Gut-liver axis calibrates intestinal stem cell fitness.

The gut and liver are recognized to mutually communicate through the biliary tract, portal vein, and systemic circulation. However, it remains unclear how this gut-liver axis regulates intestinal physiology. Through hepatectomy and transcriptomic and proteomic profiling, we identified pigment epithelium-derived factor (PEDF), a liver-derived soluble Wnt inhibitor, which restrains intestinal stem cell (ISC) hyperproliferation to maintain gut homeostasis by suppressing the Wnt/ß-catenin signaling pathway. Furthermore, we found that microbial danger signals resulting from intestinal inflammation can be sensed by the liver, leading to the repression of PEDF production through peroxisome proliferator-activated receptor-α (PPARα). This repression liberates ISC proliferation to accelerate tissue repair in the gut. Additionally, treating mice with fenofibrate, a clinical PPARα agonist used for hypolipidemia, enhances colitis susceptibility due to PEDF activity. Therefore, we have identified a distinct role for PEDF in calibrating ISC expansion for intestinal homeostasis through reciprocal interactions between the gut and liver.

Targeted alveolar regeneration with Frizzled-specific agonists.

Wnt ligands oligomerize Frizzled (Fzd) and Lrp5/6 receptors to control the specification and activity of stem cells in many species. How Wnt signaling is selectively activated in different stem cell populations, often within the same organ, is not understood. In lung alveoli, we show that distinct Wnt receptors are expressed by epithelial (Fzd5/6), endothelial (Fzd4), and stromal (Fzd1) cells. Fzd5 is uniquely required for alveolar epithelial stem cell activity, whereas fibroblasts utilize distinct Fzd receptors. Using an expanded repertoire of Fzd-Lrp agonists, we could activate canonical Wnt signaling in alveolar epithelial stem cells via either Fzd5 or, unexpectedly, non-canonical Fzd6. A Fzd5 agonist (Fzd5ag) or Fzd6ag stimulated alveolar epithelial stem cell activity and promoted survival in mice after lung injury, but only Fzd6ag promoted an alveolar fate in airway-derived progenitors. Therefore, we identify a potential strategy for promoting regeneration without exacerbating fibrosis during lung injury.

Molecular basis of Wnt biogenesis, secretion, and Wnt7-specific signaling.

Wnt proteins are enzymatically lipidated by Porcupine (PORCN) in the ER and bind to Wntless (WLS) for intracellular transport and secretion. Mechanisms governing the transfer of these low-solubility Wnts from the ER to the extracellular space remain unclear. Through structural and functional analyses of Wnt7a, a crucial Wnt involved in central nervous system angiogenesis and blood-brain barrier maintenance, we have elucidated the principles of Wnt biogenesis and Wnt7-specific signaling. The Wnt7a-WLS complex binds to calreticulin (CALR), revealing that CALR functions as a chaperone to facilitate Wnt transfer from PORCN to WLS during Wnt biogenesis. Our structures, functional analyses, and molecular dynamics simulations demonstrate that a phospholipid in the core of Wnt-bound WLS regulates the association and dissociation between Wnt and WLS, suggesting a lipid-mediated Wnt secretion mechanism. Finally, the structure of Wnt7a bound to RECK, a cell-surface Wnt7 co-receptor, reveals how RECKCC4 engages the N-terminal domain of Wnt7a to activate Wnt7-specific signaling.

Dual-specificity RNA aptamers enable manipulation of target-specific O-GlcNAcylation and unveil functions of O-GlcNAc on ß-catenin.

O-GlcNAc is a dynamic post-translational modification (PTM) that regulates protein functions. In studying the regulatory roles of O-GlcNAc, a major roadblock is the inability to change O-GlcNAcylation on a single protein at a time. Herein, we developed a dual RNA-aptamer-based approach that simultaneously targeted O-GlcNAc transferase (OGT) and ß-catenin, the key transcription factor of the Wnt signaling pathway, to selectively increase O-GlcNAcylation of the latter without affecting other OGT substrates. Using the OGT/ß-catenin dual-specificity aptamers, we found that O-GlcNAcylation of ß-catenin stabilizes the protein by inhibiting its interaction with ß-TrCP. O-GlcNAc also increases ß-catenin's interaction with EZH2, recruits EZH2 to promoters, and dramatically alters the transcriptome. Further, by coupling riboswitches or an inducible expression system to aptamers, we enabled inducible regulation of protein-specific O-GlcNAcylation. Together, our findings demonstrate the efficacy and versatility of dual-specificity aptamers for regulating O-GlcNAcylation on individual proteins.

Humanized mouse liver reveals endothelial control of essential hepatic metabolic functions.

Hepatocytes, the major metabolic hub of the body, execute functions that are human-specific, altered in human disease, and currently thought to be regulated through endocrine and cell-autonomous mechanisms. Here, we show that key metabolic functions of human hepatocytes are controlled by non-parenchymal cells (NPCs) in their microenvironment. We developed mice bearing human hepatic tissue composed of human hepatocytes and NPCs, including human immune, endothelial, and stellate cells. Humanized livers reproduce human liver architecture, perform vital human-specific metabolic/homeostatic processes, and model human pathologies, including fibrosis and non-alcoholic fatty liver disease (NAFLD). Leveraging species mismatch and lipidomics, we demonstrate that human NPCs control metabolic functions of human hepatocytes in a paracrine manner. Mechanistically, we uncover a species-specific interaction whereby WNT2 secreted by sinusoidal endothelial cells controls cholesterol uptake and bile acid conjugation in hepatocytes through receptor FZD5. These results reveal the essential microenvironmental regulation of hepatic metabolism and its human-specific aspects.

The Wnt Pathway: From Signaling Mechanisms to Synthetic Modulators.

The Wnt pathway is central to a host of developmental and disease-related processes. The remarkable conservation of this intercellular signaling cascade throughout metazoan lineages indicates that it coevolved with multicellularity to regulate the generation and spatial arrangement of distinct cell types. By regulating cell fate specification, mitotic activity, and cell polarity, Wnt signaling orchestrates development and tissue homeostasis, and its dysregulation is implicated in developmental defects, cancer, and degenerative disorders. We review advances in our understanding of this key pathway, from Wnt protein production and secretion to relay of the signal in the cytoplasm of the receiving cell. We discuss the evolutionary history of this pathway as well as endogenous and synthetic modulators of its activity. Finally, we highlight remaining gaps in our knowledge of Wnt signal transduction and avenues for future research.

Structural Basis of WLS/Evi-Mediated Wnt Transport and Secretion.

Wnts are evolutionarily conserved ligands that signal at short range to regulate morphogenesis, cell fate, and stem cell renewal. The first and essential steps in Wnt secretion are their O-palmitoleation and subsequent loading onto the dedicated transporter Wntless/evenness interrupted (WLS/Evi). We report the 3.2 Å resolution cryogenic electron microscopy (cryo-EM) structure of palmitoleated human WNT8A in complex with WLS. This is accompanied by biochemical experiments to probe the physiological implications of the observed association. The WLS membrane domain has close structural homology to G protein-coupled receptors (GPCRs). A Wnt hairpin inserts into a conserved hydrophobic cavity in the GPCR-like domain, and the palmitoleate protrudes between two helices into the bilayer. A conformational switch of highly conserved residues on a separate Wnt hairpin might contribute to its transfer to receiving cells. This work provides molecular-level insights into a central mechanism in animal body plan development and stem cell biology.

Distinct Processing of lncRNAs Contributes to Non-conserved Functions in Stem Cells.

Long noncoding RNAs (lncRNAs) evolve more rapidly than mRNAs. Whether conserved lncRNAs undergo conserved processing, localization, and function remains unexplored. We report differing subcellular localization of lncRNAs in human and mouse embryonic stem cells (ESCs). A significantly higher fraction of lncRNAs is localized in the cytoplasm of hESCs than in mESCs. This turns out to be important for hESC pluripotency. FAST is a positionally conserved lncRNA but is not conserved in its processing and localization. In hESCs, cytoplasm-localized hFAST binds to the WD40 domain of the E3 ubiquitin ligase ß-TrCP and blocks its interaction with phosphorylated ß-catenin to prevent degradation, leading to activated WNT signaling, required for pluripotency. In contrast, mFast is nuclear retained in mESCs, and its processing is suppressed by the splicing factor PPIE, which is highly expressed in mESCs but not hESCs. These findings reveal that lncRNA processing and localization are previously under-appreciated contributors to the rapid evolution of function.

Beyond Casual Resemblance: Rigorous Frameworks for Comparing Regeneration Across Species.

The majority of animal phyla have species that can regenerate. Comparing regeneration across animals can reconstruct the molecular and cellular evolutionary history of this process. Recent studies have revealed some similarity in regeneration mechanisms, but rigorous comparative methods are needed to assess whether these resemblances are ancestral pathways (homology) or are the result of convergent evolution (homoplasy). This review aims to provide a framework for comparing regeneration across animals, focusing on gene regulatory networks (GRNs), which are substrates for assessing process homology. The homology of the wound-induced activation of Wnt signaling and of adult stem cells provides examples of ongoing studies of regeneration that enable comparisons in a GRN framework. Expanding the study of regeneration GRNs in currently studied species and broadening taxonomic sampling for these approaches will identify processes that are unifying principles of regeneration biology across animals. These insights are important both for evolutionary studies of regeneration and for human regenerative medicine.

Cell Biology of Canonical Wnt Signaling.

Wnt signaling has multiple functions beyond the transcriptional effects of ß-catenin stabilization. We review recent investigations that uncover new cell physiological effects through the regulation of Wnt receptor endocytosis, Wnt-induced stabilization of proteins (Wnt-STOP), macropinocytosis, increase in lysosomal activity, and metabolic changes. Many of these growth-promoting effects of canonical Wnt occur within minutes and are independent of new protein synthesis. A key element is the sequestration of glycogen synthase kinase 3 (GSK3) inside multivesicular bodies and lysosomes. Twenty percent of human proteins contain consecutive GSK3 phosphorylation motifs, which in the absence of Wnt can form phosphodegrons for polyubiquitination and proteasomal degradation. Wnt signaling by either the pharmacological inhibition of GSK3 or the loss of tumor-suppressor proteins, such as adenomatous polyposis coli (APC) and Axin1, increases lysosomal acidification, anabolic metabolites, and macropinocytosis, which is normally repressed by the GSK3-Axin1-APC destruction complex. The combination of these cell physiological effects drives cell growth.

Quiescence Modulates Stem Cell Maintenance and Regenerative Capacity in the Aging Brain.

The function of somatic stem cells declines with age. Understanding the molecular underpinnings of this decline is key to counteract age-related disease. Here, we report a dramatic drop in the neural stem cells (NSCs) number in the aging murine brain. We find that this smaller stem cell reservoir is protected from full depletion by an increase in quiescence that makes old NSCs more resistant to regenerate the injured brain. Once activated, however, young and old NSCs show similar proliferation and differentiation capacity. Single-cell transcriptomics of NSCs indicate that aging changes NSCs minimally. In the aging brain, niche-derived inflammatory signals and the Wnt antagonist sFRP5 induce quiescence. Indeed, intervention to neutralize them increases activation of old NSCs during homeostasis and following injury. Our study identifies quiescence as a key feature of old NSCs imposed by the niche and uncovers ways to activate NSCs to repair the aging brain.

Human Pluripotency Is Initiated and Preserved by a Unique Subset of Founder Cells.

The assembly of organized colonies is the earliest manifestation in the derivation or induction of pluripotency in vitro. However, the necessity and origin of this assemblance is unknown. Here, we identify human pluripotent founder cells (hPFCs) that initiate, as well as preserve and establish, pluripotent stem cell (PSC) cultures. PFCs are marked by N-cadherin expression (NCAD+) and reside exclusively at the colony boundary of primate PSCs. As demonstrated by functional analysis, hPFCs harbor the clonogenic capacity of PSC cultures and emerge prior to commitment events or phenotypes associated with pluripotent reprogramming. Comparative single-cell analysis with pre- and post-implantation primate embryos revealed hPFCs share hallmark properties with primitive endoderm (PrE) and can be regulated by non-canonical Wnt signaling. Uniquely informed by primate embryo organization in vivo, our study defines a subset of founder cells critical to the establishment pluripotent state.

Divergent Routes toward Wnt and R-spondin Niche Independency during Human Gastric Carcinogenesis.

Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers.

The Mitochondrial Unfolded Protein Response Is Mediated Cell-Non-autonomously by Retromer-Dependent Wnt Signaling.

The mitochondrial unfolded protein response (UPRmt) can be triggered in a cell-non-autonomous fashion across multiple tissues in response to mitochondrial dysfunction. The ability to communicate information about the presence of mitochondrial stress enables a global response that can ultimately better protect an organism from local mitochondrial challenges. We find that animals use retromer-dependent Wnt signaling to propagate mitochondrial stress signals from the nervous system to peripheral tissues. Specifically, the polyQ40-triggered activation of mitochondrial stress or reduction of cco-1 (complex IV subunit) in neurons of C. elegans results in the Wnt-dependent induction of cell-non-autonomous UPRmt in peripheral cells. Loss-of-function mutations of retromer complex components that are responsible for recycling the Wnt secretion-factor/MIG-14 prevent Wnt secretion and thereby suppress cell-non-autonomous UPRmt. Neuronal expression of the Wnt ligand/EGL-20 is sufficient to induce cell-non-autonomous UPRmt in a retromer complex-, Wnt signaling-, and serotonin-dependent manner, clearly implicating Wnt signaling as a strong candidate for the "mitokine" signal.

Inflammatory Cytokine TNFα Promotes the Long-Term Expansion of Primary Hepatocytes in 3D Culture.

In the healthy adult liver, most hepatocytes proliferate minimally. However, upon physical or chemical injury to the liver, hepatocytes proliferate extensively in vivo under the direction of multiple extracellular cues, including Wnt and pro-inflammatory signals. Currently, liver organoids can be generated readily in vitro from bile-duct epithelial cells, but not hepatocytes. Here, we show that TNFα, an injury-induced inflammatory cytokine, promotes the expansion of hepatocytes in 3D culture and enables serial passaging and long-term culture for more than 6 months. Single-cell RNA sequencing reveals broad expression of hepatocyte markers. Strikingly, in vitro-expanded hepatocytes engrafted, and significantly repopulated, the injured livers of Fah-/- mice. We anticipate that tissue repair signals can be harnessed to promote the expansion of otherwise hard-to-culture cell-types, with broad implications.

Modulation of Phase Shift between Wnt and Notch Signaling Oscillations Controls Mesoderm Segmentation.

How signaling dynamics encode information is a central question in biology. During vertebrate development, dynamic Notch signaling oscillations control segmentation of the presomitic mesoderm (PSM). In mouse embryos, this molecular clock comprises signaling oscillations of several pathways, i.e., Notch, Wnt, and FGF signaling. Here, we directly address the role of the relative timing between Wnt and Notch signaling oscillations during PSM patterning. To this end, we developed a new experimental strategy using microfluidics-based entrainment that enables specific control of the rhythm of segmentation clock oscillations. Using this approach, we find that Wnt and Notch signaling are coupled at the level of their oscillation dynamics. Furthermore, we provide functional evidence that the oscillation phase shift between Wnt and Notch signaling is critical for PSM segmentation. Our work hence reveals that dynamic signaling, i.e., the relative timing between oscillatory signals, encodes essential information during multicellular development.

Nervous System Regionalization Entails Axial Allocation before Neural Differentiation.

Neural induction in vertebrates generates a CNS that extends the rostral-caudal length of the body. The prevailing view is that neural cells are initially induced with anterior (forebrain) identity; caudalizing signals then convert a proportion to posterior fates (spinal cord). To test this model, we used chromatin accessibility to define how cells adopt region-specific neural fates. Together with genetic and biochemical perturbations, this identified a developmental time window in which genome-wide chromatin-remodeling events preconfigure epiblast cells for neural induction. Contrary to the established model, this revealed that cells commit to a regional identity before acquiring neural identity. This "primary regionalization" allocates cells to anterior or posterior regions of the nervous system, explaining how cranial and spinal neurons are generated at appropriate axial positions. These findings prompt a revision to models of neural induction and support the proposed dual evolutionary origin of the vertebrate CNS.

Distinct Mesenchymal Lineages and Niches Promote Epithelial Self-Renewal and Myofibrogenesis in the Lung.

The lung is an architecturally complex organ comprising a heterogeneous mixture of various epithelial and mesenchymal lineages. We use single-cell RNA sequencing and signaling lineage reporters to generate a spatial and transcriptional map of the lung mesenchyme. We find that each mesenchymal lineage has a distinct spatial address and transcriptional profile leading to unique niche regulatory functions. The mesenchymal alveolar niche cell is Wnt responsive, expresses Pdgfrα, and is critical for alveolar epithelial cell growth and self-renewal. In contrast, the Axin2+ myofibrogenic progenitor cell preferentially generates pathologically deleterious myofibroblasts after injury. Analysis of the secretome and receptome of the alveolar niche reveals functional pathways that mediate growth and self-renewal of alveolar type 2 progenitor cells, including IL-6/Stat3, Bmp, and Fgf signaling. These studies define the cellular and molecular framework of lung mesenchymal niches and reveal the functional importance of developmental pathways in promoting self-renewal versus a pathological response to tissue injury.

Wnt/ß-Catenin Signaling, Disease, and Emerging Therapeutic Modalities.

The WNT signal transduction cascade is a main regulator of development throughout the animal kingdom. Wnts are also key drivers of most types of tissue stem cells in adult mammals. Unsurprisingly, mutated Wnt pathway components are causative to multiple growth-related pathologies and to cancer. Here, we describe the core Wnt/ß-catenin signaling pathway, how it controls stem cells, and contributes to disease. Finally, we discuss strategies for Wnt-based therapies.

Anatomically and Functionally Distinct Lung Mesenchymal Populations Marked by Lgr5 and Lgr6.

The diversity of mesenchymal cell types in the lung that influence epithelial homeostasis and regeneration is poorly defined. We used genetic lineage tracing, single-cell RNA sequencing, and organoid culture approaches to show that Lgr5 and Lgr6, well-known markers of stem cells in epithelial tissues, are markers of mesenchymal cells in the adult lung. Lgr6+ cells comprise a subpopulation of smooth muscle cells surrounding airway epithelia and promote airway differentiation of epithelial progenitors via Wnt-Fgf10 cooperation. Genetic ablation of Lgr6+ cells impairs airway injury repair in vivo. Distinct Lgr5+ cells are located in alveolar compartments and are sufficient to promote alveolar differentiation of epithelial progenitors through Wnt activation. Modulating Wnt activity altered differentiation outcomes specified by mesenchymal cells. This identification of region- and lineage-specific crosstalk between epithelium and their neighboring mesenchymal partners provides new understanding of how different cell types are maintained in the adult lung.