Production Of Wnt Ligands Research Articles

Wnt Signaling in the Phenotype and Function of Tumor-Associated Macrophages.

Tumor-associated macrophages (TAM) play an important role in supporting tumor growth and suppressing antitumor immune responses, and TAM infiltration has been associated with poor patient prognosis in various cancers. TAMs can be classified as pro-inflammatory, M1-like, or anti-inflammatory, M2-like. While multiple factors within the tumor microenvironment affect the recruitment, polarization, and functions of TAMs, accumulating evidence suggests that Wnt signaling represents an important, targetable driver of an immunosuppressive, M2-like TAM phenotype. TAM production of Wnt ligands mediates TAM-tumor cross-talk to support cancer cell proliferation, invasion, and metastasis. Targeting TAM polarization and the protumorigenic functions of TAMs through inhibitors of Wnt signaling may prove a beneficial treatment strategy in cancers where macrophages are prevalent in the microenvironment.

Positive feedback regulation of frizzled-7 expression robustly shapes a steep Wnt gradient in Xenopus heart development, together with sFRP1 and heparan sulfate.

Secreted molecules called morphogens govern tissue patterning in a concentration-dependent manner. However, it is still unclear how reproducible patterning can be achieved with diffusing molecules, especially when that patterning concerns differentiation of thin tissues. Wnt is a morphogen that organizes cardiac development. Wnt6 patterns cardiogenic mesoderm to induce differentiation of a thin tissue, the pericardium, in Xenopus. In this study, we revealed that a Wnt receptor, frizzled-7, is expressed in a Wnt-dependent manner. With a combination of experiments and mathematical modeling, this receptor-feedback appears essential to shape a steep gradient of Wnt signaling. In addition, computer simulation revealed that this feedback imparts robustness against variations of Wnt ligand production and allows the system to reach a steady state quickly. We also found that a Wnt antagonist sFRP1, which is expressed on the opposite side of the Wnt source, accumulates on N-acetyl-rich heparan sulfate (HS). N-acetyl-rich HS concentration is high between the sources of Wnt and sFRP1, achieving local inhibition of Wnt signaling via restriction of sFRP1 spreading. These integrated regulatory systems restrict the Wnt signaling range and ensure reproducible patterning of the thin pericardium.

Enteric glia bring fresh WNT to the intestinal stem cell niche

Intestinal stem cells continuously self-renew and differentiate into a variety of specialized epithelial cells that maintain gut health. New research in this issue of Cell Stem Cell (Baghdadi etal., 2022) shows that enteric glial cells regulate the intestinal stem cell niche during regeneration and disease through the production of WNT ligands.

Liver Endothelial Heg Regulates Vascular/Biliary Network Patterning and Metabolic Zonation Via Wnt Signaling.

Background & AimsThe liver has complex interconnecting blood vessel and biliary networks; however, how the vascular and biliary network form and regulate each other and liver function are not well-understood. We aimed to examine the role of Heg in mammalian liver development and functional maintenance.MethodsGlobal (Heg-/-) or liver endothelial cell (EC)-specific deletion of Heg (Lyve1-Cre;Hegfl/fl ) mice were used to study the in vivo function of Heg in the liver. Carbon-ink anterograde and retrograde injection were used to visualize the 3-dimensional patterning of liver portal and biliary networks, respectively. RNA sequencing, histology, and molecular and biochemical assays were used to assess liver gene expression, protein distribution, liver injury response, and function.ResultsHeg deficiency in liver ECs led to a sparse liver vascular and biliary network. This network paucity does not compromise liver function under baseline conditions but did alter liver zonation. Molecular analysis revealed that endothelial Heg deficiency decreased expression of Wnt ligands/agonists including Wnt2, Wnt9b, and Rspo3 in ECs, which limits Axin2 mediated canonical Wnt signaling and the expression of cytochrome P450 enzymes in hepatocytes. Under chemical-induced stressed conditions, Heg-deficiency in liver ECs protected mice from drug-induced liver injuries.ConclusionOur study found that endothelial Heg is essential for the 3-D patterning of the liver vascular and indirectly regulates biliary networks and proper liver zonation via its regulation of Wnt ligand production in liver endothelial cells. The endothelial Heg-initiated changes of the liver metabolic zonation and metabolic enzyme expression in hepatocytes was functionally relevant to xenobiotic metabolism and drug induced liver toxicity.

Abstract 14787: Loss of Wnt7a is Associated With Reduced Vegf-a/vegfr2 Mediated Endothelial Tip Cell Formation and Angiogenesis in Pulmonary Arterial Hypertension

Rationale: Pulmonary arterial hypertension (PAH) is characterized by loss of microvessels. The Wnt pathways control pulmonary angiogenesis but their role in PAH is incompletely understood. Objective: We hypothesized that Wnt activation in pulmonary microvascular endothelial cells (PMVECs) is required for pulmonary angiogenesis and its loss contributes to PAH. Methods: Lung tissue and PMVECs from healthy donors and PAH patients were screened for Wnt ligand production. Tip cell dynamics were analyzed using 3D spheroid assays and in vivo studies were carried out with tamoxifen-inducible Wnt knockout mice exposed to chronic hypoxia and Sugen-hypoxia (SuHx). Results: Healthy PMVECs demonstrated ~6-fold Wnt7a expression that was absent in PAH PMVECs and vascular lesions. Wnt7a expression correlated with formation of tip cells, an endothelial phenotype critical for angiogenesis. To analyze PMVEC tip cell formation, we developed a 3D PMVEC spheroid model combined with live imaging to monitor the behavior of healthy and PAH PMVECs in response to VEGF-A and Wnt7a. We found that PAH PMVECs demonstrated reduced tip cell formation as evidenced by reduced filopodia formation and random motility, which could be partially rescued by recombinant Wnt7a. The mechanism by which Wnt7a promotes VEGF signaling activity is dependent on its capacity to activate the small GTPase cdc42 and facilitate phosphorylation of the Y951 and Y1175 tyrosine residues in VEGFR2 following VEGF-A stimulation. While there was no phenotypic difference between wild type and mice with endothelial-specific Wnt7a knockout (Wnt7a EC/EC ) under chronic hypoxia and SuHx, we found that global Wnt7a +/- mice demonstrated higher pressures and vascular remodeling compared to wild type mice. Interestingly, we found that global Wnt7a knockout (Wnt7a KO/KO ) mice succumbed 10 days after tamoxifen injection demonstrating interstitial thickening associated with expansion of the pulmonary capillaries, an unexpected phenotype resembling alveolar capillary dysplasia. Conclusions: We have identified Wnt7a as a novel angiogenic factor that plays a pivotal role in pulmonary angiogenesis by promoting VEGF-A mediated tip cell formation. Targeting Wnt7a could promote recovery of small vessel loss in PAH.

Distinct Spatiotemporally Dynamic Wnt-Secreting Niches Regulate Proximal Airway Regeneration and Aging.

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 invivo. 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 invivo. Collectively, these data underscore multiple spatiotemporally dynamic Wnt-secreting niches that regulate functionally distinct phases of airway regeneration and aging.

Inhibiting WNT Ligand Production for Improved Immune Recognition in the Ovarian Tumor Microenvironment.

In ovarian cancer, upregulation of the Wnt/β–catenin pathway leads to chemoresistance and correlates with T cell exclusion from the tumor microenvironment (TME). Our objectives were to validate these findings in an independent cohort of ovarian cancer subjects and determine whether inhibiting the Wnt pathway in a syngeneic ovarian cancer murine model could create a more T-cell-inflamed TME, which would lead to decreased tumor growth and improved survival. We preformed RNA sequencing in a cohort of human high grade serous ovarian carcinoma subjects. We used CGX1321, an inhibitor to the porcupine (PORCN) enzyme that is necessary for secretion of WNT ligand, in mice with established ID8 tumors, a murine ovarian cancer cell line. In order to investigate the effect of decreased Wnt/β–catenin pathway activity in the dendritic cells (DCs), we injected ID8 cells in mice that lacked β–catenin specifically in DCs. Furthermore, to understand how much the effects of blocking the Wnt/β–catenin pathway are dependent on CD8+ T cells, we injected ID8 cells into mice with CD8+ T cell depletion. We confirmed a negative correlation between Wnt activity and T cell signature in our cohort. Decreasing WNT ligand production resulted in increases in T cell, macrophage and dendritic cell functions, decreased tumor burden and improved survival. Reduced tumor growth was found in mice that lacked β–catenin specifically in DCs. When CD8+ T cells were depleted, CGX1321 treatment did not have the same magnitude of effect on tumor growth. Our investigation confirmed an increase in Wnt activity correlated with a decreased T-cell-inflamed environment; a relationship that was further supported in our pre-clinical model that suggests inhibiting the Wnt/β–catenin pathway was associated with decreased tumor growth and improved survival via a partial dependence on CD8+ T cells.

Non-canonical Wnt signalling regulates scarring in biliary disease via the planar cell polarity receptors

The number of patients diagnosed with chronic bile duct disease is increasing and in most cases these diseases result in chronic ductular scarring, necessitating liver transplantation. The formation of ductular scaring affects liver function; however, scar-generating portal fibroblasts also provide important instructive signals to promote the proliferation and differentiation of biliary epithelial cells. Therefore, understanding whether we can reduce scar formation while maintaining a pro-regenerative microenvironment will be essential in developing treatments for biliary disease. Here, we describe how regenerating biliary epithelial cells express Wnt-Planar Cell Polarity signalling components following bile duct injury and promote the formation of ductular scars by upregulating pro-fibrogenic cytokines and positively regulating collagen-deposition. Inhibiting the production of Wnt-ligands reduces the amount of scar formed around the bile duct, without reducing the development of the pro-regenerative microenvironment required for ductular regeneration, demonstrating that scarring and regeneration can be uncoupled in adult biliary disease and regeneration.

Synergy with TGFβ ligands switches WNT pathway dynamics from transient to sustained during human pluripotent cell differentiation

WNT/β-catenin signaling is crucial to all stages of life. It controls early morphogenetic events in embryos, maintains stem cell niches in adults, and is dysregulated in many types of cancer. Despite its ubiquity, little is known about the dynamics of signal transduction or whether it varies across contexts. Here we probe the dynamics of signaling by monitoring nuclear accumulation of β-catenin, the primary transducer of canonical WNT signals, using quantitative live cell imaging. We show that β-catenin signaling responds adaptively to constant WNT signaling in pluripotent stem cells, and that these dynamics become sustained on differentiation. Varying dynamics were also observed in the response to WNT in commonly used mammalian cell lines. Signal attenuation in pluripotent cells is observed even at saturating doses, where ligand stability does not affect the dynamics. TGFβ superfamily ligands Activin and BMP, which coordinate with WNT signaling to pattern the gastrula, increase the β-catenin response in a manner independent of their ability to induce new WNT ligand production. Our results reveal how variables external to the pathway, including differentiation status and cross-talk with other pathways, dramatically alter WNT/β-catenin dynamics.

Cross-talk in the mammary gland

Stem Cells Macrophages engulf damaged and dead cells to clear infection, but they also participate in tissue regeneration. Chakrabarti et al. expand the macrophage repertoire for mammary gland development (see the Perspective by Kannan and Eaves). Mammary gland stem cells secrete the Notch ligand Dll1 and activate Notch signaling, which promotes survival of adjacent macrophages. This stimulates production of Wnt ligands, which signal back to the mammary gland stem cells. This cross-talk plays an important role in coordinating mammary gland development, tissue homeostasis, and, not least, breast cancer. Science , this issue p. [eaan4153][1]; see also p. [1401][2] [1]: /lookup/doi/10.1126/science.aan4153 [2]: /lookup/doi/10.1126/science.aau1394

Notch ligand Dll1 mediates cross-talk between mammary stem cells and the macrophageal niche.

The stem cell niche is a specialized environment that dictates stem cell function during development and homeostasis. We show that Dll1, a Notch pathway ligand, is enriched in mammary gland stem cells (MaSCs) and mediates critical interactions with stromal macrophages in the surrounding niche in mouse models. Conditional deletion of Dll1 reduced the number of MaSCs and impaired ductal morphogenesis in the mammary gland. Moreover, MaSC-expressed Dll1 activates Notch signaling in stromal macrophages, increasing their expression of Wnt family ligands such as Wnt3, Wnt10A, and Wnt16, thereby initiating a feedback loop that promotes the function of Dll1-expressing MaSCs. Together, these findings reveal functionally important cross-talk between MaSCs and their macrophageal niche through Dll1-mediated Notch signaling.

Subepithelial telocytes are an important source of Wnts that supports intestinal crypts.

Tissues with rapid cellular turnover, such as the mammalian hematopoietic system or the intestinal epithelium, are dependent upon stem and progenitor cells, which through proliferation provide differentiated cells to maintain organismal health. Stem and progenitor cells, in turn, are thought to rely upon signals and growth factors provided by local niche cells to support their function and self-renewal. Several cell types have been proposed to provide the signals required for the proliferation and differentiation of the ISC in the crypt1–6. Here, we identify subepithelial telocytes as an important source of Wnt proteins, without which intestinal stem cells cannot proliferate and support epithelial renewal. Telocytes are large but rare mesenchymal cells that are marked by Foxl1 and PDGFRα expression and form a subepithelial plexus that extends from the stomach to the colon. While supporting the entire epithelium, Foxl1+ telocytes compartmentalize the production of Wnt ligands and inhibitors to enable localized pathway activation. Conditional gene ablation of Porcupine (Porcn), which is required for functional maturation of all Wnt proteins, in Foxl1+ telocytes causes rapid cessation of Wnt signaling to intestinal crypts, followed by loss of stem and transit amplifying cell proliferation and impaired epithelial renewal. Thus, Foxl1+ telocytes are an important source of niche signals to intestinal stem cells.

Top-Down Inhibition of BMP Signaling Enables Robust Induction of hPSCs Into Neural Crest in Fully Defined, Xeno-free Conditions.

SummaryDefects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. However, the protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have now found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can also affect the outcome of differentiation protocols.

Osteoblastic heparan sulfate glycosaminoglycans control bone remodeling by regulating Wnt signaling and the crosstalk between bone surface and marrow cells

Stimulating bone formation is an important challenge for bone anabolism in osteoporotic patients or to repair bone defects. The osteogenic properties of matrix glycosaminoglycans (GAGs) have been explored; however, the functions of GAGs at the surface of bone-forming cells are less documented. Syndecan-2 is a membrane heparan sulfate proteoglycan that is associated with osteoblastic differentiation. We used a transgenic mouse model with high syndecan-2 expression in osteoblasts to enrich the bone surface with cellular GAGs. Bone mass was increased in these transgenic mice. Syndecan-2 overexpression reduced the expression of receptor activator of NF-kB ligand (RANKL) in bone marrow cells and strongly inhibited bone resorption. Osteoblast activity was not modified in the transgenic mice, but bone formation was decreased in 4-month-old transgenic mice because of reduced osteoblast number. Increased proteoglycan expression at the bone surface resulted in decreased osteoblastic and osteoclastic precursors in bone marrow. Indeed, syndecan-2 overexpression increased apoptosis of mesenchymal precursors within the bone marrow. However, syndecan-2 specifically promoted the vasculature characterized by high expression of CD31 and Endomucin in 6-week-old transgenic mice, but this was reduced in 12-week-old transgenic mice. Finally, syndecan-2 functions as an inhibitor of Wnt-β-catenin–T-cell factor signaling pathway, activating glycogen synthase kinase 3 and then decreasing the Wnt-dependent production of Wnt ligands and R-spondin. In conclusion, our results show that GAG supply may improve osteogenesis, but also interfere with the crosstalk between the bone surface and marrow cells, altering the supporting function of osteoblasts.

Abstract 1042: Suppression of colorectal cancer cell proliferation by resveratrol involves upregulation of the non-canonical ligand Wnt9a

Abstract Our previous studies showed that the suppression of colorectal cancer (CRC) cell proliferation by resveratrol (RV) was accompanied by a disproportionate increase of the non-canonical Wnt signaling pathway (N-CAN) components relative to the canonical Wnt signaling pathway (CAN) components. Wnt signaling, which consists of CAN and N-CAN is an intricate ensemble of signaling components involved with various cellular processes. The CAN utilizes β-catenin and stimulates cell proliferation whereas the N-CAN is independent of β-catenin and is instrumental in cell differentiation. These opposing mechanisms act in concert to maintain homeostasis in healthy tissues which is dysregulated in cancer. These observations have led us to propose a novel hypothesis that suppression of CRC proliferation may be mediated by the activation of the non-canonical Wnt pathway by nutritional supplements (NNS) such as RV. As far as we know, no known studies have determined the effects of RV on the non-canonical pathways in human CRC. The aim of this study was to determine whether suppression of CRC cell proliferation by RV involves non-canonical Wnt pathway components. CRC cells derived from surgical specimens (n = 5) were treated with RV (5ug/ml) for 72h. Cell proliferation was determined using MTS assay and gene expression was performed using commercially available quantitative RT-PCR Wnt pathway gene expression arrays. RV significantly increased N-CAN ligand Wnt A and Wnt 11(p<0.01) and CAN ligand Wnt 10A (p<0.05) by 18, 9 and 4-folds, respectively. RV treatment resulted in a significant decrease of CRC cell proliferation (56% inhibition; p<0.01) compared to untreated cells. This inhibition was partially reversed by IWP-2, a Wnt ligand production inhibitor. Conditioned media from CRC cells treated with RV significantly suppressed CRC cell proliferation (p<0.05) compared to cells treated with conditioned media from untreated cells and that from cells treated with conditioned media from RV+IWP treated cells. Proliferation was not different in cells treated with conditioned media from untreated cells, IWP-2 or RV+IWP-2-treated cells. Wnt 9A antibody partially reversed the RV-mediated suppression of CRC cell proliferation relative to cells treated with IgG controls. To the best of our knowledge this is the first known study to show the involvement of a non-canonical Wnt pathway ligand, Wnt9A, in the suppression of CRC proliferation mediated by a NNS, resveratrol. This study suggests the presence of a novel mechanism for the suppression of CRC proliferation which entails activation of the non-canonical pathway. Given the excellent tolerance profile of NNS such as RV, the capacity of such agents to activate the non-canonical pathway to inhibit tumor proliferation, offers the opportunity to investigate the effects of chronic administration of these agents on tumor growth and progression in patients. Citation Format: Irshad Ali, Bani M. Fagla, Donald P. Braun. Suppression of colorectal cancer cell proliferation by resveratrol involves upregulation of the non-canonical ligand Wnt9a. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1042. doi:10.1158/1538-7445.AM2015-1042

High-resolution molecular validation of self-renewal and spontaneous differentiation in clinical-grade adipose-tissue derived human mesenchymal stem cells.

Improving the effectiveness of adipose-tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi-potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non-proliferative state. Flow cytometry combined with high-throughput RNA sequencing (RNASeq) and RT-qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1, and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self-renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto-architectural enlargement, and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle-related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone-related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10-fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while upregulating WNT-related genes (WISP2, SFRP2, and SFRP4). Furthermore, post-proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic, and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self-renewing and multi-potent AMSCs by providing high-resolution quality control data that support their clinical versatility.

The β-adrenoceptor agonist isoproterenol rescues acetaminophen-injured livers through increasing progenitor numbers by Wnt in mice.

Acetaminophen (APAP)-induced acute liver injury (AILI) is a major health problem. Accumulating evidence suggests that the sympathetic nervous system (SNS) regulates neuronal and hematopoietic progenitors. SNS signaling affects hepatic progenitor/oval cells (HPCs) and β-adrenoceptor agonism will expand HPCs to reduce AILI. Dopamine β-hydroxylase-deficient mice (Dbh-/-), lacking catecholamine SNS neurotransmitters, isolated HPCs, and immature ductular 603B cells were initially used to investigate SNS involvement in HPC physiology. Subsequently, control mice were treated with APAP (350 mg/kg) followed by the β-adrenoceptor agonist, isoproterenol (ISO), or the β-adrenoceptor antagonist, propranolol. Mechanistic studies examined effects of non-SNS HPC expansion on AILI, involvement of the canonical Wnt/β-catenin pathway (CWP) in the action of ISO on HPC expansion and comparison of ISO with the current standard of care, N-acetylcysteine (NAC). Dbh-/- mice lacking catecholamines had low HPC numbers, reconstituted by ISO. In vitro, ISO-induced proliferation of 603B cells was CWP dependent. In control mice, AILI raised HPC numbers, further increased by ISO, with attenuation of liver injury. Delayed administration of NAC did not, but delayed ISO did, reverse AILI. Propranolol worsened AILI. AILI activated the CWP, and ISO enhanced Wnt-ligand production. HPCs were the major source of Wnt ligands. Recombinant Wnt3a and ISO-603B-conditioned media, but not ISO alone, protected isolated hepatocytes from death, reversed by DKK1-a Wnt antagonist. Additionally, tumor-associated weak inducer of apoptosis expanded HPCs and protected against AILI. Furthermore, allotransplantation of HPCs from APAP+ISO-treated mice to other APAP-injured mice improved AILI, an effect antagonized by DKK1. SNS catecholamines expand HPCs, which are both targets and sources of Wnt ligands. Hepatoprotection by ISO is mediated by para- and autocrine effects of Wnt signaling. ISO represents novel pharmacotherapy for AILI.

Wnt/beta‐catenin signaling in patterning and differentiation of the vertebrate endoderm

The endoderm gives rise to organs as different in anatomy and function as the lungs, colon and liver, and we want to understand the signaling processes controlling this diversification. Our primary interest is in the pancreas, and our work indicates multiple roles for the Wnt/beta (β)‐catenin signaling pathway in its development. Early, Wnt signaling inhibits pancreas specification within the foregut; later, it promotes expansion of multipotent progenitor cells and their differentiation into exocrine acinar tissue. Until recently, our studies have focused on response to Wnt ligands, mediated by the non‐redundant functions of β‐catenin. To better understand the role of Wnt ligand production, while bypassing redundancy between the ligand‐encoding genes, we have targeted the mouse Porcn gene, homologous to Drosophila porcupine. Porcupine is required for wingless secretion and bioactivity, and we find that mouse cells lacking Porcn cannot produce any of several Wnt ligands tested, but retain normal responsiveness to exogenous Wnts. The Porcn mutant defect thus closely resembles that of fly porcupine, indicating a conserved and non‐redundant role for this gene in Wnt production. Wnt production by Porcn mutant cells is rescued by wildtype human PORCN, but not a PORCN mutant recently identified in a pleiotropic congenital syndrome, Focal Dermal Hypoplasia. Mice carrying a conditional Porcn allele are viable, and will provide a novel genetic tool to understand Wnt function in endoderm development and beyond.Supported by the NIH (R01‐DK075072) and Searle Scholars Program (06‐B‐116).

High-Density Gene Expression Analysis of Tumor-Associated Macrophages from Mouse Mammary Tumors

Clinical and experimental evidence indicates that tumor-associated macrophages (TAMs) promote malignant progression. In breast cancer, TAMs enhance tumor angiogenesis, tumor cell invasion, matrix remodeling, and immune suppression against the tumor. In this study, we examined late-stage mammary tumors from a transgenic mouse model of breast cancer. We used flow cytometry under conditions that minimized gene expression changes to isolate a rigorously defined TAM population previously shown to be associated with invasive carcinoma cells. The gene expression signature of this population was compared with a similar population derived from spleens of non-tumor-bearing mice using high-density oligonucleotide arrays. Using stringent selection criteria, transcript abundance of 460 genes was shown to be differentially regulated between the two populations. Bioinformatic analyses of known functions of these genes indicated that formerly ascribed TAM functions, including suppression of immune activation and matrix remodeling, as well as multiple mediators of tumor angiogenesis, were elevated in TAMs. Further bioinformatic analyses confirmed that a pure and valid TAM gene expression signature in mouse tumors could be used to assess expression of TAMs in human breast cancer. The data derived from these more physiologically relevant autochthonous tumors compared with previous studies in tumor xenografts suggest tactics by which TAMs may regulate tumor angiogenesis and thus provide a basis for exploring other transcriptional mediators of TAM trophic functions within the tumor microenvironment.

Wnt signal from multiple tissues and lin-3 /EGF signal from the gonad maintain vulval precursor cell competence in Caenorhabditis elegans

The Caenorhabditis elegans vulva has been a valuable paradigm for defining components of signaling pathways and elucidating how signaling events are coordinated to generate a developmental pattern. Vulval precursor cells (VPCs) are induced to adopt vulval fates in the third larval stage by LIN-3, an EGF-like signal produced by the gonad. Competence to respond to the inductive signal requires that the VPCs do not fuse to the major hypodermal syncytium, hyp7. We found that two Wnt-encoding genes, cwn-1 and egl-20, play a major role in preventing fusion of VPCs with hyp7 in the second larval stage. By using tissue-specific rescue of mig-14/Wntless, which is required for the production of Wnt ligands, we found that Wnt signal produced by multiple tissues, including neurons and muscles, promotes or maintains VPC competence before vulval induction. In addition, through laser ablation and genetic analysis, we provide evidence that LIN-3 signal from the gonad also plays a significant role in preventing VPCs from fusing with hyp7. We propose that Wnt signaling plays a permissive role in preventing VPCs from fusing with hyp7 and reevaluate the roles of Wnt and LIN-3/EGF signaling in competence and induction.