Live Zebrafish Embryos Research Articles

Structural effects of naphthalimide-based fluorescent sensor for hydrogen sulfide and imaging in live zebrafish.

Hydrogen sulfide (H2S) is an important biological messenger, but few biologically-compatible methods are available for its detection in aqueous solution. Herein, we report a highly water-soluble naphthalimide-based fluorescent probe (L1), which is a highly versatile building unit that absorbs and emits at long wavelengths and is selective for hydrogen sulfide over cysteine, glutathione, and other reactive sulfur, nitrogen, and oxygen species in aqueous solution. We describe turn-on fluorescent probes based on azide group reduction on the fluorogenic ‘naphthalene’ moiety to fluorescent amines and intracellular hydrogen sulfide detection without the use of an organic solvent. L1 and L2 were synthetically modified to functional groups with comparable solubility on the N-imide site, showing a marked change in turn-on fluorescent intensity in response to hydrogen sulfide in both PBS buffer and living cells. The probes were readily employed to assess intracellular hydrogen sulfide level changes by imaging endogenous hydrogen sulfide signal in RAW264.7 cells incubated with L1 and L2. Expanding the use of L1 to complex and heterogeneous biological settings, we successfully visualized hydrogen sulfide detection in the yolk, brain and spinal cord of living zebrafish embryos, thereby providing a powerful approach for live imaging for investigating chemical signaling in complex multicellular systems.

Extracellular interactions and ligand degradation shape the nodal morphogen gradient.

The correct distribution and activity of secreted signaling proteins called morphogens is required for many developmental processes. Nodal morphogens play critical roles in embryonic axis formation in many organisms. Models proposed to generate the Nodal gradient include diffusivity, ligand processing, and a temporal activation window. But how the Nodal morphogen gradient forms in vivo remains unclear. Here, we have measured in vivo for the first time, the binding affinity of Nodal ligands to their major cell surface receptor, Acvr2b, and to the Nodal inhibitor, Lefty, by fluorescence cross-correlation spectroscopy. We examined the diffusion coefficient of Nodal ligands and Lefty inhibitors in live zebrafish embryos by fluorescence correlation spectroscopy. We also investigated the contribution of ligand degradation to the Nodal gradient. We show that ligand clearance via degradation shapes the Nodal gradient and correlates with its signaling range. By computational simulations of gradient formation, we demonstrate that diffusivity, extra-cellular interactions, and selective ligand destruction collectively shape the Nodal morphogen gradient.

Single neuron morphology in vivo with confined primed conversion.

Unraveling the structural organization of neurons can provide fundamental insights into brain function. However, visualizing neurite morphology in vivo remains difficult due to the high density and complexity of neural packing in the nervous system. Detailed analysis of neural morphology requires distinction of closely neighboring, highly intricate cellular structures such as neurites with high contrast. Green-to-red photoconvertible fluorescent proteins have become powerful tools to optically highlight molecular and cellular structures for developmental and cell biological studies. Yet, selective labeling of single cells of interest in vivo has been precluded due to inefficient photoconversion when using high intensity, pulsed, near-infrared laser sources that are commonly applied for achieving axially confined two-photon (2P) fluorescence excitation. Here we describe a novel optical mechanism, "confined primed conversion,"which employs continuous dual-wave illumination to achieve confined green-to-red photoconversion of single cells in live zebrafish embryos. Confined primed conversion exhibits wide applicability and this chapter specifically elaborates on employing this imaging modality to analyze neural morphology of optically targeted single neurons in the developing zebrafish brain.

Assembly and Maintenance of Myofibrils in Striated Muscle.

In this chapter, we present the current knowledge on de novo assembly, growth, and dynamics of striated myofibrils, the functional architectural elements developed in skeletal and cardiac muscle. The data were obtained in studies of myofibrils formed in cultures of mouse skeletal and quail myotubes, in the somites of living zebrafish embryos, and in mouse neonatal and quail embryonic cardiac cells. The comparative view obtained revealed that the assembly of striated myofibrils is a three-step process progressing from premyofibrils to nascent myofibrils to mature myofibrils. This process is specified by the addition of new structural proteins, the arrangement of myofibrillar components like actin and myosin filaments with their companions into so-called sarcomeres, and in their precise alignment. Accompanying the formation of mature myofibrils is a decrease in the dynamic behavior of the assembling proteins. Proteins are most dynamic in the premyofibrils during the early phase and least dynamic in mature myofibrils in the final stage of myofibrillogenesis. This is probably due to increased interactions between proteins during the maturation process. The dynamic properties of myofibrillar proteins provide a mechanism for the exchange of older proteins or a change in isoforms to take place without disassembling the structural integrity needed for myofibril function. An important aspect of myofibril assembly is the role of actin-nucleating proteins in the formation, maintenance, and sarcomeric arrangement of the myofibrillar actin filaments. This is a very active field of research. We also report on several actin mutations that result in human muscle diseases.

Compact plane illumination plugin device to enable light sheet fluorescence imaging of multi-cellular organisms on an inverted wide-field microscope.

We developed a compact plane illumination plugin (PIP) device which enabled plane illumination and light sheet fluorescence imaging on a conventional inverted microscope. The PIP device allowed the integration of microscope with tunable laser sheet profile, fast image acquisition, and 3-D scanning. The device is both compact, measuring approximately 15 by 5 by 5 cm, and cost-effective, since we employed consumer electronics and an inexpensive device molding method. We demonstrated that PIP provided significant contrast and resolution enhancement to conventional microscopy through imaging different multi-cellular fluorescent structures, including 3-D branched cells in vitro and live zebrafish embryos. Imaging with the integration of PIP greatly reduced out-of-focus contamination and generated sharper contrast in acquired 2-D plane images when compared with the stand-alone inverted microscope. As a result, the dynamic fluid domain of the beating zebrafish heart was clearly segmented and the functional monitoring of the heart was achieved. Furthermore, the enhanced axial resolution established by thin plane illumination of PIP enabled the 3-D reconstruction of the branched cellular structures, which leads to the improvement on the functionality of the wide field microscopy.

3D Light-Sheet Fluorescence Microscopy of Cranial Neurons and Vasculature during Zebrafish Embryogenesis.

Precise 3D spatial mapping of cells and their connections within living tissues is required to fully understand developmental processes and neural activities. Zebrafish embryos are relatively small and optically transparent, making them the vertebrate model of choice for live in vivo imaging. However, embryonic brains cannot be imaged in their entirety by confocal or two-photon microscopy due to limitations in optical range and scanning speed. Here, we use light-sheet fluorescence microscopy to overcome these limitations and image the entire head of live transgenic zebrafish embryos. We simultaneously imaged cranial neurons and blood vessels during embryogenesis, generating comprehensive 3D maps that provide insight into the coordinated morphogenesis of the nervous system and vasculature during early development. In addition, blood cells circulating through the entire head, vagal and cardiac vasculature were also visualized at high resolution in a 3D movie. These data provide the foundation for the construction of a complete 4D atlas of zebrafish embryogenesis and neural activity.

The caveolin-cavin system plays a conserved and critical role in mechanoprotection of skeletal muscle.

Dysfunction of caveolae is involved in human muscle disease, although the underlying molecular mechanisms remain unclear. In this paper, we have functionally characterized mouse and zebrafish models of caveolae-associated muscle disease. Using electron tomography, we quantitatively defined the unique three-dimensional membrane architecture of the mature muscle surface. Caveolae occupied around 50% of the sarcolemmal area predominantly assembled into multilobed rosettes. These rosettes were preferentially disassembled in response to increased membrane tension. Caveola-deficient cavin-1(-/-) muscle fibers showed a striking loss of sarcolemmal organization, aberrant T-tubule structures, and increased sensitivity to membrane tension, which was rescued by muscle-specific Cavin-1 reexpression. In vivo imaging of live zebrafish embryos revealed that loss of muscle-specific Cavin-1 or expression of a dystrophy-associated Caveolin-3 mutant both led to sarcolemmal damage but only in response to vigorous muscle activity. Our findings define a conserved and critical role in mechanoprotection for the unique membrane architecture generated by the caveolin-cavin system.

Quinoxaline-Based Polymer Dots with Ultrabright Red to Near-Infrared Fluorescence for In Vivo Biological Imaging.

This article describes the design and synthesis of quinoxaline-based semiconducting polymer dots (Pdots) that exhibit near-infrared fluorescence, ultrahigh brightness, large Stokes shifts, and excellent cellular targeting capability. We also introduced fluorine atoms and long alkyl chains into polymer backbones and systematically investigated their effect on the fluorescence quantum yields of Pdots. These new series of quinoxaline-based Pdots have a fluorescence quantum yield as high as 47% with a Stokes shift larger than 150 nm. Single-particle analysis reveals that the average per-particle brightness of the Pdots is at least 6 times higher than that of the commercially available quantum dots. We further demonstrated the use of this new class of quinoxaline-based Pdots for effective and specific cellular and subcellular labeling without any noticeable nonspecific binding. Moreover, the cytotoxicity of Pdots were evaluated on HeLa cells and zebrafish embryos to demonstrate their great biocompatibility. By taking advantage of their extreme brightness and minimal cytotoxicity, we performed, for the first time, in vivo microangiography imaging on living zebrafish embryos using Pdots. These quinoxaline-based NIR-fluorescent Pdots are anticipated to find broad use in a variety of in vitro and in vivo biological research.

Systemic Fluorescence Imaging of Zebrafish Glycans with Bioorthogonal Chemistry

AbstractVertebrate glycans constitute a large, important, and dynamic set of post‐translational modifications that are notoriously difficult to manipulate and image. Although the chemical reporter strategy has been used in conjunction with bioorthogonal chemistry to image the external glycosylation state of live zebrafish and detect tumor‐associated glycans in mice, the ability to image glycans systemically within a live organism has remained elusive. Here, we report a method that combines the metabolic incorporation of a cyclooctyne‐functionalized sialic acid derivative with a ligation reaction of a fluorogenic tetrazine, allowing for the imaging of sialylated glycoconjugates within live zebrafish embryos.

Systemic Fluorescence Imaging of Zebrafish Glycans with Bioorthogonal Chemistry.

Vertebrate glycans constitute a large, important, and dynamic set of post-translational modifications that are notoriously difficult to manipulate and image. Although the chemical reporter strategy has been used in conjunction with bioorthogonal chemistry to image the external glycosylation state of live zebrafish and detect tumor-associated glycans in mice, the ability to image glycans systemically within a live organism has remained elusive. Here, we report a method that combines the metabolic incorporation of a cyclooctyne-functionalized sialic acid derivative with a ligation reaction of a fluorogenic tetrazine, allowing for the imaging of sialylated glycoconjugates within live zebrafish embryos.

Fluorescent Probe HKSOX-1 for Imaging and Detection of Endogenous Superoxide in Live Cells and In Vivo.

Superoxide anion radical (O2(•-)) is undoubtedly the most important primary reactive oxygen species (ROS) found in cells, whose formation and fate are intertwined with diverse physiological and pathological processes. Here we report a highly sensitive and selective O2(•-) detecting strategy involving O2(•-) cleavage of an aryl trifluoromethanesulfonate group to yield a free phenol. We have synthesized three new O2(•-) fluorescent probes (HKSOX-1, HKSOX-1r for cellular retention, and HKSOX-1m for mitochondria-targeting) which exhibit excellent selectivity and sensitivity toward O2(•-) over a broad range of pH, strong oxidants, and abundant reductants found in cells. In confocal imaging, flow cytometry, and 96-well microplate assay, HKSOX-1r has been robustly applied to detect O2(•-) in multiple cellular models, such as inflammation and mitochondrial stress. Additionally, our probes can be efficiently applied to visualize O2(•-) in intact live zebrafish embryos. These probes open up exciting opportunities for unmasking the roles of O2(•-) in health and disease.

BioSpotlight / Citations

BioTechniquesVol. 58, No. 5 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsNathan S. Blow & Nijsje DormanNathan S. BlowSearch for more papers by this author & Nijsje DormanSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000114282AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Fade resistant culturingWhen it comes to fluorescent proteins (FP) and their usage in cell biology, photostability is a key factor. If fluorescence decreases rapidly or is low to begin with, obtaining biologically relevant data from an experiment can be difficult. In an effort to better understand photostability issues associated with cells in culture, Konstantin Lukyanov and colleagues from the Institute of Bioorganic Chemistry in Moscow, Russia systematically evaluated the influence of growth conditions and media composition on green fluorescent protein (GFP) stability. The authors examined the components in Ham's F12 culture medium that might enhance GFP photostability, finding that decreased concentrations of riboflavin and pyridoxine but increased concentrations of other components compared to DMEM were key to generating greater FP photostability. They also showed that cell density and serum concentration have profound effects on photostability. Taken together, Lukyanov and his co-authors provide a set of guidelines for researchers looking to optimize FP photostability for their cell culture experiments and ultimately get the most meaningful data out of FP experiments.See “Influence of cell growth conditions and medium composition on EGFP photostability in live cells”Finding that better mateAlthough the number of sequenced genomes is quickly rising, there are still many species for which a reference genome remains missing. When it comes to de novo genome sequencing in these cases, mate-pair reads are essential to obtaining the necessary scaffolding data that will ultimately result in a genome assembly, but generating good mate-pair libraries for this purpose remains a challenging task. In this issue of BioTechniques, a team of researchers from the RIKEN Center for Developmental Biology, led by Shigehiro Kuraku, describe a series of modifications for one of the most popular mate-pair library kits, the Nextera Mate Pair Sample Prep Kit, that should enhance both yield and efficiency. Through intensive DNA shearing to decrease insert sizes, sequencing with >150 cycles, and the use of variable tagmentation conditions, Kuraku and his colleagues were able to demonstrate clear increases in overall sequence continuity while at the same time reducing the costs associated with generating mate-pair libraries.See “Optimization and cost-saving in tagmentation-based mate-pair library preparation and sequencing”Visualizing proteome-level metabolic dynamicsMost methods for monitoring global protein metabolism in cell culture lack spatial information or require cell fixation. Previously, Wei et al. described a live imaging technique based on stimulated Raman scattering (SRS) detection of the carbon-deuterium bond in cells metabolically labeled with deuterated amino acids. While promising, the method was relatively insensitive and could only assay protein synthesis. Now the authors have improved the sensitivity and, by examining the vibrational signature of methyl groups in pre-existing protein pools, their approach can also monitor protein degradation. Even pulse-chase experiments are possible, by sequentially exposing cells to non-branched and then branched deuterated amino acids (the two subtypes can be distinguished by SRS). The new method works in cell culture, live brain tissue slices, zebrafish embryos, and mice. It should be especially well-suited for exploring protein synthesis related to memory formation and protein metabolism in animal models of disease.L. Wei et al. 2015. Imaging complex protein metabolism in live organisms by stimulated Raman scattering microscopy with isotope labeling. ACS Chem Biol. 10(3):901-8.Tissue-specific genome editing in zebrafishLoss-of-function studies in zebrafish have come a long way from random mutagenesis screens. However, CRISPR/Cas9-based genome editing results in global inactivation of the target gene. Ablain et al. now report a vector system for tissue-specific gene inactivation in zebrafish. In their integratable vector, which is microinjected into one-cell-stage embryos, the guide RNA is ubiquitously expressed, while Cas9 is driven by a tissue-specific promoter. Tests with an erythrocyte-specific promoter showed blood-lineage knockout of an enzyme involved in heme biosynthesis in both F0 embryos and F1 offspring selected for presence of the integrated vector. Moreover, experiments involving a vector targeting p53 confirmed the system is also applicable to genetic suppressor screens. The authors anticipate that their vector will be compatible with targeting multiple genes (via incorporation of several guide RNA units) and that the overall approach may be transferable to other vertebrate models such as Xenopus and medaka.J. Albain et al. 2015. A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish. Dev Cell. 32(6):756-64.FiguresReferencesRelatedDetails Vol. 58, No. 5 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print May 2015 Information© 2015 Author(s)PDF download

Bayesian model selection applied to the analysis of fluorescence correlation spectroscopy data of fluorescent proteins in vitro and in vivo.

Fluorescence correlation spectroscopy (FCS) is a powerful technique to investigate molecular dynamics with single molecule sensitivity. In particular, in the life sciences it has found widespread application using fluorescent proteins as molecularly specific labels. However, FCS data analysis and interpretation using fluorescent proteins remains challenging due to typically low signal-to-noise ratio of FCS data and correlated noise in autocorrelated data sets. As a result, naive fitting procedures that ignore these important issues typically provide similarly good fits for multiple competing models without clear distinction of which model is preferred given the signal-to-noise ratio present in the data. Recently, we introduced a Bayesian model selection procedure to overcome this issue with FCS data analysis. The method accounts for the highly correlated noise that is present in FCS data sets and additionally penalizes model complexity to prevent over interpretation of FCS data. Here, we apply this procedure to evaluate FCS data from fluorescent proteins assayed in vitro and in vivo. Consistent with previous work, we demonstrate that model selection is strongly dependent on the signal-to-noise ratio of the measurement, namely, excitation intensity and measurement time, and is sensitive to saturation artifacts. Under fixed, low intensity excitation conditions, physical transport models can unambiguously be identified. However, at excitation intensities that are considered moderate in many studies, unwanted artifacts are introduced that result in nonphysical models to be preferred. We also determined the appropriate fitting models of a GFP tagged secreted signaling protein, Wnt3, in live zebrafish embryos, which is necessary for the investigation of Wnt3 expression and secretion in development. Bayes model selection therefore provides a robust procedure to determine appropriate transport and photophysical models for fluorescent proteins when appropriate models are provided, to help detect and eliminate experimental artifacts in solution, cells, and in living organisms.

Investigation of the Cellular and Molecular Mechanisms of Facial Branchiomotor Neuron Migration

A fundamental feature of brain development is the migration of neurons from their place of birth to the location where they will assemble and integrate into a neural circuit. We have recently reported that one facial branchiomotor neuron (FBMN) can influence the migration of another FBMN, defining this cell movement as a collective migration. As with any collective migration, cell‐to‐cell contact is required for one cell's influence on another. To determine if this was the case, we imaged the migration of membrane‐mCherry‐expressing FBMNs in live zebrafish embryos. We find that some neurons display persistent, sustained migration in one direction (posterior), whereas other FBMNs stall for short periods of time. We noticed that FBMNs stalled in migration shortly after cell‐to‐cell contact with neuron in their migratory path. This is reminiscent of contact inhibition of locomotion (CIL) observed for many other cell types including neural crest cells. We therefore, quantified the speed and direction of neuron movement before and after neuron‐to‐neuron cell contacts. Consistent with this, centrosomes, which often re‐orient in the direction of migration, were often found on the anterior side of migrating FBMNs. Previous work has shown that planar cell polarity (PCP) genes are essential for FBMN migration. Here we show that PCP mutant neurons migrate in random directions suggesting that PCP proteins are not required for migration per se, but are required for persistent directed movements. Taken together these data suggest the possibility that PCP proteins control contact‐dependent cell polarization in FBMN migration.

Intraciliary Calcium Oscillations Initiate Vertebrate Left-Right Asymmetry

SummaryBackgroundBilateral symmetry during vertebrate development is broken at the left-right organizer (LRO) by ciliary motility and the resultant directional flow of extracellular fluid. However, how ciliary motility is perceived and transduced into asymmetrical intracellular signaling at the LRO remains controversial. Previous work has indicated that sensory cilia and polycystin-2 (Pkd2), a cation channel, are required for sensing ciliary motility, yet their function and the molecular mechanism linking both to left-right signaling cascades is unknown.ResultsHere, we report novel intraciliary calcium oscillations (ICOs) at the LRO that connect ciliary sensation of ciliary motility to downstream left-right signaling. Utilizing cilia-targeted genetically-encoded calcium indicators in live zebrafish embryos, we show that ICOs depend on Pkd2 and are left-biased at the LRO in response to ciliary motility. Asymmetric ICOs occur with onset of LRO ciliary motility, thus representing the earliest known LR asymmetric molecular signal. Suppression of ICOs using a cilia-targeted calcium sink demonstrates that they are essential for LR development.ConclusionsThese findings demonstrate that intraciliary calcium initiates LR development and identify cilia as a functional ion signaling compartment connecting ciliary motility and flow to molecular LR signaling.

Nanoscale Imaging of Caveolin-1 Membrane Domains In Vivo

Light microscopy enables noninvasive imaging of fluorescent species in biological specimens, but resolution is generally limited by diffraction to ~200–250 nm. Many biological processes occur on smaller length scales, highlighting the importance of techniques that can image below the diffraction limit and provide valuable single-molecule information. In recent years, imaging techniques have been developed which can achieve resolution below the diffraction limit. Utilizing one such technique, fluorescence photoactivation localization microscopy (FPALM), we demonstrated its ability to construct super-resolution images from single molecules in a living zebrafish embryo, expanding the realm of previous super-resolution imaging to a living vertebrate organism. We imaged caveolin-1 in vivo, in living zebrafish embryos. Our results demonstrate the successful image acquisition of super-resolution images in a living vertebrate organism, opening several opportunities to answer more dynamic biological questions in vivo at the previously inaccessible nanoscale.

Biocompatible photoresistant far-red emitting, fluorescent polymer probes, with near-infrared two-photon absorption, for living cell and zebrafish embryo imaging

Exogenous probes with far-red or near-infrared (NIR) two-photon absorption and fluorescence emission are highly desirable for deep tissue imaging while limiting autofluorescence. However, molecular probes exhibiting such properties are often hydrophobic. As an attractive alternative, we synthesized water-soluble polymer probes carrying multiple far-red fluorophores and demonstrated here their potential for live cell and zebrafish embryo imaging. First, at concentrations up to 10 μm, these polymer probes were not cytotoxic. They could efficiently label living HeLa cells, T lymphocytes and neurons at an optimal concentration of 0.5 μm. Moreover, they exhibited a high resistance to photobleaching in usual microscopy conditions. In addition, these polymer probes could be successfully used for in toto labeling and in vivo two-photon microscopy imaging of developing zebrafish embryos, with remarkable properties in terms of biocompatibility, internalization, diffusion, stability and wavelength emission range. The near-infrared two-photon absorption peak at 910 nm is particularly interesting since it does not excite the zebrafish endogenous fluorescence and is likely to enable long-term time-lapse imaging with limited photodamage.

High Resolution Imaging Reveals Hematopoietic Stem Cells in the Perivascular Niche Are Anchored to Mesenchymal Stromal Cells That Orient Their Divisions

Hematopoietic stem cells (HSC) reside in a highly structured microenvironment called the niche. There is two-way communication between a stem cell and its niche that determines important cell fate decisions. HSC must remain quiescent to persist throughout life but also divide and contribute progenitors that will replenish the blood supply. Although there have been a number of elegant studies that have imaged the mammalian bone marrow, we still lack a high-resolution real-time view of endogenous HSC behaviors and interactions within the niche. To overcome these challenges, we developed a transgenic zebrafish line that expresses GFP or mCherry in HSC. We generated this line using the previously described mouse Runx1 +23 kb intronic enhancer. We confirmed the purity of these stem cells by adult-to-adult limiting dilution transplantation with as few as one cell. Based on long-term multi-lineage engraftment, we estimated a stem cell purity of approximately 1/35, which is similar to the KSL (Kit+Sca1+Lin-) population in mouse. Using a novel embryo-to-embryo transplantation assay that is unique to zebrafish, we estimated an even higher stem cell purity of 1/2. These experiments have defined the most pure HSC population in the zebrafish. Using this novel transgenic reporter we have tracked HSC as they migrate in the live zebrafish embryo. This allowed us to image HSC as they interact with other cell types in their microenvironment, including endothelial cells and mesenchymal stromal cells. We have shown that a small group of endothelial cells remodel around a single HSC soon after it lodges in the niche. Recently, we have also found that a single stromal cell can anchor an HSC as it divides. In most cases, we observed that an HSC divides perpendicular to the stromal cell, with one daughter cell remaining attached to the stromal cell and the other migrating away. To gain a much higher resolution view of these cellular events than is possible with confocal microscopy we looked for an alternative approach. A combined method is called “Correlative Light and Electron Microscopy” (CLEM), and involves identification of cells by confocal microscopy, followed by processing of the same sample for EM scanning. We have applied this method by: 1) tracking endogenous HSC in the live embryo; 2) fixing the same embryo for serial block-face scanning EM; 3) reconstructing 3D models from high resolution serial EM sections. We used easily visible blood vessels as anatomical markers that allowed us to pinpoint a single cell in a relatively large block of scanned tissue. As expected, the identified HSC was round, had a distinctive large nucleus, scant cytoplasm, and ruffled membrane. The HSC was surrounded by a small group of 5-6 endothelial cells, as predicted from our confocal live imaging. However at this very high resolution (10 nm/pixel), we could see that only part of the HSC surface was contacted and wrapped by an endothelial cell. Other regions of the HSC surface were contacted by small endothelial cell protrusions. Much of the HSC surface was surrounded by a narrow extracellular space with endothelial and stromal cells lying opposite. Strikingly, we were able to identify the firm anchored attachment between a single stromal cell and HSC that we showed previously oriented the plane of division. By combining confocal live imaging of a novel zebrafish HSC reporter, and serial block-face scanning EM, we have created the first high-resolution 3D model of an endogenous stem cell in its niche. DisclosuresTamplin:Boston Children’s Hospital: Patents & Royalties. Zon:FATE Therapeutics, Inc: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Other; Scholar Rock: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Other; Stemgent: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Abstract 745: A non-redox reactive allosteric inhibitor of MAPK phosphatases with selective toxicity to human cancer cells

Abstract Mitogen-activated protein kinase phosphatases (MKPs) are members of a family of dual specificity phosphatases (DUSPs) that regulate cellular signaling homeostasis by dephosphorylating mitogen-activated kinases, key mediators of cell death and survival. The archetype MKP, DUSP1 (also termed MKP-1/CL100), is overexpressed in ovarian, breast, and prostate cancers and found elevated in the “oncogenic signature” of RAS mutant tumor cells. DUSP1 mediates cancer cell migration and invasion, and protects cells from apoptosis by clinically used antineoplastic agents and gamma-irradiation. Despite the evidence that DUSP1 is a promising anticancer target, efforts to discover small molecule inhibitors have been largely unsuccessful. The MKP active site is shallow and contains a catalytic cysteine that is sensitive to oxidation. Hence, prior discovery screens have mostly identified redox-reactive structures with promiscuous biological activities. Using a zebrafish chemical screen, we recently discovered a new DUSP inhibitory chemotype that has no redox-reactive structural features and that is thought to bind to an allosteric site adjacent to the catalytic cavity. Structure-activity relationship studies in transgenic zebrafish identified an analog (VNK-215) that retained DUSP1 and DUSP6 inhibitory activity but was well tolerated by the developing embryo. Here we hypothesized that VNK-215 could be selectively cytotoxic to cancer cells due to its ability to inhibit DUSP1 and lack of redox-reactive features. We found that in MDA-MB-231 human breast cancer cells, VNK-215 had apoptotic and antimigratory activity at concentrations that inhibited DUSP1 in mammalian cells, and induced phosphorylation of the DUSP1 substrates ERK, p38, and JNK. Studies in primary rat hepatocytes and live zebrafish embryos revealed that VNK-215, unlike previously described DUSP inhibitors, did not induce reactive oxygen species, mitochondrial damage, or toxicity. Our data suggest that the promiscuous activities of existing DUSP inhibitors are the cause of redox reactivity rather than target inhibition, and posit that DUSP1 is a druggable anticancer target for small molecule allosteric inhibitors. Citation Format: Laura Vollmer, Lawrence Vernetti, Ahmet Bakan, Vasiliy Korotchenko, Ivet Bahar, Billy Day, Michael Tsang, Andreas Vogt. A non-redox reactive allosteric inhibitor of MAPK phosphatases with selective toxicity to human cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 745. doi:10.1158/1538-7445.AM2014-745

In vivo analysis of formation and endocytosis of the Wnt/β-Catenin signaling complex in zebrafish embryos

After activation by Wnt/β-Catenin ligands, a multi-protein complex assembles at the clustering membrane-bound receptors and intracellular signal transducers into the so-called Lrp6-signalosome. 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/β-catenin signaling in vertebrates.