Academic Appointments


Administrative Appointments


  • Chair, Developmental Biology (2007 - 2012)

Honors & Awards


  • Member, US National Academy of Sciences (2010)
  • Member, American Academy of Arts and Sciences (2001)
  • Member, Royal Dutch Academy of Sciences (1997)
  • Member, European Molecular Biology Organization (1988)

Current Research and Scholarly Interests


Our laboratory is interested in the growth, development and integrity of animal tissues. We study multiple different organs, trying to identify common principles, and we extend these investigations to cancer and injury repair. In most organs, different cell types are generated by stem cells - cells that also make copies of themselves and thereby maintain the tissue. An optimal balance between the number of stem and differentiated cells is essential for the proper function of the organs. Locally-acting signals are important to maintain this balance in a spatially-organized manner and these signals are key to understanding the regulation of growth.

A common theme linking our work together are Wnt signals. Work from many laboratories, including our own, has shown that Wnt proteins are essential for the control over stem cells. How this is achieved is far from clear and is the subject of studies in the lab, both in vivo and in cell culture. In vivo, a particular question we address is how physiological changes, such as those occurring during hormonal stimuli, injury or programmed tissue degeneration have an impact on the self-renewal signals and on stem cell biology.

2013-14 Courses


Postdoctoral Advisees


Journal Articles


  • 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-?-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 ?-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

  • 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/?-catenin-responsive cells throughout mammary gland development. This reveals a switch in Wnt/?-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/?-catenin-responsive adult stem cell. Our study uncovers dynamic changes in Wnt/?-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

  • 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

  • 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

  • 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

  • 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

  • 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 Web of Science ID 000322441500050

    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/?-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/?-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/?-catenin-responsive stem cells in both of these sites. These results demonstrate the continued importance of Wnt/?-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

  • 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

  • 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

  • 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

  • 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 ?-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 Web of Science ID 000303193100012

    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 ?-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

  • 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

  • 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-?B protein during embryonic dorsal/ventral patterning and the adult innate immune response, independent of the well-studied Armadillo/?-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/?-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 ?-catenin nuclear translocation in glioblastoma, suggesting a novel mechanism for glioblastoma progression in the absence of conventional Wnt/?-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

  • 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

  • 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

  • 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

  • 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 Web of Science ID 000260913800033

    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

  • 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

  • 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

  • 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

  • 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

  • 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 Web of Science ID 000253170400005

    View details for PubMedID 18245445

  • Wnt Signaling and Stem Cell Control CONTROL AND REGULATION OF STEM CELLS Nusse, R., Fuerer, C., Ching, W., Harnish, K., Logan, C., Zeng, A., Ten Berge, D., Kalani, Y. 2008; 73: 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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 SYMPOSIA ON QUANTITATIVE BIOLOGY Nusse, R., Samos, C. H., Brink, M., Willert, K., Cadigan, K. M., Wodarz, A., Fish, M., Rulifson, E. 1997; 62: 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 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

  • 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

  • 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

  • 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

  • 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

Conference Proceedings


  • Interactions between wingless and frizzled molecules in Drosophila 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

  • CELL PATTERNING IN THE DROSOPHILA SEGMENT - ENGRAILED AND WINGLESS ANTIGEN DISTRIBUTIONS IN SEGMENT POLARITY MUTANT EMBRYOS 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

  • THE WNT GENE FAMILY IN TUMORIGENESIS AND IN NORMAL DEVELOPMENT 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