatment with an autotaxin inhibitor ameliorated EAE severity, decreased the number of CNS infiltrating T and B cells, and suppressed relapses, suggesting autotaxin may be a promising therapeutic target in the treatment of MS.

MicroRNAs targeting TGF-ß signaling exacerbate central nervous system autoimmunity by disrupting regulatory T cell development and function.

Transforming growth factor beta (TGF-ß) signaling is essential for a balanced immune response by mediating the development and function of regulatory T cells (Tregs) and suppressing autoreactive T cells. Disruption of this balance can result in autoimmune diseases, including multiple sclerosis (MS). MicroRNAs (miRNAs) targeting TGF-ß signaling have been shown to be upregulated in naïve CD4 T cells in MS patients, resulting in a limited in vitro generation of human Tregs. Utilizing the murine model experimental autoimmune encephalomyelitis, we show that perinatal administration of miRNAs, which target the TGF-ß signaling pathway, enhanced susceptibility to central nervous system (CNS) autoimmunity. Neonatal mice administered with these miRNAs further exhibited reduced Treg frequencies with a loss in T cell receptor repertoire diversity following the induction of experimental autoimmune encephalomyelitis in adulthood. Exacerbated CNS autoimmunity as a result of miRNA overexpression in CD4 T cells was accompanied by enhanced Th1 and Th17 cell frequencies. These findings demonstrate that increased levels of TGF-ß-associated miRNAs impede the development of a diverse Treg population, leading to enhanced effector cell activity, and contributing to an increased susceptibility to CNS autoimmunity. Thus, TGF-ß-targeting miRNAs could be a risk factor for MS, and recovering optimal TGF-ß signaling may restore immune homeostasis in MS patients.

Colonic healing requires Wnt produced by epithelium as well as Tagln+ and Acta2+ stromal cells.

Although Wnt signaling is clearly important for the intestinal epithelial homeostasis, the relevance of various sources of Wnt ligands themselves remains incompletely understood. Blocking the release of Wnt in distinct stromal cell types suggests obligatory functions of several stromal cell sources and yields different observations. The physiological contribution of epithelial Wnt to tissue homeostasis remains unclear. We show here that blocking epithelial Wnts affects colonic Reg4+ epithelial cell differentiation and impairs colonic epithelial regeneration after injury in mice. Single-cell RNA analysis of intestinal stroma showed that the majority of Wnt-producing cells were contained in transgelin (Tagln+) and smooth muscle actin α2 (Acta2+) expressing populations. We genetically attenuated Wnt production from these stromal cells using Tagln-Cre and Acta2-CreER drivers, and found that blockage of Wnt release from either epithelium or Tagln+ and Acta2+ stromal cells impaired colonic epithelial healing after chemical-induced injury. Aggregated blockage of Wnt release from both epithelium and Tagln+ or Acta2+ stromal cells drastically diminished epithelial repair, increasing morbidity and mortality. These results from two uncharacterized stromal populations suggested that colonic recovery from colitis-like injury depends on multiple Wnt-producing sources.