Microscopic multifrequency MR elastography for mapping viscoelasticity in zebrafish.

PURPOSE: The zebrafish (Danio rerio) has become an important animal model in a wide range of biomedical research disciplines. Growing awareness of the role of biomechanical properties in tumor progression and neuronal development has led to an increasing interest in the noninvasive mapping of the viscoelastic properties of zebrafish by elastography methods applicable to bulky and nontranslucent tissues. METHODS: Microscopic multifrequency MR elastography is introduced for mapping shear wave speed (SWS) and loss angle (φ) as markers of stiffness and viscosity of muscle, brain, and neuroblastoma tumors in postmortem zebrafish with 60 µm in-plane resolution. Experiments were performed in a 7 Tesla MR scanner at 1, 1.2, and 1.4 kHz driving frequencies. RESULTS: Detailed zebrafish viscoelasticity maps revealed that the midbrain region (SWS = 3.1 ± 0.7 m/s, φ = 1.2 ± 0.3 radian [rad]) was stiffer and less viscous than telencephalon (SWS = 2.6 ± 0. 5 m/s, φ = 1.4 ± 0.2 rad) and optic tectum (SWS = 2.6 ± 0.5 m/s, φ = 1.3 ± 0.4 rad), whereas the cerebellum (SWS = 2.9 ± 0.6 m/s, φ = 0.9 ± 0.4 rad) was stiffer but less viscous than both (all p < .05). Overall, brain tissue (SWS = 2.9 ± 0.4 m/s, φ = 1.2 ± 0.2 rad) had similar stiffness but lower viscosity values than muscle tissue (SWS = 2.9 ± 0.5 m/s, φ = 1.4 ± 0.2 rad), whereas neuroblastoma (SWS = 2.4 ± 0.3 m/s, φ = 0.7 ± 0.1 rad, all p < .05) was the softest and least viscous tissue. CONCLUSION: Microscopic multifrequency MR elastography-generated maps of zebrafish show many details of viscoelasticity and resolve tissue regions, of great interest in neuromechanical and oncological research and for which our study provides first reference values.

Secreted Frizzled-related Protein 2 (sFRP2) Redirects Non-canonical Wnt Signaling from Fz7 to Ror2 during Vertebrate Gastrulation.

Convergent extension movements during vertebrate gastrulation require a balanced activity of non-canonical Wnt signaling pathways, but the factors regulating this interplay on the molecular level are poorly characterized. Here we show that sFRP2, a member of the secreted frizzled-related protein (sFRP) family, is required for morphogenesis and papc expression during Xenopus gastrulation. We further provide evidence that sFRP2 redirects non-canonical Wnt signaling from Frizzled 7 (Fz7) to the receptor tyrosine kinase-like orphan receptor 2 (Ror2). During this process, sFRP2 promotes Ror2 signal transduction by stabilizing Wnt5a-Ror2 complexes at the membrane, whereas it inhibits Fz7 signaling, probably by blocking Fz7 receptor endocytosis. The cysteine-rich domain of sFRP2 is sufficient for Ror2 activation, and related sFRPs can substitute for this function. Notably, direct interaction of the two receptors via their cysteine-rich domains also promotes Ror2-mediated papc expression but inhibits Fz7 signaling. We propose that sFRPs can act as a molecular switch, channeling the signal input for different non-canonical Wnt pathways during vertebrate gastrulation.

In vivo analysis of formation and endocytosis of the Wnt/ß-catenin signaling complex in zebrafish embryos.

After activation by Wnt/ß-Catenin ligands, a multi-protein complex assembles at the plasma membrane as membrane-bound receptors and intracellular signal transducers are clustered into the so-called Lrp6-signalosome [Corrected]. However, the mechanism of signalosome formation and dissolution is yet not clear. Our imaging studies of live zebrafish embryos show that the signalosome is a highly dynamic structure. It is continuously assembled by Dvl2-mediated recruitment of the transducer complex to the activated receptors and partially disassembled by endocytosis. We find that, after internalization, the ligand-receptor complex and the transducer complex take separate routes. The Wnt-Fz-Lrp6 complex follows a Rab-positive endocytic path. However, when still bound to the transducer complex, Dvl2 forms intracellular aggregates. We show that this endocytic process is not only essential for ligand-receptor internalization but also for signaling. The µ2-subunit of the endocytic Clathrin adaptor Ap2 interacts with Dvl2 to maintain its stability during endocytosis. Blockage of Ap2µ2 function leads to Dvl2 degradation, inhibiton of signalosome formation at the plasma membrane and, consequently, reduction of signaling. We conclude that Ap2µ2-mediated endocytosis is important to maintain Wnt/ß-catenin signaling in vertebrates.

Tyrosine phosphorylation of LRP6 by Src and Fer inhibits Wnt/ß-catenin signalling.

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) function as transmembrane receptors to transduce Wnt signals. A key mechanism for signalling is Wnt-induced serine/threonine phosphorylation at conserved PPPSPxS motifs in the LRP6 cytoplasmic domain, which promotes pathway activation. Conserved tyrosine residues are positioned close to all PPPSPxS motifs, which suggests they have a functional significance. Using a cell culture-based cDNA expression screen, we identified the non-receptor tyrosine kinases Src and Fer as novel LRP6 modifiers. Both Src and Fer associate with LRP6 and phosphorylate LRP6 directly. In contrast to the known PPPSPxS Ser/Thr kinases, tyrosine phosphorylation by Src and Fer negatively regulates LRP6-Wnt signalling. Epistatically, they function upstream of ß-catenin to inhibit signalling and in agreement with a negative role in regulating LRP6, MEF cells lacking these kinases show enhanced Wnt signalling. Wnt3a treatment of cells enhances tyrosine phosphorylation of endogenous LRP6 and, mechanistically, Src reduces cell surface LRP6 levels and disrupts LRP6 signalosome formation. Interestingly, CK1γ inhibits Fer-induced LRP6 phosphorylation, suggesting a mechanism whereby CK1γ acts to de-represses inhibitory LRP6 tyrosine phosphorylation. We propose that LRP6 tyrosine phosphorylation by Src and Fer serves a negative regulatory function to prevent over-activation of Wnt signalling at the level of the Wnt receptor, LRP6.

Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer.

DNA amplifications in cancer do not only harbor oncogenes. We sought to determine whether passenger coamplifications could create collateral therapeutic vulnerabilities. Through an analysis of >3,000 cancer genomes followed by the interrogation of CRISPR-Cas9 loss-of-function screens across >700 cancer cell lines, we determined that passenger coamplifications are accompanied by distinct dependency profiles. In a proof-of-principle study, we demonstrate that the coamplification of the bona fide passenger gene DEAD-Box Helicase 1 (DDX1) creates an increased dependency on the mTOR pathway. Interaction proteomics identified tricarboxylic acid (TCA) cycle components as previously unrecognized DDX1 interaction partners. Live-cell metabolomics highlighted that this interaction could impair TCA activity, which in turn resulted in enhanced mTORC1 activity. Consequently, genetic and pharmacologic disruption of mTORC1 resulted in pronounced cell death in vitro and in vivo. Thus, structurally linked coamplification of a passenger gene and an oncogene can result in collateral vulnerabilities. SIGNIFICANCE: We demonstrate that coamplification of passenger genes, which were largely neglected in cancer biology in the past, can create distinct cancer dependencies. Because passenger coamplifications are frequent in cancer, this principle has the potential to expand target discovery in oncology. This article is featured in Selected Articles from This Issue, p. 384.

Rab5-mediated endocytosis of activin is not required for gene activation or long-range signalling in Xenopus.

Morphogen gradients provide positional cues for cell fate specification and tissue patterning during embryonic development. One important aspect of morphogen function, the mechanism by which long-range signalling occurs, is still poorly understood. In Xenopus, members of the TGF-beta family such as the nodal-related proteins and activin act as morphogens to induce mesoderm and endoderm. In an effort to understand the mechanisms and dynamics of morphogen gradient formation, we have used fluorescently labelled activin to study ligand distribution and Smad2/Smad4 bimolecular fluorescence complementation (BiFC) to analyse, in a quantitative manner, the cellular response to induction. Our results indicate that labelled activin travels exclusively through the extracellular space and that its range is influenced by numbers of type II activin receptors on responding cells. Inhibition of endocytosis, by means of a dominant-negative form of Rab5, blocks internalisation of labelled activin, but does not affect the ability of cells to respond to activin and does not significantly influence signalling range. Together, our data indicate that long-range signalling in the early Xenopus embryo, in contrast to some other developmental systems, occurs through extracellular movement of ligand. Signalling range is not regulated by endocytosis, but is influenced by numbers of cognate receptors on the surfaces of responding cells.

A Reproducible Bioprinted 3D Tumor Model Serves as a Preselection Tool for CAR T Cell Therapy Optimization.

Chimeric antigen receptor (CAR) T cell performance against solid tumors in mouse models and clinical trials is often less effective than predicted by CAR construct selection in two-dimensional (2D) cocultures. Three-dimensional (3D) solid tumor architecture is likely to be crucial for CAR T cell efficacy. We used a three-dimensional (3D) bioprinting approach for large-scale generation of highly reproducible 3D human tumor models for the test case, neuroblastoma, and compared these to 2D cocultures for evaluation of CAR T cells targeting the L1 cell adhesion molecule, L1CAM. CAR T cells infiltrated the model, and both CAR T and tumor cells were viable for long-term experiments and could be isolated as single-cell suspensions for whole-cell assays quantifying CAR T cell activation, effector function and tumor cell cytotoxicity. L1CAM-specific CAR T cell activation by neuroblastoma cells was stronger in the 3D model than in 2D cocultures, but neuroblastoma cell lysis was lower. The bioprinted 3D neuroblastoma model is highly reproducible and allows detection and quantification of CAR T cell tumor infiltration, representing a superior in vitro analysis tool for preclinical CAR T cell characterization likely to better select CAR T cells for in vivo performance than 2D cocultures.

Fast, In Vivo Model for Drug-Response Prediction in Patients with B-Cell Precursor Acute Lymphoblastic Leukemia.

Only half of patients with relapsed B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) currently survive with standard treatment protocols. Predicting individual patient responses to defined drugs prior to application would help therapy stratification and could improve survival. With the purpose to aid personalized targeted treatment approaches, we developed a human-zebrafish xenograft (ALL-ZeFiX) assay to predict drug response in a patient in 5 days. Leukemia blast cells were pericardially engrafted into transiently immunosuppressed Danio rerio embryos, and engrafted embryos treated for the test case, venetoclax, before single-cell dissolution for quantitative whole blast cell analysis. Bone marrow blasts from patients with newly diagnosed or relapsed BCP-ALL were successfully expanded in 60% of transplants in immunosuppressed zebrafish embryos. The response of BCP-ALL cell lines to venetoclax in ALL-ZeFiX assays mirrored responses in 2D cultures. Venetoclax produced varied responses in patient-derived BCP-ALL grafts, including two results mirroring treatment responses in two refractory BCP-ALL patients treated with venetoclax. Here we demonstrate proof-of-concept for our 5-day ALL-ZeFiX assay with primary patient blasts and the test case, venetoclax, which after expanded testing for further targeted drugs could support personalized treatment decisions within the clinical time window for decision-making.

Visualizing protein interactions by bimolecular fluorescence complementation in Xenopus.

Bimolecular fluorescence complementation (BiFC) provides a simple and direct way to visualise protein-protein interactions in vivo and in real-time. In this article, we describe methods by which one can implement this approach in embryos of the South African claw-toed frog Xenopus laevis. We have made use of Venus, an improved version of yellow fluorescent protein (YFP), so as to achieve rapid detection of protein interactions. To suppress spontaneous interactions between the N- and C-terminal fragments of Venus, a point mutation (T153M) was introduced into the N-terminal fragment. We have used this reagent to monitor signalling by members of the transforming growth factor type beta family in cells of the Xenopus embryo.

Visualizing long-range movement of the morphogen Xnr2 in the Xenopus embryo.

One way in which cells acquire positional information during embryonic development is by measuring the local concentration of a signaling factor, or morphogen, that is secreted by an organizing center . The ways in which morphogen gradients are established, particularly in vertebrates, remain obscure, although various suggestions have been made for the mechanisms by which signaling molecules traverse fields of cells. These include simple diffusion, "cytonemes", filopodia, "argosomes", and "transcytosis". In this study, we use a functional EGFP-tagged ligand to visualize long-range signaling in the Xenopus embryo in real time. Our results show that the TGF-beta family member Xnr2 is secreted efficiently from embryonic cells, and a new method of tissue recombination allows us to investigate the way in which the morphogen traverses multiple cell diameters. This reveals that Xnr2 exerts long-range effects by diffusing rapidly through the extracellular milieu of nonexpressing cells. No evidence has been obtained for long-range signaling through cytonemes, filopodia, argosomes, or transcytosis. In demonstrating that long-range signaling in the early Xenopus embryo occurs by diffusion rather than by these alternative routes, our results suggest that different morphogens in different developmental contexts use different means of transport.

A phase II investigator-initiated pilot study with low-dose cyclosporine A for the treatment of articular involvement in primary Sjögren's syndrome.

UNLABELLED: This study aims to investigate the efficacy and safety of low-dose cyclosporine A (CyA) in patients with primary Sjögren's syndrome (pSS) and articular involvement. This phase II open-label clinical study included 30 patients meeting the American-European Consensus group criteria for pSS with active joint involvement under stable symptomatic therapy. Treatment consisted of approximately 2 mg kg(-1) body weight of CyA day(-1) over a period of 16 weeks. The primary endpoint was defined as a reduction in the number of painful and/or swollen joints at end of treatment (EOT). Secondary endpoints included the changes in general health, sicca symptoms, European League Against Rheumatism (EULAR) Sjögren's Syndrome Disease Activity Index (ESSDAI), arthrosonography, and safety profile. At baseline (BL), the mean number of tender joints (68 count) was 16.2 (±13.2) and at EOT 10.4 (±11.9; p = 0.002). The mean number of swollen joints (66 counts) was reduced from 3.2 (±3.3) at BL to 1.3 (±3.2) at EOT (p < 0.001). Overall, 21 (70 %) and 13 (43.3 %) patients had a reduction of two or more tender and swollen joints, respectively, in the 68/66 joint counts. The disease activity score (DAS28) showed a statistically and clinically meaningful decrease over the 16-week period of treatment. Treatment was well tolerated, and adverse events were consistent with the known safety profile of CyA (e.g., hypertension, headache). In this pilot study, promising effects of low-dose CyA treatment on articular involvement were observed in patients with pSS justifying further controlled studies in this indication. No new or unexpected safety observations were made. TRIAL REGISTRATION: Low-Dose Cyclosporin A in Primary Sjögren Syndrome (CYPRESS), ClinicalTrials.gov Identifier: NCT01693393 .

Filopodia-based Wnt transport during vertebrate tissue patterning.

Paracrine Wnt/ß-catenin signalling is important during developmental processes, tissue regeneration and stem cell regulation. Wnt proteins are morphogens, which form concentration gradients across responsive tissues. Little is known about the transport mechanism for these lipid-modified signalling proteins in vertebrates. Here we show that Wnt8a is transported on actin-based filopodia to contact responding cells and activate signalling during neural plate formation in zebrafish. Cdc42/N-Wasp regulates the formation of these Wnt-positive filopodia. Enhanced formation of filopodia increases the effective signalling range of Wnt by facilitating spreading. Consistently, reduction in filopodia leads to a restricted distribution of the ligand and a limited signalling range. Using a simulation, we provide evidence that such a short-range transport system for Wnt has a long-range signalling function. Indeed, we show that a filopodia-based transport system for Wnt8a controls anteroposterior patterning of the neural plate during vertebrate gastrulation.

The Tale of the Three Brothers - Shh, Wnt, and Fgf during Development of the Thalamus.

The thalamic complex is an essential part of the brain that requires a combination of specialized activities to attain its final complexity. In the following review we will describe the induction process of the mid-diencephalic organizer (MDO) where three different signaling pathways merge: Wnt, Shh, and Fgf. Here, we dissect the function of each signaling pathway in the thalamus in chronological order of their appearance. First we describe the Wnt mediated induction of the MDO and compartition of the caudal forebrain, then the Shh mediated determination of proneural gene expression before discussing recent progress in characterizing Fgf function during thalamus development. Then, we focus on transcription factors, which are regulated by these pathways and which play a pivotal role in neurogenesis in the thalamus. The three signaling pathways act together in a strictly regulated chronology to orchestrate the development of the entire thalamus.

Nuclear accumulation of Smad complexes occurs only after the midblastula transition in Xenopus.

Activin and the Nodal-related proteins induce mesendodermal tissues during Xenopus development. These signals act through specific receptors to cause the phosphorylation, at their carboxyl termini, of Smad2 and Smad3. The phosphorylated Smad proteins form heteromeric complexes with Smad4 and translocate into the nucleus to activate the transcription, after the midblastula transition, of target genes such as Xbra and goosecoid (gsc). In this paper we use bimolecular fluorescence complementation (BiFC) to study complex formation between Smad proteins both in vivo and in response to exogenous proteins. The technique has allowed us to detect Smad2-Smad4 heteromeric interactions during normal Xenopus development and Smad2 and Smad4 homo- and heteromers in isolated Xenopus blastomeres. Smad2-Smad2 and Smad2-Smad4 complexes accumulate rapidly in the nuclei of responding cells following Activin treatment, whereas Smad4 homomeric complexes remain cytoplasmic. When cells divide, Smad2-Smad4 complexes associate with chromatin, even in the absence of ligand. Our observation that Smad2-Smad4 complexes accumulate in the nucleus only after the midblastula transition, irrespective of the stage at which cells were treated with Activin, may shed light on the mechanisms of developmental timing.