Roeland Nusse

All Publications

• Inflammatory Cytokine TNF alpha Promotes the Long-Term Expansion of Primary Hepatocytes in 3D Culture CELL Peng, W., Logan, C. Y., Fish, M., Anbarchian, T., Aguisanda, F., Alvarez-Varela, A., Wu, P., Jin, Y., Zhu, J., Li, B., Grompe, M., Wang, B., Nusse, R. 2018; 175 (6): 1607-+

  View details for DOI 10.1016/j.cell.2018.11.012

  View details for Web of Science ID 000451771700019

• Generating Cellular Diversity and Spatial Form: Wnt Signaling and the Evolution of Multicellular Animals. Developmental cell Loh, K. M., van Amerongen, R., Nusse, R. 2016; 38 (6): 643-655

  Abstract

  There were multiple prerequisites to the evolution of multicellular animal life, including the generation of multiple cell fates ("cellular diversity") and their patterned spatial arrangement ("spatial form"). Wnt proteins operate as primordial symmetry-breaking signals. By virtue of their short-range nature and their capacity to activate both lineage-specifying and cell-polarizing intracellular signaling cascades, Wnts can polarize cells at their site of contact, orienting the axis of cell division while simultaneously programming daughter cells to adopt diverging fates in a spatially stereotyped way. By coupling cell fate to position, symmetry-breaking Wnt signals were pivotal in constructing the metazoan body by generating cellular diversity and spatial form.

  View details for DOI 10.1016/j.devcel.2016.08.011

  View details for PubMedID 27676437

• Self-renewing diploid Axin2(+) cells fuel homeostatic renewal of the liver NATURE Wang, B., Zhao, L., Fish, M., Logan, C. Y., Nusse, R. 2015; 524 (7564): 180-?

  Abstract

  The source of new hepatocytes in the uninjured liver has remained an open question. By lineage tracing using the Wnt-responsive gene Axin2 in mice, we identify a population of proliferating and self-renewing cells adjacent to the central vein in the liver lobule. These pericentral cells express the early liver progenitor marker Tbx3, are diploid, and thereby differ from mature hepatocytes, which are mostly polyploid. The descendants of pericentral cells differentiate into Tbx3-negative, polyploid hepatocytes, and can replace all hepatocytes along the liver lobule during homeostatic renewal. Adjacent central vein endothelial cells provide Wnt signals that maintain the pericentral cells, thereby constituting the niche. Thus, we identify a cell population in the liver that subserves homeostatic hepatocyte renewal, characterize its anatomical niche, and identify molecular signals that regulate its activity.

  View details for DOI 10.1038/nature14863

  View details for Web of Science ID 000359386900023

  View details for PubMedID 26245375

  View details for PubMedCentralID PMC4589224

• An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control SCIENCE Clevers, H., Loh, K. M., Nusse, R. 2014; 346 (6205): 54-?

  View details for DOI 10.1126/science.1248012

  View details for Web of Science ID 000342446900046

• Interfollicular Epidermal Stem Cells Self-Renew via Autocrine Wnt Signaling SCIENCE Lim, X., Tan, S. H., Koh, W. L., Chau, R. M., Yan, K. S., Kuo, C. J., van Amerongen, R., Klein, A. M., Nusse, R. 2013; 342 (6163): 1226-1230

  Abstract

  The skin is a classical example of a tissue maintained by stem cells. However, the identity of the stem cells that maintain the interfollicular epidermis and the source of the signals that control their activity remain unclear. Using mouse lineage tracing and quantitative clonal analyses, we showed that the Wnt target gene Axin2 marks interfollicular epidermal stem cells. These Axin2-expressing cells constitute the majority of the basal epidermal layer, compete neutrally, and require Wnt/β-catenin signaling to proliferate. The same cells contribute robustly to wound healing, with no requirement for a quiescent stem cell subpopulation. By means of double-labeling RNA in situ hybridization in mice, we showed that the Axin2-expressing cells themselves produce Wnt signals as well as long-range secreted Wnt inhibitors, suggesting an autocrine mechanism of stem cell self-renewal.

  View details for DOI 10.1126/science.1239730

  View details for Web of Science ID 000327857900046

  View details for PubMedID 24311688

• A Localized Wnt Signal Orients Asymmetric Stem Cell Division in Vitro SCIENCE Habib, S. J., Chen, B., Tsai, F., Anastassiadis, K., Meyer, T., Betzig, E., Nusse, R. 2013; 339 (6126): 1445-1448

  Abstract

  Developmental signals such as Wnts are often presented to cells in an oriented manner. To examine the consequences of local Wnt signaling, we immobilized Wnt proteins on beads and introduced them to embryonic stem cells in culture. At the single-cell level, the Wnt-bead induced asymmetric distribution of Wnt-β-catenin signaling components, oriented the plane of mitotic division, and directed asymmetric inheritance of centrosomes. Before cytokinesis was completed, the Wnt-proximal daughter cell expressed high levels of nuclear β-catenin and pluripotency genes, whereas the distal daughter cell acquired hallmarks of differentiation. We suggest that a spatially restricted Wnt signal induces an oriented cell division that generates distinct cell fates at predictable positions relative to the Wnt source.

  View details for DOI 10.1126/science.1231077

  View details for Web of Science ID 000316740700046

  View details for PubMedID 23520113

• Wnt Proteins Are Self-Renewal Factors for Mammary Stem Cells and Promote Their Long-Term Expansion in Culture CELL STEM CELL Zeng, Y. A., Nusse, R. 2010; 6 (6): 568-577

  Abstract

  Adult stem cells have the ability to self-renew and to generate specialized cells. Self-renewal is dependent on extrinsic niche factors but few of those signals have been identified. In addition, stem cells tend to differentiate in the absence of the proper signals and are therefore difficult to maintain in cell culture. The mammary gland provides an excellent system to study self-renewal signals, because the organ develops postnatally, arises from stem cells, and is readily generated from transplanted cells. We show here that adult mammary glands contain a Wnt-responsive cell population that is enriched for stem cells. In addition, stem cells mutant for the negative-feedback regulator Axin2 and therefore sensitized to Wnt signals have a competitive advantage in mammary gland reconstitution assays. In cell culture experiments, exposure to purified Wnt protein clonally expands mammary stem cells for many generations and maintains their ability to generate functional glands in transplantation assays. We conclude that Wnt proteins serve as rate-limiting self-renewal signals acting directly on mammary stem cells.

  View details for DOI 10.1016/j.stem.2010.03.020

  View details for Web of Science ID 000278840700017

  View details for PubMedID 20569694

  View details for PubMedCentralID PMC2917779

• Purified Wnt5a protein activates or inhibits beta-catenin-TCF signaling depending on receptor context PLOS BIOLOGY Mikels, A. J., Nusse, R. 2006; 4 (4): 570-582

  Abstract

  The Wnts comprise a large class of secreted proteins that control essential developmental processes such as embryonic patterning, cell growth, migration, and differentiation. In the most well-understood "canonical" Wnt signaling pathway, Wnt binding to Frizzled receptors induces beta-catenin protein stabilization and entry into the nucleus, where it complexes with T-cell factor/lymphoid enhancer factor transcription factors to affect the transcription of target genes. In addition to the canonical pathway, evidence for several other Wnt signaling pathways has accumulated, in particular for Wnt5a, which has therefore been classified as a noncanonical Wnt family member. To study the alternative mechanisms by which Wnt proteins signal, we purified the Wnt5a protein to homogeneity. We find that purified Wnt5a inhibits Wnt3a protein-induced canonical Wnt signaling in a dose-dependent manner, not by influencing beta-catenin levels but by downregulating beta-catenin-induced reporter gene expression. The Wnt5a signal is mediated by the orphan tyrosine kinase Ror2, is pertussis toxin insensitive, and does not influence cellular calcium levels. We show that in addition to its inhibitory function, Wnt5a can also activate beta-catenin signaling in the presence of the appropriate Frizzled receptor, Frizzled 4. Thus, this study shows for the first time that a single Wnt ligand can initiate discrete signaling pathways through the activation of two distinct receptors. Based on these and additional observations, we propose a model wherein receptor context dictates Wnt signaling output. In this model, signaling by different Wnt family members is not intrinsically regulated by the Wnt proteins themselves but by receptor availability.

  View details for DOI 10.1371/journal.pbio.0040115

  View details for Web of Science ID 000237066500011

  View details for PubMedID 16602827

• Wnt proteins are lipid-modified and can act as stem cell growth factors NATURE Willert, K., BROWN, J. D., Danenberg, E., Duncan, A. W., Weissman, I. L., Reya, T., Yates, J. R., Nusse, R. 2003; 423 (6938): 448-452

  Abstract

  Wnt signalling is involved in numerous events in animal development, including the proliferation of stem cells and the specification of the neural crest. Wnt proteins are potentially important reagents in expanding specific cell types, but in contrast to other developmental signalling molecules such as hedgehog proteins and the bone morphogenetic proteins, Wnt proteins have never been isolated in an active form. Although Wnt proteins are secreted from cells, secretion is usually inefficient and previous attempts to characterize Wnt proteins have been hampered by their high degree of insolubility. Here we have isolated active Wnt molecules, including the product of the mouse Wnt3a gene. By mass spectrometry, we found the proteins to be palmitoylated on a conserved cysteine. Enzymatic removal of the palmitate or site-directed and natural mutations of the modified cysteine result in loss of activity, and indicate that the lipid is important for signalling. The purified Wnt3a protein induces self-renewal of haematopoietic stem cells, signifying its potential use in tissue engineering.

  View details for DOI 10.1038/nature01611

  View details for Web of Science ID 000183012000044

  View details for PubMedID 12717451

• APEX2-Mediated Proximity Labeling of Wnt Receptor Interactors Upon Pathway Activation. microPublication biology Rim, E. Y., Nusse, R. 2023; 2023

  Abstract

  The Wnt signaling pathway regulates metazoan development, tissue homeostasis, and regeneration. Many outstanding questions in Wnt signal transduction revolve around the molecular events immediately following Wnt-receptor interactions. To identify binding partners of the Wnt receptor Frizzled 7 (Fzd7) upon pathway activation, we tagged Fzd7 with APEX2, an enzyme that allows biotinylation of proximal interactors with high temporal and spatial resolution. Upon confirming proper localization and signaling activity of APEX2-tagged Fzd7, we labeled proximal interactors of Fzd7 with or without Wnt3a stimulation. Mass spectrometry analysis of biotinylated interactors identified several known Wnt pathway proteins. Top interactors enriched upon Wnt treatment were involved in actin cytoskeleton regulation, vesicle trafficking, or phospholipid modification. Proteins enriched in the Wnt-activated Fzd7 interactome that are without established roles in Wnt signaling warrant further examination.

  View details for DOI 10.17912/micropub.biology.000817

  View details for PubMedID 37260921

  View details for PubMedCentralID PMC10227642

• PATIENT-DERIVED HEPATOBLASTOMA TUMOROIDS DEPEND ON BOTH RTK SIGNALING AND WNT ACTIVATION Wu, P., Bucher, S., Burhan, D., Nijagal, A., Rangaswami, A., Wang, B., Nusse, R. WILEY. 2021: S69

  View details for Web of Science ID 000642514400137

• WNT signaling in pre-granulosa cells is required for ovarian folliculogenesis and female fertility. Development (Cambridge, England) Habara, O., Logan, C. Y., Kanai-Azuma, M., Nusse, R., Takase, H. M. 2021; 148 (9)

  Abstract

  In mammalian ovaries, immature oocytes are reserved in primordial follicles until their activation for potential ovulation. Precise control of primordial follicle activation (PFA) is essential for reproduction, but how this is achieved is unclear. Here, we show that canonical wingless-type MMTV integration site family (WNT) signaling is pivotal for pre-granulosa cell (pre-GC) activation during PFA. We identified several WNT ligands expressed in pre-GCs that act in an autocrine manner. Inhibition of WNT secretion from pre-GCs/GCs by conditional knockout (cKO) of the wntless (Wls) gene led to female infertility. In Wls cKO mice, GC layer thickness was greatly reduced in growing follicles, which resulted in impaired oocyte growth with both an abnormal, sustained nuclear localization of forkhead box O3 (FOXO3) and reduced phosphorylation of ribosomal protein S6 (RPS6). Constitutive stabilization of beta-catenin (CTNNB1) in pre-GCs/GCs induced morphological changes of pre-GCs from a squamous into a cuboidal form, though it did not influence oocyte activation. Our results reveal that canonical WNT signaling plays a permissive role in the transition of pre-GCs to GCs, which is an essential step to support oocyte growth.

  View details for DOI 10.1242/dev.198846

  View details for PubMedID 33914868

• Running Against the Wnt: How Wnt/beta-Catenin Suppresses Adipogenesis. Frontiers in cell and developmental biology de Winter, T. J., Nusse, R. 2021; 9: 627429

  Abstract

  Mesenchymal stem cells (MSCs) give rise to adipocytes, osteocytes, and chondrocytes and reside in various tissues, including bone marrow and adipose tissue. The differentiation choices of MSCs are controlled by several signaling pathways, including the Wnt/beta-catenin signaling. When MSCs undergo adipogenesis, they first differentiate into preadipocytes, a proliferative adipocyte precursor cell, after which they undergo terminal differentiation into mature adipocytes. These two steps are controlled by the Wnt/beta-catenin pathway, in such a way that when signaling is abrogated, the next step in adipocyte differentiation can start. This sequence suggests that the main role of Wnt/beta-catenin signaling is to suppress differentiation while increasing MSC and preadipocytes cell mass. During later steps of MSC differentiation, however, active Wnt signaling can promote osteogenesis instead of keeping the MSCs undifferentiated and proliferative. The exact mechanisms behind the various functions of Wnt signaling remain elusive, although recent research has revealed that during lineage commitment of MSCs into preadipocytes, Wnt signaling is inactivated by endogenous Wnt inhibitors. In part, this process is regulated by histone-modifying enzymes, which can lead to increased or decreased Wnt gene expression. The role of Wnt in adipogenesis, as well as in osteogenesis, has implications for metabolic diseases since Wnt signaling may serve as a therapeutic target.

  View details for DOI 10.3389/fcell.2021.627429

  View details for PubMedID 33634128

• Beta-catenin-mediated Wnt signal transduction proceeds through an endocytosis-independent mechanism. Molecular biology of the cell Rim, E. Y., Kinney, L. K., Nusse, R. 2020: mbcE20020114

  Abstract

  The Wnt pathway is a key intercellular signaling cascade that regulates development, tissue homeostasis, and regeneration. However, gaps remain in our understanding of the molecular events that take place between ligand-receptor binding and target gene transcription. We used a novel tool for quantitative, real-time assessment of endogenous pathway activation, measured in single cells, to answer an unresolved question in the field - whether receptor endocytosis is required for Wnt signal transduction. We combined knockdown or knockout of essential components of Clathrin-mediated endocytosis with quantitative assessment of Wnt signal transduction in mouse embryonic stem cells (mESCs). Disruption of Clathrin-mediated endocytosis did not affect accumulation and nuclear translocation of beta-catenin, as measured by single-cell live imaging of endogenous beta-catenin, and subsequent target gene transcription. Disruption of another receptor endocytosis pathway, Caveolin-mediated endocytosis, did not affect Wnt pathway activation in mESCs. Additional results in multiple cell lines support that endocytosis is not a requirement for Wnt signal transduction. We show that off-target effects of a drug used to inhibit endocytosis may be one source of the discrepancy among reports on the role of endocytosis in Wnt signaling.

  View details for DOI 10.1091/mbc.E20-02-0114

  View details for PubMedID 32320321

• Next-Generation Surrogate Wnts Support Organoid Growth and Deconvolute Frizzled Pleiotropy In Vivo. Cell stem cell Miao, Y. n., Ha, A. n., de Lau, W. n., Yuki, K. n., Santos, A. J., You, C. n., Geurts, M. H., Puschhof, J. n., Pleguezuelos-Manzano, C. n., Peng, W. C., Senlice, R. n., Piani, C. n., Buikema, J. W., Gbenedio, O. M., Vallon, M. n., Yuan, J. n., de Haan, S. n., Hemrika, W. n., Rösch, K. n., Dang, L. T., Baker, D. n., Ott, M. n., Depeille, P. n., Wu, S. M., Drost, J. n., Nusse, R. n., Roose, J. P., Piehler, J. n., Boj, S. F., Janda, C. Y., Clevers, H. n., Kuo, C. J., Garcia, K. C. 2020

  Abstract

  Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocyte, ovarian, and breast. NGS Wnts are superior to Wnt3a conditioned media in organoid expansion and single-cell organoid outgrowth. Administration of Fzd subtype-specific NGS Wnt in vivo reveals that adult intestinal crypt proliferation can be promoted by agonism of Fzd5 and/or Fzd8 receptors, while a broad spectrum of Fzd receptors can induce liver zonation. Thus, NGS Wnts offer a unified organoid expansion protocol and a laboratory "tool kit" for dissecting the functions of Fzd subtypes in stem cell biology.

  View details for DOI 10.1016/j.stem.2020.07.020

  View details for PubMedID 32818433

• A ZNRF3-dependent Wnt/beta-catenin signaling gradient is required for adrenal homeostasis GENES & DEVELOPMENT Basham, K. J., Rodriguez, S., Turcu, A. F., Lerario, A. M., Logan, C. Y., Rysztak, M. R., Gomez-Sanchez, C. E., Breault, D. T., Koo, B., Clevers, H., Nusse, R., Val, P., Hammer, G. D. 2019; 33 (3-4): 209-220

  View details for DOI 10.1101/gad.317412.118

  View details for Web of Science ID 000457457100007

• A ZNRF3-dependent Wnt/beta-catenin signaling gradient is required for adrenal homeostasis. Genes & development Basham, K. J., Rodriguez, S., Turcu, A. F., Lerario, A. M., Logan, C. Y., Rysztak, M. R., Gomez-Sanchez, C. E., Breault, D. T., Koo, B., Clevers, H., Nusse, R., Val, P., Hammer, G. D. 2019

  Abstract

  Spatiotemporal control of Wnt signaling is essential for the development and homeostasis of many tissues. The transmembrane E3 ubiquitin ligases ZNRF3 (zinc and ring finger 3) and RNF43 (ring finger protein 43) antagonize Wnt signaling by promoting degradation of frizzled receptors. ZNRF3 and RNF43 are frequently inactivated in human cancer, but the molecular and therapeutic implications remain unclear. Here, we demonstrate that adrenocortical-specific loss of ZNRF3, but not RNF43, results in adrenal hyperplasia that depends on Porcupine-mediated Wnt ligand secretion. Furthermore, we discovered a Wnt/beta-catenin signaling gradient in the adrenal cortex that is disrupted upon loss of ZNRF3. Unlike beta-catenin gain-of-function models, which induce high Wnt/beta-catenin activation and expansion of the peripheral cortex, ZNRF3 loss triggers activation of moderate-level Wnt/beta-catenin signaling that drives proliferative expansion of only the histologically and functionally distinct inner cortex. Genetically reducing beta-catenin dosage significantly reverses the ZNRF3-deficient phenotype. Thus, homeostatic maintenance of the adrenal cortex is dependent on varying levels of Wnt/beta-catenin activation, which is regulated by ZNRF3.

  View details for PubMedID 30692207

• Wnt/beta-catenin signaling regulates ependymal cell development and adult homeostasis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Xing, L., Anbarchian, T., Tsai, J. M., Plant, G. W., Nusse, R. 2018; 115 (26): E5954–E5962

  View details for DOI 10.1073/pnas.1803297115

  View details for Web of Science ID 000436245000017

• Wnt/β-catenin signaling regulates ependymal cell development and adult homeostasis. Proceedings of the National Academy of Sciences of the United States of America Xing, L. n., Anbarchian, T. n., Tsai, J. M., Plant, G. W., Nusse, R. n. 2018

  Abstract

  In the adult mouse spinal cord, the ependymal cell population that surrounds the central canal is thought to be a promising source of quiescent stem cells to treat spinal cord injury. Relatively little is known about the cellular origin of ependymal cells during spinal cord development, or the molecular mechanisms that regulate ependymal cells during adult homeostasis. Using genetic lineage tracing based on the Wnt target gene Axin2, we have characterized Wnt-responsive cells during spinal cord development. Our results revealed that Wnt-responsive progenitor cells are restricted to the dorsal midline throughout spinal cord development, which gives rise to dorsal ependymal cells in a spatially restricted pattern. This is contrary to previous reports that suggested an exclusively ventral origin of ependymal cells, suggesting that ependymal cells may retain positional identities in relation to their neural progenitors. Our results further demonstrated that in the postnatal and adult spinal cord, all ependymal cells express the Wnt/β-catenin signaling target gene Axin2, as well as Wnt ligands. Genetic elimination of β-catenin or inhibition of Wnt secretion in Axin2-expressing ependymal cells in vivo both resulted in impaired proliferation, indicating that Wnt/β-catenin signaling promotes ependymal cell proliferation. These results demonstrate the continued importance of Wnt/β-catenin signaling for both ependymal cell formation and regulation. By uncovering the molecular signals underlying the formation and regulation of spinal cord ependymal cells, our findings thus enable further targeting and manipulation of this promising source of quiescent stem cells for therapeutic interventions.

  View details for PubMedID 29891676

• Stromal R-spondin orchestrates gastric epithelial stem cells and gland homeostasis. Nature Sigal, M. n., Logan, C. Y., Kapalczynska, M. n., Mollenkopf, H. J., Berger, H. n., Wiedenmann, B. n., Nusse, R. n., Amieva, M. R., Meyer, T. F. 2017; 548 (7668): 451–55

  Abstract

  The constant regeneration of stomach epithelium is driven by long-lived stem cells, but the mechanism that regulates their turnover is not well understood. We have recently found that the gastric pathogen Helicobacter pylori can activate gastric stem cells and increase epithelial turnover, while Wnt signalling is known to be important for stem cell identity and epithelial regeneration in several tissues. Here we find that antral Wnt signalling, marked by the classic Wnt target gene Axin2, is limited to the base and lower isthmus of gastric glands, where the stem cells reside. Axin2 is expressed by Lgr5+ cells, as well as adjacent, highly proliferative Lgr5- cells that are able to repopulate entire glands, including the base, upon depletion of the Lgr5+ population. Expression of both Axin2 and Lgr5 requires stroma-derived R-spondin 3 produced by gastric myofibroblasts proximal to the stem cell compartment. Exogenous R-spondin administration expands and accelerates proliferation of Axin2+/Lgr5- but not Lgr5+ cells. Consistent with these observations, H. pylori infection increases stromal R-spondin 3 expression and expands the Axin2+ cell pool to cause hyperproliferation and gland hyperplasia. The ability of stromal niche cells to control and adapt epithelial stem cell dynamics constitutes a sophisticated mechanism that orchestrates epithelial regeneration and maintenance of tissue integrity.

  View details for PubMedID 28813421

• Paracrine Wnt/beta-catenin signaling mediates proliferation of undifferentiated spermatogonia in the adult mouse testis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Takase, H. M., Nusse, R. 2016; 113 (11): E1489-E1497

  Abstract

  Spermatogonial stem cells (SSCs) fuel the production of male germ cells but the mechanisms behind SSC self-renewal, proliferation, and differentiation are still poorly understood. Using the Wnt target gene Axin2 and genetic lineage-tracing experiments, we found that undifferentiated spermatogonia, comprising SSCs and transit amplifying progenitor cells, respond to Wnt/β-catenin signals. Genetic elimination of β-catenin indicates that Wnt/β-catenin signaling promotes the proliferation of these cells. Signaling is likely initiated by Wnt6, which is uniquely expressed by neighboring Sertoli cells, the only somatic cells in the seminiferous tubule that support germ cells and act as a niche for SSCs. Therefore, unlike other stem cell systems where Wnt/β-catenin signaling is implicated in self-renewal, the Wnt pathway in the testis specifically contributes to the proliferation of SSCs and progenitor cells.

  View details for DOI 10.1073/pnas.1601461113

  View details for Web of Science ID 000372014200013

  View details for PubMedCentralID PMC4801309

• Axin2 marks quiescent hair follicle bulge stem cells that are maintained by autocrine Wnt/β-catenin signaling. Proceedings of the National Academy of Sciences of the United States of America Lim, X., Tan, S. H., Yu, K. L., Lim, S. B., Nusse, R. 2016; 113 (11): E1498-505

  Abstract

  How stem cells maintain their identity and potency as tissues change during growth is not well understood. In mammalian hair, it is unclear how hair follicle stem cells can enter an extended period of quiescence during the resting phase but retain stem cell potential and be subsequently activated for growth. Here, we use lineage tracing and gene expression mapping to show that the Wnt target gene Axin2 is constantly expressed throughout the hair cycle quiescent phase in outer bulge stem cells that produce their own Wnt signals. Ablating Wnt signaling in the bulge cells causes them to lose their stem cell potency to contribute to hair growth and undergo premature differentiation instead. Bulge cells express secreted Wnt inhibitors, including Dickkopf (Dkk) and secreted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 (Dkk3) protein becomes localized to the Wnt-inactive inner bulge that contains differentiated cells. We find that Axin2 expression remains confined to the outer bulge, whereas Dkk3 continues to be localized to the inner bulge during the hair cycle growth phase. Our data suggest that autocrine Wnt signaling in the outer bulge maintains stem cell potency throughout hair cycle quiescence and growth, whereas paracrine Wnt inhibition of inner bulge cells reinforces differentiation.

  View details for DOI 10.1073/pnas.1601599113

  View details for PubMedID 26903625

  View details for PubMedCentralID PMC4801317

• Paracrine Wnt/β-catenin signaling mediates proliferation of undifferentiated spermatogonia in the adult mouse testis. Proceedings of the National Academy of Sciences of the United States of America Takase, H. M., Nusse, R. 2016; 113 (11): E1489-97

  Abstract

  Spermatogonial stem cells (SSCs) fuel the production of male germ cells but the mechanisms behind SSC self-renewal, proliferation, and differentiation are still poorly understood. Using the Wnt target gene Axin2 and genetic lineage-tracing experiments, we found that undifferentiated spermatogonia, comprising SSCs and transit amplifying progenitor cells, respond to Wnt/β-catenin signals. Genetic elimination of β-catenin indicates that Wnt/β-catenin signaling promotes the proliferation of these cells. Signaling is likely initiated by Wnt6, which is uniquely expressed by neighboring Sertoli cells, the only somatic cells in the seminiferous tubule that support germ cells and act as a niche for SSCs. Therefore, unlike other stem cell systems where Wnt/β-catenin signaling is implicated in self-renewal, the Wnt pathway in the testis specifically contributes to the proliferation of SSCs and progenitor cells.

  View details for DOI 10.1073/pnas.1601461113

  View details for PubMedID 26929341

  View details for PubMedCentralID PMC4801309

• Axin2 marks quiescent hair follicle bulge stem cells that are maintained by autocrine Wnt/beta-catenin signaling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lim, X., Tan, S. H., Yu, K. L., Lim, S. B., Nusse, R. 2016; 113 (11): E1498-E1505

  Abstract

  How stem cells maintain their identity and potency as tissues change during growth is not well understood. In mammalian hair, it is unclear how hair follicle stem cells can enter an extended period of quiescence during the resting phase but retain stem cell potential and be subsequently activated for growth. Here, we use lineage tracing and gene expression mapping to show that the Wnt target gene Axin2 is constantly expressed throughout the hair cycle quiescent phase in outer bulge stem cells that produce their own Wnt signals. Ablating Wnt signaling in the bulge cells causes them to lose their stem cell potency to contribute to hair growth and undergo premature differentiation instead. Bulge cells express secreted Wnt inhibitors, including Dickkopf (Dkk) and secreted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 (Dkk3) protein becomes localized to the Wnt-inactive inner bulge that contains differentiated cells. We find that Axin2 expression remains confined to the outer bulge, whereas Dkk3 continues to be localized to the inner bulge during the hair cycle growth phase. Our data suggest that autocrine Wnt signaling in the outer bulge maintains stem cell potency throughout hair cycle quiescence and growth, whereas paracrine Wnt inhibition of inner bulge cells reinforces differentiation.

  View details for DOI 10.1073/pnas.1601599113

  View details for Web of Science ID 000372014200014

  View details for PubMedCentralID PMC4801317

• Fibrosis of the Neonatal Mouse Heart After Cryoinjury Is Accompanied by Wnt Signaling Activation and Epicardial-to-Mesenchymal Transition. Journal of the American Heart Association Mizutani, M., Wu, J. C., Nusse, R. 2016; 5 (3)

  Abstract

  The adult mammalian heart responds to cardiac injury by formation of persistent fibrotic scar that eventually leads to heart failure. In contrast, the neonatal mammalian heart reacts to injury by the development of transient fibrotic tissue that is eventually replaced by regenerated cardiomyocytes. How fibrosis occurs in the neonatal mammalian heart remains unknown. To start elucidating the molecular underpinnings of neonatal cardiac fibrosis, we investigated Wnt signaling in the neonatal heart after cryoinjury.Using expression of the Wnt target gene Axin2 as an indicator of Wnt/β-catenin signaling activation, we discovered that epicardial cells in the ventricles are responsive to Wnt in the uninjured neonatal heart. Lineage-tracing studies of these Wnt-responsive epicardial cells showed that they undergo epithelial-to-mesenchymal transition and infiltrate into the subepicardial space and exhibit fibroblast phenotypes after injury. In addition, we showed that-similar to adult ischemic injury-neonatal cryoinjury results in activation of Wnt signaling in cardiac fibroblasts near injured areas. Furthermore, through in situ hybridization of all 19 Wnt ligands in injured neonatal hearts, we observed upregulation of Wnt ligands (Wnt2b, Wnt5a, and Wnt9a) that had not been implicated in the adult cardiac injury response.These results demonstrate that cryoinjury in neonatal heart leads to the formation of fibrotic tissue that involves Wnt-responsive epicardial cells undergoing epithelial-to-mesenchymal transition to give rise to fibroblasts and activation of Wnt signaling in resident cardiac fibroblasts.

  View details for DOI 10.1161/JAHA.115.002457

  View details for PubMedID 27068625

• Wnt/beta-Catenin-Responsive Cells in Prostatic Development and Regeneration STEM CELLS Lee, S. H., Johnson, D. T., Luong, R., Yu, E. J., Cunha, G. R., Nusse, R., Sun, Z. 2015; 33 (11): 3356-3367

  Abstract

  The precise role of Wnt/β-catenin signaling during prostatic development and tumorigenesis is unclear. Axin2 is a direct transcriptional target of β-catenin. Recent studies have shown that Axin2-expressing cells have stem/progenitor cell properties in a variety of mouse tissues. Here, we genetically labeled Axin2-expressing cells at various time points and tracked their cellular behavior at different developmental and mature stages. We found that prostatic Axin2-expressing cells mainly express luminal epithelial cell markers and are able to expand luminal cell lineages during prostatic development and maturation. They can also survive androgen withdrawal and regenerate prostatic luminal epithelial cells following androgen replacement. Deletion of β-catenin or expression of stabilized β-catenin in these Axin2-expressing cells results in abnormal development or oncogenic transformation, respectively. Our study uncovers a critical role of Wnt/β-catenin-responsive cells in prostatic development and regeneration, and that dysregulation of Wnt/β-catenin signaling in these cells contributes to prostatic developmental defects and tumorigenesis. Stem Cells 2015.

  View details for DOI 10.1002/stem.2096

  View details for PubMedID 26220362

• A distinct regulatory region of the Bmp5 locus activates gene expression following adult bone fracture or soft tissue injury. Bone Guenther, C. A., Wang, Z., Li, E., Tran, M. C., Logan, C. Y., Nusse, R., Pantalena-Filho, L., Yang, G. P., Kingsley, D. M. 2015; 77: 31-41

  Abstract

  Bone morphogenetic proteins (BMPs) are key signaling molecules required for normal development of bones and other tissues. Previous studies have shown that null mutations in the mouse Bmp5 gene alter the size, shape and number of multiple bone and cartilage structures during development. Bmp5 mutations also delay healing of rib fractures in adult mutants, suggesting that the same signals used to pattern embryonic bone and cartilage are also reused during skeletal regeneration and repair. Despite intense interest in BMPs as agents for stimulating bone formation in clinical applications, little is known about the regulatory elements that control developmental or injury-induced BMP expression. To compare the DNA sequences that activate gene expression during embryonic bone formation and following acute injuries in adult animals, we assayed regions surrounding the Bmp5 gene for their ability to stimulate lacZ reporter gene expression in transgenic mice. Multiple genomic fragments, distributed across the Bmp5 locus, collectively coordinate expression in discrete anatomic domains during normal development, including in embryonic ribs. In contrast, a distinct regulatory region activated expression following rib fracture in adult animals. The same injury control region triggered gene expression in mesenchymal cells following tibia fracture, in migrating keratinocytes following dorsal skin wounding, and in regenerating epithelial cells following lung injury. The Bmp5 gene thus contains an "injury response" control region that is distinct from embryonic enhancers, and that is activated by multiple types of injury in adult animals.

  View details for DOI 10.1016/j.bone.2015.04.010

  View details for PubMedID 25886903

  View details for PubMedCentralID PMC4447581

• A distinct regulatory region of the Bmp5 locus activates gene expression following adult bone fracture or soft tissue injury BONE Guenther, C. A., Wang, Z., Li, E., Tran, M. C., Logan, C. Y., Nusse, R., Pantalena-Filho, L., Yang, G. P., Kingsley, D. M. 2015; 77: 31-41

  Abstract

  Bone morphogenetic proteins (BMPs) are key signaling molecules required for normal development of bones and other tissues. Previous studies have shown that null mutations in the mouse Bmp5 gene alter the size, shape and number of multiple bone and cartilage structures during development. Bmp5 mutations also delay healing of rib fractures in adult mutants, suggesting that the same signals used to pattern embryonic bone and cartilage are also reused during skeletal regeneration and repair. Despite intense interest in BMPs as agents for stimulating bone formation in clinical applications, little is known about the regulatory elements that control developmental or injury-induced BMP expression. To compare the DNA sequences that activate gene expression during embryonic bone formation and following acute injuries in adult animals, we assayed regions surrounding the Bmp5 gene for their ability to stimulate lacZ reporter gene expression in transgenic mice. Multiple genomic fragments, distributed across the Bmp5 locus, collectively coordinate expression in discrete anatomic domains during normal development, including in embryonic ribs. In contrast, a distinct regulatory region activated expression following rib fracture in adult animals. The same injury control region triggered gene expression in mesenchymal cells following tibia fracture, in migrating keratinocytes following dorsal skin wounding, and in regenerating epithelial cells following lung injury. The Bmp5 gene thus contains an "injury response" control region that is distinct from embryonic enhancers, and that is activated by multiple types of injury in adult animals.

  View details for DOI 10.1016/j.bone.2015.04.010

  View details for Web of Science ID 000355717800006

  View details for PubMedID 25886903

  View details for PubMedCentralID PMC4447581

• Helicobacter pylori Activates and Expands Lgr5(+) Stem Cells Through Direct Colonization of the Gastric Glands. Gastroenterology Sigal, M., Rothenberg, M. E., Logan, C. Y., Lee, J. Y., Honaker, R. W., Cooper, R. L., Passarelli, B., Camorlinga, M., Bouley, D. M., Alvarez, G., Nusse, R., Torres, J., Amieva, M. R. 2015; 148 (7): 1392-404 e21

  Abstract

  Helicobacter pylori infection is the main risk factor for gastric cancer. We characterized the interactions of H pylori with gastric epithelial progenitor and stem cells in humans and mice and investigated how these interactions contribute to H pylori-induced pathology.We used quantitative confocal microscopy and 3-dimensional reconstruction of entire gastric glands to determine the localizations of H pylori in stomach tissues from humans and infected mice. Using lineage tracing to mark cells derived from leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells (Lgr5-eGFP-IRES-CreERT2/Rosa26-TdTomato mice) and in situ hybridization, we analyzed gastric stem cell responses to infection. Isogenic H pylori mutants were used to determine the role of specific virulence factors in stem cell activation and pathology.H pylori grow as distinct bacterial microcolonies deep in the stomach glands and interact directly with gastric progenitor and stem cells in tissues from mice and humans. These gland-associated bacteria activate stem cells, increasing the number of stem cells, accelerating Lgr5(+) stem cell proliferation, and up-regulating expression of stem cell-related genes. Mutant bacteria with defects in chemotaxis that are able to colonize the stomach surface but not the antral glands in mice do not activate stem cells. In addition, bacteria that are unable to inject the contact-dependent virulence factor CagA into the epithelium colonized stomach glands in mice, but did not activate stem cells or produce hyperplasia to the same extent as wild-type H pylori.H pylori colonize and manipulate the progenitor and stem cell compartments, which alters turnover kinetics and glandular hyperplasia. Bacterial ability to alter the stem cells has important implications for gastrointestinal stem cell biology and H pylori-induced gastric pathology.

  View details for DOI 10.1053/j.gastro.2015.02.049

  View details for PubMedID 25725293

• Helicobacter pylori Activates and Expands Lgr5(+) Stem Cells Through Direct Colonization of the Gastric Glands GASTROENTEROLOGY Sigal, M., Rothenberg, M. E., Logan, C. Y., Lee, J. Y., Honaker, R. W., Cooper, R. L., Passarelli, B., Camorlinga, M., Bouley, D. M., Alvarez, G., Nusse, R., Torres, J., Amieva, M. R. 2015; 148 (7): 1392-?

  Abstract

  Helicobacter pylori infection is the main risk factor for gastric cancer. We characterized the interactions of H pylori with gastric epithelial progenitor and stem cells in humans and mice and investigated how these interactions contribute to H pylori-induced pathology.We used quantitative confocal microscopy and 3-dimensional reconstruction of entire gastric glands to determine the localizations of H pylori in stomach tissues from humans and infected mice. Using lineage tracing to mark cells derived from leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells (Lgr5-eGFP-IRES-CreERT2/Rosa26-TdTomato mice) and in situ hybridization, we analyzed gastric stem cell responses to infection. Isogenic H pylori mutants were used to determine the role of specific virulence factors in stem cell activation and pathology.H pylori grow as distinct bacterial microcolonies deep in the stomach glands and interact directly with gastric progenitor and stem cells in tissues from mice and humans. These gland-associated bacteria activate stem cells, increasing the number of stem cells, accelerating Lgr5(+) stem cell proliferation, and up-regulating expression of stem cell-related genes. Mutant bacteria with defects in chemotaxis that are able to colonize the stomach surface but not the antral glands in mice do not activate stem cells. In addition, bacteria that are unable to inject the contact-dependent virulence factor CagA into the epithelium colonized stomach glands in mice, but did not activate stem cells or produce hyperplasia to the same extent as wild-type H pylori.H pylori colonize and manipulate the progenitor and stem cell compartments, which alters turnover kinetics and glandular hyperplasia. Bacterial ability to alter the stem cells has important implications for gastrointestinal stem cell biology and H pylori-induced gastric pathology.

  View details for DOI 10.1053/j.gastro.2015.02.049

  View details for Web of Science ID 000355014700031

  View details for PubMedID 25725293

• Wnts produced by Osterix-expressing osteolineage cells regulate their proliferation and differentiation. Proceedings of the National Academy of Sciences of the United States of America Tan, S. H., Senarath-Yapa, K., Chung, M. T., Longaker, M. T., Wu, J. Y., Nusse, R. 2014; 111 (49): E5262-71

  Abstract

  Wnt signaling is a critical regulator of bone development, but the identity and role of the Wnt-producing cells are still unclear. We addressed these questions through in situ hybridization, lineage tracing, and genetic experiments. First, we surveyed the expression of all 19 Wnt genes and Wnt target gene Axin2 in the neonatal mouse bone by in situ hybridization, and demonstrated-to our knowledge for the first time-that Osterix-expressing cells coexpress Wnt and Axin2. To track the behavior and cell fate of Axin2-expressing osteolineage cells, we performed lineage tracing and showed that they sustain bone formation over the long term. Finally, to examine the role of Wnts produced by Osterix-expressing cells, we inhibited Wnt secretion in vivo, and observed inappropriate differentiation, impaired proliferation, and diminished Wnt signaling response. Therefore, Osterix-expressing cells produce their own Wnts that in turn induce Wnt signaling response, thereby regulating their proliferation and differentiation.

  View details for DOI 10.1073/pnas.1420463111

  View details for PubMedID 25422448

• Wnts produced by Osterix-expressing osteolineage cells regulate their proliferation and differentiation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tan, S. H., Senarath-Yapa, K., Chung, M. T., Longaker, M. T., Wu, J. Y., Nusse, R. 2014; 111 (49): E5262-E5271

  Abstract

  Wnt signaling is a critical regulator of bone development, but the identity and role of the Wnt-producing cells are still unclear. We addressed these questions through in situ hybridization, lineage tracing, and genetic experiments. First, we surveyed the expression of all 19 Wnt genes and Wnt target gene Axin2 in the neonatal mouse bone by in situ hybridization, and demonstrated-to our knowledge for the first time-that Osterix-expressing cells coexpress Wnt and Axin2. To track the behavior and cell fate of Axin2-expressing osteolineage cells, we performed lineage tracing and showed that they sustain bone formation over the long term. Finally, to examine the role of Wnts produced by Osterix-expressing cells, we inhibited Wnt secretion in vivo, and observed inappropriate differentiation, impaired proliferation, and diminished Wnt signaling response. Therefore, Osterix-expressing cells produce their own Wnts that in turn induce Wnt signaling response, thereby regulating their proliferation and differentiation.

  View details for DOI 10.1073/pnas.1420463111

  View details for Web of Science ID 000345921500004

  View details for PubMedID 25422448

• In Vivo clonal analysis reveals lineage-restricted progenitor characteristics in Mammalian kidney development, maintenance, and regeneration. Cell reports Rinkevich, Y., Montoro, D. T., Contreras-Trujillo, H., Harari-Steinberg, O., Newman, A. M., Tsai, J. M., Lim, X., Van-Amerongen, R., Bowman, A., Januszyk, M., Pleniceanu, O., Nusse, R., Longaker, M. T., Weissman, I. L., Dekel, B. 2014; 7 (4): 1270-1283

  Abstract

  The mechanism and magnitude by which the mammalian kidney generates and maintains its proximal tubules, distal tubules, and collecting ducts remain controversial. Here, we use long-term in vivo genetic lineage tracing and clonal analysis of individual cells from kidneys undergoing development, maintenance, and regeneration. We show that the adult mammalian kidney undergoes continuous tubulogenesis via expansions of fate-restricted clones. Kidneys recovering from damage undergo tubulogenesis through expansions of clones with segment-specific borders, and renal spheres developing in vitro from individual cells maintain distinct, segment-specific fates. Analysis of mice derived by transfer of color-marked embryonic stem cells (ESCs) into uncolored blastocysts demonstrates that nephrons are polyclonal, developing from expansions of singly fated clones. Finally, we show that adult renal clones are derived from Wnt-responsive precursors, and their tracing in vivo generates tubules that are segment specific. Collectively, these analyses demonstrate that fate-restricted precursors functioning as unipotent progenitors continuously maintain and self-preserve the mouse kidney throughout life.

  View details for DOI 10.1016/j.celrep.2014.04.018

  View details for PubMedID 24835991

  View details for PubMedCentralID PMC4425291

• The Role of Ryk and Ror Receptor Tyrosine Kinases in Wnt Signal Transduction COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Green, J., Nusse, R., van Amerongen, R. 2014; 6 (2)

  Abstract

  Receptor tyrosine kinases of the Ryk and Ror families were initially classified as orphan receptors because their ligands were unknown. They are now known to contain functional extracellular Wnt-binding domains and are implicated in Wnt-signal transduction in multiple species. Although their signaling mechanisms still remain to be resolved in detail, both Ryk and Ror control important developmental processes in different tissues. However, whereas many other Wnt-signaling responses affect cell proliferation and differentiation, Ryk and Ror are mostly associated with controlling processes that rely on the polarized migration of cells. Here we discuss what is currently known about the involvement of this exciting class of receptors in development and disease.

  View details for DOI 10.1101/cshperspect.a009175

  View details for Web of Science ID 000333118800001

  View details for PubMedID 24370848

• Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sugiyama, T., Benitez, C. M., Ghodasara, A., Liu, L., McLean, G. W., Lee, J., Blauwkamp, T. A., Nusse, R., Wright, C. V., Gu, G., Kim, S. K. 2013; 110 (31): 12691-12696

  Abstract

  Developmental biology is challenged to reveal the function of numerous candidate genes implicated by recent genome-scale studies as regulators of organ development and diseases. Recapitulating organogenesis from purified progenitor cells that can be genetically manipulated would provide powerful opportunities to dissect such gene functions. Here we describe systems for reconstructing pancreas development, including islet β-cell and α-cell differentiation, from single fetal progenitor cells. A strict requirement for native genetic regulators of in vivo pancreas development, such as Ngn3, Arx, and Pax4, revealed the authenticity of differentiation programs in vitro. Efficient genetic screens permitted by this system revealed that Prdm16 is required for pancreatic islet development in vivo. Discovering the function of genes regulating pancreas development with our system should enrich strategies for regenerating islets for treating diabetes mellitus.

  View details for DOI 10.1073/pnas.1304507110

  View details for PubMedID 23852729

• Structural Studies of Wnts and Identification of an LRP6 Binding Site STRUCTURE Chu, M. L., Ahn, V. E., Choi, H., Daniels, D. L., Nusse, R., Weis, W. I. 2013; 21 (7): 1235-1242

  Abstract

  Wnts are secreted growth factors that have critical roles in cell fate determination and stem cell renewal. The Wnt/β-catenin pathway is initiated by binding of a Wnt protein to a Frizzled (Fzd) receptor and a coreceptor, LDL receptor-related protein 5 or 6 (LRP5/6). We report the 2.1 Å resolution crystal structure of a Drosophila WntD fragment encompassing the N-terminal domain and the linker that connects it to the C-terminal domain. Differences in the structures of WntD and Xenopus Wnt8, including the positions of a receptor-binding β hairpin and a large solvent-filled cavity in the helical core, indicate conformational plasticity in the N-terminal domain that may be important for Wnt-Frizzled specificity. Structure-based mutational analysis of mouse Wnt3a shows that the linker between the N- and C-terminal domains is required for LRP6 binding. These findings provide important insights into Wnt function and evolution.

  View details for DOI 10.1016/j.str.2013.05.006

  View details for Web of Science ID 000321681600020

  View details for PubMedID 23791946

• Lineage tracing with Axin2 reveals distinct developmental and adult populations of Wnt/ß-catenin-responsive neural stem cells. Proceedings of the National Academy of Sciences of the United States of America Bowman, A. N., van Amerongen, R., Palmer, T. D., Nusse, R. 2013; 110 (18): 7324-7329

  Abstract

  Since the discovery of neural stem cells in the mammalian brain, there has been significant interest in understanding their contribution to tissue homeostasis at both the cellular and molecular level. Wnt/β-catenin signaling is crucial for development of the central nervous system and has been implicated in stem cell maintenance in multiple tissues. Based on this, we hypothesized that the Wnt pathway likely controls neural stem cell maintenance and differentiation along the entire developmental continuum. To test this, we performed lineage tracing experiments using the recently developed tamoxifen-inducible Cre at Axin2 mouse strain to follow the developmental fate of Wnt/β-catenin-responsive cells in both the embryonic and postnatal mouse brain. From as early as embryonic day 8.5 onwards, Axin2(+) cells can give rise to spatially and functionally restricted populations of adult neural stem cells in the subventricular zone. Similarly, progeny from Axin2(+) cells labeled from E12.5 contribute to both the subventricular zone and the dentate gyrus of the hippocampus. Labeling in the postnatal brain, in turn, demonstrates the persistence of long-lived, Wnt/β-catenin-responsive stem cells in both of these sites. These results demonstrate the continued importance of Wnt/β-catenin signaling for neural stem and progenitor cell formation and function throughout developmental time.

  View details for DOI 10.1073/pnas.1305411110

  View details for PubMedID 23589866

  View details for PubMedCentralID PMC3645553

• Paracrine Wnt signaling both promotes and inhibits human breast tumor growth PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Green, J. L., La, J., Yum, K. W., Desai, P., Rodewald, L., Zhang, X., LeBlanc, M., Nusse, R., Lewis, M. T., Wahl, G. M. 2013; 110 (17): 6991-6996

  Abstract

  Wnt signaling in mouse mammary development and tumorigenesis has been heavily studied and characterized, but its role in human breast cancer remains elusive. Although Wnt inhibitors are in early clinical development, it is unclear whether they will be of therapeutic benefit to breast cancer patients, and subsequently, to which ones. To address this, we generated a panel of Wnt reporting human breast cancer cell lines and identified a previously unrecognized enrichment for the ability to respond to Wnt in the basal B or claudin-low subtype, which has a poor prognosis and no available targeted therapies. By co-injecting Wnt3A expressing human mammary fibroblasts with human breast cancer cell lines into mouse mammary fat pads, we showed that elevated paracrine Wnt signaling was correlated with accelerated tumor growth. Using this heterotypic system and a dual lentiviral reporter system that enables simultaneous real-time measurement of both Wnt-responsive cells and bulk tumor cells, we analyzed the outcome of elevated Wnt signaling in patient-derived xenograft (PDX) models. Interestingly, the PDX models exhibited responses not observed in the cell lines analyzed. Exogenous WNT3A promoted tumor growth in one human epidermal growth factor receptor 2-overexpressing PDX line but inhibited growth in a second PDX line obtained from a patient with triple-negative breast cancer. Tumor suppression was associated with squamous differentiation in the latter. Thus, our work suggests that paracrine Wnt signaling can either fuel or repress the growth of human breast cancers depending on yet to be determined aspects of the molecular pathways they express.

  View details for DOI 10.1073/pnas.1303671110

  View details for Web of Science ID 000318677300082

  View details for PubMedID 23559372

  View details for PubMedCentralID PMC3637696

• Tympanic border cells are Wnt-responsive and can act as progenitors for postnatal mouse cochlear cells DEVELOPMENT Jan, T. A., Chai, R., Sayyid, Z. N., van Amerongen, R., Xia, A., Wang, T., Sinkkonen, S. T., Zeng, Y. A., Levin, J. R., Heller, S., Nusse, R., Cheng, A. G. 2013; 140 (6): 1196-1206

  Abstract

  Permanent hearing loss is caused by the irreversible damage of cochlear sensory hair cells and nonsensory supporting cells. In the postnatal cochlea, the sensory epithelium is terminally differentiated, whereas tympanic border cells (TBCs) beneath the sensory epithelium are proliferative. The functions of TBCs are poorly characterized. Using an Axin2(lacZ) Wnt reporter mouse, we found transient but robust Wnt signaling and proliferation in TBCs during the first 3 postnatal weeks, when the number of TBCs decreases. In vivo lineage tracing shows that a subset of hair cells and supporting cells is derived postnatally from Axin2-expressing TBCs. In cochlear explants, Wnt agonists stimulated the proliferation of TBCs, whereas Wnt inhibitors suppressed it. In addition, purified Axin2(lacZ) cells were clonogenic and self-renewing in culture in a Wnt-dependent manner, and were able to differentiate into hair cell-like and supporting cell-like cells. Taken together, our data indicate that Axin2-positive TBCs are Wnt responsive and can act as precursors to sensory epithelial cells in the postnatal cochlea.

  View details for DOI 10.1242/dev.087528

  View details for Web of Science ID 000315445800006

  View details for PubMedID 23444352

  View details for PubMedCentralID PMC3585657

• Prospective isolation of human embryonic stem cell-derived cardiovascular progenitors that integrate into human fetal heart tissue PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ardehali, R., Ali, S. R., Inlay, M. A., Abilez, O. J., Chen, M. Q., Blauwkamp, T. A., Yazawa, M., Gong, Y., Nusse, R., Drukker, M., Weissman, I. L. 2013; 110 (9): 3405-3410

  Abstract

  A goal of regenerative medicine is to identify cardiovascular progenitors from human ES cells (hESCs) that can functionally integrate into the human heart. Previous studies to evaluate the developmental potential of candidate hESC-derived progenitors have delivered these cells into murine and porcine cardiac tissue, with inconclusive evidence regarding the capacity of these human cells to physiologically engraft in xenotransplantation assays. Further, the potential of hESC-derived cardiovascular lineage cells to functionally couple to human myocardium remains untested and unknown. Here, we have prospectively identified a population of hESC-derived ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells that give rise to cardiomyocytes, endothelial cells, and vascular smooth muscle cells in vitro at a clonal level. We observed rare clusters of ROR2(+) cells and diffuse expression of KDR and PDGFRα in first-trimester human fetal hearts. We then developed an in vivo transplantation model by transplanting second-trimester human fetal heart tissues s.c. into the ear pinna of a SCID mouse. ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells were delivered into these functioning fetal heart tissues: in contrast to traditional murine heart models for cell transplantation, we show structural and functional integration of hESC-derived cardiovascular progenitors into human heart.

  View details for DOI 10.1073/pnas.1220832110

  View details for Web of Science ID 000315841900046

  View details for PubMedID 23391730

  View details for PubMedCentralID PMC3587189

• Wnt Signaling in Skin Development, Homeostasis, and Disease COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Lim, X., Nusse, R. 2013; 5 (2)

  Abstract

  The skin and its appendages constitute the largest organ of the body. Its stratified epithelia offer protection from environmental stresses such as dehydration, irradiation, mechanical trauma, and pathogenic infection, whereas its appendages, like hair and sebaceous glands, help regulate body temperature as well as influence animal interaction and social behavior through camouflage and sexual signaling. To respond to and function effectively in a dynamic external environment, the skin and its appendages possess a remarkable ability to regenerate in a carefully controlled fashion. When this finely tuned homeostatic process is disrupted, skin diseases such as cancers may result. At present, the molecular signals that orchestrate cell proliferation, differentiation, and patterning in the skin remain incompletely understood. It is increasingly apparent that many morphogenetic pathways with key roles in development are also important in regulating skin biology. Of these, Wnt signaling has emerged as the dominant pathway controlling the patterning of skin and influencing the decisions of embryonic and adult stem cells to adopt the various cell lineages of the skin and its appendages, as well as subsequently controlling the function of differentiated skin cells. Here we will review established concepts and present recent advances in our understanding of the diverse roles that Wnt signaling plays in skin development, homeostasis, and disease.

  View details for DOI 10.1101/cshperspect.a008029

  View details for Web of Science ID 000315984100009

  View details for PubMedID 23209129

• Developmental Stage and Time Dictate the Fate of Wnt/beta-Catenin-Responsive Stem Cells in the Mammary Gland CELL STEM CELL van Amerongen, R., Bowman, A. N., Nusse, R. 2012; 11 (3): 387-400

  Abstract

  The mammary epithelium undergoes extensive growth and remodeling during pregnancy, suggesting a role for stem cells. Yet their origin, identity, and behavior in the intact tissue remain unknown. Using an Axin2(CreERT2) allele, we labeled and traced Wnt/β-catenin-responsive cells throughout mammary gland development. This reveals a switch in Wnt/β-catenin signaling around birth and shows that, depending on the developmental stage, Axin2(+) cells contribute differently to basal and luminal epithelial cell lineages of the mammary epithelium. Moreover, an important difference exists between the developmental potential tested in transplantation assays and that displayed by the same cell population in situ. Finally, Axin2(+) cells in the adult build alveolar structures during multiple pregnancies, demonstrating the existence of a Wnt/β-catenin-responsive adult stem cell. Our study uncovers dynamic changes in Wnt/β-catenin signaling in the mammary epithelium and offers insights into the developmental fate of mammary gland stem and progenitor cells.

  View details for DOI 10.1016/j.stem.2012.05.023

  View details for Web of Science ID 000309641300012

  View details for PubMedID 22863533

• Endogenous Wnt signalling in human embryonic stem cells generates an equilibrium of distinct lineage-specified progenitors NATURE COMMUNICATIONS Blauwkamp, T. A., Nigam, S., Ardehali, R., Weissman, I. L., Nusse, R. 2012; 3

  Abstract

  The pluripotent nature of human embryonic stem cells (hESCs) makes them convenient for deriving therapeutically relevant cells. Here we show using Wnt reporter hESC lines that the cells are heterogeneous with respect to endogenous Wnt signalling activity. Moreover, the level of Wnt signalling activity in individual cells correlates with differences in clonogenic potential and lineage-specific differentiation propensity. The addition of Wnt protein or, conversely, a small-molecule Wnt inhibitor (IWP2) reduces heterogeneity, allowing stable expansion of Wnt(high) or Wnt(low) hESC populations, respectively. On differentiation, the Wnt(high) hESCs predominantly form endodermal and cardiac cells, whereas the Wnt(low) hESCs generate primarily neuroectodermal cells. Thus, heterogeneity with respect to endogenous Wnt signalling underlies much of the inefficiency in directing hESCs towards specific cell types. The relatively uniform differentiation potential of the Wnt(high) and Wnt(low) hESCs leads to faster and more efficient derivation of targeted cell types from these populations.

  View details for DOI 10.1038/ncomms2064

  View details for Web of Science ID 000309338100037

  View details for PubMedID 22990866

  View details for PubMedCentralID PMC3657997

• Wnt Proteins COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Willert, K., Nusse, R. 2012; 4 (9)

  Abstract

  Wnt proteins comprise a major family of signaling molecules that orchestrate and influence a myriad of cell biological and developmental processes. Although our understanding of the role of Wnt signaling in regulating development and affecting disease, such as cancer, has been ever increasing, the study of the Wnt proteins themselves has been painstaking and slow moving. Despite advances in the biochemical characterization of Wnt proteins, many mysteries remain unsolved. In contrast to other developmental signaling molecules, such as fibroblast growth factors (FGF), transforming growth factors (TGFβ), and Sonic hedgehog (Shh), Wnt proteins have not conformed to many standard methods of protein production, such as bacterial overexpression, and analysis, such as ligand-receptor binding assays. The reasons for their recalcitrant nature are likely a consequence of the complex set of posttranslational modifications involving several highly specialized and poorly characterized processing enzymes. With the recent description of the first Wnt protein structure, the time is ripe to uncover and possibly resolve many of the remaining issues surrounding Wnt proteins and their interactions. Here we describe the process of maturation of Wnt from its initial translation to its eventual release from a cell and interactions in the extracellular environment.

  View details for DOI 10.1101/cshperspect.a007864

  View details for Web of Science ID 000308739800004

  View details for PubMedID 22952392

• Wnt5a can both activate and repress Wnt/beta-catenin signaling during mouse embryonic development DEVELOPMENTAL BIOLOGY van Amerongen, R., Fuerer, C., Mizutani, M., Nusse, R. 2012; 369 (1): 101-114

  Abstract

  Embryonic development is controlled by a small set of signal transduction pathways, with vastly different phenotypic outcomes depending on the time and place of their recruitment. How the same molecular machinery can elicit such specific and distinct responses, remains one of the outstanding questions in developmental biology. Part of the answer may lie in the high inherent genetic complexity of these signaling cascades, as observed for the Wnt-pathway. The mammalian genome encodes multiple Wnt proteins and receptors, each of which show dynamic and tightly controlled expression patterns in the embryo. Yet how these components interact in the context of the whole organism remains unknown. Here we report the generation of a novel, inducible transgenic mouse model that allows spatiotemporal control over the expression of Wnt5a, a protein implicated in many developmental processes and multiple Wnt-signaling responses. We show that ectopic Wnt5a expression from E10.5 onwards results in a variety of developmental defects, including loss of hair follicles and reduced bone formation in the skull. Moreover, we find that Wnt5a can have dual signaling activities during mouse embryonic development. Specifically, Wnt5a is capable of both inducing and repressing β-catenin/TCF signaling in vivo, depending on the time and site of expression and the receptors expressed by receiving cells. These experiments show for the first time that a single mammalian Wnt protein can have multiple signaling activities in vivo, thereby furthering our understanding of how signaling specificity is achieved in a complex developmental context.

  View details for DOI 10.1016/j.ydbio.2012.06.020

  View details for Web of Science ID 000307368600009

  View details for PubMedID 22771246

• Three decades of Wnts: a personal perspective on how a scientific field developed EMBO JOURNAL Nusse, R., Varmus, H. 2012; 31 (12): 2670-2684

  Abstract

  Wnt genes and components of Wnt signalling pathways have been implicated in a wide spectrum of important biological phenomena, ranging from early organismal development to cell behaviours to several diseases, especially cancers. Emergence of the field of Wnt signalling can be largely traced back to the discovery of the first mammalian Wnt gene in 1982. In this essay, we mark the thirtieth anniversary of that discovery by describing some of the critical scientific developments that led to the flowering of this field of research.

  View details for DOI 10.1038/emboj.2012.146

  View details for Web of Science ID 000305299200002

  View details for PubMedID 22617420

• ROR2 is a novel prognostic biomarker and a potential therapeutic target in leiomyosarcoma and gastrointestinal stromal tumour JOURNAL OF PATHOLOGY Edris, B., Espinosa, I., Muehlenberg, T., Mikels, A., Lee, C., Steigen, S. E., Zhu, S., Montgomery, K. D., Lazar, A. J., Lev, D., Fletcher, J. A., Beck, A. H., West, R. B., Nusse, R., van de Rijn, M. 2012; 227 (2): 223-233

  Abstract

  Soft-tissue sarcomas are a group of malignant tumours whose clinical management is complicated by morphological heterogeneity, inadequate molecular markers and limited therapeutic options. Receptor tyrosine kinases (RTKs) have been shown to play important roles in cancer, both as therapeutic targets and as prognostic biomarkers. An initial screen of gene expression data for 48 RTKs in 148 sarcomas showed that ROR2 was expressed in a subset of leiomyosarcoma (LMS), gastrointestinal stromal tumour (GIST) and desmoid-type fibromatosis (DTF). This was further confirmed by immunohistochemistry (IHC) on 573 tissue samples from 59 sarcoma tumour types. Here we provide evidence that ROR2 expression plays a role in the invasive abilities of LMS and GIST cells in vitro. We also show that knockdown of ROR2 significantly reduces tumour mass in vivo using a xenotransplantation model of LMS. Lastly, we show that ROR2 expression, as measured by IHC, predicts poor clinical outcome in patients with LMS and GIST, although it was not independent of other clinico-pathological features in a multivariate analysis, and that ROR2 expression is maintained between primary tumours and their metastases. Together, these results show that ROR2 is a useful prognostic indicator in the clinical management of these soft-tissue sarcomas and may represent a novel therapeutic target.

  View details for DOI 10.1002/path.3986

  View details for PubMedID 22294416

• Wnt signaling induces proliferation of sensory precursors in the postnatal mouse cochlea PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chai, R., Kuo, B., Wang, T., Liaw, E. J., Xia, A., Jan, T. A., Liu, Z., Taketo, M. M., Oghalai, J. S., Nusse, R., Zuo, J., Cheng, A. G. 2012; 109 (21): 8167-8172

  Abstract

  Inner ear hair cells are specialized sensory cells essential for auditory function. Previous studies have shown that the sensory epithelium is postmitotic, but it harbors cells that can behave as progenitor cells in vitro, including the ability to form new hair cells. Lgr5, a Wnt target gene, marks distinct supporting cell types in the neonatal cochlea. Here, we tested the hypothesis that Lgr5(+) cells are Wnt-responsive sensory precursor cells. In contrast to their quiescent in vivo behavior, Lgr5(+) cells isolated by flow cytometry from neonatal Lgr5(EGFP-CreERT2/+) mice proliferated and formed clonal colonies. After 10 d in culture, new sensory cells formed and displayed specific hair cell markers (myo7a, calretinin, parvalbumin, myo6) and stereocilia-like structures expressing F-actin and espin. In comparison with other supporting cells, Lgr5(+) cells were enriched precursors to myo7a(+) cells, most of which formed without mitotic division. Treatment with Wnt agonists increased proliferation and colony-formation capacity. Conversely, small-molecule inhibitors of Wnt signaling suppressed proliferation without compromising the myo7a(+) cells formed by direct differentiation. In vivo lineage tracing supported the idea that Lgr5(+) cells give rise to myo7a(+) hair cells in the neonatal Lgr5(EGFP-CreERT2/+) cochlea. In addition, overexpression of β-catenin initiated proliferation and led to transient expansion of Lgr5(+) cells within the cochlear sensory epithelium. These results suggest that Lgr5 marks sensory precursors and that Wnt signaling can promote their proliferation and provide mechanistic insights into Wnt-responsive progenitor cells during sensory organ development.

  View details for DOI 10.1073/pnas.1202774109

  View details for Web of Science ID 000304445800053

  View details for PubMedID 22562792

  View details for PubMedCentralID PMC3361451

• Wnt Signaling COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Nusse, R. 2012; 4 (5)

  View details for DOI 10.1101/cshperspect.a011163

  View details for Web of Science ID 000308024100013

  View details for PubMedID 22550232

• The Receptor Tyrosine Kinase ROR2 Is a Novel Marker for TSC-Associated Lesions and a Potential Therapeutic Target Independent of the TSC/mTOR Pathway 101st Annual Meeting of United-States-and-Canadian-Academy-of-Pathology (USCAP) SWEENEY, R. T., Badreddin, E., Montgomery, K. D., Nusse, R., van de Rijn, M. NATURE PUBLISHING GROUP. 2012: 491A–491A

  View details for Web of Science ID 000299986902559

• Secreted Wingless-interacting molecule (Swim) promotes long-range signaling by maintaining Wingless solubility PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mulligan, K. A., Fuerer, C., Ching, W., Fish, M., Willert, K., Nusse, R. 2012; 109 (2): 370-377

  Abstract

  Lipid-modified Wnt/Wingless (Wg) proteins can signal to their target cells in a short- or long-range manner. How these hydrophobic proteins travel through the extracellular environment remains an outstanding question. Here, we report on a Wg binding protein, Secreted Wg-interacting molecule (Swim), that facilitates Wg diffusion through the extracellular matrix. Swim, a putative member of the Lipocalin family of extracellular transport proteins, binds to Wg with nanomolar affinity in a lipid-dependent manner. In quantitative signaling assays, Swim is sufficient to maintain the solubility and activity of purified Wg. In Drosophila, swim RNAi phenotypes resemble wg loss-of-function phenotypes in long-range signaling. We propose that Swim is a cofactor that promotes long-range Wg signaling in vivo by maintaining the solubility of Wg.

  View details for DOI 10.1073/pnas.1119197109

  View details for Web of Science ID 000298950200014

  View details for PubMedID 22203956

  View details for PubMedCentralID PMC3258625

• Identification of Cardiovascular Progenitors From Human Embryonic Stem Cells Scientific Sessions of the American-Heart-Association/Resuscitation Science Symposium Ardehali, R., Ali, S., Drukker, M., Abilez, O., Blauwkamp, T., Nusse, R., Weissman, I. LIPPINCOTT WILLIAMS & WILKINS. 2011

  View details for Web of Science ID 000299738707322

• A Suppressor/Enhancer Screen in Drosophila Reveals a Role for Wnt-Mediated Lipid Metabolism in Primordial Germ Cell Migration PLOS ONE McElwain, M. A., Ko, D. C., Gordon, M. D., Fyrst, H., Saba, J. D., Nusse, R. 2011; 6 (11)

  Abstract

  Wnt proteins comprise a large family of secreted ligands implicated in a wide variety of biological roles. WntD has previously been shown to inhibit the nuclear accumulation of Dorsal/NF-κB protein during embryonic dorsal/ventral patterning and the adult innate immune response, independent of the well-studied Armadillo/β-catenin pathway. In this paper, we present a novel phenotype for WntD mutant embryos, suggesting that this gene is involved in migration of primordial germ cells (PGC) to the embryonic gonad. Additionally, we describe a genetic suppressor/enhancer screen aimed at identifying genes required for WntD signal transduction, based on the previous observation that maternal overexpression of WntD results in lethally dorsalized embryos. Using an algorithm to narrow down our hits from the screen, we found two novel WntD signaling components: Fz4, a member of the Frizzled family, and the Drosophila Ceramide Kinase homolog, Dcerk. We show here that Dcerk and Dmulk (Drosophila Multi-substrate lipid kinase) redundantly mediate PGC migration. Our data are consistent with a model in which the activity of lipid phosphate phosphatases shapes a concentration gradient of ceramide-1-phosphate (C1P), the product of Dcerk, allowing proper PGC migration.

  View details for DOI 10.1371/journal.pone.0026993

  View details for Web of Science ID 000297150900042

  View details for PubMedID 22069480

• Location, Location, Location: FoxM1 Mediates beta-Catenin Nuclear Translocation and Promotes Glioma Tumorigenesis CANCER CELL Bowman, A., Nusse, R. 2011; 20 (4): 415-416

  Abstract

  Genetic alterations in the Wnt/β-catenin/TCF-signaling pathway are commonly found in human tumors, but not in glioblastomas. In this issue of Cancer Cell, Zhang et al. report that FoxM1 mediates β-catenin nuclear translocation in glioblastoma, suggesting a novel mechanism for glioblastoma progression in the absence of conventional Wnt/β-catenin pathway activation.

  View details for DOI 10.1016/j.ccr.2011.10.003

  View details for Web of Science ID 000296121300001

  View details for PubMedID 22014565

• Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells NATURE CELL BIOLOGY Ten Berge, D., Kurek, D., Blauwkamp, T., Koole, W., Maas, A., Eroglu, E., Siu, R. K., Nusse, R. 2011; 13 (9): 1070-U88

  Abstract

  Pluripotent stem cells exist in naive and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref. 1). In the naive state of ESCs, the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks. In contrast, EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types. The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs, and are required to inhibit their differentiation into EpiSCs. Moreover, we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors, and support the derivation of new ESC lines, including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition, but also identify Wnt as an essential and limiting ESC self-renewal factor.

  View details for DOI 10.1038/ncb2314

  View details for Web of Science ID 000294487000011

  View details for PubMedID 21841791

• A crucial role for lipid phosphorylation in WntD-mediated primordial germ cell migration McElwain, M. A., Ko, D. C., Gordon, M. D., Nusse, R. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2011: 192

  View details for DOI 10.1016/j.ydbio.2011.05.248

  View details for Web of Science ID 000292784400283

• Wnt Proteins Promote Bone Regeneration SCIENCE TRANSLATIONAL MEDICINE Minear, S., Leucht, P., Jiang, J., Liu, B., Zeng, A., Fuerer, C., Nusse, R., Helms, J. A. 2010; 2 (29)

  Abstract

  The Wnt signaling pathway plays a central role in bone development and homeostasis. In most cases, Wnt ligands promote bone growth, which has led to speculation that Wnt factors could be used to stimulate bone healing. We gained insights into the mechanism by which Wnt signaling regulates adult bone repair through the use of the mouse strain Axin2(LacZ/LacZ) in which the cellular response to Wnt is increased. We found that bone healing after injury is accelerated in Axin2(LacZ/LacZ) mice, a consequence of more robust proliferation and earlier differentiation of skeletal stem and progenitor cells. In parallel, we devised a biochemical strategy to increase the duration and strength of Wnt signaling at the sites of skeletal injury. Purified Wnt3a was packaged in liposomal vesicles and delivered to skeletal defects, where it stimulated the proliferation of skeletal progenitor cells and accelerated their differentiation into osteoblasts, cells responsible for bone growth. The end result was faster bone regeneration. Because Wnt signaling is conserved in mammalian tissue repair, this protein-based approach may have widespread applications in regenerative medicine.

  View details for DOI 10.1126/scitranslmed.3000231

  View details for Web of Science ID 000277304700001

  View details for PubMedID 20427820

• Lentiviral Vectors to Probe and Manipulate the Wnt Signaling Pathway PLOS ONE Fuerer, C., Nusse, R. 2010; 5 (2)

  Abstract

  The Wnt signaling pathway plays key roles in development, adult tissue homeostasis and stem cell maintenance. Further understanding of the function of Wnt signaling in specific cell types could benefit from lentiviral vectors expressing reporters for the Wnt pathway or vectors interfering with signaling.We have developed a set of fluorescent and luminescent lentiviral vectors that report Wnt signaling activity and discriminate between negative and uninfected cells. These vectors possess a 7xTcf-eGFP or 7xTcf-FFluc (Firefly Luciferase) reporter cassette followed by either an SV40-mCherry or SV40-Puro(R) (puromycin N-acetyltransferase) selection cassette. We have also constructed a vector that allows drug-based selection of cells with activated Wnt signaling by placing Puro(R) under the control of the 7xTcf promoter. Lastly, we have expressed dominant-negative Tcf4 (dnTcf4) or constitutively active beta-catenin (beta-catenin(4A)) from the hEF1alpha promoter in a SV40-Puro(R) or SV40-mCherry backbone to create vectors that inhibit or activate the Wnt signaling pathway. These vectors will be made available to the scientific community through Addgene.These novel lentiviruses are efficient tools to probe and manipulate Wnt signaling. The use of a selection cassette in Wnt-reporter viruses enables discriminating between uninfected and non-responsive cells, an important requirement for experiments where selection of clones is not possible. The use of a chemiluminescent readout enables quantification of signaling. Finally, selectable vectors can be used to either inhibit or activate the Wnt signaling pathway. Altogether, these vectors can probe and modulate the Wnt signaling pathway in experimental settings where persistence of the transgene or gene transfer cannot be accomplished by non-viral techniques.

  View details for DOI 10.1371/journal.pone.0009370

  View details for Web of Science ID 000274924000013

  View details for PubMedID 20186325

• A Study on the Interactions Between Heparan Sulfate Proteoglycans and Wnt Proteins DEVELOPMENTAL DYNAMICS Fuerer, C., Habib, S. J., Nusse, R. 2010; 239 (1): 184-190

  Abstract

  The Wnt signaling pathway plays key roles in development and adult homeostasis. Wnt proteins are secreted, lipid-modified glycoproteins. They can form morphogen gradients that are regulated at the level of protein secretion, diffusion, and internalization. These gradients can only exist if the hydrophobic Wnt proteins are prevented from aggregating in the extracellular environment. Heparan sulfate proteoglycans (HSPGs) are necessary for proper activity of Wnt proteins and influence their distribution along the morphogenetic gradient. In this study, we show that HSPGs are able to maintain the solubility of Wnt proteins, thus stabilizing their signaling activity. Our results suggest that the role of HSPGs is not only to concentrate Wnt molecules at the cell surface but also to prevent them from aggregating in the extracellular environment.

  View details for DOI 10.1002/dvdy.22067

  View details for Web of Science ID 000273703900016

  View details for PubMedID 19705435

• Ror2 Receptor Requires Tyrosine Kinase Activity to Mediate Wnt5A Signaling JOURNAL OF BIOLOGICAL CHEMISTRY Mikels, A., Minami, Y., Nusse, R. 2009; 284 (44): 30167-30176

  Abstract

  The Wnts include a large family of secreted proteins that serve as important signals during embryonic development and adult homeostasis. In the most well understood Wnt signaling pathway, Wnt binding to Frizzled and low density lipoprotein receptor-related protein induces beta-catenin protein stabilization and entry into the nucleus, resulting in changes in target gene transcription. Emerging evidence suggests that Wnt5a can inhibit Wnt/beta-catenin signaling through interaction with the receptor Ror2. The Ror2 protein belongs to the receptor tyrosine kinase superfamily and contains several recognizable structural motifs. However, limited information is available regarding which specific domains are required for the inhibitory signaling activity of Wnt5a. Through mutation and deletion analysis, we have analyzed which specific domains and residues, including those necessary for tyrosine kinase activity, mediate the Wnt5a signal. To determine whether Ror2 can inhibit canonical Wnt signaling in vivo, we examined the effect of Ror2 loss on the expression of the Wnt reporter Axin2(LacZ), finding increased reporter activity in Ror2 null mice, demonstrating that Ror2 can also inhibit Wnt/beta-catenin signaling in the context of intact tissues.

  View details for DOI 10.1074/jbc.M109.041715

  View details for Web of Science ID 000271090000020

  View details for PubMedID 19720827

• Towards an integrated view of Wnt signaling in development DEVELOPMENT van Amerongen, R., Nusse, R. 2009; 136 (19): 3205-3214

  Abstract

  Wnt signaling is crucial for embryonic development in all animal species studied to date. The interaction between Wnt proteins and cell surface receptors can result in a variety of intracellular responses. A key remaining question is how these specific responses take shape in the context of a complex, multicellular organism. Recent studies suggest that we have to revise some of our most basic ideas about Wnt signal transduction. Rather than thinking about Wnt signaling in terms of distinct, linear, cellular signaling pathways, we propose a novel view that considers the integration of multiple, often simultaneous, inputs at the level of both Wnt-receptor binding and the downstream, intracellular response.

  View details for DOI 10.1242/dev.033910

  View details for Web of Science ID 000269568400001

  View details for PubMedID 19736321

• Ror2, a developmentally regulated kinase, promotes tumor growth potential in renal cell carcinoma ONCOGENE Wright, T. M., Brannon, A. R., Gordan, J. D., Mikels, A. J., Mitchell, C., Chen, S., Espinosa, I., van de Rijn, M., Pruthi, R., Wallen, E., Edwards, L., Nusse, R., Rathmell, W. K. 2009; 28 (27): 2513-2523

  Abstract

  Inappropriate kinase expression and subsequent promiscuous activity defines the transformation of many solid tumors including renal cell carcinoma (RCC). Thus, the expression of novel tumor-associated kinases has the potential to dramatically shape tumor cell behavior. Further, identifying tumor-associated kinases can lend insight into patterns of tumor growth and characteristics. Here, we report the identification of the RTK-like orphan receptor 2 (Ror2), a new tumor-associated kinase in RCC cell lines and primary tumors. Ror2 is an orphan receptor tyrosine kinase with physiological expression normally seen in the embryonic kidney. However, in RCC, Ror2 expression correlated with expression of genes involved at the extracellular matrix, including Twist and matrix metalloprotease-2 (MMP2). Expression of MMP2 in RCC cells was suppressed by Ror2 knockdown, placing Ror2 as a mediator of MMP2 regulation in RCC and a potential regulator of extracellular matrix remodeling. The suppression of Ror2 not only inhibited cell migration, but also inhibited anchorage-independent growth in soft agar and growth in an orthotopic xenograft model. These findings suggest a novel pathway of tumor-promoting activity by Ror2 within a subset of renal carcinomas, with significant implications for unraveling the tumorigenesis of RCC.

  View details for DOI 10.1038/onc.2009.116

  View details for Web of Science ID 000267806800004

  View details for PubMedID 19448672

  View details for PubMedCentralID PMC2771692

• Telomerase modulates Wnt signalling by association with target gene chromatin NATURE Park, J., Venteicher, A. S., Hong, J. Y., Choi, J., Jun, S., Shkreli, M., Chang, W., Meng, Z., Cheung, P., Ji, H., McLaughlin, M., Veenstra, T. D., Nusse, R., McCrea, P. D., Artandi, S. E. 2009; 460 (7251): 66-U77

  Abstract

  Stem cells are controlled, in part, by genetic pathways frequently dysregulated during human tumorigenesis. Either stimulation of Wnt/beta-catenin signalling or overexpression of telomerase is sufficient to activate quiescent epidermal stem cells in vivo, although the mechanisms by which telomerase exerts these effects are not understood. Here we show that telomerase directly modulates Wnt/beta-catenin signalling by serving as a cofactor in a beta-catenin transcriptional complex. The telomerase protein component TERT (telomerase reverse transcriptase) interacts with BRG1 (also called SMARCA4), a SWI/SNF-related chromatin remodelling protein, and activates Wnt-dependent reporters in cultured cells and in vivo. TERT serves an essential role in formation of the anterior-posterior axis in Xenopus laevis embryos, and this defect in Wnt signalling manifests as homeotic transformations in the vertebrae of Tert(-/-) mice. Chromatin immunoprecipitation of the endogenous TERT protein from mouse gastrointestinal tract shows that TERT physically occupies gene promoters of Wnt-dependent genes. These data reveal an unanticipated role for telomerase as a transcriptional modulator of the Wnt/beta-catenin signalling pathway.

  View details for DOI 10.1038/nature08137

  View details for Web of Science ID 000267545200030

  View details for PubMedID 19571879

  View details for PubMedCentralID PMC4349391

• Wnt Signaling Mediates Self-Organization and Axis Formation in Embryoid Bodies CELL STEM CELL ten Berge, D., Koole, W., Fuerer, C., Fish, M., Eroglu, E., Nusse, R. 2008; 3 (5): 508-518

  Abstract

  Embryonic stem cells (ESCs) form descendants of all three germ layers when differentiated as aggregates, termed embryoid bodies. In vivo, differentiation of cells depends on signals and morphogen gradients that provide instructive and positional cues, but do such gradients exist in embryoid bodies? We report here the establishment of anteroposterior polarity and the formation of a primitive streak-like region in the embryoid body, dependent on local activation of the Wnt pathway. In this region, cells undergo an epithelial-to-mesenchymal transition and differentiate into mesendodermal progenitors. Exogenous Wnt3a protein posteriorizes the embryoid body, resulting in predominantly mesendodermal differentiation. Conversely, inhibiting Wnt signaling promotes anterior character and results in neurectodermal differentiation. The activation of Wnt signaling and primitive streak formation requires external signals but is self-reinforcing after initiation. Our findings show that the Wnt pathway mediates the local execution of a gastrulation-like process in the embryoid body, which displays an unexpected degree of self-organization.

  View details for DOI 10.1016/j.stem.2008.09.013

  View details for Web of Science ID 000260895100010

  View details for PubMedID 18983966

  View details for PubMedCentralID PMC2683270

• Wnt-mediated self-renewal of neural stem/progenitor cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kalani, M. Y., Cheshier, S. H., Cord, B. J., Bababeygy, S. R., Vogel, H., Weissman, I. L., Palmer, T. D., Nusse, R. 2008; 105 (44): 16970-16975

  Abstract

  In this work we have uncovered a role for Wnt signaling as an important regulator of stem cell self-renewal in the developing brain. We identified Wnt-responsive cells in the subventricular zone of the developing E14.5 mouse brain. Responding cell populations were enriched for self-renewing stem cells in primary culture, suggesting that Wnt signaling is a hallmark of self-renewing activity in vivo. We also tested whether Wnt signals directly influence neural stem cells. Using inhibitors of the Wnt pathway, we found that Wnt signaling is required for the efficient cloning and expansion of single-cell derived populations that are able to generate new stem cells as well as neurons, astrocytes, and oligodendrocytes. The addition of exogenous Wnt3a protein enhances clonal outgrowth, demonstrating not only a critical role for the Wnt pathway for the regulation of neurogenesis but also its use for the expansion of neural stem cells in cell culture and in tissue engineering.

  View details for DOI 10.1073/pnas.0808616105

  View details for PubMedID 18957545

• Translating insights from development into regenerative medicine: The function of Wnts in bone biology SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY Leucht, P., Minear, S., Ten Berge, D., Nusse, R., Helms, J. A. 2008; 19 (5): 434-443

  Abstract

  The Wnt pathway constitutes one of the most attractive candidates for modulating skeletal tissue regeneration based on its functions during skeletal development and homeostasis. Wnts participate in every stage of skeletogenesis, from the self-renewal and proliferation of skeletal stem cells to the specification of osteochondroprogenitor cells and the maturation of chondrocytes and osteoblasts. We propose that the function of Wnts depend upon a skeletogenic cell's state of differentiation. In this review we summarize recent data with a focus on the roles of Wnt signaling in mesenchymal stem cell fate, osteoprogenitor cell differentiation, chondrocyte maturation, bone remodeling, and bone regeneration.

  View details for DOI 10.1016/j.semcdb.2008.09.002

  View details for Web of Science ID 000262670500003

  View details for PubMedID 18824114

• Wnt and FGF signals interact to coordinate growth with cell fate specification during limb development DEVELOPMENT ten Berge, D., Brugmann, S. A., Helms, J. A., Nusse, R. 2008; 135 (19): 3247-3257

  Abstract

  A fundamental question in developmental biology is how does an undifferentiated field of cells acquire spatial pattern and undergo coordinated differentiation? The development of the vertebrate limb is an important paradigm for understanding these processes. The skeletal and connective tissues of the developing limb all derive from a population of multipotent progenitor cells located in its distal tip. During limb outgrowth, these progenitors segregate into a chondrogenic lineage, located in the center of the limb bud, and soft connective tissue lineages located in its periphery. We report that the interplay of two families of signaling proteins, fibroblast growth factors (FGFs) and Wnts, coordinate the growth of the multipotent progenitor cells with their simultaneous segregation into these lineages. FGF and Wnt signals act together to synergistically promote proliferation while maintaining the cells in an undifferentiated, multipotent state, but act separately to determine cell lineage specification. Withdrawal of both signals results in cell cycle withdrawal and chondrogenic differentiation. Continued exposure to Wnt, however, maintains proliferation and re-specifies the cells towards the soft connective tissue lineages. We have identified target genes that are synergistically regulated by Wnts and FGFs, and show how these factors actively suppress differentiation and promote growth. Finally, we show how the spatial restriction of Wnt and FGF signals to the limb ectoderm, and to a specialized region of it, the apical ectodermal ridge, controls the distribution of cell behaviors within the growing limb, and guides the proper spatial organization of the differentiating tissues.

  View details for DOI 10.1242/dev.023176

  View details for Web of Science ID 000258989400009

  View details for PubMedID 18776145

  View details for PubMedCentralID PMC2756806

• Alternative Wnt Signaling Is Initiated by Distinct Receptors SCIENCE SIGNALING van Amerongen, R., Mikels, A., Nusse, R. 2008; 1 (35)

  Abstract

  An unanswered question in the field of signal transduction research is how different signaling pathways are activated with strict specificity in a temporally and spatially controlled manner. Because extracellular ligands and membrane receptors constitute the first signaling modalities for most pathways, selectivity in ligand-receptor binding likely dictates the outcome of downstream signaling events. Unfortunately, possible complexities underlying ligand-receptor interactions are often overlooked. Here, we discuss basic principles of signal transduction initiated at the cell membrane, with the Wnt pathway, which harbors a multitude of ligands and receptors, as an example.

  View details for DOI 10.1126/scisignal.135re9

  View details for Web of Science ID 000207496300005

  View details for PubMedID 18765832

• Liposomal Packaging Generates Wnt Protein with In Vivo Biological Activity PLOS ONE Morrell, N. T., Leucht, P., Zhao, L., Kim, J., Ten Berge, D., Ponnusamy, K., Carre, A. L., Dudek, H., Zachlederova, M., McElhaney, M., Brunton, S., Gunzner, J., Callow, M., Polakis, P., Costa, M., Zhang, X. M., Helms, J. A., Nusse, R. 2008; 3 (8)

  Abstract

  Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context.

  View details for DOI 10.1371/journal.pone.0002930

  View details for Web of Science ID 000264412600016

  View details for PubMedID 18698373

  View details for PubMedCentralID PMC2515347

• Pathogenesis of Listeria-infected Drosophila wntD mutants is associated with elevated levels of the novel immunity gene edin PLOS PATHOGENS Gordon, M. D., Ayres, J. S., Schneider, D. S., Nusse, R. 2008; 4 (7)

  Abstract

  Drosophila melanogaster mount an effective innate immune response against invading microorganisms, but can eventually succumb to persistent pathogenic infections. Understanding of this pathogenesis is limited, but it appears that host factors, induced by microbes, can have a direct cost to the host organism. Mutations in wntD cause susceptibility to Listeria monocytogenes infection, apparently through the derepression of Toll-Dorsal target genes, some of which are deleterious to survival. Here, we use gene expression profiling to identify genes that may mediate the observed susceptibility of wntD mutants to lethal infection. These genes include the TNF family member eiger and the novel immunity gene edin (elevated during infection; synonym CG32185), both of which are more strongly induced by infection of wntD mutants compared to controls. edin is also expressed more highly during infection of wild-type flies with wild-type Salmonella typhimurium than with a less pathogenic mutant strain, and its expression is regulated in part by the Imd pathway. Furthermore, overexpression of edin can induce age-dependent lethality, while loss of function in edin renders flies more susceptible to Listeria infection. These results are consistent with a model in which the regulation of host factors, including edin, must be tightly controlled to avoid the detrimental consequences of having too much or too little activity.

  View details for DOI 10.1371/journal.ppat.1000111

  View details for Web of Science ID 000259783000017

  View details for PubMedID 18654628

  View details for PubMedCentralID PMC2453329

• Lipid-independent secretion of a Drosophila Wnt protein JOURNAL OF BIOLOGICAL CHEMISTRY Ching, W., Hang, H. C., Nusse, R. 2008; 283 (25): 17092-17098

  Abstract

  Wnt proteins comprise a large class of secreted signaling molecules with key roles during embryonic development and throughout adult life. Recently, much effort has been focused on understanding the factors that regulate Wnt signal production. For example, Porcupine and Wntless/Evi/Sprinter have been identified as being required in Wnt-producing cells for the processing and secretion of many Wnt proteins. Interestingly, in this study we find that WntD, a recently characterized Drosophila Wnt family member, does not require Porcupine or Wntless/Evi/Sprinter for its secretion or signaling activity. Because Porcupine is involved in post-translational lipid modification of Wnt proteins, we used a novel labeling method and mass spectrometry to ask whether WntD undergoes lipid modification and found that it does not. Although lipid modification is also hypothesized to be required for Wnt secretion, we find that WntD is secreted very efficiently. WntD secretion does, however, maintain a requirement for the secretory pathway component Rab1. Our results show that not all Wnt family members require lipid modification, Porcupine, or Wntless/Evi/Sprinter for secretion and suggest that different modes of secretion may exist for different Wnt proteins.

  View details for DOI 10.1074/jbc.M802059200

  View details for Web of Science ID 000256720600016

  View details for PubMedID 18430724

  View details for PubMedCentralID PMC2427328

• Asymmetric homotypic interactions of the atypical cadherin Flamingo mediate intercellular polarity signaling CELL Chen, W., Antic, D., Matis, M., Logan, C. Y., Povelones, M., Anderson, G. A., Nusse, R., Axelrod, J. D. 2008; 133 (6): 1093-1105

  Abstract

  Acquisition of planar cell polarity (PCP) in epithelia involves intercellular communication, during which cells align their polarity with that of their neighbors. The transmembrane proteins Frizzled (Fz) and Van Gogh (Vang) are essential components of the intercellular communication mechanism, as loss of either strongly perturbs the polarity of neighboring cells. How Fz and Vang communicate polarity information between neighboring cells is poorly understood. The atypical cadherin, Flamingo (Fmi), is implicated in this process, yet whether Fmi acts permissively as a scaffold or instructively as a signal is unclear. Here, we provide evidence that Fmi functions instructively to mediate Fz-Vang intercellular signal relay, recruiting Fz and Vang to opposite sides of cell boundaries. We propose that two functional forms of Fmi, one of which is induced by and physically interacts with Fz, bind each other to create cadherin homodimers that signal bidirectionally and asymmetrically, instructing unequal responses in adjacent cell membranes to establish molecular asymmetry.

  View details for DOI 10.1016/j.cell.2008.04.048

  View details for Web of Science ID 000256693400023

  View details for PubMedID 18555784

  View details for PubMedCentralID PMC2446404

• A dermal HOX transcriptional program regulates site-specific epidermal fate GENES & DEVELOPMENT Rinn, J. L., Wang, J. K., Allen, N., Brugmann, S. A., Mikels, A. J., Liu, H., Ridky, T. W., Stadler, H. S., Nusse, R., Helms, J. A., Chang, H. Y. 2008; 22 (3): 303-307

  Abstract

  Reciprocal epithelial-mesenchymal interactions shape site-specific development of skin. Here we show that site-specific HOX expression in fibroblasts is cell-autonomous and epigenetically maintained. The distal-specific gene HOXA13 is continually required to maintain the distal-specific transcriptional program in adult fibroblasts, including expression of WNT5A, a morphogen required for distal development. The ability of distal fibroblasts to induce epidermal keratin 9, a distal-specific gene, is abrogated by depletion of HOXA13, but rescued by addition of WNT5A. Thus, maintenance of appropriate HOX transcriptional program in adult fibroblasts may serve as a source of positional memory to differentially pattern the epithelia during homeostasis and regeneration.

  View details for DOI 10.1101/gad.1610508

  View details for PubMedID 18245445

• Wnt Signaling and Stem Cell Control 73rd Cold Spring Harbor Symposium on Quantitative Biology Nusse, R., Fuerer, C., Ching, W., Harnish, K., Logan, C., Zeng, A., Ten Berge, D., Kalani, Y. COLD SPRING HARBOR LABORATORY PRESS. 2008: 59–66

  Abstract

  In many contexts, self-renewal and differentiation of stem cells are influenced by signals from their environment, constituting a niche. It is postulated that stem cells compete for local growth factors in the niche, thereby maintaining a balance between the numbers of self-renewing and differentiated cells. A critical aspect of the niche model for stem cell regulation is that the availability of self-renewing factors is limited and that stem cells compete for these factors (Fig. 1). Consequently, the range and concentrations of the niche factors are of critical importance. Now that some of the few self-renewing factors have become identified, aspects of the niche models can be tested experimentally. In this chapter, we address mechanisms of signal regulation that take place at the level of signal-producing cells, constituting a niche for stem cells. We emphasize the biochemical properties and posttranslational modifications of the signals, all in the context of Wnt signaling. We propose that these modifications control the range of Wnt signaling and have critical roles in establishing niches for stem cells in various tissues.

  View details for Web of Science ID 000267135700008

  View details for PubMedID 19028988

• Wnt/beta-catenin signaling in murine hepatic transit amplifying progenitor cells GASTROENTEROLOGY Hu, M., Kurobe, M., Jeong, Y. J., Fuerer, C., Ghole, S., Nusse, R., Sylvester, K. G. 2007; 133 (5): 1579-1591

  Abstract

  Oval cells are postnatal hepatic progenitors with high proliferative potential and bipotent differentiation ability to become hepatocytes and cholangiocytes. Because Wnt/beta-catenin signaling is a known regulatory pathway for liver development and regeneration, we studied the role of Wnt signaling in oval cells using a mouse model of chronic liver injury.A 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-enriched diet was used to stimulate oval cell proliferation. Livers were harvested for histologic analysis and determination of Wnt family gene expression by quantitative reverse transcription-polymerase chain reaction and in situ hybridization. The transgenic beta-catenin reporter mouse (TOPGAL) was use to confirm canonical Wnt/beta-catenin signal transduction in proliferating oval cells within atypical ductal proliferations (ADPs). Confocal fluorescence microscopy and immunohistochemistry was used to confirm colocalization of beta-catenin with the oval cell antigen A-6.Several Wnt ligands were significantly induced in the liver of DDC-fed mice and localized to proliferating cells in and adjacent to the ADPs. Oval cells isolated from DDC-fed mouse livers showed the presence of active beta-catenin in the nucleus along with cell-cycle entry in response to purified Wnt3a in vitro. Moreover, Wnt3a-induced beta-catenin/T-cell factor/lymphoid enhancer factor (TCF/LEF) transcriptional activation was quantified by TCF/LEF luciferase reporter assays.From these data, we conclude that oval cells respond to Wnt ligands (Wnt3a) in vitro with an increase in amino-terminus dephosphorylated beta-catenin and cell-cycle entry and that canonical Wnt/beta-catenin/TCF signaling is active in proliferating facultative hepatic progenitor cells in vivo. These findings may lend insight to the consequences of increased canonical Wnt signaling during periods of chronic liver injury.

  View details for DOI 10.1053/j.gastro.2007.08.036

  View details for Web of Science ID 000250820100025

  View details for PubMedID 17983805

• Transcriptional Program Induced by Wnt Protein in Human Fibroblasts Suggests Mechanisms for Cell Cooperativity in Defining Tissue Microenvironments PLOS ONE Klapholz-Brown, Z., Walmsley, G. G., Nusse, Y. M., Nusse, R., Brown, P. O. 2007; 2 (9)

  Abstract

  The Wnt signaling system plays key roles in development, regulation of stem cell self-renewal and differentiation, cell polarity, morphogenesis and cancer. Given the multifaceted roles of Wnt signaling in these processes, its transcriptional effects on the stromal cells that make up the scaffold and infrastructure of epithelial tissues are of great interest.To begin to investigate these effects, we used DNA microarrays to identify transcriptional targets of the Wnt pathway in human lung fibroblasts. Cells were treated with active Wnt3a protein in culture, and RNA was harvested at 4 hours and 24 hours. Nuclear accumulation of ss-Catenin, as shown by immunofluorescence, and induction of AXIN2 demonstrate that fibroblasts are programmed to respond to extracellular Wnt signals. In addition to several known Wnt targets, we found many new Wnt induced genes, including many transcripts encoding regulatory proteins. Transcription factors with important developmental roles, including HOX genes, dominated the early transcriptional response. Furthermore, we found differential expression of several genes that play direct roles in the Wnt signaling pathway, as well as genes involved in other cell signaling pathways including fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signaling. The gene most highly induced by Wnt3a was GREMLIN2, which encodes a secreted BMP antagonist.Elevated expression of GREMLIN2 suggests a new role for Wnt signals in the maintenance of stem cell niches, whereby Wnt signals induce nearby fibroblasts to produce a BMP antagonist, inhibiting differentiation and promoting expansion of stem cells in their microenvironment. We suggest that Wnt-induced changes in the gene expression program of local stromal cells may play an important role in the establishment of specialized niches hospitable to the self-renewal of normal or malignant epithelial stem cells in vivo.

  View details for DOI 10.1371/journal.pone.0000945

  View details for Web of Science ID 000207455800024

  View details for PubMedID 17895986

  View details for PubMedCentralID PMC1976560

• Wnt signaling mediates regional specification in the vertebrate face DEVELOPMENT Brugmann, S. A., Goodnough, L. H., Gregorieff, A., Leucht, P., Ten Berge, D., Fuerer, C., Clevers, H., Nusse, R., Helms, J. A. 2007; 134 (18): 3283-3295

  Abstract

  At early stages of development, the faces of vertebrate embryos look remarkably similar, yet within a very short timeframe they adopt species-specific facial characteristics. What are the mechanisms underlying this regional specification of the vertebrate face? Using transgenic Wnt reporter embryos we found a highly conserved pattern of Wnt responsiveness in the developing mouse face that later corresponded to derivatives of the frontonasal and maxillary prominences. We explored the consequences of disrupting Wnt signaling, first using a genetic approach. Mice carrying compound null mutations in the nuclear mediators Lef1 and Tcf4 exhibited radically altered facial features that culminated in a hyperteloric appearance and a foreshortened midface. We also used a biochemical approach to perturb Wnt signaling and found that in utero delivery of a Wnt antagonist, Dkk1, produced similar midfacial malformations. We tested the hypothesis that Wnt signaling is an evolutionarily conserved mechanism controlling facial morphogenesis by determining the pattern of Wnt responsiveness in avian faces, and then by evaluating the consequences of Wnt inhibition in the chick face. Collectively, these data elucidate a new role for Wnt signaling in regional specification of the vertebrate face, and suggest possible mechanisms whereby species-specific facial features are generated.

  View details for DOI 10.1242/dev.005132

  View details for Web of Science ID 000249013700006

  View details for PubMedID 17699607

• Mutants in the Mouse NuRD/Mi2 Component P66 alpha Are Embryonic Lethal PLOS ONE Marino, S., Nusse, R. 2007; 2 (6)

  Abstract

  The NuRD/Mi2 chromatin complex is involved in histone modifications and contains a large number of subunits, including the p66 protein. There are two mouse and human p66 paralogs, p66alpha and p66beta. The functions of these genes are not clear, in part because there are no mutants available, except in invertebrate model systems.We made loss of function mutants in the mouse p66alpha gene (mp66alpha, official name Gatad2a, MGI:2384585). We found that mp66alpha is essential for development, as mutant embryos die around day 10 of embryogenesis. The gene is not required for normal blastocyst development or for implantation. The phenotype of mutant embryos and the pattern of gene expression in mutants are consistent with a role of mp66alpha in gene silencing.mp66alpha is an essential gene, required for early mouse development. The lethal phenotype supports a role in execution of methylated DNA silencing.

  View details for DOI 10.1371/journal.pone.0000519

  View details for Web of Science ID 000207451600007

  View details for PubMedID 17565372

• Converging on beta-catenin in Wilms tumor SCIENCE Nusse, R. 2007; 316 (5827): 988-989

  View details for DOI 10.1126/science.1143337

  View details for Web of Science ID 000246554000031

  View details for PubMedID 17510350

• Wnt signaling regulates pancreatic beta cell proliferation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rulifson, I. C., Karnik, S. K., Heiser, P. W., Ten Berge, D., Chen, H., Gu, X., Taketo, M. M., Nusse, R., Hebrok, M., Kim, S. K. 2007; 104 (15): 6247-6252

  Abstract

  There is widespread interest in defining factors and mechanisms that stimulate proliferation of pancreatic islet cells. Wnt signaling is an important regulator of organ growth and cell fates, and genes encoding Wnt-signaling factors are expressed in the pancreas. However, it is unclear whether Wnt signaling regulates pancreatic islet proliferation and differentiation. Here we provide evidence that Wnt signaling stimulates islet beta cell proliferation. The addition of purified Wnt3a protein to cultured beta cells or islets promoted expression of Pitx2, a direct target of Wnt signaling, and Cyclin D2, an essential regulator of beta cell cycle progression, and led to increased beta cell proliferation in vitro. Conditional pancreatic beta cell expression of activated beta-catenin, a crucial Wnt signal transduction protein, produced similar phenotypes in vivo, leading to beta cell expansion, increased insulin production and serum levels, and enhanced glucose handling. Conditional beta cell expression of Axin, a potent negative regulator of Wnt signaling, led to reduced Pitx2 and Cyclin D2 expression by beta cells, resulting in reduced neonatal beta cell expansion and mass and impaired glucose tolerance. Thus, Wnt signaling is both necessary and sufficient for islet beta cell proliferation, and our study provides previously unrecognized evidence of a mechanism governing endocrine pancreas growth and function.

  View details for DOI 10.1073/pnas.0701509104

  View details for Web of Science ID 000245737500029

  View details for PubMedID 17404238

  View details for PubMedCentralID PMC1847455

• Creating transgenic Drosophila by microinjecting the site-specific phi C31 integrase mRNA and a transgene-containing donor plasmid NATURE PROTOCOLS Fish, M. P., Groth, A. C., Calos, M. P., Nusse, R. 2007; 2 (10): 2325-2331

  Abstract

  We describe a microinjection-based phiC31 integrase mRNA-mediated method for creating transgenic Drosophila strains. This approach is more efficient than traditional methods and ensures that the transgene is targeted to a precise genomic position. The method involves targeting integration of an exogenous plasmid (containing the transgene and sequences to facilitate integration) to a preplaced recipient site in the Drosophila genome. The plasmid is coinjected into embryos with mRNA encoding the phiC31 integrase, the enzyme that catalyzes the integration reaction. Using the protocol described here, transgenic lines can be established from, on average, 46% of fertile adults obtained after injection, and all integrations should be targeted to the chosen genomic insertion site. The whole procedure, from injection to established transgenic stocks, can be completed in three generations (approximately 1 month) and can be adapted for other types of transgenesis and mRNA injections in Drosophila.

  View details for DOI 10.1038/nprot.2007.328

  View details for Web of Science ID 000253139900004

  View details for PubMedID 17947973

• Highly enriched mesenchymal stem cells (MSC) can initiate sarcoma formation in vivo 61st Annual Session of the Surgical Forum 2006 Clinical Congress Tataria, M., Kuscuoglu, U., Lauire, A., Cheshier, S., Nusse, R., Weissman, I., Karl, S. ELSEVIER SCIENCE INC. 2006: S39–S40

  View details for Web of Science ID 000240406800071

• Wingless signaling modulates cadherin-mediated cell adhesion in Drosophila imaginal disc cells JOURNAL OF CELL SCIENCE Wodarz, A., Stewart, D. B., Nelson, W. J., Nusse, R. 2006; 119 (12): 2425-2434

  Abstract

  Armadillo, the Drosophila homolog of beta-catenin, plays a crucial role in both the Wingless signal transduction pathway and cadherin-mediated cell-cell adhesion, raising the possibility that Wg signaling affects cell adhesion. Here, we use a tissue culture system that allows conditional activation of the Wingless signaling pathway and modulation of E-cadherin expression levels. We show that activation of the Wingless signaling pathway leads to the accumulation of hypophosphorylated Armadillo in the cytoplasm and in cellular processes, and to a concomitant reduction of membrane-associated Armadillo. Activation of the Wingless pathway causes a loss of E-cadherin from the cell surface, reduced cell adhesion and increased spreading of the cells on the substratum. After the initial loss of E-cadherin from the cell surface, E-cadherin gene expression is increased by Wingless. We suggest that Wingless signaling causes changes in Armadillo levels and subcellular localization that result in a transient reduction of cadherin-mediated cell adhesion, thus facilitating cell shape changes, division and movement of cells in epithelial tissues.

  View details for DOI 10.1242/jcs.02973

  View details for Web of Science ID 000238413500004

  View details for PubMedID 16720643

  View details for PubMedCentralID PMC3372910

• Differential inhibition of Wnt-3a by Sfrp-1, Sfrp-2, and Sfrp-3 DEVELOPMENTAL DYNAMICS Galli, L. M., Barnes, T., Cheng, T., Acosta, L., Anglade, A., Willert, K., Nusse, R., Burrus, L. W. 2006; 235 (3): 681-690

  Abstract

  Secreted frizzled related proteins (Sfrps) are extracellular attenuators of Wnt signaling that play important roles in both embryogenesis and oncogenesis. Although Sfrps are generally thought to bind and sequester Wnts away from active receptor complexes, very little is known about the specificity of Sfrp family members for various Wnts. In the developing chick neural tube, sfrp-1, 2, and 3 transcripts are expressed in and adjacent to the dorsal neural tube, where Wnt-1 and Wnt-3a are expressed. To better define the possible roles of Sfrp-1, 2, and 3 in the neural tube, we first tested the ability of purified Sfrps to inhibit Wnt-3a-induced accumulation of beta-catenin in L cells. We find that both Sfrp-1 and Sfrp-2 can inhibit Wnt-3a activity while Sfrp-3 cannot. To determine where Sfrp-1 and Sfrp-2 impinge on the Wnt signaling pathway, we tested the ability of these Sfrps to inhibit Wnt signaling induced by the addition of LiCl, an inhibitor of GSK-3. Sfrp-1 and Sfrp-2 are unable to inhibit the accumulation of beta-catenin in LiCl-treated cells, suggesting that the ability of Sfrps to inhibit the accumulation of beta-catenin is GSK-3 dependent. We have further shown that Sfrp-2 inhibits the ability of ectopic Wnt-3a to stimulate proliferation in the developing chick neural tube. These results provide the framework for understanding how Sfrps function to regulate Wnt-3a activity in developing embryos and in cancer.

  View details for DOI 10.1002/dvdy.20681

  View details for Web of Science ID 000236385500011

  View details for PubMedID 16425220

• Genetic evidence that drosophila frizzled controls planar cell polarity and armadillo signaling by a common mechanism GENETICS Povelones, M., Howes, R., Fish, M., Nusse, R. 2005; 171 (4): 1643-1654

  Abstract

  The frizzled (fz) gene in Drosophila controls two distinct signaling pathways: it directs the planar cell polarization (PCP) of epithelia and it regulates cell fate decisions through Armadillo (Arm) by acting as a receptor for the Wnt protein Wingless (Wg). With the exception of dishevelled (dsh), the genes functioning in these two pathways are distinct. We have taken a genetic approach, based on a series of new and existing fz alleles, for identifying individual amino acids required for PCP or Arm signaling. For each allele, we have attempted to quantify the strength of signaling by phenotypic measurements. For PCP signaling, the defect was measured by counting the number of cells secreting multiple hairs in the wing. We then examined each allele for its ability to participate in Arm signaling by the rescue of fz mutant embryos with maternally provided fz function. For both PCP and Arm signaling we observed a broad range of phenotypes, but for every allele there is a strong correlation between its phenotypic strength in each pathway. Therefore, even though the PCP and Arm signaling pathways are genetically distinct, the set of signaling-defective fz alleles affected both pathways to a similar extent. This suggests that fz controls these two different signaling activities by a common mechanism. In addition, this screen yielded a set of missense mutations that identify amino acids specifically required for fz signaling function.

  View details for DOI 10.1534/genetics.105.045245

  View details for Web of Science ID 000234407100019

  View details for PubMedID 16085697

  View details for PubMedCentralID PMC1456092

• The role of the cysteine-rich domain of Frizzled in Wingless-Armadillo signaling EMBO JOURNAL Povelones, M., Nusse, R. 2005; 24 (19): 3493-3503

  Abstract

  The Frizzled (Fz) receptors contain seven transmembrane helices and an amino-terminal cysteine-rich domain (CRD) that is sufficient and necessary for binding of the ligands, the Wnts. Recent genetic experiments have suggested, however, that the CRD is dispensable for signaling. We engineered fz CRD mutant transgenes and tested them for Wg signaling activity. None of the mutants was functional in cell culture or could fully replace fz in vivo. We also show that replacing the CRD with a structurally distinct Wnt-binding domain, the Wnt inhibitory factor, reconstitutes a functional Wg receptor. We therefore hypothesized that the function of the CRD is to bring Wg in close proximity with the membrane portion of the receptor. We tested this model by substituting Wg itself for the CRD, a manipulation that results in a constitutively active receptor. We propose that Fz activates signaling in two steps: Fz uses its CRD to capture Wg, and once bound Wg interacts with the membrane portion of the receptor to initiate signaling.

  View details for DOI 10.1038/sj.emboj.7600817

  View details for Web of Science ID 000232551800014

  View details for PubMedID 16163385

• WntD is a feedback inhibitor of Dorsal/NF-kappa B in Drosophila development and immunity NATURE Gordon, M. D., Dionne, M. S., Schneider, D. S., Nusse, R. 2005; 437 (7059): 746-749

  Abstract

  Regulating the nuclear factor-kappaB (NF-kappaB) family of transcription factors is of critical importance to animals, with consequences of misregulation that include cancer, chronic inflammatory diseases and developmental defects. Studies in Drosophila melanogaster have proved fruitful in determining the signals used to control NF-kappaB proteins, beginning with the discovery that the Toll/NF-kappaB pathway, in addition to patterning the dorsal-ventral axis of the fly embryo, defines a major component of the innate immune response in both Drosophila and mammals. Here, we characterize the Drosophila wntD (Wnt inhibitor of Dorsal) gene. We show that WntD acts as a feedback inhibitor of the NF-kappaB homologue Dorsal during both embryonic patterning and the innate immune response to infection. wntD expression is under the control of Toll/Dorsal signalling, and increased levels of WntD block Dorsal nuclear accumulation, even in the absence of the IkappaB homologue Cactus. The WntD signal is independent of the common Wnt signalling component Armadillo (beta-catenin). By engineering a gene knockout, we show that wntD loss-of-function mutants have immune defects and exhibit increased levels of Toll/Dorsal signalling. Furthermore, the wntD mutant phenotype is suppressed by loss of zygotic dorsal. These results describe the first secreted feedback antagonist of Toll signalling, and demonstrate a novel Wnt activity in the fly.

  View details for Web of Science ID 000232157900055

  View details for PubMedID 16107793

• Developmental roles of the Mi-2/NURD-Associated protein p66 in Drosophila GENETICS Kon, C., Cadigan, K. M., da Silva, S. L., Nusse, R. 2005; 169 (4): 2087-2100

  Abstract

  The NURD and Sin3 histone deacetylase complexes are involved in transcriptional repression through global deacetylation of chromatin. Both complexes contain many different components that may control how histone deacetylase complexes are regulated and interact with other transcription factors. In a genetic screen for modifiers of wingless signaling in the Drosophila eye, we isolated mutations in the Drosophila homolog of p66, a protein previously purified as part of the Xenopus NURD/Mi-2 complex. p66 encodes a highly conserved nuclear zinc-finger protein that is required for development and we propose that the p66 protein acts as a regulatory component of the NURD complex. Animals homozygous mutant for p66 display defects during metamorphosis possibly caused by misregulation of ecdysone-regulated expression. Although heterozygosity for p66 enhances a wingless phenotype in the eye, loss-of-function clones in the wing and the eye discs do not have any detectable phenotype, possibly due to redundancy with the Sin3 complex. Overexpression of p66, on the other hand, can repress wingless-dependent phenotypes. Furthermore, p66 expression can repress multiple reporters in a cell culture assay, including a Wnt-responsive TCF reporter construct, implicating the NURD complex in repression of Wnt target genes. By co-immunoprecipitation, p66 associates with dMi-2, a known NURD complex member.

  View details for DOI 10.1534/genetics.104.034595

  View details for Web of Science ID 000229263700026

  View details for PubMedID 15695365

  View details for PubMedCentralID PMC1449583

• Gene expression profile of the WNT family in cranial suture development 11th International Congress of the International-Society-of-Craniofacial-Surgery Malladi, P., Xu, Y., Nusse, R., Longaker, M. MEDIMOND S R L. 2005: 263–263

  View details for Web of Science ID 000243381600065

• LRP5 and Wnt signaling: A union made for bone JOURNAL OF BONE AND MINERAL RESEARCH Johnson, M. L., Harnish, K., Nusse, R., Van Hul, W. 2004; 19 (11): 1749-1757

  View details for DOI 10.1359/JBMR.040816

  View details for Web of Science ID 000224696600001

  View details for PubMedID 15476573

• A proliferative role for Wnt-3a in chick somites DEVELOPMENTAL BIOLOGY Galli, L. M., Willert, K., Nusse, R., Yablonka-Reuveni, Z., Nohno, T., Denetclaw, W., Burrus, L. W. 2004; 269 (2): 489-504

  Abstract

  The proper patterning of somites to give rise to sclerotome, dermomyotome, and myotome involves the coordination of many different cellular processes, including lineage specification, cell proliferation, cell death, and differentiation, by intercellular signals. One such family of secreted signaling proteins known to influence somite patterning is the Wnt family. Although the participation of Wnt-3a in the patterning of dorsal structures in the somite is well established, no clear consensus has emerged about the cellular processes that are governed by Wnt-3a in the somite. The recent demonstration that Wnt-3a has a proliferative role in the neural tube [Development 129 (2002) 2087] suggested that Wnt-3a might also act to regulate proliferation in somites. To test this hypothesis, we first analyzed the effects of Wnt-3a on segmental plate and somite explants (from Hamburger and Hamilton stage 10 chick embryos) grown in culture. These studies indicate that Wnt-3a is capable of maintaining and/or inducing expression of both Pax-3 and Pax-7, transcription factors that have been implicated in proliferation. To directly test for a role in proliferation, explants were immunostained with antibodies against phospho-histone H3. Explants treated with Wnt-3a show an increase in the percentage of cells expressing phospho-histone H3 as compared to controls. To test the proliferative effect of Wnt-3a in vivo, we ectopically expressed Wnt-3a in chick neural tubes via electroporation. Consistent with previous studies, ectopic expression of Wnt-3a in vivo results in a mediolateral expansion of the dermomyotome and myotome. We now show that proliferation of dorsal/dermomyotomal cells is significantly enhanced by ectopic Wnt-3a. Collectively, our explant and in vivo studies indicate that an increase in proliferation plays an important role in the expansion of the dermomyotome and myotome in Wnt-3a-treated embryos. Furthermore, our results demonstrate that small changes in proliferation can dramatically influence patterning and morphogenesis.

  View details for DOI 10.1016/j.ydbio.2004.01.041

  View details for Web of Science ID 000221158300013

  View details for PubMedID 15110715

• Construction of transgenic Drosophila by using the site-specific integrase from phage phi C31 GENETICS Groth, A. C., Fish, M., Nusse, R., Calos, M. P. 2004; 166 (4): 1775-1782

  Abstract

  The phiC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites. Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos. Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47%. In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells. Two lines of Drosophila were created by integrating an attP site into the genome with a P element. phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring. A total of 100% of these progeny carried a precise integration event at the genomic attP site. These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the phiC31 integrase system and will likely benefit research in Drosophila and other insects.

  View details for Web of Science ID 000221377700016

  View details for PubMedID 15126397

• Convergence of Wnt, beta-catenin, and cadherin pathways SCIENCE Nelson, W. J., Nusse, R. 2004; 303 (5663): 1483-1487

  Abstract

  The specification and proper arrangements of new cell types during tissue differentiation require the coordinated regulation of gene expression and precise interactions between neighboring cells. Of the many growth factors involved in these events, Wnts are particularly interesting regulators, because a key component of their signaling pathway, beta-catenin, also functions as a component of the cadherin complex, which controls cell-cell adhesion and influences cell migration. Here, we assemble evidence of possible interrelations between Wnt and other growth factor signaling, beta-catenin functions, and cadherin-mediated adhesion.

  View details for Web of Science ID 000220000100031

  View details for PubMedID 15001769

• Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4 SCIENCE Chen, W., Ten Berge, D., Brown, J., Ahn, S., Hu, L. A., Miller, W. E., Caron, M. G., Barak, L. S., Nusse, R., Lefkowitz, R. J. 2003; 301 (5638): 1391-1394

  Abstract

  Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membrane.Frizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients. Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells was dependent on added Wnt5A protein and was accomplished by the multifunctional adaptor protein beta-arrestin 2 (betaarr2), which was recruited to Fz4 by binding to phosphorylated Dvl2. These findings provide a previously unrecognized mechanism for receptor recruitment of beta-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling.

  View details for Web of Science ID 000185116400045

  View details for PubMedID 12958364

• A role for Wnt signalling in self-renewal of haematopoietic stem cells NATURE Reya, T., Duncan, A. W., Ailles, L., Domen, J., Scherer, D. C., Willert, K., Hintz, L., Nusse, R., Weissman, I. L. 2003; 423 (6938): 409-414

  Abstract

  Haematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood; however, the signals that regulate HSC self-renewal remain unclear. Here we show that the Wnt signalling pathway has an important role in this process. Overexpression of activated beta-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore, HSCs in their normal microenvironment activate a LEF-1/TCF reporter, which indicates that HCSs respond to Wnt signalling in vivo. To demonstrate the physiological significance of this pathway for HSC proliferation we show that the ectopic expression of axin or a frizzled ligand-binding domain, inhibitors of the Wnt signalling pathway, leads to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore, activation of Wnt signalling in HSCs induces increased expression of HoxB4 and Notch1, genes previously implicated in self-renewal of HSCs. We conclude that the Wnt signalling pathway is critical for normal HSC homeostasis in vitro and in vivo, and provide insight into a potential molecular hierarchy of regulation of HSC development.

  View details for DOI 10.1038/nature01593

  View details for Web of Science ID 000183012000034

  View details for PubMedID 12717450

• Ligand receptor interactions in the Wnt signaling pathway in Drosophila JOURNAL OF BIOLOGICAL CHEMISTRY Wu, C. H., Nusse, R. 2002; 277 (44): 41762-41769

  Abstract

  Secreted Wnt proteins have numerous signaling functions during development, mediated by Frizzled molecules that act as Wnt receptors on the cell surface. In the genome of Drosophila, seven Wnt genes (including wingless; wg), and five frizzled genes have been identified. Relatively little is known about signaling and binding specificities of different Wnt and Frizzled proteins. We have developed an assay to determine the strength of binding between membrane-tethered Wnts and ligand binding domains of the Frizzled receptors. We found a wide spectrum of binding affinities, reflecting known genetic interactions. Most Wnt proteins can bind to multiple Frizzleds and vice versa, suggesting redundancy in vivo. In an extension of these experiments, we tested whether two different subdomains of the Wg protein would by themselves bind to Frizzled and generate a biological response. Whereas these two separate domains are secreted from cells, suggesting that they form independently folded parts of the protein, they were only able to evoke a response when co-transfected, indicating that both are required for function. In addition to the Frizzleds, members of the LRP family (represented by the arrow gene in Drosophila) are also necessary for Wnt signal transduction and have been postulated to act as co-receptors. We have therefore examined whether a soluble form of the Arrow molecule can bind to Wingless and Frizzled, but no interactions were detected.

  View details for DOI 10.1074/jbc.M207850200

  View details for Web of Science ID 000178985300060

  View details for PubMedID 12205098

• A transcriptional response to Wnt protein in human embryonic carcinoma cells. BMC developmental biology Willert, J., Epping, M., Pollack, J. R., Brown, P. O., Nusse, R. 2002; 2: 8-?

  Abstract

  Wnt signaling is implicated in many developmental decisions, including stem cell control, as well as in cancer. There are relatively few target genes known of the Wnt pathway.We have identified target genes of Wnt signaling using microarray technology and human embryonic carcinoma cells stimulated with active Wnt protein. The ~50 genes upregulated early after Wnt addition include the previously known Wnt targets Cyclin D1, MYC, ID2 and betaTRCP. The newly identified targets, which include MSX1, MSX2, Nucleophosmin, Follistatin, TLE/Groucho, Ubc4/5E2, CBP/P300, Frizzled and REST/NRSF, have important implications for understanding the roles of Wnts in development and cancer. The protein synthesis inhibitor cycloheximide blocks induction by Wnt, consistent with a requirement for newly synthesized beta-catenin protein prior to target gene activation. The promoters of nearly all the target genes we identified have putative TCF binding sites, and we show that the TCF binding site is required for induction of Follistatin. Several of the target genes have a cooperative response to a combination of Wnt and BMP.Wnt signaling activates genes that promote stem cell fate and inhibit cellular differentiation and regulates a remarkable number of genes involved in its own signaling system.

  View details for PubMedID 12095419

• Wingless blocks bristle formation and morphogenetic furrow progression in the eye through repression of Daughterless DEVELOPMENT Cadigan, K. M., Jou, A. D., Nusse, R. 2002; 129 (14): 3393-3402

  Abstract

  In the developing eye, wingless activity represses proneural gene expression (and thus interommatidial bristle formation) and positions the morphogenetic furrow by blocking its initiation in the dorsal and ventral regions of the presumptive eye. We provide evidence that wingless mediates both effects, at least in part, through repression of the basic helix-loop-helix protein Daughterless. daughterless is required for high proneural gene expression and furrow progression. Ectopic expression of wingless blocks Daughterless expression in the proneural clusters. This repression, and that of furrow progression, can be mimicked by an activated form of armadillo and blocked by a dominant negative form of pangolin/TCF. Placing daughterless under the control of a heterologous promoter blocks the ability of ectopic wingless to inhibit bristle formation and furrow progression. hedgehog and decapentapleigic could not rescue the wingless furrow progression block, indicating that wingless acts downstream of these genes. In contrast, Atonal and Scute, which are thought to heterodimerize with Daughterless to promote furrow progression and bristle formation, respectively, can block ectopic wingless action. These results are summarized in a model where daughterless is a major, but probably not the only, target of wingless action in the eye.

  View details for Web of Science ID 000177325500010

  View details for PubMedID 12091309

• A mutational analysis of dishevelled in Drosophila defines novel domains in the dishevelled protein as well as novel suppressing alleles of axin GENETICS Penton, A., Wodarz, A., Nusse, R. 2002; 161 (2): 747-762

  Abstract

  Drosophila dishevelled (dsh) functions in two pathways: it is necessary to transduce Wingless (Wg) signaling and it is required in planar cell polarity. To learn more about how Dsh can discriminate between these functions, we performed genetic screens to isolate additional dsh alleles and we examined the potential role of protein phosphorylation by site-directed mutagenesis. We identified two alleles with point mutations in the Dsh DEP domain that specifically disrupt planar polarity signaling. When positioned in the structure of the DEP domain, these mutations are located close to each other and to a previously identified planar polarity mutation. In addition to the requirement for the DEP domain, we found that a cluster of potential phosphorylation sites in a binding domain for the protein kinase PAR-1 is also essential for planar polarity signaling. To identify regions of dsh that are necessary for Wg signaling, we screened for mutations that modified a GMR-GAL4;UAS-dsh overexpression phenotype in the eye. We recovered many alleles of the transgene containing missense mutations, including mutations in the DIX domain and in the DEP domain, the latter group mapping separately from the planar polarity mutations. In addition, several transgenes had mutations within a domain containing a consensus sequence for an SH3-binding protein. We also recovered second-site-suppressing mutations in axin, mapping at a region that may specifically interact with overexpressed Dsh.

  View details for Web of Science ID 000176374600024

  View details for PubMedID 12072470

• Ablation of insulin-producing neurons in flies: Growth and diabetic phenotypes SCIENCE Rulifson, E. J., Kim, S. K., Nusse, R. 2002; 296 (5570): 1118-1120

  Abstract

  In the fruit fly Drosophila, four insulin genes are coexpressed in small clusters of cells [insulin-producing cells (IPCs)] in the brain. Here, we show that ablation of these IPCs causes developmental delay, growth retardation, and elevated carbohydrate levels in larval hemolymph. All of the defects were reversed by ectopic expression of a Drosophila insulin transgene. On the basis of these functional data and the observation that IPCs release insulin into the circulatory system, we conclude that brain IPCs are the main systemic supply of insulin during larval growth. We propose that IPCs and pancreatic islet beta cells are functionally analogous and may have evolved from a common ancestral insulin-producing neuron. Interestingly, the phenotype of flies lacking IPCs includes certain features of diabetes mellitus.

  View details for Web of Science ID 000175565000053

  View details for PubMedID 12004130

• Direct flight muscles in Drosophila develop from cells with characteristics of founders and depend on DWnt-2 for their correct patterning DEVELOPMENTAL BIOLOGY Kozopas, K. M., Nusse, R. 2002; 243 (2): 312-325

  Abstract

  The direct flight muscles (DFMs) of Drosophila allow for the fine control of wing position necessary for flight. In DWnt-2 mutant flies, certain DFMs are either missing or fail to attach to the correct epithelial sites. Using a temperature-sensitive allele, we show that DWnt-2 activity is required only during pupation for correct DFM patterning. DWnt-2 is expressed in the epithelium of the wing hinge primordium during pupation. This expression is in the vicinity of the developing DFMs, as revealed by expression of the muscle founder cell-specific gene dumbfounded in DFM precursors. The observation that a gene necessary for embryonic founder cell function is expressed in the DFM precursors suggests that these cells may have a similar founder cell role. Although the expression pattern of DWnt-2 suggests that it could influence epithelial cells to differentiate into attachment sites for muscle, the expression of stripe, a transcription factor necessary for epithelial cells to adopt an attachment cell fate, is unaltered in the mutant. Ectopic expression of DWnt-2 in the wing hinge during pupation can also create defects in muscle patterning without alterations in stripe expression. We conclude that DWnt-2 promotes the correct patterning of DFMs through a mechanism that is independent of the attachment site differentiation initiated by stripe.

  View details for DOI 10.1006/dbio.2002.0572

  View details for Web of Science ID 000174465500010

  View details for PubMedID 11884040

• Are Wnts ligands for planar polarity signaling? Logan, C. Y., Wu, C. H., Aurora, A., Cadigan, K., Nusse, R. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2001: 253–53

  View details for Web of Science ID 000169701100342

• The status of Wnt signalling regulates neural and epidermal fates in the chick embryo NATURE Wilson, S. I., Rydstrom, A., Trimborn, T., Willert, K., Nusse, R., Jessell, T. M., Edlund, T. 2001; 411 (6835): 325-330

  Abstract

  The acquisition of neural fate by embryonic ectodermal cells is a fundamental step in the formation of the vertebrate nervous system. Neural induction seems to involve signalling by fibroblast growth factors (FGFs) and attenuation of the activity of bone morphogenetic protein (BMP). But FGFs, either alone or in combination with BMP antagonists, are not sufficient to induce neural fate in prospective epidermal ectoderm of amniote embryos. These findings suggest that additional signals are involved in the specification of neural fate. Here we show that the state of Wnt signalling is a critical determinant of neural and epidermal fates in the chick embryo. Continual Wnt signalling blocks the response of epiblast cells to FGF signals, permitting the expression and signalling of BMP to direct an epidermal fate. Conversely, a lack of exposure of epiblast cells to Wnt signals permits FGFs to induce a neural fate.

  View details for Web of Science ID 000168710000052

  View details for PubMedID 11357137

• naked cuticle targets dishevelled to antagonize Wnt signal transduction GENES & DEVELOPMENT Rousset, R., Mack, J. A., Wharton, K. A., Axelrod, J. D., Cadigan, K. M., Fish, M. P., Nusse, R., Scott, M. P. 2001; 15 (6): 658-671

  Abstract

  In Drosophila embryos the protein Naked cuticle (Nkd) limits the effects of the Wnt signal Wingless (Wg) during early segmentation. nkd loss of function results in segment polarity defects and embryonic death, but how nkd affects Wnt signaling is unknown. Using ectopic expression, we find that Nkd affects, in a cell-autonomous manner, a transduction step between the Wnt signaling components Dishevelled (Dsh) and Zeste-white 3 kinase (Zw3). Zw3 is essential for repressing Wg target-gene transcription in the absence of a Wg signal, and the role of Wg is to relieve this inhibition. Our double-mutant analysis shows that, in contrast to Zw3, Nkd acts when the Wg pathway is active to restrain signal transduction. Yeast two hybrid and in vitro experiments indicate that Nkd directly binds to the basic-PDZ region of Dsh. Specially timed Nkd overexpression is capable of abolishing Dsh function in a distinct signaling pathway that controls planar-cell polarity. Our results suggest that Nkd acts directly through Dsh to limit Wg activity and thus determines how efficiently Wnt signals stabilize Armadillo (Arm)/beta-catenin and activate downstream genes.

  View details for Web of Science ID 000167821300003

  View details for PubMedID 11274052

• Pathway specificity by the bifunctional receptor frizzled is determined by affinity for wingless MOLECULAR CELL Rulifson, E. J., Wu, C. H., Nusse, R. 2000; 6 (1): 117-126

  Abstract

  The Frizzled (Fz) protein in Drosophila is a bifunctional receptor that acts through a GTPase pathway in planar polarity signaling and as a receptor for Wingless (Wg) using the canonical Wnt pathway. We found that the ligand-binding domain (CRD) of Fz has an approximately 10-fold lower affinity for Wg than the CRD of DFz2, a Wg receptor without polarity activity. When the Fz CRD is replaced by the high-affinity CRD of DFz2, the resulting chimeric protein gains Wg signaling activity, yet also retains polarity signaling activity. In contrast, the reciprocal exchange of the Fz CRD onto DFz2 is not sufficient to confer polarity activity to DFz2. This suggests that Fz has an intrinsic capacity for polarity signaling and that high-affinity interaction with Wg couples it to the Wnt pathway.

  View details for Web of Science ID 000088799400012

  View details for PubMedID 10949033

• Wnt signaling regulates B lymphocyte proliferation through a LEF-1 dependent mechanism IMMUNITY Reya, T., O'Riordan, M., Okamura, R., Devaney, E., Willert, K., Nusse, R., Grosschedl, R. 2000; 13 (1): 15-24

  Abstract

  Lymphocyte enhancer factor-1 (LEF-1) is a member of the LEF-1/TCF family of transcription factors, which have been implicated in Wnt signaling and tumorigenesis. LEF-1 was originally identified in pre-B and T cells, but its function in B lymphocyte development remains unknown. Here we report that LEF-1-deficient mice exhibit defects in pro-B cell proliferation and survival in vitro and in vivo. We further show that Lef1-/- pro-B cells display elevated levels of fas and c-myc transcription, providing a potential mechanism for their increased sensitivity to apoptosis. Finally, we establish a link between Wnt signaling and normal B cell development by demonstrating that Wnt proteins are mitogenic for pro-B cells and that this effect is mediated by LEF-1.

  View details for Web of Science ID 000088463600003

  View details for PubMedID 10933391

• Interactions between wingless and frizzled molecules in Drosophila 29th Ernst-Schering-Research-Foundation Workshop Nusse, R., Rulifson, E., Fish, M., Harryman-Samos, C., Brink, M., Wu, C. H., Cadigan, K. SPRINGER-VERLAG BERLIN. 2000: 1–11

  View details for Web of Science ID 000086852400001

  View details for PubMedID 10943301

• A Drosophila Axin homolog, Daxin, inhibits Wnt signaling DEVELOPMENT Willert, K., Logan, C. Y., Arora, A., Fish, M., Nusse, R. 1999; 126 (18): 4165-4173

  Abstract

  The vertebrate Axin protein, the product of the mouse fused gene, binds to beta-catenin to inhibit Wnt signaling. We have identified a homolog of Axin in Drosophila, Daxin. Using double-stranded RNA interference, we generated loss-of-function phenotypes that are similar to overexpression of the Drosophila Wnt gene wingless (wg). Overexpression of Daxin produces phenotypes similar to loss of wg. In addition, we show that Daxin overexpression can modify phenotypes elicited by wg and another Drosophila Wnt gene, DWnt-2. Using immunoprecipitation of endogenous Daxin protein from embryos we show that Daxin interacts with Armadillo and Zeste-white 3. The loss-of-function and overexpression phenotypes show that Daxin, like its mammalian counterpart, acts as a negative regulator of wg/Wnt signaling.

  View details for Web of Science ID 000082965700019

  View details for PubMedID 10457025

• Frizzled and DFrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development DEVELOPMENT Bhanot, P., Fish, M., Jemison, J. A., Nusse, R., Nathans, J., Cadigan, K. M. 1999; 126 (18): 4175-4186

  Abstract

  In cell culture assays, Frizzled and Dfrizzled2, two members of the Frizzled family of integral membrane proteins, are able to bind Wingless and transduce the Wingless signal. To address the role of these proteins in the intact organism and to explore the question of specificity of ligand-receptor interactions in vivo, we have conducted a genetic analysis of frizzled and Dfrizzled2 in the embryo. These experiments utilize a small gamma-ray-induced deficiency that uncovers Dfrizzled2. Mutants lacking maternal frizzled and zygotic frizzled and Dfrizzled2 exhibit defects in the embryonic epidermis, CNS, heart and midgut that are indistinguishable from those observed in wingless mutants. Epidermal patterning defects in the frizzled, Dfrizzled2 double-mutant embryos can be rescued by ectopic expression of either gene. In frizzled, Dfrizzled2 mutant embryos, ectopic production of Wingless does not detectably alter the epidermal patterning defect, but ectopic production of an activated form of Armadillo produces a naked cuticle phenotype indistinguishable from that produced by ectopic production of activated Armadillo in wild-type embryos. These experiments indicate that frizzled and Dfrizzled2 function downstream of wingless and upstream of armadillo, consistent with their proposed roles as Wingless receptors. The lack of an effect on epidermal patterning of ectopic Wingless in a frizzled, Dfrizzled2 double mutant argues against the existence of additional Wingless receptors in the embryo or a model in which Frizzled and Dfrizzled2 act simply to present the ligand to its bona fide receptor. These data lead to the conclusion that Frizzled and Dfrizzled2 function as redundant Wingless receptors in multiple embryonic tissues and that this role is accurately reflected in tissue culture experiments. The redundancy of Frizzled and Dfrizzled2 explains why Wingless receptors were not identified in earlier genetic screens for mutants defective in embryonic patterning.

  View details for Web of Science ID 000082965700020

  View details for PubMedID 10457026

• Wnt-induced dephosphorylation of Axin releases beta-catenin from the Axin complex GENES & DEVELOPMENT Willert, K., Shibamoto, S., Nusse, R. 1999; 13 (14): 1768-1773

  Abstract

  The stabilization of beta-catenin is a key regulatory step during cell fate changes and transformations to tumor cells. Several interacting proteins, including Axin, APC, and the protein kinase GSK-3beta are implicated in regulating beta-catenin phosphorylation and its subsequent degradation. Wnt signaling stabilizes beta-catenin, but it was not clear whether and how Wnt signaling regulates the beta-catenin complex. Here we show that Axin is dephosphorylated in response to Wnt signaling. The dephosphorylated Axin binds beta-catenin less efficiently than the phosphorylated form. Thus, Wnt signaling lowers Axin's affinity for beta-catenin, thereby disengaging beta-catenin from the degradation machinery.

  View details for Web of Science ID 000081711100002

  View details for PubMedID 10421629

• A new secreted protein that binds to Wnt proteins and inhibits their activities NATURE Hsieh, J. C., Kodjabachian, L., Rebbert, M. L., Rattner, A., Smallwood, P. M., Samos, C. H., Nusse, R., Dawid, I. B., Nathans, J. 1999; 398 (6726): 431-436

  Abstract

  The Wnt proteins constitute a large family of extracellular signalling molecules that are found throughout the animal kingdom and are important for a wide variety of normal and pathological developmental processes. Here we describe Wnt-inhibitory factor-1 (WIF-1), a secreted protein that binds to Wnt proteins and inhibits their activities. WIF-1 is present in fish, amphibia and mammals, and is expressed during Xenopus and zebrafish development in a complex pattern that includes paraxial presomitic mesoderm, notochord, branchial arches and neural crest derivatives. We use Xenopus embryos to show that WIF-1 overexpression affects somitogenesis (the generation of trunk mesoderm segments), in agreement with its normal expression in paraxial mesoderm. In vitro, WIF-1 binds to Drosophila Wingless and Xenopus Wnt8 produced by Drosophila S2 cells. Together with earlier results obtained with the secreted Frizzled-related proteins, our results indicate that Wnt proteins interact with structurally diverse extracellular inhibitors, presumably to fine-tune the spatial and temporal patterns of Wnt activity.

  View details for Web of Science ID 000079508200054

  View details for PubMedID 10201374

• WNT signaling in the control of hair growth and structure DEVELOPMENTAL BIOLOGY Millar, S. E., Willert, K., Salinas, P. C., Roelink, H., Nusse, R., Sussman, D. J., Barsh, G. S. 1999; 207 (1): 133-149

  Abstract

  Characterization of the molecular pathways controlling differentiation and proliferation in mammalian hair follicles is central to our understanding of the regulation of normal hair growth, the basis of hereditary hair loss diseases, and the origin of follicle-based tumors. We demonstrate that the proto-oncogene Wnt3, which encodes a secreted paracrine signaling molecule, is expressed in developing and mature hair follicles and that its overexpression in transgenic mouse skin causes a short-hair phenotype due to altered differentiation of hair shaft precursor cells, and cyclical balding resulting from hair shaft structural defects and associated with an abnormal profile of protein expression in the hair shaft. A putative effector molecule for WNT3 signaling, the cytoplasmic protein Dishevelled 2 (DVL2), is normally present at high levels in a subset of cells in the outer root sheath and in precursor cells of the hair shaft cortex and cuticle which lie immediately adjacent to Wnt3-expressing cells. Overexpression of Dvl2 in the outer root sheath mimics the short-hair phenotype produced by overexpression of Wnt3, supporting the hypothesis that Wnt3 and Dvl2 have the potential to act in the same pathway in the regulation of hair growth. These experiments demonstrate a previously unrecognized role for WNT signaling in the control of hair growth and structure, as well as presenting the first example of a mammalian phenotype resulting from overexpression of a Dvl gene and providing an accessible in vivo system for analysis of mammalian WNT signaling pathways.

  View details for Web of Science ID 000079085400011

  View details for PubMedID 10049570

• WNT targets - repression and activation TRENDS IN GENETICS Nusse, R. 1999; 15 (1): 1-3

  Abstract

  Several puzzling observations made previously suggested that target genes that are activated by WNT signaling during development were actively repressed in the absence of the signal. Recent work sheds light on how this switch between repression and activation is regulated.

  View details for Web of Science ID 000079419200001

  View details for PubMedID 10087922

• The Frizzled CRD domain is conserved in diverse proteins including several receptor tyrosine kinases CURRENT BIOLOGY Xu, Y. K., Nusse, R. 1998; 8 (12): R405-R406

  View details for Web of Science ID 000074122900005

  View details for PubMedID 9637908

• Wingless repression of Drosophila frizzled 2 expression shapes the wingless morphogen gradient in the wing CELL Cadigan, K. M., Fish, M. P., Rulifson, E. J., Nusse, R. 1998; 93 (5): 767-777

  Abstract

  In Drosophila wing imaginal discs, the Wingless (Wg) protein acts as a morphogen, emanating from the dorsal/ventral (D/V) boundary of the disc to directly define cell identities along the D/V axis at short and long range. Here, we show that high levels of a Wg receptor, Drosophila frizzled 2 (Dfz2), stabilize Wg, allowing it to reach cells far from its site of synthesis. Wg signaling represses Dfz2 expression, creating a gradient of decreasing Wg stability moving toward the D/V boundary. This repression of Dfz2 is crucial for the normal shape of Wg morphogen gradient as well as the response of cells to the Wg signal. In contrast to other ligand-receptor relationships where the receptor limits diffusion of the ligand, Dfz2 broadens the range of Wg action by protecting it from degradation.

  View details for Web of Science ID 000073956700013

  View details for PubMedID 9630221

• DWnt-2, a Drosophila Wnt gene required for the development of the male reproductive tract, specifies a sexually dimorphic cell fate GENES & DEVELOPMENT Kozopas, K. M., Samos, C. H., Nusse, R. 1998; 12 (8): 1155-1165

  Abstract

  The sexually dimorphic characteristics of the reproductive tract in Drosophila require that cells of the gonad and the genital disc be assigned sex-specific fates. We report here that DWnt-2, a secreted glycoprotein related to wingless, is a signal required for cell fate determination and morphogenesis in the developing male reproductive tract. Testes from DWnt-2 null mutant flies lack the male-specific pigment cells of the reproductive tract sheath and the muscle precursors of the sheath fail to migrate normally. However, other cell types of the testis are unaffected. DWnt-2 is expressed in somatic cells of the gonad throughout development, implicating it as a signal that can influence pigment cell fate directly. Indeed, the ectopic expression of DWnt-2 in females results in the appearance of male-specific pigment cells in otherwise morphologically normal ovaries. Thus, the presence of pigment cells is a sexually dimorphic trait that is controlled by DWnt-2 expression. DWnt-2 is also expressed in regions of the male genital disc and gonad, which we have identified as sites of contact with muscle precursor cells, suggesting that secreted DWnt-2 protein is a signal for the migration or attachment of these cells.

  View details for Web of Science ID 000073299000009

  View details for PubMedID 9553045

• Expression of wingless in the Drosophila embryo: a conserved cis-acting element lacking conserved Ci-binding sites is required for patched-mediated repression DEVELOPMENT Lessing, D., Nusse, R. 1998; 125 (8): 1469-1476

  Abstract

  Patterning of the Drosophila embryo depends on the accurate expression of wingless (wg), which encodes a secreted signal required for segmentation and many other processes. Early expression of wg is regulated by the nuclear proteins of the gap and pair-rule gene classes but, after gastrulation, wg transcription is also dependent on cell-cell communication. Signaling to the Wg-producing cells is mediated by the secreted protein, Hedgehog (Hh), and by Cubitus interruptus (Ci), a transcriptional effector of the Hh signal transduction pathway. The transmembrane protein Patched (Ptc) acts as a negative regulator of wg expression; ptc- embryos have ectopic wg expression. According to the current models, Ptc is a receptor for Hh. The default activity of Ptc is to inhibit Ci function; when Ptc binds Hh, this inhibition is released and Ci can control wg transcription. We have investigated cis-acting sequences that regulate wg during the time that wg expression depends on Hh signaling. We show that approximately 4.5 kb immediately upstream of the wg transcription unit can direct expression of the reporter gene lacZ in domains similar to the normal wg pattern in the embryonic ectoderm. Expression of this reporter construct expands in ptc mutants and responds to hh activity. Within this 4.5 kb, a 150 bp element, highly conserved between D. melanogaster and Drosophila virilis, is required to spatially restrict wg transcription. Activity of this element depends on ptc, but it contains no consensus Ci-binding sites. The discovery of an element that is likely to bind a transcriptional repressor was unexpected, since the prevailing model suggests that wg expression is principally controlled by Hh signaling acting through the Ci activator. We show that wg regulatory DNA can drive lacZ in a proper wg-like pattern without any conserved Ci-binding sites and suggest that Ci can not be the sole endpoint of the Hh pathway.

  View details for Web of Science ID 000073772800011

  View details for PubMedID 9502727

• beta-catenin: a key mediator of Wnt signaling CURRENT OPINION IN GENETICS & DEVELOPMENT Willert, K., Nusse, R. 1998; 8 (1): 95-102

  Abstract

  Beta-catenin is a pivotal player in the signaling pathway initiated by Wnt proteins, mediators of several developmental processes. beta-catenin activity is controlled by a large number of binding partners that affect the stability and the localization of beta-catenin and is thereby able to participate in such varying processes as gene expression and cell adhesion. Activating mutations in beta-catenin and in components regulating its stability can contribute to the formation of certain tumors.

  View details for Web of Science ID 000072681300014

  View details for PubMedID 9529612

• Mechanisms of Wnt signaling in development ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY Wodarz, A., Nusse, R. 1998; 14: 59-88

  Abstract

  Wnt genes encode a large family of secreted, cysteine-rich proteins that play key roles as intercellular signaling molecules in development. Genetic studies in Drosophila and Caenorhabditis elegans, ectopic gene expression in Xenopus, and gene knockouts in the mouse have demonstrated the involvement of Wnts in processes as diverse as segmentation, CNS patterning, and control of asymmetric cell divisions. The transduction of Wnt signals between cells proceeds in a complex series of events including post-translational modification and secretion of Wnts, binding to transmembrane receptors, activation of cytoplasmic effectors, and, finally, transcriptional regulation of target genes. Over the past two years our understanding of Wnt signaling has been substantially improved by the identification of Frizzled proteins as cell surface receptors for Wnts and by the finding that beta-catenin, a component downstream of the receptor, can translocate to the nucleus and function as a transcriptional activator. Here we review recent data that have started to unravel the mechanisms of Wnt signaling.

  View details for Web of Science ID 000077471400004

  View details for PubMedID 9891778

• Wnt signaling: a common theme in animal development GENES & DEVELOPMENT Cadigan, K. M., Nusse, R. 1997; 11 (24): 3286-3305

  View details for Web of Science ID 000071209500002

  View details for PubMedID 9407023

• Casein kinase 2 associates with and phosphorylates dishevelled EMBO JOURNAL Willert, K., Brink, M., Wodarz, A., Varmus, H., Nusse, R. 1997; 16 (11): 3089-3096

  Abstract

  The dishevelled (dsh) gene of Drosophila melanogaster encodes a phosphoprotein whose phosphorylation state is elevated by Wingless stimulation, suggesting that the phosphorylation of Dsh and the kinase(s) responsible for this phosphorylation are integral parts of the Wg signaling pathway. We found that immunoprecipitated Dsh protein from embryos and from cells in tissue culture is associated with a kinase activity that phosphorylates Dsh in vitro. Purification and peptide sequencing of a 38 kDa protein co-purifying with this kinase activity showed it to be identical to Drosophila Casein Kinase 2 (CK2). Tryptic phosphopeptide mapping indicates that identical peptides are phosphorylated by CK2 in vitro and in vivo, suggesting that CK2 is at least one of the kinases that phosphorylates Dsh. Overexpression of Dfz2, a Wingless receptor, also stimulated phosphorylation of Dsh, Dsh-associated kinase activity, and association of CK2 with Dsh, thus suggesting a role for CK2 in the transduction of the Wg signal.

  View details for Web of Science ID A1997XE74800012

  View details for PubMedID 9214626

• A versatile transcriptional effector of wingless signaling CELL Nusse, R. 1997; 89 (3): 321-323

  View details for Web of Science ID A1997WX89900001

  View details for PubMedID 9150130

• Hedgehog signaling regulates transcription through cubitus interruptus, a sequence-specific DNA binding protein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA VONOHLEN, T., Lessing, D., Nusse, R., Hooper, J. E. 1997; 94 (6): 2404-2409

  Abstract

  Hedgehog (Hh) is a member of a family of secreted proteins that direct patterning at multiple stages in both Drosophila and vertebrate development. During Drosophila embryogenesis, Hh protein is secreted by the cells of the posterior compartment of each segment. hh activates transcription of wingless (wg), gooseberry (gsb), and patched (ptc) in the cells immediately adjacent to Hh-secreting cells. Hh signaling is thought to involve the segment polarity gene cubitus interruptus (ci). ci encodes a zinc finger protein of the Gli family of sequence-specific DNA binding proteins. ci mRNA is expressed in all non-Hh expressing cells. Here we demonstrate ci activity is both necessary and sufficient to drive expression of Hh-responsive genes in the Drosophila embryos. We show that Ci is a sequence-specific DNA binding protein that drives transcription from the wg promoter in transiently transfected cells. We demonstrate that Ci binding sites in the wg promoter are necessary for this transcriptional activation. These data taken together provide strong evidence that Ci is a transcriptional effector of Hh signaling.

  View details for Web of Science ID A1997WP33400058

  View details for PubMedID 9122207

  View details for PubMedCentralID PMC20100

• A novel human homologue of the Drosophila frizzled wnt receptor gene binds wingless protein and is in the Williams syndrome deletion at 7q11.23 HUMAN MOLECULAR GENETICS Wang, Y. K., Samos, C. H., Peoples, R., PEREZJURADO, L. A., Nusse, R., FRANCKE, U. 1997; 6 (3): 465-472

  Abstract

  Williams syndrome (WS) is a developmental disorder with a characteristic personality and cognitive profile that is associated, in most cases, with a 2 Mb deletion of part of chromosome band 7q11.23. By applying CpG island cloning methods to cosmids from the deletion region, we have identified a new gene, called FZD3. Dosage blotting of DNA from 11 WS probands confirmed that it is located within the commonly deleted region. Sequence comparisons revealed that FZD3, encoding a 591 amino acid protein, is a novel member of a seven transmembrane domain receptor family that are mammalian homologs of the Drosophila tissue polarity gene frizzled. FZD3 is expressed predominantly in brain, testis, eye, skeletal muscle and kidney. Recently, frizzled has been identified as the receptor for the wingless (wg) protein in Drosophila. We show that Drosophila as well as human cells, when transfected with FZD3 expression constructs, bind Wg protein. In mouse, the wg homologous Wnt1 gene is involved in early development of a large domain of the central nervous system encompassing much of the midbrain and rostral metencephalon. The potential function of FZD3 in transmitting a Wnt protein signal in the human brain and other tissues suggests that heterozygous deletion of the FZD3 gene could contribute to the WS phenotype.

  View details for Web of Science ID A1997WN31600016

  View details for PubMedID 9147651

• Wnt Meeting 1996 BIOCHIMICA ET BIOPHYSICA ACTA-REVIEWS ON CANCER Cadigan, K. M., Nusse, R. 1997; 1332 (1): R1-R5

  View details for Web of Science ID A1997WK29700007

  View details for PubMedID 9061012

• Cell culture and whole animal approaches to understanding signalling by Wnt proteins in Drosophila Cold Spring Harbor Symposium on Quantitative Biology - Pattern Formation During Development Nusse, R., Samos, C. H., Brink, M., Willert, K., Cadigan, K. M., Wodarz, A., Fish, M., Rulifson, E. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 1997: 185–190

  View details for Web of Science ID 000073570200023

  View details for PubMedID 9598351

• wingless signaling in the Drosophila eye and embryonic epidermis DEVELOPMENT Cadigan, K. M., Nusse, R. 1996; 122 (9): 2801-2812

  Abstract

  After the onset of pupation, sensory organ precursors, the progenitors of the interommatidial bristles, are selected in the developing Drosophila eye. We have found that wingless, when expressed ectopically in the eye via the sevenless promoter, blocks this process. Transgenic eyes have reduced expression of acheate, suggesting that wingless acts at the level of the proneural genes to block bristle development. This is in contrast to the wing, where wingless positively regulates acheate to promote bristle formation. The sevenless promoter is not active in the acheate-positive cells, indicating that the wingless is acting in a paracrine manner. Clonal analysis revealed a requirement for the genes porcupine, dishevelled and armadillo in mediating the wingless effect. Overexpression of zeste white-3 partially blocks the ability of wingless to inhibit bristle formation, consistent with the notion that wingless acts in opposition to zeste white-3. Thus the wingless signaling pathway in the eye appears to be very similar to that described in the embryo and wing. The Notch gene product has also been suggested to play a role in wingless signaling (J. P. Couso and A. M. Martinez Arias (1994) Cell 79, 259-72). Because Notch has many functions during eye development, including its role in inhibiting bristle formation through the neurogenic pathway, it is difficult to assess the relationship of Notch to wingless in the eye. However, we present evidence that wingless signaling still occurs normally in the complete absence of Notch protein in the embryonic epidermis. Thus, in the simplest model for wingless signalling, a direct role for Notch is unlikely.

  View details for Web of Science ID A1996VJ07500023

  View details for PubMedID 8787754

• A new member of the frizzled family from Drosophila functions as a Wingless receptor NATURE Bhanot, P., Brink, M., Samos, C. H., Hsieh, J. C., Wang, Y. S., Macke, J. P., Andrew, D., Nathans, J., Nusse, R. 1996; 382 (6588): 225-230

  Abstract

  Receptors for Wingless and other signalling molecules of the Wnt gene family have yet to be identified. We show here that cultured Drosophila cells transfected with a novel member of the frizzled gene family in Drosophila, Dfz2, respond to added Wingless protein by elevating the level of the Armadillo protein. Moreover, Wingless binds to Drosophila or human cells expressing Dfz2. These data demonstrate that Dfz2 functions as a Wingless receptor, and they imply, in general, that Frizzled proteins are receptors for the Wnt signalling molecules.

  View details for Web of Science ID A1996UX79000040

  View details for PubMedID 8717036

• A new member of the frizzled family from Drosophila functions as a Wingless receptor Nature Nusse, R., Bhanot, P., Brink, M., Harryman Samos, C., Hsieh, J. C., Wang, Y. S., Macke, J. P., Andrew, D., Nathans, J. 1996; 382
• DIFFERENTIAL REQUIREMENTS FOR SEGMENT POLARITY GENES IN WINGLESS SIGNALING MECHANISMS OF DEVELOPMENT Noordermeer, J., Klingensmith, J., Nusse, R. 1995; 51 (2-3): 145-155

  Abstract

  The segment polarity genes wingless and engrailed are required throughout development of Drosophila. During early embryogenesis, these two genes are expressed in adjacent domains, in an inter-dependent way. Later, their expression is regulated by different mechanisms and becomes maintained by auto-regulation. To dissect the genetic requirements for the initial signaling between wingless and engrailed expressing cells, we have previously used a transgenic Drosophila strain that expresses wingless under the control of the heat shock promoter (HS-wg). Focusing on the later phases of wingless and engrailed regulation, we have now extended these studies, using embryos carrying various combinations of segment polarity mutations and the HS-wg transgene. We confirm some of the existing models of regulation of the expression of wingless and engrailed. In addition, we find that HS-wg embryos require engrailed for induction of ectopic endogenous wingless expression. Signaling from engrailed cells to this novel wingless expression domain is dependent on hedgehog but also on porcupine. We further demonstrate a novel requirement for hedgehog in maintenance of expression of engrailed itself.

  View details for Web of Science ID A1995RL68100001

  View details for PubMedID 7547463

• ONCOGENE ACTIVATION AND ONCOGENE COOPERATION IN MMTV-INDUCED MOUSE MAMMARY-CANCER SEMINARS IN CANCER BIOLOGY VANLEEUWEN, F., Nusse, R. 1995; 6 (3): 127-133

  Abstract

  In this short review, we will give an overview of the various genes that can be activated by insertion of proviral DNA of the mouse mammary tumor virus during the formation of mammary cancer. These genes fall within three families: Wnt genes, FGF genes and Notch-related genes. We will summarize our current understanding of the roles of these genes in tumorigenesis and in normal development, and the mechanisms of action of their gene products. Finally, we will give some examples of cooperation between these genes in various biological settings.

  View details for Web of Science ID A1995RJ82100003

  View details for PubMedID 7495980

• THE DISHEVELLED PROTEIN IS MODIFIED BY WINGLESS SIGNALING IN DROSOPHILA GENES & DEVELOPMENT Yanagawa, S. I., VANLEEUWEN, F., Wodarz, A., Klingensmith, J., Nusse, R. 1995; 9 (9): 1087-1097

  Abstract

  Wingless (Wg) is an important signaling molecule in the development of Drosophila, but little is known about its signal transduction pathway. Genetic evidence indicates that another segment polarity gene, dishevelled (dsh) is required for Wg signaling. We have recently developed a cell culture system for Wg protein activity, and using this in vitro system as well as intact Drosophila embryos, we have analyzed biochemical changes in the Dsh protein as a consequence of Wg signaling. We find that Dsh is a phosphoprotein, normally present in the cytoplasm. Wg signaling generates a hyperphosphorylated form of Dsh, which is associated with a membrane fraction. Overexpressed Dsh becomes hyperphosphorylated in the absence of extracellular Wg and increases levels of the Armadillo protein, thereby mimicking the Wg signal. A deletional analysis of Dsh identifies several conserved domains essential for activity, among which is a so-called GLGF/DHR motif. We conclude that dsh, a highly conserved gene, is not merely a permissive factor in Wg signaling but encodes a novel signal transduction molecule, which may function between the Wg receptor and more downstream signaling molecules.

  View details for Web of Science ID A1995QX37000006

  View details for PubMedID 7744250

• THE DROSOPHILA WNT PROTEIN DWNT-3 IS A SECRETED GLYCOPROTEIN LOCALIZED ON THE AXON TRACTS OF THE EMBRYONIC CNS DEVELOPMENTAL BIOLOGY Fradkin, L. G., Noordermeer, J. N., Nusse, R. 1995; 168 (1): 202-213

  Abstract

  The Wnt gene family encodes highly conserved cysteine-rich proteins which appear to act as secreted developmental signals. Both the mouse Wnt-1 gene and the Drosophila wingless (wg) gene play important roles in central nervous system (CNS) development. wg is also required earlier, in the development of the embryonic metameric body pattern. We have begun to characterize the developmental expression and role of another member of the Drosophila Wnt gene family, DWnt-3. Using antisera raised to the DWnt-3 protein, we show that the protein is secreted in vivo. The early protein expression domains include the limb and appendage primordia. Late expression domains comprise the ventral cord and supraesophageal ganglia of the CNS. Notably, DWnt-3 protein accumulates on the commissural and longitudinal axon tracts of the CNS. Ectopic expression of DWnt-3 in transgenic embryos bearing a HS-DWnt-3 construct leads to specific disruption of the commissural axon tracts of the CNS. We also show that DWnt-3 does not functionally replace wg in an in vivo assay. Experiments with a tissue culture cell line transfected with a construct encoding the DWnt-3 gene show that DWnt-3 protein is efficiently synthesized, glycosylated, proteolytically processed, and transported to the extracellular matrix and medium. DWnt-3, therefore, encodes a secreted protein, which is likely to play a role in development of the Drosophila CNS.

  View details for Web of Science ID A1995QK99100017

  View details for PubMedID 7883074

• SIGNALING BY WINGLESS IN DROSOPHILA DEVELOPMENTAL BIOLOGY Klingensmith, J., Nusse, R. 1994; 166 (2): 396-414

  Abstract

  Wingless, a member of the Wnt gene family, is an essential gene for segmentation in Drosophila, and is also involved in many other patterning events. The gene encodes a secreted protein that can regulate gene expression in adjacent cells. Recently, significant progress has been made in elucidating the signal transduction pathway of wingless, mainly by genetic experiments but increasingly also at the biochemical level. While many components of wingless signaling, in particular a receptor, remain to be identified, our current understanding of wingless pathway is more advanced than that of other Wnt genes. We will give an overview of the various roles of wingless in development, and we will then summarize the wingless signaling pathway as it has emerged from genetic and biochemical studies. Where appropriate, wingless signaling will be compared to the activity of vertebrate Wnt proteins.

  View details for Web of Science ID A1994QA70700004

  View details for PubMedID 7813765

• ISOLATION AND CHARACTERIZATION OF A MOUSE HOMOLOG OF THE DROSOPHILA SEGMENT POLARITY GENE DISHEVELLED DEVELOPMENTAL BIOLOGY Sussman, D. J., Klingensmith, J., Salinas, P., Adams, P. S., Nusse, R., Perrimon, N. 1994; 166 (1): 73-86

  Abstract

  In the Drosophila embryo dishevelled (dsh) function is required by target cells in order to respond to wingless (wg, the homolog of Wnt-1), demonstrating a role for dsh in Wnt signal transduction. We have isolated a mouse homolog of the Drosophila dsh segment polarity gene. The 695-amino-acid protein encoded by the mouse dishevelled gene (Dvl-1) shares 50% identity (65% similarity) with dsh. Similarity searches of protein and DNA data bases revealed that Dvl-1 encodes an otherwise novel polypeptide. While no functional motifs were identified, one region of Dvl-1 was found to be similar to a domain of discs large-1 (dlg), a Drosophila tumor suppressor gene. In the embryo, Dvl-1 is expressed in most tissues, with uniformly high levels in the central nervous system. From 7.5 days postcoitum Dvl-1 is expressed throughout the developing brain and spinal cord, including those regions expressing Wnt-1 and En. Expression of Dvl-1 in adult mice was found to be widespread, with brain and testis exhibiting the highest levels. The majority of Dvl-1 expression in the adult cerebellum is in the granular cell layer, similar to the pattern seen for engrailed-2 (En-2). Throughout postnatal development of the brain Dvl-1 is highly expressed in areas of high neuronal cell density.

  View details for Web of Science ID A1994PT49200006

  View details for PubMedID 7958461

• MAINTENANCE OF WNT-3 EXPRESSION IN PURKINJE-CELLS OF THE MOUSE CEREBELLUM DEPENDS ON INTERACTIONS WITH GRANULE CELLS DEVELOPMENT Salinas, P. C., Fletcher, C., Copeland, N. G., Jenkins, N. A., Nusse, R. 1994; 120 (5): 1277-1286

  Abstract

  Wnt genes encode secreted proteins implicated in cell fate changes during development. To define specific cell populations in which Wnt genes act, we have examined Wnt expression in the cerebellum. This part of the brain has a relatively simple structure and contains well-characterized cell populations. We found that Wnt-3 is expressed during development of the cerebellum and that expression is restricted to the Purkinje cell layer in the adult. Wnt-3 expression in Purkinje cells increases postnatally as granule cells start to make contacts with Purkinje cells. To investigate whether interactions with granule cells influence Wnt-3 expression in Purkinje cells, we examined gene expression in several mouse mutants, using the expression of En-2 to follow the fate of granule cells. In the weaver mutant, in which granule cells fail to migrate and subsequently die in the external granular layer, Wnt-3 expression was normal at postnatal day 15 (P15). At that time, some granule cells are still present in the external granular layer. At P28, however, when granule cells could no longer be detected, Wnt-3 expression was almost absent. In the meander tail mutant, in which the anterior cerebellar lobes lack granule cells, Wnt-3 expression was only detected in the normal posterior lobes. Since En genes are implicated in cell-cell interactions mediated by Wnt genes, we examined En-2/En-2 mutant mice, finding normal Wnt-3 expression, indicating that the effect of granule cells on the maintenance of Wnt-3 is not mediated by En-2. Our results show that Wnt-3 expression in Purkinje cells is modulated by their presynaptic granule cells at the time of neuronal maturation.

  View details for Web of Science ID A1994NM70200022

  View details for PubMedID 8026336

• BIOLOGICAL-ACTIVITY OF SOLUBLE WINGLESS PROTEIN IN CULTURED DROSOPHILA IMAGINAL DISC CELLS NATURE VANLEEUWEN, F., Samos, C. H., Nusse, R. 1994; 368 (6469): 342-344

  Abstract

  The phenotypes caused by mutations in Wnt genes suggest that their gene products are involved in cell-to-cell communication. Wnt genes indeed encode secreted molecules, but soluble active Wnt protein has not been found. We have developed a novel cell culture assay for the Drosophila Wnt gene wingless, using a Drosophila imaginal disc cell line (cl-8; ref. 13), and measured effects on the adherens junction protein armadillo, a known genetic target of wingless. Transfection of a temperature-sensitive wingless complementary DNA into cl-8 cells increases the levels of the armadillo protein. The wingless protein does not affect the rate of synthesis of armadillo, but leads to increased stability of an otherwise rapidly decaying armadillo protein. The wingless protein in the extracellular matrix and soluble medium from donor cells also increases the levels of armadillo protein. The protein in the medium acts fast and is inhibited by an antibody to wingless protein, demonstrating that Wnt products can act as soluble extracellular signalling molecules.

  View details for Web of Science ID A1994NB98500048

  View details for PubMedID 8127369

• DISHEVELLED AND ARMADILLO ACT IN THE WINGLESS SIGNALING PATHWAY IN DROSOPHILA NATURE Noordermeer, J., Klingensmith, J., Perrimon, N., Nusse, R. 1994; 367 (6458): 80-83

  Abstract

  The Wnt genes encode conserved secreted proteins that play a role in normal development and tumorigenesis. Little is known about the signal transduction pathways of Wnt gene products. One of the best characterized Wnt family members is the Drosophila segment polarity gene wingless. We have investigated whether segment polarity genes with a wingless-like phenotype mediate the wingless signal. We used a wingless transgene controlled by a heat-shock promoter for genetic epistasis experiments. We show that wingless acts through dishevelled and armadillo to affect the expression of the homeobox gene engrailed and cuticle differentiation.

  View details for Web of Science ID A1994MP86500064

  View details for PubMedID 7906389

• THE DROSOPHILA SEGMENT POLARITY GENE DISHEVELLED ENCODES A NOVEL PROTEIN REQUIRED FOR RESPONSE TO THE WINGLESS SIGNAL GENES & DEVELOPMENT Klingensmith, J., Nusse, R., Perrimon, N. 1994; 8 (1): 118-130

  Abstract

  The Drosophila Wnt-1 homolog, wingless (wg), is involved in the signaling of patterning information in several contexts. In the embryonic epidermis, Wg protein is secreted and taken up by neighboring cells, in which it is required for maintenance of engrailed transcription and accumulation of Armadillo protein. The dishevelled (dsh) gene mediates these signaling events as well as wg-dependent induction across tissue layers in the embryonic midgut. dsh is also required for the development processes in which wg functions in adult development. Overall, cells lacking dsh are unable to adopt fates specified by Wg. dsh functions cell autonomously, indicating that it is involved in the response of target cells to the Wg signal. dsh is expressed uniformly in the embryo and encodes a novel protein with no known catalytic motifs, although it shares a domain of homology with several junction-associated proteins. Our results demonstrate that dsh encodes a specific component of Wg signaling and illustrate that Wnt proteins may utilize a novel mechanism of extracellular signal transduction.

  View details for Web of Science ID A1994MR21500011

  View details for PubMedID 8288125

• MUTATIONS IN THE SEGMENT POLARITY GENES WINGLESS AND PORCUPINE IMPAIR SECRETION OF THE WINGLESS PROTEIN EMBO JOURNAL VANDENHEUVEL, M., HARRYMANSAMOS, C., Klingensmith, J., Perrimon, N., Nusse, R. 1993; 12 (13): 5293-5302

  Abstract

  We have characterized the molecular nature of mutations in wingless (wg), a segment polarity gene acting during various stages of Drosophila development. Embryo-lethal alleles have undergone mutations in the protein-encoding domain of the gene, including deletions and point mutations of conserved residues. In a temperature sensitive mutation, a conserved cysteine residue is replaced by a serine. In embryo-viable alleles, the wg transcriptional unit is not affected. Immunostaining of mutant embryos shows that the embryo-lethal alleles produce either no wg antigen or a form of the protein that is retained within cells. Interestingly, embryos mutant for the segment polarity gene porcupine show a similar retention of the wg antigen. We have also transfected wild type wg alleles into Drosophila tissue culture cells, which then display wg protein on the cell surface and in the extracellular matrix. In similar experiments with mutant alleles, the proteins are retained in intracellular compartments and appear not to be secreted. These data provide further evidence that wg acts as a secreted factor and suggest that porcupine provides an accessory function for wg protein secretion or transport.

  View details for Web of Science ID A1993MM12000042

  View details for PubMedID 8262072

• MOLECULAR-CLONING AND CHROMOSOMAL LOCALIZATION TO 17Q21 OF THE HUMAN WNT3-GENE GENOMICS Roelink, H., Wang, J., Black, D. M., Solomon, E., Nusse, R. 1993; 17 (3): 790-792

  Abstract

  In mouse mammary tumors, the Wnt-3 gene can be activated by proviral insertion. Here we report on the isolation of a human homolog, WNT3. A genomic clone was isolated by use of mouse Wnt-3 sequences as a probe, after which cDNA containing most of the protein-encoding domain of the human gene was obtained by PCR. Comparison between the deduced mouse and human WNT-3 protein sequences showed four changes in 333 amino acids. WNT3 is located on chromosome 17q21. The gene was not found to be amplified or rearranged in a collection of human breast tumors.

  View details for Web of Science ID A1993LX23300045

  View details for PubMedID 8244403

• CELL PATTERNING IN THE DROSOPHILA SEGMENT - ENGRAILED AND WINGLESS ANTIGEN DISTRIBUTIONS IN SEGMENT POLARITY MUTANT EMBRYOS Meeting of the British-Society-for-Developmental-Biology: Signals, Polarity and Adhesion in Development VANDENHEUVEL, M., Klingensmith, J., Perrimon, N., Nusse, R. COMPANY OF BIOLOGISTS LTD. 1993: 105–114

  Abstract

  By a complex and little understood mechanism, segment polarity genes control patterning in each segment of the Drosophila embryo. During this process, cell to cell communication plays a pivotal role and is under direct control of the products of segment polarity genes. Many of the cloned segment polarity genes have been found to be highly conserved in evolution, providing a model system for cellular interactions in other organisms. In Drosophila, two of these genes, engrailed and wingless, are expressed on either side of the parasegment border. wingless encodes a secreted molecule and engrailed a nuclear protein with a homeobox. Maintenance of engrailed expression is dependent on wingless and vice versa. To investigate the role of other segment polarity genes in the mutual control between these two genes, we have examined wingless and engrailed protein distribution in embryos mutant for each of the segment polarity genes. In embryos mutant for armadillo, dishevelled and porcupine, the changes in engrailed expression are identical to those in wingless mutant embryos, suggesting that their gene products act in the wingless pathway. In embryos mutant for hedgehog, fused, cubitus interruptus Dominant and gooseberry, expression of engrailed is affected to varying degrees. However wingless expression in the latter group decays in a similar way earlier than engrailed expression, indicating that these gene products might function in the maintenance of wingless expression. Using double mutant embryos, epistatic relationships between some segment polarity genes have been established. We present a model showing a current view of segment polarity gene interactions.

  View details for Web of Science ID A1993NP85500013

  View details for PubMedID 8049466

• REGIONAL EXPRESSION OF THE WNT-3 GENE IN THE DEVELOPING MOUSE FOREBRAIN IN RELATIONSHIP TO DIENCEPHALIC NEUROMERES MECHANISMS OF DEVELOPMENT Salinas, P. C., Nusse, R. 1992; 39 (3): 151-160

  Abstract

  During early vertebrate development, a series of neuromeres divides the central nervous system from the forebrain to the spinal cord. Here we examine in more detail the expression of Wnt-3, a member of the Wnt gene family of secreted proteins, in the developing diencephalon, in comparison to the expression of the homeobox gene Dlx-1. In 9.5-day mouse embryos, Wnt-3 is expressed in a restricted area of the diencephalon before any morphological signs of subdivisions appear. Around embryonic day 11.5, Wnt-3 expression becomes restricted to one of the neuromeres of the diencephalon, the dorsal thalamus. Dlx-1 is expressed in a non-overlapping area immediately anterior to and abutting the Wnt-3 expressing domain, corresponding to the ventral thalamus. In addition, Wnt-3 is expressed in the midbrain-hindbrain region. In the adult mouse, Wnt-3 and Dlx-1 are expressed in subsets of neural cells derived from the original areas of expression in the diencephalon. Taken together, our results suggest that Wnt-3 and Dlx-1 provide positional information for the regional specification of neuromeres in the forebrain. The continued expression of these genes in the adult mouse brain suggests a distinct role in the mature CNS.

  View details for Web of Science ID A1992KG00100003

  View details for PubMedID 1363370

• THE CONSEQUENCES OF UBIQUITOUS EXPRESSION OF THE WINGLESS GENE IN THE DROSOPHILA EMBRYO DEVELOPMENT Noordermeer, J., Johnston, P., Rijsewijk, F., Nusse, R., Lawrence, P. A. 1992; 116 (3): 711-?

  Abstract

  The segment polarity gene wingless has an essential function in cell-to-cell communication during various stages of Drosophila development. The wingless gene encodes a secreted protein that affects gene expression in surrounding cells but does not spread far from the cells where it is made. In larvae, wingless is necessary to generate naked cuticle in a restricted part of each segment. To test whether the local accumulation of wingless is essential for its function, we made transgenic flies that express wingless under the control of a hsp70 promoter (HS-wg flies). Uniform wingless expression results in a complete naked cuticle, uniform armadillo accumulation and broadening of the engrailed domain. The expression patterns of patched, cubitus interruptus Dominant and Ultrabithorax follow the change in engrailed. The phenotype of heatshocked HS-wg embryos resembles the segment polarity mutant naked, suggesting that embryos that overexpress wingless or lack the naked gene enter similar developmental pathways. The ubiquitous effects of ectopic wingless expression may indicate that most cells in the embryo can receive and interpret the wingless signal. For the development of the wild-type pattern, it is required that wingless is expressed in a subset of these cells.

  View details for Web of Science ID A1992KB02500018

  View details for PubMedID 1289061

• THE WNT GENE FAMILY IN TUMORIGENESIS AND IN NORMAL DEVELOPMENT 4TH INTERNATIONAL CONGRESS ON HORMONES AND CANCER Nusse, R. PERGAMON-ELSEVIER SCIENCE LTD. 1992: 9–12

  Abstract

  Various members of the Wnt gene family have been identified as activated oncogenes in mouse mammary tumors. We show that some tumors are oligoclonal for activation of a Wnt gene, and clonal variation when those tumors are transplanted to become hormone-independent. The normal function of many Wnt genes is to control pattern formation in early embryos, as shown by expression profiles and by mutant analysis.

  View details for Web of Science ID A1992JM32400003

  View details for PubMedID 1388050

• WNT GENES CELL Nusse, R., Varmus, H. E. 1992; 69 (7): 1073-1087

  View details for Web of Science ID A1992JA43100003

  View details for PubMedID 1617723

• ISOLATION AND EXPRESSION OF 2 NOVEL WNT WINGLESS GENE HOMOLOGS IN DROSOPHILA DEVELOPMENT Russell, J., GENNISSEN, A., Nusse, R. 1992; 115 (2): 475-485

  Abstract

  Wingless (wg), the Drosophila homologue of the mouse Wnt-1 proto-oncogene, is a segment polarity gene essential in each segment for normal Drosophila development. We here report the isolation of two novel Drosophila Wnt homologues, DWnt-2 and DWnt-3, and thus the existence of a Wnt/wingless gene family in Drosophila. DWnt-2 and DWnt-3 map to chromosome 2 position 45E and chromosome X position 17A/B, respectively. DWnt-2 and DWnt-3, like the other known Wnt genes, encode amino-terminal signal peptides suggesting that the gene products are secreted proteins. The putative translation product of DWnt-2 and the carboxy-terminal half of the deduced DWnt-3 product are both rich in conserved cysteine residues. In comparison with other Wnt gene products, mostly about 40 x 10(3) relative molecular mass, the DWnt-3 protein has an extended amino terminus and a long internal insert, and its predicted relative molecular mass is 113 x 10(3). The expression patterns of these two Wnt/wg homologues are dynamic during Drosophila embryogenesis. The distribution of DWnt-2 transcripts is predominantly segmented, with the additional presence of transcripts in the presumptive gonads. Transcripts of both DWnt-2 and DWnt-3 appear to be associated with limb primordia in the embryo and may therefore specify limb development. DWnt-3 is also expressed in mesodermal and neurogenic regions. The distribution of DWnt-3 transcripts in cells of the central nervous system (CNS) during Drosophila embryogenesis suggests that DWnt-3 could be involved in CNS development.

  View details for Web of Science ID A1992JB71800010

  View details for PubMedID 1425336

• AMPLIFICATION AND PROVIRAL ACTIVATION OF SEVERAL WNT GENES DURING PROGRESSION AND CLONAL VARIATION OF MOUSE MAMMARY-TUMORS ONCOGENE Roelink, H., Wagenaar, E., Nusse, R. 1992; 7 (3): 487-492

  Abstract

  Mammary tumors in the GR strain are caused by a dominant locus containing an endogenous mouse mammary tumor provirus. Expression of this locus results in high virus titers, inducing tumors that progress from a hormone-dependent to a hormone-independent tumor state. We previously studied the activation of the Wnt-1 and int-2 oncogenes in several series of transplanted GR tumors and found that hormone-dependent early passages are generally oligoclonal for proviral integration at these genes. We have now re-examined several such tumor series for activation of other Wnt genes. In one series, the transition to hormone-independent growth was marked by the loss of the oligoclonal genotype and outgrowth of a hormone-independent cell population, clonal for the activation of Wnt-3. We show two examples of series of transplanted tumors that in later hormone-independent passages contain an amplified and overexpressed Wnt-2 gene, a novel mode of activation of these genes.

  View details for Web of Science ID A1992HK00500013

  View details for PubMedID 1549363

• EXPRESSION OF 2 MEMBERS OF THE WNT FAMILY DURING MOUSE DEVELOPMENT - RESTRICTED TEMPORAL AND SPATIAL PATTERNS IN THE DEVELOPING NEURAL-TUBE GENES & DEVELOPMENT Roelink, H., Nusse, R. 1991; 5 (3): 381-388

  Abstract

  The Wnt gene family encodes a group of cysteine-rich proteins implicated in intercellular signaling during several stages of vertebrate development. This family includes Wnt-1 and Wnt-3, both discovered as activated oncogenes in mouse mammary tumors. Here we describe the molecular cloning of an additional member of the Wnt family, called Wnt-3A, and the spatial and temporal expression pattern of this gene as well as that of its close relative Wnt-3. The putative amino acid sequences of both proteins are almost 90% identical, but in situ hybridization to mouse embryo sections showed highly restricted patterns of expression of Wnt-3 and Wnt-3A, largely in separate areas in the developing nervous system. In the spinal cord Wnt-3 was expressed at low levels in the alar laminae and in the ventral horns, whereas Wnt-3A expression was confined to the roof plate. In the developing brain Wnt-3 was expressed broadly across the dorsal portion of the neural tube with a rostral boundary of expression at the diencephalon. In contrast, Wnt-3A was expressed in a narrow region very close to the midline; expression extended into the bifurcating telencephalon, in a highly localized fashion. Both Wnt-3 and Wnt-3A were expressed in the ectoderm, and Wnt-3A was also expressed in the periumbilical mesenchyme. Characteristic expression patterns of these two closely related genes suggest that Wnt-3 and Wnt-3A play distinct roles in cell-cell signaling during morphogenesis of the developing neural tube.

  View details for Web of Science ID A1991FB28000005

  View details for PubMedID 2001840

• THE WNT-1 (INT-1) ONCOGENE PROMOTER AND ITS MECHANISM OF ACTIVATION BY INSERTION OF PROVIRAL DNA OF THE MOUSE MAMMARY-TUMOR VIRUS MOLECULAR AND CELLULAR BIOLOGY Nusse, R., Theunissen, H., Wagenaar, E., Rijsewijk, F., GENNISSEN, A., Otte, A., Schuuring, E., VANOOYEN, A. 1990; 10 (8): 4170-4179

  Abstract

  Wnt-1 (int-1) is a cellular oncogene often activated by insertion of proviral DNA of the mouse mammary tumor virus. We have mapped the 5' end and the promoter area of the Wnt-1 gene by nuclease protection and primer extension assays. In differentiating P19 embryonal carcinoma cells, in which Wnt-1 is naturally expressed, two start sites of transcription were found, one preceded by two TATA boxes and one preceded by several GC boxes. In P19 cells, a 1-kilobase upstream sequence of Wnt-1 was able to confer differentiation-specific expression on a heterologous gene. We have investigated how Wnt-1 transcription was affected by mouse mammary tumor virus proviral integrations in various configurations near the promoters of the gene. One provirus has been inserted in the 5' nontranslated part of Wnt-1, in the same transcriptional orientation, and has functionally replaced the Wnt-1 promoters. Wnt-1 transcription in this tumor starts in the right long terminal repeat of the provirus, with considerable readthrough transcription from the left long terminal repeat. Another provirus has been inserted in the orientation opposite that of Wnt-1 into a GC box, disrupting the first Wnt-1 transcription start site but not the downstream start site. Most insertions have not structurally altered the Wnt-1 transcripts and have enhanced the activity of the normal two promoters.

  View details for Web of Science ID A1990DP92000034

  View details for PubMedID 1695322

• WNT-3, A GENE ACTIVATED BY PROVIRAL INSERTION IN MOUSE MAMMARY-TUMORS, IS HOMOLOGOUS TO INT-1/WNT-1 AND IS NORMALLY EXPRESSED IN MOUSE EMBRYOS AND ADULT BRAIN PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Roelink, H., Wagenaar, E., DASILVA, S. L., Nusse, R. 1990; 87 (12): 4519-4523

  Abstract

  We have isolated a common insertion site, Wnt-3, for proviruses of the mouse mammary tumor virus (MMTV). Of mammary tumors induced by the GR variant of MMTV, 5% contains a provirus at Wnt-3, which is located on mouse chromosome 11. The gene is transcribed into a 3.8-kilobase (kb) mRNA in tumors with nearby proviral insertions but not in tumors with proviruses at other loci or in most adult tissues. Normal expression of Wnt-3 is detected in mouse embryos (with a peak around day 12 of gestation) and at low levels in adult brain. The transcriptional unit of the Wnt-3 gene spans approximately 55 kb, with a first intron of 36 kb. The deduced amino acid sequence of the Wnt-3 protein is 47% identical to the int-1/Wnt-1 gene product.

  View details for Web of Science ID A1990DK27300024

  View details for PubMedID 2162045

• THE INT GENES IN MOUSE MAMMARY TUMORIGENESIS AND IN NORMAL DEVELOPMENT 48TH ANNUAL SYMP OF THE SOC FOR DEVELOPMENTAL BIOLOGY : GENETICS OF PATTERN FORMATION AND GROWTH CONTROL Nusse, R., VANDENHEUVEL, M., Roelink, H. WILEY-LISS, INC. 1990: 157–163

  View details for Web of Science ID A1990BR82E00010

• PHORBOL ESTER-INDUCIBLE T-CELL-SPECIFIC EXPRESSION OF VARIANT MOUSE MAMMARY-TUMOR VIRUS LONG TERMINAL REPEATS JOURNAL OF VIROLOGY Theunissen, H. J., PAARDEKOOPER, M., MADURO, L. J., Michalides, R. J., Nusse, R. 1989; 63 (8): 3466-3471

  Abstract

  Acquired proviruses of mouse mammary tumor virus (MMTV) in T-cell leukemias of male GR mice have rearrangements in the U3 region of their long terminal repeats (LTR). In contrast to the endogenous nonrearranged MMTV proviruses, these mutated copies are highly expressed in leukemic T cells. To investigate whether the sequence alterations in the LTR are responsible for the high expression of rearranged MMTV proviruses, we made constructs in which normal and variant LTRs drive the bacterial reporter gene chloramphenicol acetyltransferase (CAT). Two different rearranged LTRs were used, one containing a 420-base-pair (bp) deletion (L13) and another carrying a 456-bp deletion plus an 82-bp insertion (L42). These constructs were transfected into murine (GRSL) and human (MOLT-4) T-cell lines that either had or had not been treated with phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA]). In GRSL cells, the L13-LTR-CAT construct showed transcriptional activity that was further enhanced by TPA. In MOLT-4 cells, both variant LTRs were active, but only after stimulation with TPA. In contrast, normal(N)-LTR-CAT constructs were not expressed, irrespective of TPA addition. In XC rat fibrosarcoma cells, neither normal nor variant LTRs gave rise to detectable CAT activity, either in the presence or in the absence of TPA, but dexamethasone strongly stimulated CAT activity driven by N and L42 LTRs. The L13 LTR was considerably less active, probably caused by the deletion of the distal part of the glucocorticoid responsive element. We conclude that the LTR rearrangements generate TPA responsiveness and contribute to T-cell-specific expression of MMTV variants.

  View details for Web of Science ID A1989AF40600034

  View details for PubMedID 2545916

  View details for PubMedCentralID PMC250923

• THE DROSOPHILA HOMOLOG OF THE MOUSE MAMMARY ONCOGENE INT-1 IS IDENTICAL TO THE SEGMENT POLARITY GENE WINGLESS CELL Rijsewijk, F., Schuermann, M., Wagenaar, E., Parren, P., Weigel, D., Nusse, R. 1987; 50 (4): 649-657

  Abstract

  We have isolated the Drosophila melanogaster homolog (Dint-1) of int-1, a conserved cellular oncogene implicated in viral mammary tumorigenesis in mice. The deduced Dint-1 protein sequence contains 468 amino acids and starts with a hydrophobic leader; it is 54% identical to the int-1 sequence, and all 23 cysteine residues are conserved. The putative Drosophila protein has an extra sequence of 85 amino acids, encoded on an additional exon. Dint-1 is expressed throughout development, but transcripts are barely detectable in adult flies. Hybridization in situ to embryos reveals a segmented pattern of expression. We show that Dint-1 and the segment polarity gene wingless are identical and map to the same location. The sequence of the gene suggests that the Dint-1/wingless protein functions in morphogenesis as a signal in cell-cell communication.

  View details for Web of Science ID A1987J746100016

  View details for PubMedID 3111720

• TRANSFECTION OF THE INT-1 MAMMARY ONCOGENE IN CUBOIDAL RAC MAMMARY CELL-LINE RESULTS IN MORPHOLOGICAL TRANSFORMATION AND TUMORIGENICITY EMBO JOURNAL Rijsewijk, F., VANDEEMTER, L., Wagenaar, E., Sonnenberg, A., Nusse, R. 1987; 6 (1): 127-131

  Abstract

  The int-1 gene is often activated by proviral insertion in mouse mammary tumors. Direct evidence for the normal function of this gene and its role in tumorigenesis has therefore been lacking. To examine possible biological effects of int-1 activation in in vitro cell systems, we have constructed recombinant molecules of genomic int-1 DNA, transcriptionally activated by retroviral promoters. Transfection of these constructs into cuboidal RAC311C mammary cells leads to morphological transformation of the cells and rapid tumorigenicity.

  View details for Web of Science ID A1987F878300020

  View details for PubMedID 3034569

• ONCOGENE EXPRESSION DURING PROGRESSION OF MOUSE MAMMARY-TUMOR CELLS - ACTIVITY OF A PROVIRAL ENHANCER AND THE RESULTING EXPRESSION OF INT-2 IS INFLUENCED BY THE STATE OF DIFFERENTIATION EMBO JOURNAL Sonnenberg, A., VANBALEN, P., Hilgers, J., Schuuring, E., Nusse, R. 1987; 6 (1): 121-125

  Abstract

  The RAC cell lines are derived from a mammary tumor induced by the mouse mammary tumor virus (MMTV). Polygonal cells have retained many characteristics of epithelial cells and induce adenocarcinomas; cuboidal cells are poorly tumorigenic; and elongated cells produce highly malignant sarcoma-like tumors. MMTV proviral integrations, one of which has cis-activated the int-2 gene, demonstrated that the lines are clonal descendents from a single tumor cell. Polygonal cells have a high constitutive expression of MMTV and contain int-2 RNA. In contrast, the cuboidal and the elongated cells show no detectable expression of int-2, even in the presence of glucocorticoid hormone. Transcription of MMTV in these cells is also low but can be stimulated by dexamethasone, albeit to levels lower than in polygonal cells. Thus, expression of int-2 seems to be caused by an enhancing activity on the MMTV provirus which is not dependent on steroid hormone and is specific for mammary tumor cells with epithelial characteristics. Progression in this cell system does not require sustained expression of int-2.

  View details for Web of Science ID A1987F878300019

  View details for PubMedID 3034568

• CONSTRUCTION OF A RETROVIRAL CDNA VERSION OF THE INT-1 MAMMARY ONCOGENE AND ITS EXPRESSION INVITRO NUCLEIC ACIDS RESEARCH Rijsewijk, F. A., VANLOHUIZEN, M., VANOOYEN, A., Nusse, R. 1986; 14 (2): 693-702

  Abstract

  The int-1 mammary oncogene is activated by proviruses of the Mouse Mammary Tumor Virus in many different mammary tumors. We have inserted a genomic fragment containing the protein-encoding domain of the gene into the retroviral shuttle vector pZIPneoSV(X)1. After one round of virus replication we recovered recombinant proviral DNA containing a correctly spliced copy of int-1. In vitro transcription of this cDNA version of int-1 using SP6 polymerase and translation in a reticulocyte lysate yielded a protein of approximately 37,000 daltons. High expression of int-1 in NIH-3T3 cells infected with recombinant virus did not lead to morphological transformation.

  View details for Web of Science ID A1986AZK7700006

  View details for PubMedID 3003692

• RETROVIRAL INSERTIONAL MUTAGENESIS IN MURINE MAMMARY-CANCER PROCEEDINGS OF THE ROYAL SOCIETY SERIES B-BIOLOGICAL SCIENCES Nusse, R., VANOOYEN, A., Rijsewijk, F., VANLOHUIZEN, M., Schuuring, E., VANTVEER, L. 1985; 226 (1242): 3-13

  Abstract

  We are attempting to identify cellular oncogenes activated in mammary tumours by using the mouse mammary tumour virus (MMTV) as an insertional mutagen. MMTV, a retrovirus lacking a host cell-derived viral oncogene, induces adenocarcinomas of the mammary gland after a long latency period. The tumours are clonal outgrowths of cells carrying one or more integrated MMTV proviral copies. We have cloned an integrated MMTV provirus with its adjacent chromosomal DNA and we have established that the insertion site was part of a domain of the mouse genome in which MMTV proviruses are inserted in many different tumours. A gene within this domain, called int-1 is transcriptionally activated as a consequence of proviral integration. We have proposed that int-1 is a cellular oncogene for mammary tumours. Proviral activation of int-1 occurs in cis, over distances of up to 10 kilobases and is presumably caused by the transcriptional enhancer present on the MMTV long terminal repeat. The putative int-1 mammary oncogene has been subjected to a detailed structural analysis by S1 mapping and DNA sequencing. It encodes a protein that is highly conserved between mouse and man. The protein encoding domain of the gene is distributed over four exons which are demarcated by the insertion sites of MMTV proviruses found in mammary tumours. Some insertions, however, are found in the transcriptional unit of int-1, but these insertions do not disrupt the protein encoding domain of the gene.

  View details for Web of Science ID A1985AUH6000001

  View details for PubMedID 2866522

• THE NUCLEOTIDE-SEQUENCE OF THE HUMAN INT-1 MAMMARY ONCOGENE - EVOLUTIONARY CONSERVATION OF CODING AND NON-CODING SEQUENCES EMBO JOURNAL VANOOYEN, A., KWEE, V., Nusse, R. 1985; 4 (11): 2905-2909

  Abstract

  The mouse mammary tumor virus can induce mammary tumors in mice by proviral activation of an evolutionarily conserved cellular oncogene called int-1. Here we present the nucleotide sequence of the human homologue of int-1, and compare it with the mouse gene. Like the mouse gene, the human homologue contains a reading frame of 370 amino acids, with only four substitutions. The amino acid changes are all in the hydrophobic leader domain of the int-1 encoded protein, and do not significantly alter its hydropathic index. The conservation between the mouse and the human int-1 genes is not restricted to exons; extensive parts of the introns are also homologous. Thus, int-1 ranks among the most conserved genes known, a property shared with other oncogenes.

  View details for Web of Science ID A1985ATY8400027

  View details for PubMedID 2998762

• MODE OF PROVIRAL ACTIVATION OF A PUTATIVE MAMMARY ONCOGENE (INT-1) ON MOUSE CHROMOSOME-15 NATURE Nusse, R., VANOOYEN, A., Cox, D., Fung, Y. K., Varmus, H. 1984; 307 (5947): 131-136

  Abstract

  Most mammary carcinomas induced in C3H mice by the mouse mammary tumour virus (MMTV) bear a new proviral insertion within a highly conserved locus on chromosome 15 called int-1. A transcriptional unit within this locus is inactive in all tested normal tissues but expressed at low levels in mammary tumours with proviral insertions positioned on either the 5' and 3' sides of the gene. Transcription of the proviruses proceeds away from int-1; thus an indirect mechanism appears to activate expression of this putative oncogene.

  View details for Web of Science ID A1984RY26500040

  View details for PubMedID 6318122

• STRUCTURE AND NUCLEOTIDE-SEQUENCE OF THE PUTATIVE MAMMARY ONCOGENE INT-1 - PROVIRAL INSERTIONS LEAVE THE PROTEIN-ENCODING DOMAIN INTACT CELL VANOOYEN, A., Nusse, R. 1984; 39 (1): 233-240

  Abstract

  Many mammary tumors induced by mouse mammary tumor virus (MMTV) contain a provirus in the same region of the host-cell genome, leading to expression of a putative cellular oncogene called int-1. Here we present the structure and nucleotide sequence of int-1. We have established several proviral insertion sites exactly by nuclease S1 analysis or by molecular cloning and DNA sequencing. The protein-encoding domain of int-1 is distributed over four exons. At the 5' end of the gene two overlapping exons were detected, one of which is preceded by a TATA box. The deduced int-1-encoded protein has 370 amino acids, with a preponderance of hydrophobic residues at the NH2 terminus. Proviruses are found at both sides of the gene, usually oriented away from the gene. Downstream integrations occur frequently in the long 3' untranslated region of the last exon. One upstream provirus is inserted in the 5' untranslated region and, unlike the other upstream insertions, in the same orientation as the int-1 gene. Proviral integrations always leave the protein-encoding domain intact, providing further evidence that the int-1 protein contributes an essential step in mammary tumorigenesis.

  View details for Web of Science ID A1984TS61800025

  View details for PubMedID 6091914

• MOUSE MAMMARY-TUMOR VIRUS EXPRESSION AND MAMMARY-TUMOR DEVELOPMENT CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY Michalides, R., VANOOYEN, A., Nusse, R. 1983; 106: 57-78

  View details for Web of Science ID A1983RV67100003

  View details for PubMedID 6315307

• STRUCTURAL-ANALYSIS OF A 1.7-KILOBASE MOUSE MAMMARY-TUMOR VIRUS-SPECIFIC RNA JOURNAL OF VIROLOGY VANOOYEN, A. J., Michalides, R. J., Nusse, R. 1983; 46 (2): 362-370

  Abstract

  We have detected a mouse mammary tumor virus (MMTV)-specific 1.7-kilobase (kb) polyadenylated RNA in mammary glands of several mouse strains. In BALB/c mice, it is the only MMTV-specific RNA species present. C3H and GR mammary glands and tumors contain, in addition, 3.8- and 7.8-kb MMTV RNAs. Nuclease S1 analysis was performed to map 1.7-kb polyadenylated RNA. It contains predominantly long terminal repeat (LTR) sequences. The 5' end maps approximately 134 nucleotides upstream from the 3' end of the LTR. Colinearity with complete proviral DNA continues to a site about 153 nucleotides downstream from the left (5') LTR. No sequences from the middle part of proviral DNA were found. Colinearity with proviral DNA is resumed 72 nucleotides upstream from the right (3') LTR. The nucleotide sequence in this area is TTCCAGT, which is a splice acceptor consensus sequence. The anatomy of 1.7-kb RNA indicates that it may serve as a messenger for the 36,700-dalton protein encoded by the LTRs of MMTV.

  View details for Web of Science ID A1983QL86400004

  View details for PubMedID 6188860

• MANY TUMORS INDUCED BY THE MOUSE MAMMARY-TUMOR VIRUS CONTAIN A PROVIRUS INTEGRATED IN THE SAME REGION OF THE HOST GENOME CELL Nusse, R., Varmus, H. E. 1982; 31 (1): 99-109

  Abstract

  We have asked whether oncogenesis by the mouse mammary tumor virus (MMTV), a slowly oncogenic retrovirus, involves integration of viral DNA within a certain region of the host genome. We first identified a C3H mouse mammary tumor bearing a single new MMTV provirus and cloned a 19 kilobase (kb) DNA restriction fragment containing a junction of viral and host sequences. Host sequences from this clone were used to retrieve 25 kb of the uninterrupted locus (termed MMTV int1) from a bacteriophage library of normal mouse DNA. Hybridization with subcloned DNA fragments of MMTV int1 detected abnormal restriction fragments in digests of DNA from 18 of 26 C3H mammary tumors. The rearrangements all appeared to be due to the insertion of an MMTV provirus, and the integration sites were located in at least five clusters over a total distance of 19 kb. A polyadenylated 2.6 kb RNA species transcribed from int1 was found in the few tumors tested, but not in lactating mammary glands from C3H mice. Of 12 tested viral oncogenes, none exhibited homology with cloned DNA from this locus. We propose that tumorigenesis by MMTV is strongly favored by proviral insertion within the int1 locus, perhaps as a consequence of enhanced expression of a novel cellular oncogene.

  View details for Web of Science ID A1982PQ46100012

  View details for PubMedID 6297757

• QUANTIFICATION OF MOUSE MAMMARY-TUMOR VIRUS STRUCTURAL PROTEINS IN HORMONE-INDUCED MAMMARY-TUMORS OF LOW MAMMARY-TUMOR MOUSE STRAINS INTERNATIONAL JOURNAL OF CANCER Nusse, R., Michalides, R., BOOT, L. M., ROPCKE, G. 1980; 25 (3): 377-383

  Abstract

  The expression of the mouse mammary tumor virus (MMTV) in hormone-induced mammary tumors was investigated by means of a radioimmunoassay for two major MMTV proteins, gp52 and p27. MMTV proteins were isolated on lectin affinity- and ion-exchange chromatography columns. The purified viral proteins were electrophoretically homogeneous and retained immunoreactivity after labelling with 125iodine. Standard competition assays showed that group-specific antigenic determinants were reacting. Mammary tumors were induced in three strains of mice with a low natural incidence of mammary tumors, C57BL, O20 and C3Hf, by a combined hormone treatment, consisting of hypophyseal isografts and administration of progesterone and estrone. Mammary tumors and mammary glands of hormone-treated animals were extracted and used for competition radioimmunoassays. In general, the tumorigenic hormone treatment resulted in enhanced amounts of MMTV proteins in the mammary glands, compared to the amounts found in lactating mammary glands of untreated animals. The levels of MMTV proteins in the mammary tumors were lower than in the mammary glands.

  View details for Web of Science ID A1980JL36200010

  View details for PubMedID 6248469

• ANALYSIS OF SECONDARY MODIFICATIONS OF MOUSE MAMMARY-TUMOR VIRUS PROTEINS BY TWO-DIMENSIONAL GEL-ELECTROPHORESIS JOURNAL OF VIROLOGY Nusse, R., Janssen, H., DeVries, L., Michalides, R. 1980; 35 (2): 340-348

  Abstract

  The structural proteins of mouse mammary tumor virus (MMTV) were analyzed by two-dimensional electrophoresis on isoelectric focusing and sodium dodecyl sulfate gels. Many of the viral proteins displayed heterogeneity in charge due to variable contents of carbohydrates (in particular, sialic acid) and phosphate residues. Neuraminidase treatment of the virions influenced the isoelectric pattern of the envelope glycoproteins. The glycoproteins of an MMTV variant which was attenuated by replication in feline kidney cells had different isoelectric points. This suggested that the acquisition of an altered carbohydrate configuration had changed the host range of the virus. The major MMTV structural core protein, p27, consisted of two species, which had identical iodinated tryptic peptide compositions but differed in phosphate contents. Another MMTV phosphoprotein, p21, was separated into four different phosphorylated species. Phosphorylation of p21 could be performed in vitro by the MMTV virion-associated protein kinase. This enzyme also has a high affinity for MMTV p30 as a substrate. Possible functions of this enzyme are discussed.

  View details for Web of Science ID A1980KB42800008

  View details for PubMedID 6255175

  View details for PubMedCentralID PMC288818

• ANTIGENIC MODULATION OF MAMMARY-TUMOR VIRUS ENVELOPE ANTIGEN ON GR THYMIC LYMPHOMA-CELLS IN RELATION TO EXPRESSION OF H-2, TL CELL-SURFACE ANTIGENS AND THY1 BRITISH JOURNAL OF CANCER Hilgers, J., Sonnenberg, A., Nusse, R. 1980; 42 (4): 542-550

  Abstract

  The MLr antigen, a mammary tumour virus-induced antigen on the surface of GR thymic lymphoma cells (GRSL) can be modulated from the cell surface upon incubation with specific antiserum for 1-2 h at 37 degrees C, followed by washing the cells. In contrast, a number of other cell-surface antigens on these GRSL cells cannot be modulated under similar conditions. These antigens include histocompatibility antigens of the H-2 complex (H-2.8 of the K-end and H-2dx(D) of the H-2dx haplotype) and two thymic markers, TL1.2 and Thy1.2. Antigenic modulation of MLr as tested by trypan-blue exclusion and by chromium51 release does not lead to a measurable change in the expression of H-2K, H-2D, TL and Thy1.2 antigens. These results could be confirmed by absorption analysis. The latter analysis showed that the number of antigenic sites per cell are about the same for MLr and the two H-2 antigens, while TL antigens are scarcer and Thy1.2 antigens are more abundant. The procedure of antigenic modulation showed that the MLr antigen resides on MTVgp52, the major protein of the envelope. There was no evidence of internal proteins, such as MTVp27, on the surface of GRSL cells.

  View details for Web of Science ID A1980KS52300008

  View details for PubMedID 6254554

• IMPAIRED MATURATION OF MOUSE MAMMARY-TUMOR VIRUS PRECURSOR POLYPEPTIDES IN LYMPHOID LEUKEMIA-CELLS, PRODUCING INTRACYTOPLASMIC-A PARTICLES AND NO EXTRACELLULAR B-TYPE VIRIONS JOURNAL OF VIROLOGY Nusse, R., VANDERPLOEG, L., VANDUIJN, L., Michalides, R., Hilgers, J. 1979; 32 (1): 251-258

  Abstract

  Processing of polypeptides of the mouse mammary tumor virus, a type B retrovirus, was investigated in a transplanted thymic lymphoma cell line of the GR strain (GRSL). This cell line was maintained in vivo in ascites form and in vitro as a suspension culture. GRSL cells produce clusters of intracytoplasmic A particles and are virtually deficient in the production of mature extracellular B-type particles. As control, a mammary tumor cell line of the same mouse strain capable of complete virion synthesis was used. The kinetics of viral polypeptide synthesis were studied by pulse labeling with various isotopes (including (35)S and (32)P), followed by immunoprecipitation of cell lysates with monospecific antisera to the major mouse mammary tumor virus gag and env proteins, p27 and gp52, respectively. Both the primary gag and env precursor polypeptides were synthesized in the GRSL cells, but their conversion into viral proteins was impaired. The major gag precursor, Pr73(gag), was stable over a period of 8 h, and mature viral core polypeptides could not be detected. Also, the highly phosphorylated intermediates in the proteolytic processing of Pr73(gag) in virus-producing cells were absent in GRSL cells. By immunoprecipitation, Pr73(gag) was detected in a GRSL particle fraction with the density of intracytoplasmic A particles. The precursor for envelope proteins, Pr73(env), was turned over without the generation of mature viral envelope components gp52 and gp36. The in vivo-transplanted ascites GRSL cells, however, were shown to express gp52 on the cell surface together with a 73,000-dalton polypeptide, as indicated by cell surface iodination and immunoprecipitation.

  View details for Web of Science ID A1979HQ50600030

  View details for PubMedID 232176

  View details for PubMedCentralID PMC353549

• AUTOGENOUS ANTIBODIES AGAINST THE MURINE MAMMARY-TUMOR VIRUS IN STRAINS OF MICE WITH LOW INCIDENCES OF MAMMARY-TUMORS JNCI-JOURNAL OF THE NATIONAL CANCER INSTITUTE Michalides, R., Wagenaar, E., Nusse, R. 1979; 62 (4): 935-941

  Abstract

  The radioimmunoprecipitation assay for the murine mammary tumor virus (MuMTV) was used to detect naturally occurring antibodies against MuMTV in 3 groups of highly inbred mouse strains. 1) Some strains had high incidences of mammary tumors, such as strains GR and C3H. Antibodies against MuMTV were detected in the sera of females of these strains at early ages. 2) Some mouse strains had low incidences of mammary tumors with an intermediate MuMTV expression, such as strains C3Hf, RIIIf, and BALB/c. Some females of these strains developed antibodies against MuMTV. Hormone treatment of these mice resulted in an increase in the proportion of mice carrying antibodies against MuMTV. 3) Some mouse strains were MuMTV-free, such as strains O20, C57BL, and Gr-Mtv2-. No antibodies against MuMTV were detected in the sera of these mice. However, antibodies against MuMTV appeared in the sera of these animals after hormone treatment. The presence of a natural humoral immunity toward MuMTV appeared to be related to the expression of MuMTV in the animals.

  View details for Web of Science ID A1979GS77300025

  View details for PubMedID 219283

• INDUCTION OF MOUSE MAMMARY-TUMOR VIRUS-RNA IN MAMMARY-TUMORS OF BALB-C MICE TREATED WITH URETHANE, X-IRRADIATION, AND HORMONES JOURNAL OF VIROLOGY Michalides, R., VANDEEMTER, L., Nusse, R., Hageman, P. 1979; 31 (1): 63-72

  Abstract

  The involvement of mouse mammary tumor virus (MTV) in the development of mammary tumors of nonviral etiology in BALB/c mice was studied by measuring the levels of MTV RNA, MTV DNA, and MTV proteins in spontaneously arising and hormonally, chemically, and/or physically induced mammary tumors of BALB/c females. The following results were obtained. (i) Spontaneous mammary tumors contained very low levels of MTV RNA; 4 X 10(-6)% of the the cytoplasmic RNA was MTV RNA. No MTV proteins could be demonstrated by using sensitive radioimmunoassays for MTV proteins p27 and gp52. (ii) Mammary tumors induced by treatments with urethane or X-irradiation alone contained higher levels of MTV RNA; these tumors contained 3- and 19-fold more MTV RNA, respectively, compared with spontaneous mammary tumors. (iii) Mammary tumors induced by combined treatment with urethane and X-irradiation expressed high levels of MTV RNA in the mammary tumors; a 1,724-fold increase in MTV RNA content compared with spontaneous mammary tumors was observed. However, very low levels of MTV proteins gp52 and p27 were detected, suggesting some kind of impairment at the translation of the MTV RNA. MTV RNA was also induced by this treatment in mammary glands and spleens, but not in the livers of tumor-bearing animals. (iv) Balb/c females continuously exposed to prolactin contained high levels of MTV RNA and MTV proteins in stimulated mammary glands and in the hormonally induced mammary tumors. These findings suggest that MTV is not responsible for the maintenance and probably also not for the development of all murine mammary cancers.

  View details for Web of Science ID A1979HB43800008

  View details for PubMedID 228060

  View details for PubMedCentralID PMC353422

• INVOLVEMENT OF MOUSE MAMMARY-TUMOR VIRUS IN SPONTANEOUS AND HORMONE-INDUCED MAMMARY-TUMORS IN LOW-MAMMARY-TUMOR MOUSE STRAINS JOURNAL OF VIROLOGY Michalides, R., VANDEEMTER, L., Nusse, R., ROPCKE, G., BOOT, L. 1978; 27 (3): 551-559

  Abstract

  The involvement of the mouse mammary tumor virus (MTV) in spontaneous and hormone-induced mammary tumors in low-mammary-tumor mouse strains was studied by comparing the amounts of MTV RNA and MTV DNA sequences in mammary tumors and other tissues of mice with an without hormonal treatments. The following results were obtained. (i) Mammary tumors which appeared in C3H mice as a result of an infection with MTV contained more MTV DNA compared with noninfected organs; these mammary tumors also contained more MTV RNA than was present in lactating mammary gland cells. (ii) Hormonal stimulation by administration of excessive amounts of prolactin via hypophyseal isografts in C3Hf and O20 mice resulted in an increased expression of MTV RNA in the mammary glands. This elevated level of MTV RNA expression was, however, not maintained in the hormone-induced mammary tumors. (iii) Spontaneous mammary tumors in BALB/c mice contained similar levels of MTV DNA and MTV RNA sequences as were found in other cells of these animals.

  View details for Web of Science ID A1978FN21800010

  View details for PubMedID 212580

  View details for PubMedCentralID PMC525841

• IDENTIFICATION OF MTV-2 GENE RESPONSIBLE FOR EARLY APPEARANCE OF MAMMARY-TUMORS IN GR MOUSE BY NUCLEIC-ACID HYBRIDIZATION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Michalides, R., VANDEEMTER, L., Nusse, R., VANNIE, R. 1978; 75 (5): 2368-2372

  Abstract

  In the mouse strain GR, the Mtv-2 gene controls the expression of large amounts of mammary tumor virus (MTV) antigens in the milk at first lactation. It also controls the early appearance of mammary tumors. We have investigated the number of MTV proviral sequences associated with this Mtv-2 gene by nucleic acid hybridization between MTV [(3)H]cDNA and DNA from GR, B10, and GR-Mtv-2(-) mice. B10 and GR-Mtv-2(-) mice lack Mtv-2 gene expression. The molecular hybridizations revealed that the DNA of GR mice contains 12 copies of MTV proviral sequences, whereas only 4 copies are present in the DNA of B10 and GR-Mtv-2(-) mice. We therefore conclude that the Mtv-2 gene in the GR mouse strain is associated with eight additional MTV proviral sequences. The four Mtv proviral sequences in the GR-Mtv-2(-) DNA might represent another Mtv gene in the GR mouse. Different amounts of MTV RNA are detected in mammary glands at first lactation of B10 and GR-Mtv-2(-) mice, even though both contain four copies of MTV proviral sequences. This indicates a difference between these two mouse strains either in the regulation of expression of these MTV proviral sequences or in the location of these sequences in the murine genome.

  View details for Web of Science ID A1978FB94300069

  View details for PubMedID 209461

  View details for PubMedCentralID PMC392554

• RELATIONSHIP BETWEEN NUCLEIC-ACIDS ASSOCIATED WITH INTRACYTOPLASMIC-A PARTICLES AND MOUSE MAMMARY-TUMOR VIRUS-RNA JOURNAL OF GENERAL VIROLOGY Michalides, R., Nusse, R., Smith, G. H., ZOTTER, S., Muller, M. 1977; 37 (DEC): 511-521

  View details for Web of Science ID A1977ED09100006