The beauty of seeing the real thing: BMP heterodimer detection in vivo reveals dimer composition and activity.

BMP family ligands can direct cells to divide, differentiate, or die, depending on cell context and specific hetero- or homodimer combinations. In this issue of Developmental Cell, Bauer et al. detect endogenous Drosophila ligand dimers in situ and show that BMP dimer composition affects both signaling range and activity.

Wnt-frizzled planar cell polarity signaling in the regulation of cell motility.

The molecular complexes underlying planar cell polarity (PCP) were first identified in Drosophila through analysis of mutant phenotypes in the adult cuticle and the orientation of associated polarized protrusions such as wing hairs and sensory bristles. The same molecules are conserved in vertebrates and are required for the localization of polarized protrusions such as primary or sensory cilia and the orientation of hair follicles. Not only is PCP signaling required to align cellular structures across a tissue, it is also required to coordinate movement during embryonic development and adult homeostasis. PCP signaling allows cells to interpret positional cues within a tissue to move in the appropriate direction and to coordinate this movement with their neighbors. In this review we outline the molecular basis of the core Wnt-Frizzled/PCP pathway, and describe how this signaling orchestrates collective motility in Drosophila and vertebrates. Here we cover the paradigms of ommatidial rotation and border cell migration in Drosophila, and convergent extension in vertebrates. The downstream cell biological processes that underlie polarized motility include cytoskeletal reorganization, and adherens junctional and extracellular matrix remodeling. We discuss the contributions of these processes in the respective cell motility contexts. Finally, we address examples of individual cell motility guided by PCP factors during nervous system development and in cancer disease contexts.

When cells get in the flow.

New imaging approaches question a long-standing model for how the eyes of fruit flies acquire their geometric patterning.

Elevated EDAR signalling promotes mammary gland tumourigenesis with squamous metaplasia.

Ectodysplasin A receptor (EDAR) is a death receptor in the Tumour Necrosis Factor Receptor (TNFR) superfamily with roles in the development of hair follicles, teeth and cutaneous glands. Here we report that human Oestrogen Receptor (ER) negative breast carcinomas which display squamous differentiation express EDAR strongly. Using a mouse model with a high Edar copy number, we show that elevated EDAR signalling results in a high incidence of mammary tumours in breeding female mice. These tumours resemble the EDAR-high human tumours in that they are characterised by a lack of oestrogen receptor expression, contain extensive squamous metaplasia, and display strong ß-catenin transcriptional activity. In the mouse model, all of the tumours carry somatic deletions of the third exon of the CTNNB1 gene that encodes ß-catenin. Deletion of this exon yields unconstrained ß-catenin signalling activity. We also demonstrate that ß-catenin activity is required for transformed cell growth, showing that increased EDAR signalling creates an environment in which ß-catenin activity can readily promote tumourigenesis. Together, this work identifies a novel death receptor oncogene in breast cancer, whose mechanism of transformation is based on the interaction between the WNT and Ectodysplasin A (EDA) pathways.

Tissue fluidity mediated by adherens junction dynamics promotes planar cell polarity-driven ommatidial rotation.

The phenomenon of tissue fluidity-cells' ability to rearrange relative to each other in confluent tissues-has been linked to several morphogenetic processes and diseases, yet few molecular regulators of tissue fluidity are known. Ommatidial rotation (OR), directed by planar cell polarity signaling, occurs during Drosophila eye morphogenesis and shares many features with polarized cellular migration in vertebrates. We utilize in vivo live imaging analysis tools to quantify dynamic cellular morphologies during OR, revealing that OR is driven autonomously by ommatidial cell clusters rotating in successive pulses within a permissive substrate. Through analysis of a rotation-specific nemo mutant, we demonstrate that precise regulation of junctional E-cadherin levels is critical for modulating the mechanical properties of the tissue to allow rotation to progress. Our study defines Nemo as a molecular tool to induce a transition from solid-like tissues to more viscoelastic tissues broadening our molecular understanding of tissue fluidity.

Inhibition of Wnt signalling by Notch via two distinct mechanisms.

Notch and Wnt are two essential signalling pathways that help to shape animals during development and to sustain adult tissue homeostasis. Although they are often active at the same time within a tissue, they typically have opposing effects on cell fate decisions. In fact, crosstalk between the two pathways is important in generating the great diversity of cell types that we find in metazoans. Several different mechanisms have been proposed that allow Notch to limit Wnt signalling, driving a Notch-ON/Wnt-OFF state. Here we explore these different mechanisms in human cells and demonstrate two distinct mechanisms by which Notch itself, can limit the transcriptional activity of ß-catenin. At the membrane, independently of DSL ligands, Notch1 can antagonise ß-catenin activity through an endocytic mechanism that requires its interaction with Deltex and sequesters ß-catenin into the membrane fraction. Within the nucleus, the intracellular domain of Notch1 can also limit ß-catenin induced transcription through the formation of a complex that requires its interaction with RBPjκ. We believe these mechanisms contribute to the robustness of cell-fate decisions by sharpening the distinction between opposing Notch/Wnt responses.

Integrins are required for synchronous ommatidial rotation in the Drosophila eye linking planar cell polarity signalling to the extracellular matrix.

Integrins mediate the anchorage between cells and their environment, the extracellular matrix (ECM), and form transmembrane links between the ECM and the cytoskeleton, a conserved feature throughout development and morphogenesis of epithelial organs. Here, we demonstrate that integrins and components of the ECM are required during the planar cell polarity (PCP) signalling-regulated cell movement of ommatidial rotation in the Drosophila eye. The loss-of-function mutations of integrins or ECM components cause defects in rotation, with mutant clusters rotating asynchronously compared to wild-type clusters. Initially, mutant clusters tend to rotate faster, and at later stages they fail to be synchronous with their neighbours, leading to aberrant rotation angles and resulting in a disorganized ommatidial arrangement in adult eyes. We further demonstrate that integrin localization changes dynamically during the rotation process. Our data suggest that core Frizzled/PCP factors, acting through RhoA and Rho kinase, regulate the function/activity of integrins and that integrins thus contribute to the complex interaction network of PCP signalling, cell adhesion and cytoskeletal elements required for a precise and synchronous 90° rotation movement.

LZTR1 is a regulator of RAS ubiquitination and signaling.

In genetic screens aimed at understanding drug resistance mechanisms in chronic myeloid leukemia cells, inactivation of the cullin 3 adapter protein-encoding leucine zipper-like transcription regulator 1 (LZTR1) gene led to enhanced mitogen-activated protein kinase (MAPK) pathway activity and reduced sensitivity to tyrosine kinase inhibitors. Knockdown of the Drosophila LZTR1 ortholog CG3711 resulted in a Ras-dependent gain-of-function phenotype. Endogenous human LZTR1 associates with the main RAS isoforms. Inactivation of LZTR1 led to decreased ubiquitination and enhanced plasma membrane localization of endogenous KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog). We propose that LZTR1 acts as a conserved regulator of RAS ubiquitination and MAPK pathway activation. Because LZTR1 disease mutations failed to revert loss-of-function phenotypes, our findings provide a molecular rationale for LZTR1 involvement in a variety of inherited and acquired human disorders.

Prickle is phosphorylated by Nemo and targeted for degradation to maintain Prickle/Spiny-legs isoform balance during planar cell polarity establishment.

Planar cell polarity (PCP) instructs tissue patterning in a wide range of organisms from fruit flies to humans. PCP signaling coordinates cell behavior across tissues and is integrated by cells to couple cell fate identity with position in a developing tissue. In the fly eye, PCP signaling is required for the specification of R3 and R4 photoreceptors based upon their positioning relative to the dorso-ventral axis. The 'core' PCP pathway involves the asymmetric localization of two distinct membrane-bound complexes, one containing Frizzled (Fz, required in R3) and the other Van Gogh (Vang, required in R4). Inhibitory interactions between the cytosolic components of each complex reinforce asymmetric localization. Prickle (Pk) and Spiny-legs (Pk-Sple) are two antagonistic isoforms of the prickle (pk) gene and are cytoplasmic components of the Vang complex. The balance between their levels is critical for tissue patterning, with Pk-Sple being the major functional isoform in the eye. Here we uncover a post-translational role for Nemo kinase in limiting the amount of the minor isoform Pk. We identified Pk as a Nemo substrate in a genome-wide in vitro band-shift screen. In vivo, nemo genetically interacts with pkpk but not pksple and enhances PCP defects in the eye and leg. Nemo phosphorylation limits Pk levels and is required specifically in the R4 photoreceptor like the major isoform, Pk-Sple. Genetic interaction and biochemical data suggest that Nemo phosphorylation of Pk leads to its proteasomal degradation via the Cullin1/SkpA/Slmb complex. dTAK and Homeodomain interacting protein kinase (Hipk) may also act together with Nemo to target Pk for degradation, consistent with similar observations in mammalian studies. Our results therefore demonstrate a mechanism to maintain low levels of the minor Pk isoform, allowing PCP complexes to form correctly and specify cell fate.

Components of Intraflagellar Transport Complex A Function Independently of the Cilium to Regulate Canonical Wnt Signaling in Drosophila.

The development of multicellular organisms requires the precisely coordinated regulation of an evolutionarily conserved group of signaling pathways. Temporal and spatial control of these signaling cascades is achieved through networks of regulatory proteins, segregation of pathway components in specific subcellular compartments, or both. In vertebrates, dysregulation of primary cilia function has been strongly linked to developmental signaling defects, yet it remains unclear whether cilia sequester pathway components to regulate their activation or cilia-associated proteins directly modulate developmental signaling events. To elucidate this question, we conducted an RNAi-based screen in Drosophila non-ciliated cells to test for cilium-independent loss-of-function phenotypes of ciliary proteins in developmental signaling pathways. Our results show no effect on Hedgehog signaling. In contrast, our screen identified several cilia-associated proteins as functioning in canonical Wnt signaling. Further characterization of specific components of Intraflagellar Transport complex A uncovered a cilia-independent function in potentiating Wnt signals by promoting ß-catenin/Armadillo activity.

Planar polarity: converting a morphogen gradient into cellular polarity.

Epithelial cells are polarized within the apico-basal and planar axes. The latter - planar cell polarity - requires long-range regulation of orientation as well as short-range, cell-to-cell realignment through feedback loops. New insights into the long-range, gradient-type regulation reveal how a kinase translates the morphogen gradient input into cellular orientation.

Wnt-Notch signalling crosstalk in development and disease.

The Notch and Wnt pathways are two of only a handful of highly conserved signalling pathways that control cell-fate decisions during animal development (Pires-daSilva and Sommer in Nat Rev Genet 4: 39-49, 2003). These two pathways are required together to regulate many aspects of metazoan development, ranging from germ layer patterning in sea urchins (Peter and Davidson in Nature 474: 635-639, 2011) to the formation and patterning of the fly wing (Axelrod et al in Science 271:1826-1832, 1996; Micchelli et al in Development 124:1485-1495, 1997; Rulifson et al in Nature 384:72-74, 1996), the spacing of the ciliated cells in the epidermis of frog embryos (Collu et al in Development 139:4405-4415, 2012) and the maintenance and turnover of the skin, gut lining and mammary gland in mammals (Clayton et al in Nature 446:185-189, 2007; Clevers in Cell 154:274-284, 2013; Doupe et al in Dev Cell 18:317-323, 2010; Lim et al in Science 342:1226-1230, 2013; Lowell et al in Curr Biol 10:491-500, 2000; van et al in Nature 435:959-963, 2005; Yin et al in Nat Methods 11:106-112, 2013). In addition, many diseases, including several cancers, are caused by aberrant signalling through the two pathways (Bolós et al in Endocr Rev 28: 339-363, 2007; Clevers in Cell 127: 469-480, 2006). In this review, we will outline the two signalling pathways, describe the different points of interaction between them, and cover how these interactions influence development and disease.

Dishevelled limits Notch signalling through inhibition of CSL.

Notch and Wnt are highly conserved signalling pathways that are used repeatedly throughout animal development to generate a diverse array of cell types. However, they often have opposing effects on cell-fate decisions with each pathway promoting an alternate outcome. Commonly, a cell receiving both signals exhibits only Wnt pathway activity. This suggests that Wnt inhibits Notch activity to promote a Wnt-ON/Notch-OFF output; but what might underpin this Notch regulation is not understood. Here, we show that Wnt acts via Dishevelled to inhibit Notch signalling, and that this crosstalk regulates cell-fate specification in vivo during Xenopus development. Mechanistically, Dishevelled binds and directly inhibits CSL transcription factors downstream of Notch receptors, reducing their activity. Furthermore, our data suggest that this crosstalk mechanism is conserved between vertebrate and invertebrate homologues. Thus, we identify a dual function for Dishevelled as an inhibitor of Notch signalling and an activator of the Wnt pathway that sharpens the distinction between opposing Wnt and Notch responses, allowing for robust cell-fate decisions.

Is there more to Wnt signalling in breast cancer than stabilisation of beta-catenin?

Increased Wnt signalling has been implicated in the aetiology of many different human cancers, including breast cancers. In most cases, Wnt signalling is thought to drive tumourigenesis through the stabilisation of cytosolic beta-catenin and the subsequent changes in the expression of T-cell factor (TCF)-dependent genes. However, this is not necessarily the only mechanism, as Wnt proteins can signal through a number of different intracellular signalling pathways. The ongoing work from Nancy Hynes' laboratory continues to highlight this latter possibility.

Notch activation induces Akt signaling via an autocrine loop to prevent apoptosis in breast epithelial cells.

The Notch pathway is aberrantly activated in a wide range of cancers, including breast carcinoma, and is required to maintain the transformed phenotype of many of these tumors. Notch signaling contributes to the transformed phenotype, in part, by preventing apoptosis in response to many different stimuli. However, it is unclear how Notch activation can lead to a general suppression of apoptosis. We show here that Notch signaling induced an autocrine signaling loop that activates Akt in breast epithelial cells. This activation of Akt was necessary for Notch-induced protection against apoptosis in the nontransformed breast epithelial cell line MCF10A. Moreover, inhibiting Notch signaling in breast cancer cells induced a decrease in Akt activity and an increase in sensitivity to apoptosis. Finally, the inhibition of ASK1 by Akt was responsible for the protection from apoptosis induced by DNA damage, as it prevented c-Jun NH(2)-terminal kinase-mediated phosphorylation and activation of p53.

Cooperation between Wnt and Notch signalling in human breast cancer.

The Wnt and Notch signalling pathways play major roles in mammary gland development and tumourigenesis. During development, these pathways have opposing effects. However, in a recent paper Ayyanan and coworkers show that expression of Wnt1 is sufficient to transform primary human mammary epithelial cells, and that this is in part due to activation of the Notch pathway. This indicates that during tumourigenesis the two pathways cooperate. Here we ask why activation of Wnt signalling alone is sufficient to cause transformation; whether there is evidence for inhibitory crosstalk between the pathways during tumourigenesis; and whether cooperation between these pathways occurs in other forms of cancer.