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Meningiomas are associated with inactivation of NF2/Merlin, but approximately one-third of meningiomas with favorable clinical outcomes retain Merlin expression. Biochemical mechanisms underlying Merlin-intact meningioma growth are incompletely understood, and non-invasive biomarkers that may be used to guide treatment de-escalation or imaging surveillance are lacking. Here, we use single-cell RNA sequencing, proximity-labeling proteomic mass spectrometry, mechanistic and functional approaches, and magnetic resonance imaging (MRI) across meningioma xenografts and patients to define biochemical mechanisms and an imaging biomarker that underlie Merlin-intact meningiomas. We find Merlin serine 13 (S13) dephosphorylation drives meningioma Wnt signaling and tumor growth by attenuating inhibitory interactions with β-catenin and activating the Wnt pathway. MRI analyses show Merlin-intact meningiomas with S13 phosphorylation and favorable clinical outcomes are associated with high apparent diffusion coefficient (ADC). These results define mechanisms underlying a potential imaging biomarker that could be used to guide treatment de-escalation or imaging surveillance for patients with Merlin-intact meningiomas.

Activated Wnt/β-catenin pathway is a key genetic event in liver cancer development. Solute carrier (SLC) transporters are promising drug targets. Here, we identify SLC13A3 as a drug-targetable effector downstream of β-catenin in liver cancer. SLC13A3 expression is elevated in human liver cancer samples with gain of function (GOF) mutant CTNNB1, the gene encoding β-catenin. Activation of β-catenin up-regulates SLC13A3, leading to intracellular accumulation of endogenous SLC13A3 substrates. SLC13A3 is identified as a low-affinity transporter for glutathione (GSH). Silencing of SLC13A3 downregulates the leucine transporter SLC7A5 via c-MYC signaling, leading to leucine depletion and mTOR inactivation. Furthermore, silencing of SLC13A3 depletes GSH and induces autophagic ferroptosis in β-catenin-activated liver cancer cells. Importantly, both genetic inhibition of SLC13A3 and a small molecule SLC13A3 inhibitor suppress β-catenin-driven hepatocarcinogenesis in mice. Altogether, our study suggests that SLC13A3 could be a promising therapeutic target for treating human liver cancers with GOF CTNNB1 mutations.

Detailed binding experiments reveal new insights into the Norrin/Wnt signaling pathway that helps to control vascularization in the retina.

Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the β-catenin destruction complex inside multivesicular bodies (MVBs) and lysosomes. In this study, we introduce Ouabain, an inhibitor of the Na,K-ATPase pump that establishes electric potentials across membranes, as a potent inhibitor of Wnt signaling. We find that Na,K-ATPase levels are elevated in advanced colon carcinoma, that this enzyme is elevated in cancer cells with constitutively activated Wnt pathway and is activated by GSK3 inhibitors that increase macropinocytosis. Ouabain blocks macropinocytosis, which is an essential step in Wnt signaling, probably explaining the strong effects of Ouabain on this pathway. In Xenopus embryos, brief Ouabain treatment at the 32-cell stage, critical for the earliest Wnt signal in development-inhibited brains, could be reversed by treatment with Lithium chloride, a Wnt mimic. Inhibiting membrane trafficking may provide a way of targeting Wnt-driven cancers.

The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.

The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.

The Wnt/β-catenin pathway contains multiple high-confidence risk genes that are linked to neurodevelopmental disorders, including autism spectrum disorder. However, its ubiquitous roles across brain cell types and developmental stages have made it challenging to define its impact on neural circuit development and behavior. Here, we show that TCF7L2, which is a key transcriptional effector of the Wnt/β-catenin pathway, plays a cell-autonomous role in postnatal astrocyte maturation and impacts adult social behavior. TCF7L2 was the dominant Wnt effector that was expressed in both mouse and human astrocytes, with a peak during astrocyte maturation. The conditional knockout of Tcf7l2 in postnatal astrocytes led to an enlargement of astrocytes with defective tiling and gap junction coupling. These mice also exhibited an increase in the number of cortical excitatory and inhibitory synapses and a marked increase in social interaction by adulthood. These data reveal an astrocytic role for developmental Wnt/β-catenin signaling in restricting excitatory synapse numbers and regulating adult social behavior.

Wnt signaling pathways are transmitted via 10 homologous frizzled receptors (FZD1-10) in humans. Reagents broadly inhibiting Wnt signaling pathways reduce growth and metastasis of many tumors, but their therapeutic development has been hampered by the side effect. Inhibitors targeting specific Wnt-FZD pair(s) enriched in cancer cells may reduce side effect, but the therapeutic effect of narrow-spectrum Wnt-FZD inhibitors remains to be established in vivo. Here, we developed a fragment of C. difficile toxin B (TcdBFBD), which recognizes and inhibits a subclass of FZDs, FZD1/2/7, and examined whether targeting this FZD subgroup may offer therapeutic benefits for treating breast cancer models in mice. Utilizing 2 basal-like and 1 luminal-like breast cancer models, we found that TcdBFBD reduces tumor-initiating cells and attenuates growth of basal-like mammary tumor organoids and xenografted tumors, without damaging Wnt-sensitive tissues such as bones in vivo. Furthermore, FZD1/2/7-positive cells are enriched in chemotherapy-resistant cells in both basal-like and luminal mammary tumors treated with cisplatin, and TcdBFBD synergizes strongly with cisplatin in inhibiting both tumor types. These data demonstrate the therapeutic value of narrow-spectrum Wnt signaling inhibitor in treating breast cancers.

Targeting common oncogenic drivers of glioblastoma multiforme (GBM) in patients has remained largely ineffective, raising the possibility that alternative pathways may contribute to tumor aggressiveness. Here we demonstrate that Vangl1 and Fzd7, components of the non-canonical Wnt planar cell polarity (Wnt/PCP) signaling pathway, promote GBM malignancy by driving cellular proliferation, migration, and invasiveness, and engage Rho GTPases to promote cytoskeletal rearrangements and actin dynamics in migrating GBM cells. Mechanistically, we uncover the existence of a novel Vangl1/Fzd7 complex at the leading edge of migrating GBM cells and propose that this complex is critical for the recruitment of downstream effectors to promote tumor progression. Moreover, we observe that depletion of FZD7 results in a striking suppression of tumor growth and latency and extends overall survival in an intracranial mouse xenograft model. Our observations support a novel mechanism by which Wnt/PCP components Vangl1 and Fzd7 form a complex at the leading edge of migratory GBM cells to engage downstream effectors that promote actin cytoskeletal rearrangements dynamics. Our findings suggest that interference with Wnt/PCP pathway function may offer a novel therapeutic strategy for patients diagnosed with GBM.

Background and aimsGain-of-function (GOF) mutations of CTNNB1 and loss-of-function (LOF) mutations of AXIN1 are recurrent genetic alterations in hepatocellular carcinoma (HCC). We aim to investigate the functional contribution of Hippo/YAP/TAZ in GOF CTNNB1 or LOF AXIN1 mutant HCCs.Approach and resultsThe requirement of YAP/TAZ in c-Met/β-Catenin and c-Met/sgAxin1-driven HCC was analyzed using conditional Yap , Taz , and Yap;Taz knockout (KO) mice. Mechanisms of AXIN1 in regulating YAP/TAZ were investigated using AXIN1 mutated HCC cells. Hepatocyte-specific inducible TTR-CreER T2KO system was applied to evaluate the role of Yap;Taz during tumor progression. Cabozantinib and G007-LK combinational treatment were tested in vitro and in vivo . Nuclear YAP/TAZ was strongly induced in c-Met/sgAxin1 mouse HCC cells. Activation of Hippo via overexpression of Lats2 or concomitant deletion of Yap and Taz significantly inhibited c-Met/sgAxin1 driven HCC development, whereas the same approaches had mild effects in c-Met/β-Catenin HCCs. YAP is the major Hippo effector in c-Met/β-Catenin HCCs, and both YAP and TAZ are required for c-Met/sgAxin1-dependent hepatocarcinogenesis. Mechanistically, AXIN1 binds to YAP/TAZ in human HCC cells and regulates YAP/TAZ stability. Genetic deletion of YAP/TAZ suppresses already formed c-Met/sgAxin1 liver tumors, supporting the requirement of YAP/TAZ during tumor progression. Importantly, tankyrase inhibitor G007-LK, which targets Hippo and Wnt pathways, synergizes with cabozantinib, a c-MET inhibitor, leading to tumor regression in the c-Met/sgAxin1 HCC model.ConclusionsOur studies demonstrate that YAP/TAZ are major signaling molecules downstream of LOF AXIN1 mutant HCCs, and targeting YAP/TAZ is an effective treatment against AXIN1 mutant human HCCs.

Dermal adipocyte lineage cells are highly plastic and can undergo reversible differentiation and dedifferentiation in response to various stimuli. Using single-cell RNA sequencing of developing or wounded mouse skin, we classify dermal fibroblasts (dFBs) into distinct non-adipogenic and adipogenic cell states. Cell differentiation trajectory analyses identify IL-1-NF-κB and WNT-β-catenin as top signaling pathways that positively and negatively associate with adipogenesis, respectively. Upon wounding, activation of adipocyte progenitors and wound-induced adipogenesis are mediated in part by neutrophils through the IL-1R-NF-κB-CREB signaling axis. In contrast, WNT activation, by WNT ligand and/or ablation of Gsk3, inhibits the adipogenic potential of dFBs but promotes lipolysis and dedifferentiation of mature adipocytes, contributing to myofibroblast formation. Finally, sustained WNT activation and inhibition of adipogenesis is seen in human keloids. These data reveal molecular mechanisms underlying the plasticity of dermal adipocyte lineage cells, defining potential therapeutic targets for defective wound healing and scar formation.

Both the canonical Wnt and androgen receptor (AR) signaling pathways are important for prostate organogenesis and homeostasis. How they crosstalk to regulate prostate stem cell behaviors remains unclear. Here, we show in lineage-tracing mouse models that although Wnt is essential for basal stem cell multipotency, ectopic Wnt activity promotes basal cell over-proliferation and squamous phenotypes, which are counteracted by elevated levels of androgen. In prostate basal cell organoids, dihydrotestosterone (DHT) antagonizes R-spondin-stimulated growth in a concentration-dependent manner. DHT down-regulates the expressions of a Wnt reporter and target genes, and RNA sequencing (RNA-seq) analyses identify Wnt signaling as a key altered pathway. Mechanistically, DHT enhances AR and β-catenin protein binding, and CUT&RUN analyses reveal that ectopic AR sequesters β-catenin away from its Wnt-related cistrome. Our results suggest that an intermediate level of Wnt activity in prostate basal stem cells, achieved via AR-β-catenin interaction, is essential for normal prostate homeostasis.

A central factor in the maintenance of tissue integrity is the response of stem cells to variations in the levels of niche signals. In the gut, intestinal stem cells (ISCs) depend on Wnt ligands for self-renewal and proliferation. Transient increases in Wnt signaling promote regeneration after injury or in inflammatory bowel diseases, whereas constitutive activation of this pathway leads to colorectal cancer. Here, we report that Discs large 1 (Dlg1), although dispensable for polarity and cellular turnover during intestinal homeostasis, is required for ISC survival in the context of increased Wnt signaling. RNA sequencing (RNA-seq) and genetic mouse models demonstrated that DLG1 regulates the cellular response to increased canonical Wnt ligands. This occurs via the transcriptional regulation of Arhgap31, a GTPase-activating protein that deactivates CDC42, an effector of the non-canonical Wnt pathway. These findings reveal a DLG1-ARHGAP31-CDC42 axis that is essential for the ISC response to increased niche Wnt signaling.

We previously showed that the NO/cGMP/protein kinase G (PKG) signaling pathway positively regulates osteoblast proliferation, differentiation, and survival in vitro, and that cGMP-elevating agents have bone-anabolic effects in mice. Here, we generated mice with an osteoblast-specific (OB) knockout (KO) of type 2 PKG (gene name Prkg2) using a Col1a1(2.3 kb)-Cre driver. Compared to wild type (WT) littermates, 8-week-old male OB Prkg2-KO mice had fewer osteoblasts, reduced bone formation rates, and lower trabecular and cortical bone volumes. Female OB Prkg2-KO littermates showed no bone abnormalities, despite the same degree of PKG2 deficiency in bone. Expression of osteoblast differentiation- and Wnt/β-catenin-related genes was lower in primary osteoblasts and bones of male KO but not female KO mice compared to WT littermates. Osteoclast parameters were unaffected in both sexes. Since PKG2 is part of a mechano-sensitive complex in osteoblast membranes, we examined its role during mechanical loading. Cyclical compression of the tibia increased cortical thickness and induced mechanosensitive and Wnt/β-catenin-related genes to a similar extent in male and female WT mice and female OB Prkg2-KO mice, but loading had a minimal effect in male KO mice. We conclude that PKG2 drives bone acquisition and adaptation to mechanical loading via the Wnt/β-catenin pathway in male mice. The striking sexual dimorphism of OB Prkg2-KO mice suggests that current U.S. Food and Drug Administration-approved cGMP-elevating agents may represent novel effective treatment options for male osteoporosis. © 2022 American Society for Bone and Mineral Research (ASBMR).

Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here, we show that a macropinocytosis activator, the tumor promoter phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the Xenopus embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.

The relative abundance of Wnt receptors plays a crucial role in controlling Wnt signaling in tissue homeostasis and human disease. While the ubiquitin ligases that ubiquitylate Wnt receptors are well-characterized, the deubiquitylase that reverses these reactions remains unclear. Herein, we identify USP46, UAF1, and WDR20 (USP46 complex) as positive regulators of Wnt signaling in cultured human cells. We find that the USP46 complex is similarly required for Wnt signaling in Xenopus and zebrafish embryos. We demonstrate that Wnt signaling promotes the association between the USP46 complex and cell surface Wnt coreceptor, LRP6. Knockdown of USP46 decreases steady-state levels of LRP6 and increases the level of ubiquitylated LRP6. In contrast, overexpression of the USP46 complex blocks ubiquitylation of LRP6 by the ubiquitin ligases RNF43 and ZNFR3. Size exclusion chromatography studies suggest that the size of the USP46 cytoplasmic complex increases upon Wnt stimulation. Finally, we show that USP46 is essential for Wnt-dependent intestinal organoid viability, likely via its role in LRP6 receptor homeostasis. We propose a model in which the USP46 complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes via a pruning mechanism that removes sterically hindering ubiquitin chains.

BackgroundIn light of the growing appreciation for the role of collective cell motility in metastasis, a deeper understanding of the underlying signaling pathways will be critical to translating these observations to the treatment of advanced cancers. Here, we examine the contribution of Wnt/planar cell polarity (Wnt/PCP), one of the non-canonical Wnt signaling pathways and defined by the involvement of the tetraspanin-like proteins Vangl1 and Vangl2, to breast tumor cell motility, collective cell invasiveness and mammary tumor metastasis.MethodsVangl1 and Vangl2 knockdown and overexpression and Wnt5a stimulation were employed to manipulate Wnt/PCP signaling in a battery of breast cancer cell lines representing all breast cancer subtypes, and in tumor organoids from MMTV-PyMT mice. Cell migration was assessed by scratch and organoid invasion assays, Vangl protein subcellular localization was assessed by confocal fluorescence microscopy, and RhoA activation was assessed in real time by fluorescence imaging with an advanced FRET biosensor. The impact of Wnt/PCP suppression on mammary tumor growth and metastasis was assessed by determining the effect of conditional Vangl2 knockout on the MMTV-NDL mouse mammary tumor model.ResultsWe observed that Vangl2 knockdown suppresses the motility of all breast cancer cell lines examined, and overexpression drives the invasiveness of collectively migrating MMTV-PyMT organoids. Vangl2-dependent RhoA activity is localized in real time to a subpopulation of motile leader cells displaying a hyper-protrusive leading edge, Vangl protein is localized to leader cell protrusions within leader cells, and actin cytoskeletal regulator RhoA is preferentially activated in the leader cells of a migrating collective. Mammary gland-specific knockout of Vangl2 results in a striking decrease in lung metastases in MMTV-NDL mice, but does not impact primary tumor growth characteristics.ConclusionsWe conclude that Vangl-dependent Wnt/PCP signaling promotes breast cancer collective cell migration independent of breast tumor subtype and facilitates distant metastasis in a genetically engineered mouse model of breast cancer. Our observations are consistent with a model whereby Vangl proteins localized at the leading edge of leader cells in a migrating collective act through RhoA to mediate the cytoskeletal rearrangements required for pro-migratory protrusion formation.

Mutationally activated BRAF is detected in approximately 7% of human lung adenocarcinomas, with BRAFT1799A serving as a predictive biomarker for treatment of patients with FDA-approved inhibitors of BRAFV600E oncoprotein signaling. In genetically engineered mouse (GEM) models, expression of BRAFV600E in the lung epithelium initiates growth of benign lung tumors that, without additional genetic alterations, rarely progress to malignant lung adenocarcinoma. To identify genes that cooperate with BRAFV600E for malignant progression, we used Sleeping Beauty-mediated transposon mutagenesis, which dramatically accelerated the emergence of lethal lung cancers. Among the genes identified was Rbms3, which encodes an RNA-binding protein previously implicated as a putative tumor suppressor. Silencing of RBMS3 via CRISPR/Cas9 gene editing promoted growth of BRAFV600E lung organoids and promoted development of malignant lung cancers with a distinct micropapillary architecture in BRAFV600E and EGFRL858R GEM models. BRAFV600E/RBMS3Null lung tumors displayed elevated expression of Ctnnb1, Ccnd1, Axin2, Lgr5, and c-Myc mRNAs, suggesting that RBMS3 silencing elevates signaling through the WNT/β-catenin signaling axis. Although RBMS3 silencing rendered BRAFV600E-driven lung tumors resistant to the effects of dabrafenib plus trametinib, the tumors were sensitive to inhibition of porcupine, an acyltransferase of WNT ligands necessary for their secretion. Analysis of The Cancer Genome Atlas patient samples revealed that chromosome 3p24, which encompasses RBMS3, is frequently lost in non-small cell lung cancer and correlates with poor prognosis. Collectively, these data reveal the role of RBMS3 as a lung cancer suppressor and suggest that RBMS3 silencing may contribute to malignant NSCLC progression.SignificanceLoss of RBMS3 cooperates with BRAFV600E to induce lung tumorigenesis, providing a deeper understanding of the molecular mechanisms underlying mutant BRAF-driven lung cancer and potential strategies to more effectively target this disease.

The Xenopus embryo provides an advantageous model system where genes can be readily transplanted as DNA or mRNA or depleted with antisense techniques. Here, we present a protocol to culture and image the cell biological properties of explanted Xenopus cap cells in tissue culture. We illustrate how this protocol can be applied to visualize lysosomes, macropinocytosis, focal adhesions, Wnt signaling, and cell migration. For complete details on the use and execution of this protocol, please refer to Tejeda-Muñoz et al. (2022).

Lung maturation is not limited to proper structural development but also includes differentiation and functionality of various highly specialized alveolar cell types. Alveolar type 1 (AT1s) cells occupy nearly 95% of the alveolar surface and are critical for establishing efficient gas exchange in the mature lung. AT1 cells arise from progenitors specified during the embryonic stage as well as alveolar epithelial progenitors expressing surfactant protein C (Sftpcpos cells) during postnatal and adult stages. Previously, we found that Wnt5a, a non-canonical Wnt ligand, is required for differentiation of AT1 cells during the saccular phase of lung development. To further investigate the role of Wnt5a in AT1 cell differentiation, we generated and characterized a conditional Wnt5a gain-of-function mouse model. Neonatal Wnt5a gain-of-function disrupted alveologenesis through inhibition of cell proliferation. In this setting Wnt5a downregulated β-catenin-dependent canonical Wnt signaling, repressed AT2 (anti-AT2) and promoted AT1 (pro-AT1) lineage-specific gene expression. In addition, we identified 2 subpopulations of Sftpchigh and Sftpclow alveolar epithelial cells. In Sftpclow cells, Wnt5a exhibits pro-AT1 and anti-AT2 effects, concurrent with inhibition of canonical Wnt signaling. Interestingly, in the Sftpchigh subpopulation, although increasing AT1 lineage-specific gene expression, Wnt5a gain-of-function did not change AT2 gene expression, nor inhibit canonical Wnt signaling. Using primary epithelial cells isolated from human fetal lungs, we demonstrate that this property of Wnt5a is evolutionarily conserved. Wnt5a therefore serves as a selective regulator that ensures proper AT1/AT2 balance in the developing lung.

Restoring sensation after injury or disease requires a reproducible method for generating large quantities of bona fide somatosensory interneurons. Toward this goal, we assess the mechanisms by which dorsal spinal interneurons (dIs; dI1-dI6) can be derived from mouse embryonic stem cells (mESCs). Using two developmentally relevant growth factors, retinoic acid (RA) and bone morphogenetic protein (BMP) 4, we recapitulate the complete in vivo program of dI differentiation through a neuromesodermal intermediate. Transcriptional profiling reveals that mESC-derived dIs strikingly resemble endogenous dIs, with the correct molecular and functional signatures. We further demonstrate that RA specifies dI4-dI6 fates through a default multipotential state, while the addition of BMP4 induces dI1-dI3 fates and activates Wnt signaling to enhance progenitor proliferation. Constitutively activating Wnt signaling permits the dramatic expansion of neural progenitor cultures. These cultures retain the capacity to differentiate into diverse populations of dIs, thereby providing a method of increasing neuronal yield.

Modulating the number of muscle stems cells, called satellite cells, during early postnatal development produces long-term effects on muscle growth. We tested the hypothesis that high expression levels of the anti-aging protein Klotho in early postnatal myogenesis increase satellite cell numbers by influencing the epigenetic regulation of genes that regulate myogenesis. Our findings show that elevated klotho expression caused a transient increase in satellite cell numbers and slowed muscle fiber growth, followed by a period of accelerated muscle growth that leads to larger fibers. Klotho also transcriptionally downregulated the H3K27 demethylase Jmjd3, leading to increased H3K27 methylation and decreased expression of genes in the canonical Wnt pathway, which was associated with a delay in muscle differentiation. In addition, Klotho stimulation and Jmjd3 downregulation produced similar but not additive reductions in the expression of Wnt4, Wnt9a, and Wnt10a in myogenic cells, indicating that inhibition occurred through a common pathway. Together, our results identify a novel pathway through which Klotho influences myogenesis by reducing the expression of Jmjd3, leading to reductions in the expression of Wnt genes and inhibition of canonical Wnt signaling.

The recent classification of colon cancer into molecular subtypes revealed that patients with the poorest prognosis harbor tumors with the lowest levels of Wnt signaling. This is contrary to the general understanding that overactive Wnt signaling promotes tumor progression from early initiation stages through to the later stages including invasion and metastasis. Here, we directly test this assumption by reducing the activity of ß-catenin-dependent Wnt signaling in colon cancer cell lines at either an upstream or downstream step in the pathway. We determine that Wnt-reduced cancer cells exhibit a more aggressive disease phenotype, including increased mobility in vitro and disruptive invasion into mucosa and smooth muscle in an orthotopic mouse model. RNA sequencing reveals that interference with Wnt signaling leads to an upregulation of gene programs that favor cell migration and invasion and a downregulation of inflammation signatures in the tumor microenvironment. We identify a set of upregulated genes common among the Wnt perturbations that are predictive of poor patient outcomes in early-invasive colon cancer. Our findings suggest that while targeting Wnt signaling may reduce tumor burden, an inadvertent side effect is the emergence of invasive cancer.ImplicationsDecreased Wnt signaling in colon tumors leads to a more aggressive disease phenotype due to an upregulation of gene programs favoring cell migration in the tumor and downregulation of inflammation programs in the tumor microenvironment; these impacts must be carefully considered in developing Wnt-targeting therapies.

The choroid plexus secretes cerebrospinal fluid and is critical for the development and function of the brain. In the telencephalon, the choroid plexus epithelium arises from the Wnt- expressing cortical hem. Canonical Wnt signaling pathway molecules such as nuclear β-CATENIN are expressed in the mouse and human embryonic choroid plexus epithelium indicating that this pathway is active. Point mutations in human β-CATENIN are known to result in the constitutive activation of canonical Wnt signaling. In a mouse model that recapitulates this perturbation, we report a loss of choroid plexus epithelial identity and an apparent transformation of this tissue to a neuronal identity. Aspects of this phenomenon are recapitulated in human embryonic stem cell derived organoids. The choroid plexus is also disrupted when β-Catenin is conditionally inactivated. Together, our results indicate that canonical Wnt signaling is required in a precise and regulated manner for normal choroid plexus development in the mammalian brain.

De novo truncations in Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) lead to severe childhood-onset neurodegenerative disorders. To determine how loss of IRF2BPL causes neural dysfunction, we examined its function in Drosophila and zebrafish. Overexpression of either IRF2BPL or Pits, the Drosophila ortholog, represses Wnt transcription in flies. In contrast, neuronal depletion of Pits leads to increased wingless (wg) levels in the brain and is associated with axonal loss, whereas inhibition of Wg signaling is neuroprotective. Moreover, increased neuronal expression of wg in flies is sufficient to cause age-dependent axonal loss, similar to reduction of Pits. Loss of irf2bpl in zebrafish also causes neurological defects with an associated increase in wnt1 transcription and downstream signaling. WNT1 is also increased in patient-derived astrocytes, and pharmacological inhibition of Wnt suppresses the neurological phenotypes. Last, IRF2BPL and the Wnt antagonist, CKIα, physically and genetically interact, showing that IRF2BPL and CkIα antagonize Wnt transcription and signaling.

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

Recent findings suggest that the ribosome itself modulates gene expression. However, whether ribosomes change composition across cell types or control cell fate remains unknown. Here, employing quantitative mass spectrometry during human embryonic stem cell differentiation, we identify dozens of ribosome composition changes underlying cell fate specification. We observe upregulation of RPL10A/uL1-containing ribosomes in the primitive streak followed by progressive decreases during mesoderm differentiation. An Rpl10a loss-of-function allele in mice causes striking early mesodermal phenotypes, including posterior trunk truncations, and inhibits paraxial mesoderm production in culture. Ribosome profiling in Rpl10a loss-of-function mice reveals decreased translation of mesoderm regulators, including Wnt pathway mRNAs, which are also enriched on RPL10A/uL1-containing ribosomes. We further show that RPL10A/uL1 regulates canonical and non-canonical Wnt signaling during stem cell differentiation and in the developing embryo. These findings reveal unexpected ribosome composition modularity that controls differentiation and development through the specialized translation of key signaling networks.

The cnidarian model organism Hydra has long been studied for its remarkable ability to regenerate its head, which is controlled by a head organizer located near the hypostome. The canonical Wnt pathway plays a central role in head organizer function during regeneration and during bud formation, which is the asexual mode of reproduction in Hydra. However, it is unclear how shared the developmental programs of head organizer genesis are in budding and regeneration. Time-series analysis of gene expression changes during head regeneration and budding revealed a set of 298 differentially expressed genes during the 48-h head regeneration and 72-h budding time courses. In order to understand the regulatory elements controlling Hydra head regeneration, we first identified 27,137 open-chromatin elements that are open in one or more sections of the organism body or regenerating tissue. We used histone modification ChIP-seq to identify 9,998 candidate proximal promoter and 3,018 candidate enhancer-like regions respectively. We show that a subset of these regulatory elements is dynamically remodeled during head regeneration and identify a set of transcription factor motifs that are enriched in the enhancer regions activated during head regeneration. Our results show that Hydra displays complex gene regulatory structures of developmentally dynamic enhancers, which suggests that the evolution of complex developmental enhancers predates the split of cnidarians and bilaterians.

Bovine embryonic stem cells (ESC) have features associated with the primed pluripotent state including low expression of one of the core pluripotency transcription factors, NANOG. It has been reported that NANOG expression can be upregulated in porcine ESC by treatment with activin A and the WNT agonist CHIR99021. Accordingly, it was tested whether expression of NANOG and another pluripotency factor SOX2 could be stimulated by activin A and the WNT agonist CHIR99021. Immunoreactive NANOG and SOX2 were analyzed for bovine ESC lines derived under conditions in which activin A and CHIR99021 were added singly or in combination. Activin A enhanced NANOG expression but also reduced SOX2 expression. CHIR99021 depressed expression of both NANOG and SOX2. In a second experiment, activin A enhanced blastocyst development while CHIR99021 treatment impaired blastocyst formation and reduced number of blastomeres. Activin A treatment decreased blastomeres in the blastocyst that were positive for either NANOG or SOX2 but increased those that were CDX2+ and that were GATA6+ outside the inner cell mass. CHIR99021 reduced SOX2+ and NANOG+ blastomeres without affecting the number or percent of blastomeres that were CDX2+ and GATA6+. Results indicate activation of activin A signaling stimulates NANOG expression during self-renewal of bovine ESC but suppresses cells expressing pluripotency markers in the blastocyst and increases cells expressing CDX2. Actions of activin A to promote blastocyst development may involve its role in promoting trophectoderm formation. Furthermore, results demonstrate the negative role of canonical WNT signaling in cattle for pluripotency marker expression in ESC and in formation of the inner cell mass and epiblast during embryonic development. This article has an associated First Person interview with the first author of the paper.

Stem cells, including the resident lung mesenchymal stem cells (LMSCs), are critically important for injury repair. Compelling evidence links perinatal vitamin D (VD) deficiency to reactive airway disease; however, the effects of perinatal VD deficiency on LMSC function is unknown. We tested the hypothesis that perinatal VD deficiency alters LMSC proliferation, differentiation, and function, leading to an enhanced myogenic phenotype. We also determined whether LMSCs' effects on alveolar type II (ATII) cell function are paracrine. Using an established rat model of perinatal VD deficiency, we studied the effects of four dietary regimens (0, 250, 500, or 1,000 IU/kg cholecalciferol-supplemented groups). At Postnatal Day 21, LMSCs were isolated, and cell proliferation and differentiation (under basal and adipogenic induction conditions) were determined. LMSC paracrine effects on ATII cell proliferation and differentiation were determined by culturing ATII cells in LMSC-conditioned media from different experimental groups. Using flow cytometry, >95% of cells were CD45-ve, >90% were CD90 + ve, >58% were CD105 + ve, and >64% were Stro-1 + ve, indicating their stem cell phenotype. Compared with the VD-supplemented groups, LMSCs from the VD-deficient group demonstrated suppressed PPARγ, but enhanced Wnt signaling, under basal and adipogenic induction conditions. LMSCs from 250 VD- and 500 VD-supplemented groups effectively blocked the effects of perinatal VD deficiency. LMSC-conditioned media from the VD-deficient group inhibited ATII cell proliferation and differentiation compared with those from the 250 VD- and 500 VD-supplemented groups. These data support the concept that perinatal VD deficiency alters LMSC proliferation and differentiation, potentially contributing to increased respiratory morbidity seen in children born to mothers with VD deficiency.

The mechanisms of Myc-driven liver tumorigenesis are inadequately understood. Herein we show that Myc-driven hepatocellular carcinoma (HCC) is dramatically aggravated in mice with hepatocyte-specific Ptpn11/Shp2 deletion. However, Myc-induced tumors develop selectively from the rare Shp2-positive hepatocytes in Shp2-deficent liver, and Myc-driven oncogenesis depends on an intact Ras-Erk signaling promoted by Shp2 to sustain Myc stability. Despite a stringent requirement of Shp2 cell autonomously, Shp2 deletion induces an immunosuppressive environment, resulting in defective clearance of tumor-initiating cells and aggressive tumor progression. The basal Wnt/β-catenin signaling is upregulated in Shp2-deficient liver, which is further augmented by Myc transfection. Ablating Ctnnb1 suppresses Myc-induced HCC in Shp2-deficient livers, revealing an essential role of β-catenin. Consistently, Myc overexpression and CTNNB1 mutations are frequently co-detected in HCC patients with poor prognosis. These data elucidate complex mechanisms of liver tumorigenesis driven by cell-intrinsic oncogenic signaling in cooperation with a tumor-promoting microenvironment generated by disrupting the specific oncogenic pathway.

Oligodendrocyte (OL) maturation arrest in human white matter injury contributes significantly to the failure of endogenous remyelination in multiple sclerosis (MS) and newborn brain injuries such as hypoxic ischemic encephalopathy (HIE) that cause cerebral palsy. In this study, we identify an oligodendroglial-intrinsic factor that controls OL maturation specifically in the setting of injury. We find a requirement for the ring finger protein Rnf43 not in normal development but in neonatal hypoxic injury and remyelination in the adult mammalian CNS. Rnf43, but not the related Znrf3, is potently activated by Wnt signaling in OL progenitor cells (OPCs) and marks activated OPCs in human MS and HIE. Rnf43 is required in an injury-specific context, and it promotes OPC differentiation through negative regulation of Wnt signal strength in OPCs at the level of Fzd1 receptor presentation on the cell surface. Inhibition of Fzd1 using UM206 promotes remyelination following ex vivo and in vivo demyelinating injury.

BackgroundStructural birth defects occur in approximately 3% of live births; most such defects lack defined genetic or environmental causes. Despite advances in surgical approaches, pharmacologic prevention remains largely out of reach.MethodsWe queried worldwide databases of 20,248 families that included children with neurodevelopmental disorders and that were enriched for parental consanguinity. Approximately one third of affected children in these families presented with structural birth defects or microcephaly. We performed exome or genome sequencing of samples obtained from the children, their parents, or both to identify genes with biallelic pathogenic or likely pathogenic mutations present in more than one family. After identifying disease-causing variants, we generated two mouse models, each with a pathogenic variant "knocked in," to study mechanisms and test candidate treatments. We administered a small-molecule Wnt agonist to pregnant animals and assessed their offspring.ResultsWe identified homozygous mutations in WLS, which encodes the Wnt ligand secretion mediator (also known as Wntless or WLS) in 10 affected persons from 5 unrelated families. (The Wnt ligand secretion mediator is essential for the secretion of all Wnt proteins.) Patients had multiorgan defects, including microcephaly and facial dysmorphism as well as foot syndactyly, renal agenesis, alopecia, iris coloboma, and heart defects. The mutations affected WLS protein stability and Wnt signaling. Knock-in mice showed tissue and cell vulnerability consistent with Wnt-signaling intensity and individual and collective functions of Wnts in embryogenesis. Administration of a pharmacologic Wnt agonist partially restored embryonic development.ConclusionsGenetic variations affecting a central Wnt regulator caused syndromic structural birth defects. Results from mouse models suggest that what we have named Zaki syndrome is a potentially preventable disorder. (Funded by the National Institutes of Health and others.).

The signaling pathway directly controlling the maintenance of adult glutamatergic synapses has not been well understood. Planar cell polarity (PCP) signaling components were recently shown to play essential roles in the formation of glutamatergic synapses. Here, we show that they are localized in the adult synapses and are essential for their maintenance. Synapse loss at early stages of Alzheimer's disease is thought to be induced by β-amyloid (Aβ) pathology. We found that oligomeric Aβ binds to Celsr3 and assists Vangl2 in disassembling synapses. Moreover, a Wnt receptor and regulator of PCP signaling, Ryk, is also required for Aβ-induced synapse loss. In the 5XFAD mouse model of Alzheimer's disease, Ryk conditional knockout or a function-blocking monoclonal Ryk antibody protected synapses and preserved cognitive function. We propose that tipping of the fine balance of Wnt/PCP signaling components in glutamatergic synapses may cause synapse degeneration in neurodegenerative disorders with Aβ pathology.

The WNT signaling system plays an important but paradoxical role in the regulation of pluripotency. In the cow, IWR-1, which inhibits canonical WNT activation and has WNT-independent actions, promotes the derivation of primed pluripotent embryonic stem cells from the blastocyst. Here, we describe a series of experiments to determine whether derivation of embryonic stem cells could be generated by replacing IWR-1 with other inhibitors of WNT signaling. Results confirm the importance of inhibition of canonical WNT signaling for the establishment of pluripotent embryonic stem cells in cattle and indicate that the actions of IWR-1 can be mimicked by the WNT secretion inhibitor IWP2 but not by the tankyrase inhibitor XAV939 or WNT inhibitory protein dickkopf 1. The role of Janus kinase-mediated signaling pathways for the maintenance of pluripotency of embryonic stem cells was also evaluated. Maintenance of pluripotency of embryonic stem cells lines was blocked by a broad inhibitor of Janus kinase, even though the cells did not express phosphorylated signal transducer and activator of transcription 3 (pSTAT3). Further studies with blastocysts indicated that IWR-1 blocks the activation of pSTAT3. A likely explanation is that IWR-1 blocks differentiation of embryonic stem cells into a pSTAT3+ lineage. In conclusion, results presented here indicate the importance of inhibition of WNT signaling for the derivation of pluripotent bovine embryonic stem cells, the role of Janus kinase signaling for maintenance of pluripotency, and the participation of IWR-1 in the inhibition of activation of STAT3.

Background & aimsGain of function (GOF) mutations in the CTNNB1 gene are one of the most frequent genetic events in hepatocellular carcinoma (HCC). T-box transcription factor 3 (TBX3) is a liver-specific target of the Wnt/β-catenin pathway and thought to be an oncogene mediating activated β-catenin-driven HCC formation.MethodsWe evaluated the expression pattern of TBX3 in human HCC specimens. Tbx3 was conditionally knocked out in murine HCC models by hydrodynamic tail vein injection of Cre together with c-Met and ΔN90-β-catenin (c-Met/β-catenin) in Tbx3flox/flox mice. TBX3 was overexpressed in human HCC cell lines to investigate the functions of TBX3 in vitro.ResultsA bimodal expression pattern of TBX3 in human HCC samples was detected: high expression of TBX3 in GOF CTNNB1 HCC and downregulation of TBX3 in non-CTNNB1 mutant tumors. High expression of TBX3 was associated with increased differentiation and decreased expression signatures of tumor growth. Using Tbx3flox/flox mice, we found that ablation of Tbx3 significantly accelerates c-Met/β-catenin-driven HCC formation. Moreover, Tbx3(-) HCC demonstrated increased YAP/TAZ activity. The accelerated tumor growth induced by loss of TBX3 in c-Met/β-catenin mouse HCC was successfully prevented by overexpression of LATS2, which inhibited YAP/TAZ activity. In human HCC cell lines, overexpression of TBX3 inhibited HCC cell growth as well as YAP/TAZ activation. A negative correlation between TBX3 and YAP/TAZ target genes was observed in human HCC samples. Mechanistically, phospholipase D1 (PLD1), a known positive regulator of YAP/TAZ, was identified as a novel transcriptional target repressed by TBX3.ConclusionOur study suggests that TBX3 is induced by GOF CTNNB1 mutants and suppresses HCC growth by inactivating PLD1, thus leading to the inhibition of YAP/TAZ oncogenes.Lay summaryTBX3 is a liver-specific target of the Wnt/β-catenin pathway and thought to be an oncogene in promoting liver cancer development. Herein, we demonstrate that TBX3 is in fact a tumor suppressor gene that restricts liver tumor growth. Strategies which increase TBX3 expression and/or activities may be effective for HCC treatment.

Wnt5a-Ror signaling is a conserved pathway that regulates morphogenetic processes during vertebrate development [R. T. Moon et al, Development 119, 97-111 (1993); I. Oishi et al, Genes Cells 8, 645-654 (2003)], but its downstream signaling events remain poorly understood. Through a large-scale proteomic screen in mouse embryonic fibroblasts, we identified the E3 ubiquitin ligase Pdzrn3 as a regulatory target of the Wnt5a-Ror pathway. Upon pathway activation, Pdzrn3 is degraded in a β-catenin-independent, ubiquitin-proteasome system-dependent manner. We developed a flow cytometry-based reporter to monitor Pdzrn3 abundance and delineated a signaling cascade involving Frizzled, Dishevelled, Casein kinase 1, and Glycogen synthase kinase 3 that regulates Pdzrn3 stability. Epistatically, Pdzrn3 is regulated independently of Kif26b, another Wnt5a-Ror effector. Wnt5a-dependent degradation of Pdzrn3 requires phosphorylation of three conserved amino acids within its C-terminal LNX3H domain [M. Flynn, O. Saha, P. Young, BMC Evol. Biol. 11, 235 (2011)], which acts as a bona fide Wnt5a-responsive element. Importantly, this phospho-dependent degradation is essential for Wnt5a-Ror modulation of cell migration. Collectively, this work establishes a Wnt5a-Ror cell morphogenetic cascade involving Pdzrn3 phosphorylation and degradation.

Tumors often secrete wasting factors associated with atrophy and the degeneration of host tissues. If tumors were to be affected by the wasting factors, mechanisms allowing tumors to evade the adverse effects of the wasting factors must exist, and impairing such mechanisms may attenuate tumors. We use Drosophila midgut tumor models to show that tumors up-regulate Wingless (Wg) to oppose the growth-impeding effects caused by the wasting factor, ImpL2 (insulin-like growth factor binding protein [IGFBP]-related protein). Growth of Yorkie (Yki)-induced tumors is dependent on Wg while either elimination of ImpL2 or elevation of insulin/insulin-like growth factor signaling in tumors revokes this dependency. Notably, Wg augmentation could be a general mechanism for supporting the growth of tumors with elevated ImpL2 and exploited to attenuate muscle degeneration during wasting. Our study elucidates the mechanism by which tumors negate the action of ImpL2 to uphold their growth during cachexia-like wasting and implies that targeting the Wnt/Wg pathway might be an efficient treatment strategy for cancers with elevated IGFBPs.

The huge cadre of genes regulated by Myc has obstructed the identification of critical effectors that are essential for Myc-driven tumorigenesis. Here, we describe how only the lack of the receptor Fzd9, previously identified as a Myc transcriptional target, impairs sustained tumor expansion and β-cell dedifferentiation in a mouse model of Myc-driven insulinoma, allows pancreatic islets to maintain their physiological structure and affects Myc-related global gene expression. Importantly, Wnt signaling inhibition in Fzd9-competent mice largely recapitulates the suppression of proliferation caused by Fzd9 deficiency upon Myc activation. Together, our results indicate that the Wnt signaling receptor Fzd9 is essential for Myc-induced tumorigenesis in pancreatic islets.

Hepatocellular carcinoma (HCC) and hepatoblastoma are the major types of primary liver cancer in adulthood and childhood, respectively. Wnt/β-catenin signaling deregulation is one of the most frequent genetic events in hepatocarcinogenesis. APC regulator of WNT signaling pathway (APC) encodes an inhibitor of the Wnt cascade and acts as a tumor suppressor. Germline defects of the APC gene lead to familial adenomatous polyposis, and its somatic mutations occur in multiple tumor types. However, the contribution of APC in hepatocarcinogenesis remains unclear. Therefore, APC mutations and expression patterns were examined in human HCC and hepatoblastoma samples. Whether loss of Apc alone or in cooperation with other oncogenes triggers liver tumor development in vivo was also investigated. sgApc alone could not drive liver tumor formation, but synergized with activated oncogenes (YapS127A, TazS89A, and c-Met) to induce hepatocarcinogenesis. Mechanistically, Apc deletion induced the activation of β-catenin and its downstream targets in mouse liver tumors. Furthermore, Ctnnb1 ablation or TCF4-mediated transcription blockade completely prevented liver tumor formation, indicating the requirement of a functional β-catenin pathway for loss of Apc-driven hepatocarcinogenesis. This study shows that a subset of HCC patients with loss-of-function APC mutations might benefit from therapeutic strategies targeting the Wnt/β-catenin pathway.

The developing CNS is exposed to physiological hypoxia, under which hypoxia-inducible factor α (HIFα) is stabilized and plays a crucial role in regulating neural development. The cellular and molecular mechanisms of HIFα in developmental myelination remain incompletely understood. A previous concept proposes that HIFα regulates CNS developmental myelination by activating the autocrine Wnt/β-catenin signaling in oligodendrocyte progenitor cells (OPCs). Here, by analyzing a battery of genetic mice of both sexes, we presented in vivo evidence supporting an alternative understanding of oligodendroglial HIFα-regulated developmental myelination. At the cellular level, we found that HIFα was required for developmental myelination by transiently controlling upstream OPC differentiation but not downstream oligodendrocyte maturation and that HIFα dysregulation in OPCs but not oligodendrocytes disturbed normal developmental myelination. We demonstrated that HIFα played a minor, if any, role in regulating canonical Wnt signaling in the oligodendroglial lineage or in the CNS. At the molecular level, blocking autocrine Wnt signaling did not affect HIFα-regulated OPC differentiation and myelination. We further identified HIFα-Sox9 regulatory axis as an underlying molecular mechanism in HIFα-regulated OPC differentiation. Our findings support a concept shift in our mechanistic understanding of HIFα-regulated CNS myelination from the previous Wnt-dependent view to a Wnt-independent one and unveil a previously unappreciated HIFα-Sox9 pathway in regulating OPC differentiation.SIGNIFICANCE STATEMENT Promoting disturbed developmental myelination is a promising option in treating diffuse white matter injury, previously called periventricular leukomalacia, a major form of brain injury affecting premature infants. In the developing CNS, hypoxia-inducible factor α (HIFα) is a key regulator that adapts neural cells to physiological and pathologic hypoxic cues. The role and mechanism of HIFα in oligodendroglial myelination, which is severely disturbed in preterm infants affected with diffuse white matter injury, is incompletely understood. Our findings presented here represent a concept shift in our mechanistic understanding of HIFα-regulated developmental myelination and suggest the potential of intervening with an oligodendroglial HIFα-mediated signaling pathway to mitigate disturbed myelination in premature white matter injury.

During whole-body regeneration, a bisection injury can trigger two different types of regeneration. To understand the transcriptional regulation underlying this adaptive response, we characterized transcript abundance and chromatin accessibility during oral and aboral regeneration in the cnidarian Hydra vulgaris. We found that the initial response to amputation at both wound sites is identical and includes widespread apoptosis and the activation of the oral-specifying Wnt signaling pathway. By 8 hr post amputation, Wnt signaling became restricted to oral regeneration. Wnt pathway genes were also upregulated in puncture wounds, and these wounds induced the formation of ectopic oral structures if pre-existing organizers were simultaneously amputated. Our work suggests that oral patterning is activated as part of a generic injury response in Hydra, and that alternative injury outcomes are dependent on signals from the surrounding tissue. Furthermore, Wnt signaling is likely part of a conserved wound response predating the split of cnidarians and bilaterians.

The respiratory tract is a vital, intricate system for several important biological processes including mucociliary clearance, airway conductance, and gas exchange. The Wnt signaling pathway plays several crucial and indispensable roles across lung biology in multiple contexts. This review highlights the progress made in characterizing the role of Wnt signaling across several disciplines in lung biology, including development, homeostasis, regeneration following injury, in vitro directed differentiation efforts, and disease progression. We further note uncharted directions in the field that may illuminate important biology. The discoveries made collectively advance our understanding of Wnt signaling in lung biology and have the potential to inform therapeutic advancements for lung diseases.

Wound healing is a multi-step process to rapidly restore the barrier function. This process is often impaired in diabetic patients resulting in chronic wounds and amputation. We previously found that paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway via topical administration of the BRAF inhibitor vemurafenib accelerates wound healing by activating keratinocyte proliferation and reepithelialization pathways in healthy mice. Herein, we investigated whether this wound healing acceleration also occurs in impaired diabetic wounds and found that topical vemurafenib not only improves wound healing in a murine diabetic wound model but unexpectedly promotes hair follicle regeneration. Hair follicles expressing Sox-9 and K15 surrounded by CD34+ stroma were found in wounds of diabetic and non-diabetic mice, and their formation can be prevented by blocking downstream MEK signaling. Thus, topically applied BRAF inhibitors may accelerate wound healing, and promote the restoration of improved skin architecture in both normal and impaired wounds.

CCN1/Cyr61 is a dynamically expressed matricellular protein that serves regulatory functions in multiple tissues. Previous studies from our laboratory demonstrated that CCN1 regulates bone maintenance. Using an osteoblast and osteocyte conditional knockout mouse model (Ccn1OCN ), we found a significant decrease in trabecular and cortical bone mass in vivo, in part through suppression of Wnt signaling since the expression of the Wnt antagonist sclerostin (SOST) is increased in osteoblasts lacking CCN1. It has been established that parathyroid hormone (PTH) signaling also suppresses SOST expression in bone. We therefore investigated the interaction between CCN1 and PTH-mediated responses in this study. We find that loss of Ccn1 in osteoblasts leads to impaired responsiveness to anabolic intermittent PTH treatment in Ccn1OCN mice in vivo and in osteoblasts from these mice in vitro. Analysis of Ccn1OCN mice demonstrated a significant decrease in parathyroid hormone receptor-1 (PTH1R) expression in osteoblasts in vivo and in vitro. We investigated the regulatory role of a non-canonical integrin-binding domain of CCN1 because several studies indicate that specific integrins are critical to mechanotransduction, a PTH-dependent response, in bone. These data suggest that CCN1 regulates the expression of PTH1R through interaction with the αvβ3 and/or αvβ5 integrin complexes. Osteoblasts that express a mutant form of CCN1 that cannot interact with αvβ3/β5 integrin demonstrate a significant decrease in mRNA and protein expression of both PTH1R and αv integrin. Overall, these data suggest that the αvβ3/β5-binding domain of CCN1 is required to endow PTH signaling with anabolic activity in bone cells. © 2020 American Society for Bone and Mineral Research (ASBMR).

Embryogenesis is directed by morphogens that induce differentiation within a defined tissue geometry. Tissue organization is mediated by cell-cell and cell-extracellular matrix (ECM) adhesions and is modulated by cell tension and tissue-level forces. Whether cell tension regulates development by modifying morphogen signaling is less clear. Human embryonic stem cells (hESCs) exhibit an intrinsic capacity for self-organization, which motivates their use as a tractable model of early human embryogenesis. We engineered patterned substrates that recapitulate the biophysical properties of the early embryo and mediate the self-organization of "gastrulation-like" nodes in cultured hESCs. Tissue geometries that generated local nodes of high cell-adhesion tension directed the spatial patterning of the BMP4-dependent "gastrulation-like" phenotype by enhancing phosphorylation and junctional release of β-catenin to promote Wnt signaling and mesoderm specification. Furthermore, direct force application via mechanical stretching promoted BMP-dependent mesoderm specification, confirming that tissue-level forces can directly regulate cell fate specification in early human development.

Stem cells undergo differentiation in complex and dynamic environments wherein instructive signals fluctuate on various timescales. Thus, cells must be equipped to properly respond to the timing of signals, for example, to distinguish sustained signaling from transient noise. However, how stem cells respond to dynamic variations in differentiation cues is not well characterized. Here, we use optogenetic activation of β-catenin signaling to probe the dynamic responses of differentiating adult neural stem cells (NSCs). We discover that, while elevated, sustained β-catenin activation sequentially promotes proliferation and differentiation, transient β-catenin induces apoptosis. Genetic perturbations revealed that the neurogenic/apoptotic fate switch was mediated through cell-cycle regulation by Growth Arrest and DNA Damage 45 gamma (Gadd45γ). Our results thus reveal a role for β-catenin dynamics in NSC fate decisions and may suggest a role for signal timing to minimize cell-fate errors, analogous to kinetic proofreading of stem-cell differentiation.

It is known that LGR4 plays an important role in hair follicle (HF) development, but the impact of LGR4 on the hair cycle is still unclear. In this study, we have found that K14-Cre-mediated skin epithelia-specific deletion of Lgr4 results in delayed anagen entry during the physiological hair cycle and compromised HF regeneration upon transplantation. We show that, although Lgr4 deletion does not appear to affect the number of quiescent HF stem cells, it leads to reduced numbers of LGR5+ and actively proliferating stem cells in the HFs. Moreover, LGR4-deficient HFs show molecular changes consistent with decreased mTOR and Wnt signaling but upregulated BMP signaling. Importantly, the reactivation of the protein kinase B pathway by injecting the protein kinase B activator SC79 in Lgr4-/- mice can effectively reverse the hair cycle delay. Together, these data suggest that LGR4 promotes the normal hair cycle by activating HF stem cells and by influencing the activities of multiple signaling pathways that are known to regulate HF stem cells. Our study also implicates LGR4 as a potential target for treating hair disorder in the future.

Our understanding of dynamic interactions between airway basal stem cells (ABSCs) and their signaling niches in homeostasis, injury, and aging remains elusive. Using transgenic mice and pharmacologic studies, we found that Wnt/β-catenin within ABSCs was essential for proliferation post-injury in vivo. ABSC-derived Wnt ligand production was dispensable for epithelial proliferation. Instead, the PDGFRα+ lineage in the intercartilaginous zone (ICZ) niche transiently secreted Wnt ligand necessary for ABSC proliferation. Strikingly, ABSC-derived Wnt ligand later drove early progenitor differentiation to ciliated cells. We discovered additional changes in aging, as glandular-like epithelial invaginations (GLEIs) derived from ABSCs emerged exclusively in the ICZ of aged mice and contributed to airway homeostasis and repair. Further, ABSC Wnt ligand secretion was necessary for GLEI formation, and constitutive activation of β-catenin in young mice induced their formation in vivo. Collectively, these data underscore multiple spatiotemporally dynamic Wnt-secreting niches that regulate functionally distinct phases of airway regeneration and aging.

The CreER/LoxP system is widely accepted to track neural lineages and study gene functions upon tamoxifen (TAM) administration. We have observed that prenatal TAM treatment caused high rates of delayed delivery and fetal mortality. This substance could produce undesired results, leading to data misinterpretation. Here, we report that administration of TAM during early stages of cortical neurogenesis promoted precocious neural differentiation, while it inhibited neural progenitor cell (NPC) proliferation. The TAM-induced inhibition of NPC proliferation led to deficits in cortical neurogenesis, dendritic morphogenesis, synaptic formation, and cortical patterning in neonatal and postnatal offspring. Mechanistically, by employing single-cell RNA-sequencing (scRNA-seq) analysis combined with in vivo and in vitro assays, we show TAM could exert these drastic effects mainly through dysregulating the Wnt-Dmrta2 signaling pathway. In adult mice, administration of TAM significantly attenuated NPC proliferation in both the subventricular zone and the dentate gyrus. This study revealed the cellular and molecular mechanisms for the adverse effects of TAM on corticogenesis, suggesting that care must be taken when using the TAM-induced CreER/LoxP system for neural lineage tracing and genetic manipulation studies in both embryonic and adult brains.