Expanded directly binds conserved regions of Fat to restrain growth via the Hippo pathway.

The Hippo pathway is a conserved and critical regulator of tissue growth. The FERM protein Expanded is a key signaling hub that promotes activation of the Hippo pathway, thereby inhibiting the transcriptional co-activator Yorkie. Previous work identified the polarity determinant Crumbs as a primary regulator of Expanded. Here, we show that the giant cadherin Fat also regulates Expanded directly and independently of Crumbs. We show that direct binding between Expanded and a highly conserved region of the Fat cytoplasmic domain recruits Expanded to the apicolateral junctional zone and stabilizes Expanded. In vivo deletion of Expanded binding regions in Fat causes loss of apical Expanded and promotes tissue overgrowth. Unexpectedly, we find Fat can bind its ligand Dachsous via interactions of their cytoplasmic domains, in addition to the known extracellular interactions. Importantly, Expanded is stabilized by Fat independently of Dachsous binding. These data provide new mechanistic insights into how Fat regulates Expanded, and how Hippo signaling is regulated during organ growth.

RASSF8-mediated transport of Echinoid via the exocyst promotes Drosophila wing elongation and epithelial ordering.

Cell-cell junctions are dynamic structures that maintain cell cohesion and shape in epithelial tissues. During development, junctions undergo extensive rearrangements to drive the epithelial remodelling required for morphogenesis. This is particularly evident during axis elongation, where neighbour exchanges, cell-cell rearrangements and oriented cell divisions lead to large-scale alterations in tissue shape. Polarised vesicle trafficking of junctional components by the exocyst complex has been proposed to promote junctional rearrangements during epithelial remodelling, but the receptors that allow exocyst docking to the target membranes remain poorly understood. Here, we show that the adherens junction component Ras Association domain family 8 (RASSF8) is required for the epithelial re-ordering that occurs during Drosophila pupal wing proximo-distal elongation. We identify the exocyst component Sec15 as a RASSF8 interactor. Loss of RASSF8 elicits cytoplasmic accumulation of Sec15 and Rab11-containing vesicles. These vesicles also contain the nectin-like homophilic adhesion molecule Echinoid, the depletion of which phenocopies the wing elongation and epithelial packing defects observed in RASSF8 mutants. Thus, our results suggest that RASSF8 promotes exocyst-dependent docking of Echinoid-containing vesicles during morphogenesis.

Salt inducible kinases as novel Notch interactors in the developing Drosophila retina.

Developmental processes require strict regulation of proliferation, differentiation and patterning for the generation of final organ size. Aberrations in these fundamental events are critically important in tumorigenesis and cancer progression. Salt inducible kinases (Siks) are evolutionarily conserved genes involved in diverse biological processes, including salt sensing, metabolism, muscle, cartilage and bone formation, but their role in development remains largely unknown. Recent findings implicate Siks in mitotic control, and in both tumor suppression and progression. Using a tumor model in the Drosophila eye, we show that perturbation of Sik function exacerbates tumor-like tissue overgrowth and metastasis. Furthermore, we show that both Drosophila Sik genes, Sik2 and Sik3, function in eye development processes. We propose that an important target of Siks may be the Notch signaling pathway, as we demonstrate genetic interaction between Siks and Notch pathway members. Finally, we investigate Sik expression in the developing retina and show that Sik2 is expressed in all photoreceptors, basal to cell junctions, while Sik3 appears to be expressed specifically in R3/R4 cells in the developing eye. Combined, our data suggest that Sik genes are important for eye tissue specification and growth, and that their dysregulation may contribute to tumor formation.

Casein kinase 1 family proteins promote Slimb-dependent Expanded degradation.

Hippo signalling integrates diverse stimuli related to epithelial architecture to regulate tissue growth and cell fate decisions. The Hippo kinase cascade represses the growth-promoting transcription co-activator Yorkie. The FERM protein Expanded is one of the main upstream Hippo signalling regulators in Drosophila as it promotes Hippo kinase signalling and directly inhibits Yorkie. To fulfil its function, Expanded is recruited to the plasma membrane by the polarity protein Crumbs. However, Crumbs-mediated recruitment also promotes Expanded turnover via a phosphodegron-mediated interaction with a Slimb/ß-TrCP SCF E3 ligase complex. Here, we show that the Casein Kinase 1 (CKI) family is required for Expanded phosphorylation. CKI expression promotes Expanded phosphorylation and interaction with Slimb/ß-TrCP. Conversely, CKI depletion in S2 cells impairs Expanded degradation downstream of Crumbs. In wing imaginal discs, CKI loss leads to elevated Expanded and Crumbs levels. Thus, phospho-dependent Expanded turnover ensures a tight coupling of Hippo pathway activity to epithelial architecture.

Mechanical strain regulates the Hippo pathway in Drosophila.

Animal cells are thought to sense mechanical forces via the transcriptional co-activators YAP (or YAP1) and TAZ (or WWTR1), the sole Drosophila homolog of which is named Yorkie (Yki). In mammalian cells in culture, artificial mechanical forces induce nuclear translocation of YAP and TAZ. Here, we show that physiological mechanical strain can also drive nuclear localisation of Yki and activation of Yki target genes in the Drosophila follicular epithelium. Mechanical strain activates Yki by stretching the apical domain, reducing the concentration of apical Crumbs, Expanded, Kibra and Merlin, and reducing apical Hippo kinase dimerisation. Overexpressing Hippo kinase to induce ectopic activation in the cytoplasm is sufficient to prevent Yki nuclear localisation even in flattened follicle cells. Conversely, blocking Hippo signalling in warts clones causes Yki nuclear localisation even in columnar follicle cells. We find no evidence for involvement of other pathways, such as Src42A kinase, in regulation of Yki. Finally, our results in follicle cells appear generally applicable to other tissues, as nuclear translocation of Yki is also readily detectable in other flattened epithelial cells such as the peripodial epithelium of the wing imaginal disc, where it promotes cell flattening.

Stable MOB1 interaction with Hippo/MST is not essential for development and tissue growth control.

The Hippo tumor suppressor pathway is essential for development and tissue growth control, encompassing a core cassette consisting of the Hippo (MST1/2), Warts (LATS1/2), and Tricornered (NDR1/2) kinases together with MOB1 as an important signaling adaptor. However, it remains unclear which regulatory interactions between MOB1 and the different Hippo core kinases coordinate development, tissue growth, and tumor suppression. Here, we report the crystal structure of the MOB1/NDR2 complex and define key MOB1 residues mediating MOB1's differential binding to Hippo core kinases, thereby establishing MOB1 variants with selective loss-of-interaction. By studying these variants in human cancer cells and Drosophila, we uncovered that MOB1/Warts binding is essential for tumor suppression, tissue growth control, and development, while stable MOB1/Hippo binding is dispensable and MOB1/Trc binding alone is insufficient. Collectively, we decrypt molecularly, cell biologically, and genetically the importance of the diverse interactions of Hippo core kinases with the pivotal MOB1 signal transducer.The Hippo tumor suppressor pathway is essential for development and tissue growth control. Here the authors employ a multi-disciplinary approach to characterize the interactions of the three Hippo kinases with the signaling adaptor MOB1 and show how they differently affect development, tissue growth and tumor suppression.

Meru couples planar cell polarity with apical-basal polarity during asymmetric cell division.

Polarity is a shared feature of most cells. In epithelia, apical-basal polarity often coexists, and sometimes intersects with planar cell polarity (PCP), which orients cells in the epithelial plane. From a limited set of core building blocks (e.g. the Par complexes for apical-basal polarity and the Frizzled/Dishevelled complex for PCP), a diverse array of polarized cells and tissues are generated. This suggests the existence of little-studied tissue-specific factors that rewire the core polarity modules to the appropriate conformation. In Drosophila sensory organ precursors (SOPs), the core PCP components initiate the planar polarization of apical-basal determinants, ensuring asymmetric division into daughter cells of different fates. We show that Meru, a RASSF9/RASSF10 homologue, is expressed specifically in SOPs, recruited to the posterior cortex by Frizzled/Dishevelled, and in turn polarizes the apical-basal polarity factor Bazooka (Par3). Thus, Meru belongs to a class of proteins that act cell/tissue-specifically to remodel the core polarity machinery.

Zyxin antagonizes the FERM protein expanded to couple F-actin and Yorkie-dependent organ growth.

BACKGROUND: Coordinated multicellular growth during development is achieved by the sensing of spatial and nutritional boundaries. The conserved Hippo (Hpo) signaling pathway has been proposed to restrict tissue growth by perceiving mechanical constraints through actin cytoskeleton networks. The actin-associated LIM proteins Zyxin (Zyx) and Ajuba (Jub) have been linked to the control of tissue growth via regulation of Hpo signaling, but the study of Zyx has been hampered by a lack of genetic tools. RESULTS: We generated a zyx mutant in Drosophila using TALEN endonucleases and used this to show that Zyx antagonizes the FERM-domain protein Expanded (Ex) to control tissue growth, eye differentiation, and F-actin accumulation. Zyx membrane targeting promotes the interaction between the transcriptional co-activator Yorkie (Yki) and the transcription factor Scalloped (Sd), leading to activation of Yki target gene expression and promoting tissue growth. Finally, we show that Zyx's growth-promoting function is dependent on its interaction with the actin-associated protein Enabled (Ena) via a conserved LPPPP motif and is antagonized by Capping Protein (CP). CONCLUSIONS: Our results show that Zyx is a functional antagonist of Ex in growth control and establish a link between actin filament polymerization and Yki activity.

Crumbs promotes expanded recognition and degradation by the SCF(Slimb/ß-TrCP) ubiquitin ligase.

In epithelial tissues, growth control depends on the maintenance of proper architecture through apicobasal polarity and cell-cell contacts. The Hippo signaling pathway has been proposed to sense tissue architecture and cell density via an intimate coupling with the polarity and cell contact machineries. The apical polarity protein Crumbs (Crb) controls the activity of Yorkie (Yki)/Yes-activated protein, the progrowth target of the Hippo pathway core kinase cassette, both in flies and mammals. The apically localized Four-point-one, Ezrin, Radixin, Moesin domain protein Expanded (Ex) regulates Yki by promoting activation of the kinase cascade and by directly tethering Yki to the plasma membrane. Crb interacts with Ex and promotes its apical localization, thereby linking cell polarity with Hippo signaling. We show that, as well as repressing Yki by recruiting Ex to the apical membrane, Crb promotes phosphorylation-dependent ubiquitin-mediated degradation of Ex. We identify Skp/Cullin/F-box(Slimb/ß-transducin repeats-containing protein) (SCF(Slimb/ß-TrCP)) as the E3 ubiquitin ligase complex responsible for Ex degradation. Thus, Crb is part of a homeostatic mechanism that promotes Ex inhibition of Yki, but also limits Ex activity by inducing its degradation, allowing precise tuning of Yki function.

The fetal mouse is a sensitive genotoxicity model that exposes lentiviral-associated mutagenesis resulting in liver oncogenesis.

Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis.

Salt-inducible kinases regulate growth through the Hippo signalling pathway in Drosophila.

The specification of tissue size during development involves the coordinated action of many signalling pathways responding to organ-intrinsic signals, such as morphogen gradients, and systemic cues, such as nutrient status. The conserved Hippo (Hpo) pathway, which promotes both cell-cycle exit and apoptosis, is a major determinant of size control. The pathway core is a kinase cassette, comprising the kinases Hpo and Warts (Wts) and the scaffold proteins Salvador (Sav) and Mats, which inactivates the pro-growth transcriptional co-activator Yorkie (Yki). We performed a split-TEV-based genome-wide RNAi screen for modulators of Hpo signalling. We characterize the Drosophila salt-inducible kinases (Sik2 and Sik3) as negative regulators of Hpo signalling. Activated Sik kinases increase Yki target expression and promote tissue overgrowth through phosphorylation of Sav at Ser 413. As Sik kinases have been implicated in nutrient sensing, this suggests a link between the Hpo pathway and systemic growth control.

The elongation rate of RNA polymerase II in zebrafish and its significance in the somite segmentation clock.

A gene expression oscillator called the segmentation clock controls somite segmentation in the vertebrate embryo. In zebrafish, the oscillatory transcriptional repressor genes her1 and her7 are crucial for genesis of the oscillations, which are thought to arise from negative autoregulation of these genes. The period of oscillation is predicted to depend on delays in the negative-feedback loop, including, most importantly, the transcriptional delay - the time taken to make each molecule of her1 or her7 mRNA. her1 and her7 operate in parallel. Loss of both gene functions, or mutation of her1 combined with knockdown of Hes6, which we show to be a binding partner of Her7, disrupts segmentation drastically. However, mutants in which only her1 or her7 is functional show only mild segmentation defects and their oscillations have almost identical periods. This is unexpected because the her1 and her7 genes differ greatly in length. We use transgenic zebrafish to measure the RNA polymerase II elongation rate, for the first time, in the intact embryo. This rate is unexpectedly rapid, at 4.8 kb/minute at 28.5°C, implying that, for both genes, the time taken for transcript elongation is insignificant compared with other sources of delay, explaining why the mutants have similar clock periods. Our computational model shows how loss of her1 or her7 can allow oscillations to continue with unchanged period but with reduced amplitude and impaired synchrony, as manifested in the in situ hybridisation patterns of the single mutants.

Delta1 expression, cell cycle exit, and commitment to a specific secretory fate coincide within a few hours in the mouse intestinal stem cell system.

The stem cells of the small intestine are multipotent: they give rise, via transit-amplifying cell divisions, to large numbers of columnar absorptive cells mixed with much smaller numbers of three different classes of secretory cells--mucus-secreting goblet cells, hormone-secreting enteroendocrine cells, and bactericide-secreting Paneth cells. Notch signaling is known to control commitment to a secretory fate, but why are the secretory cells such a small fraction of the population, and how does the diversity of secretory cell types arise? Using the mouse as our model organism, we find that secretory cells, and only secretory cells, pass through a phase of strong expression of the Notch ligand Delta1 (Dll1). Onset of this Dll1 expression coincides with a block to further cell division and is followed in much less than a cell cycle time by expression of Neurog3--a marker of enteroendocrine fate--or Gfi1--a marker of goblet or Paneth cell fate. By conditional knock-out of Dll1, we confirm that Delta-Notch signaling controls secretory commitment through lateral inhibition. We infer that cells stop dividing as they become committed to a secretory fate, while their neighbors continue dividing, explaining the final excess of absorptive over secretory cells. Our data rule out schemes in which cells first become committed to be secretory, and then diversify through subsequent cell divisions. A simple mathematical model shows how, instead, Notch signaling may simultaneously govern the commitment to be secretory and the choice between alternative modes of secretory differentiation.

Notch signaling, the segmentation clock, and the patterning of vertebrate somites.

The Notch signaling pathway has multifarious functions in the organization of the developing vertebrate embryo. One of its most fundamental roles is in the emergence of the regular pattern of somites that will give rise to the musculoskeletal structures of the trunk. The parts it plays in the early operation of the segmentation clock and the later definition and differentiation of the somites are beginning to be understood.

Oncostatin M receptor-beta mutations underlie familial primary localized cutaneous amyloidosis.

Familial primary localized cutaneous amyloidosis (FPLCA) is an autosomal-dominant disorder associated with chronic skin itching and deposition of epidermal keratin filament-associated amyloid material in the dermis. FPLCA has been mapped to 5p13.1-q11.2, and by candidate gene analysis, we identified missense mutations in the OSMR gene, encoding oncostatin M-specific receptor beta (OSMRbeta), in three families. OSMRbeta is a component of the oncostatin M (OSM) type II receptor and the interleukin (IL)-31 receptor, and cultured FPLCA keratinocytes showed reduced activation of Jak/STAT, MAPK, and PI3K/Akt pathways after OSM or IL-31 cytokine stimulation. The pathogenic amino acid substitutions are located within the extracellular fibronectin type III-like (FNIII) domains, regions critical for receptor dimerization and function. OSM and IL-31 signaling have been implicated in keratinocyte cell proliferation, differentiation, apoptosis, and inflammation, but our OSMR data in individuals with FPLCA represent the first human germline mutations in this cytokine receptor complex and provide new insight into mechanisms of skin itching.

Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice.

Gene therapy by use of integrating vectors carrying therapeutic transgene sequences offers the potential for a permanent cure of genetic diseases by stable vector insertion into the patients' chromosomes. However, three cases of T cell lymphoproliferative disease have been identified almost 3 years after retrovirus gene therapy for X-linked severe combined immune deficiency. In two of these cases vector insertion into the LMO2 locus was implicated in leukemogenesis, demonstrating that a more profound understanding is required of the genetic and molecular effects imposed on the host by vector integration or transgene expression. In vivo models to test for retro- and lentiviral vector safety prior to clinical application are therefore needed. Here we present a high incidence of lentiviral vector-associated tumorigenesis following in utero and neonatal gene transfer in mice. This system may provide a highly sensitive model to investigate integrating vector safety prior to clinical application.

Permanent phenotypic correction of hemophilia B in immunocompetent mice by prenatal gene therapy.

Hemophilia B, also known as Christmas disease, arises from mutations in the factor IX (F9) gene. Its treatment in humans, by recombinant protein substitution, is expensive, thus limiting its application to intermittent treatment in bleeding episodes and prophylaxis during surgery; development of inhibitory antibodies is an associated hazard. This study demonstrates permanent therapeutic correction of his disease without development of immune reactions by introduction of an HIV-based lentiviral vector encoding the human factor IX protein into the fetal circulation of immunocompetent hemophiliac and normal outbred mice. Plasma factor IX antigen remained at around 9%, 13%, and 16% of normal in the 3 hemophilia B mice, respectively, until the last measurement at 14 months. Substantial improvement in blood coagulability as measured by coagulation assay was seen in all 3 mice and they rapidly stopped bleeding after venipuncture. No humoral or cellular immunity against the protein, elevation of serum liver enzymes, or vector spread to the germline or maternal circulation were detected.