Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity.

Type 1 diabetes is characterized by the destruction of pancreatic ß cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional ß-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic ß cell mass from α cells.

Systemic Inflammation and Normocytic Anemia in DOCK11 Deficiency.

BACKGROUND: Increasing evidence links genetic defects affecting actin-regulatory proteins to diseases with severe autoimmunity and autoinflammation, yet the underlying molecular mechanisms are poorly understood. Dedicator of cytokinesis 11 (DOCK11) activates the small Rho guanosine triphosphatase (GTPase) cell division cycle 42 (CDC42), a central regulator of actin cytoskeleton dynamics. The role of DOCK11 in human immune-cell function and disease remains unknown. METHODS: We conducted genetic, immunologic, and molecular assays in four patients from four unrelated families who presented with infections, early-onset severe immune dysregulation, normocytic anemia of variable severity associated with anisopoikilocytosis, and developmental delay. Functional assays were performed in patient-derived cells, as well as in mouse and zebrafish models. RESULTS: We identified rare, X-linked germline mutations in DOCK11 in the patients, leading to a loss of protein expression in two patients and impaired CDC42 activation in all four patients. Patient-derived T cells did not form filopodia and showed abnormal migration. In addition, the patient-derived T cells, as well as the T cells from Dock11-knockout mice, showed overt activation and production of proinflammatory cytokines that were associated with an increased degree of nuclear translocation of nuclear factor of activated T cell 1 (NFATc1). Anemia and aberrant erythrocyte morphologic features were recapitulated in a newly generated dock11-knockout zebrafish model, and anemia was amenable to rescue on ectopic expression of constitutively active CDC42. CONCLUSIONS: Germline hemizygous loss-of-function mutations affecting the actin regulator DOCK11 were shown to cause a previously unknown inborn error of hematopoiesis and immunity characterized by severe immune dysregulation and systemic inflammation, recurrent infections, and anemia. (Funded by the European Research Council and others.).

Optimisation of pyrazolo[1,5-a]pyrimidin-7(4H)-one derivatives as novel Hsp90 C-terminal domain inhibitors against Ewing sarcoma.

Ewing sarcoma is the second most prevalent paediatric malignant bone tumour. In most cases, it is driven by the fusion oncoprotein EWS::FLI1, which acts as an aberrant transcription factor and dysregulates gene expression. EWS::FLI1 and a large number of downstream dysregulated proteins are Hsp90 client proteins, making Hsp90 an attractive target for the treatment of Ewing sarcoma. In this article, we report a new structural class of allosteric Hsp90 C-terminal domain (CTD) inhibitors based on the virtual screening hit TVS24, which showed antiproliferative activity in the SK-N-MC Ewing sarcoma cell line with an IC50 value of 15.9 ± 0.7 µM. The optimised compounds showed enhanced anticancer activity in the SK-N-MC cell line. Exposure of Ewing sarcoma cells to the most potent analogue 11c resulted in depletion of critical Hsp90 client proteins involved in cancer pathways such as EWS::FLI1, CDK4, RAF-1 and IGF1R, without inducing a heat shock response. The results of this study highlight Hsp90 CTD inhibitors as promising new agents for the treatment of Ewing sarcoma.

In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M.

Photoswitchable reagents are powerful tools for high-precision studies in cell biology. When these reagents are globally administered yet locally photoactivated in two-dimensional (2D) cell cultures, they can exert micron- and millisecond-scale biological control. This gives them great potential for use in biologically more relevant three-dimensional (3D) models and in vivo, particularly for studying systems with inherent spatiotemporal complexity, such as the cytoskeleton. However, due to a combination of photoswitch isomerization under typical imaging conditions, metabolic liabilities, and insufficient water solubility at effective concentrations, the in vivo potential of photoswitchable reagents addressing cytosolic protein targets remains largely unrealized. Here, we optimized the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based on the styrylbenzothiazole (SBT) scaffold that are nonresponsive to typical fluorescent protein imaging wavelengths and so enable multichannel imaging studies. We applied these reagents both to 3D organoids and tissue explants and to classic model organisms (zebrafish, clawed frog) in one- and two-protein imaging experiments, in which spatiotemporally localized illuminations allowed them to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in vivo with cellular precision and second-level resolution. These nanomolar, in vivo capable photoswitchable reagents should open up new dimensions for high-precision cytoskeleton research in cargo transport, cell motility, cell division, and development. More broadly, their design can also inspire similarly capable optical reagents for a range of cytosolic protein targets, thus bringing in vivo photopharmacology one step closer to general realization.

Microbiota promote secretory cell determination in the intestinal epithelium by modulating host Notch signaling.

Resident microbes promote many aspects of host development, although the mechanisms by which microbiota influence host tissues remain unclear. We showed previously that the microbiota is required for allocation of appropriate numbers of secretory cells in the zebrafish intestinal epithelium. Because Notch signaling is crucial for secretory fate determination, we conducted epistasis experiments to establish whether the microbiota modulates host Notch signaling. We also investigated whether innate immune signaling transduces microbiota cues via the Myd88 adaptor protein. We provide the first evidence that microbiota-induced, Myd88-dependent signaling inhibits host Notch signaling in the intestinal epithelium, thereby promoting secretory cell fate determination. These results connect microbiota activity via innate immune signaling to the Notch pathway, which also plays crucial roles in intestinal homeostasis throughout life and when impaired can result in chronic inflammation and cancer.

Photocaged Hoechst Enables Subnuclear Visualization and Cell Selective Staining of DNA in vivo.

Selective targeting of DNA by means of fluorescent labeling has become a mainstay in the life sciences. While genetic engineering serves as a powerful technique and allows the visualization of nucleic acid by using DNA-targeting fluorescent fusion proteins in a cell-type- and subcellular-specific manner, it relies on the introduction of foreign genes. On the other hand, DNA-binding small fluorescent molecules can be used without genetic engineering, but they are not spatially restricted. Herein, we report a photocaged version of the DNA dye Hoechst33342 (pcHoechst), which can be uncaged by using UV to blue light for the selective staining of chromosomal DNA in subnuclear regions of live cells. Expanding its application to a vertebrate model organism, we demonstrate uncaging in epithelial cells and short-term cell tracking in vivo in zebrafish. We envision pcHoechst as a valuable tool for targeting and interrogating DNA with precise spatiotemporal resolution in living cells and wild-type organisms.

Studying the Tumor Microenvironment in Zebrafish.

The tumor microenvironment significantly contributes to tumor initiation, progression, neo-angiogenesis, and metastasis, and a better understanding of the role of the different cellular players would facilitate the development of novel therapeutic strategies for cancer treatment. Towards this goal, intravital imaging is a powerful method to unravel interaction partners of tumor cells. Among vertebrate model organisms, zebrafish is uniquely suited for in vivo imaging studies. In recent years zebrafish has also become a valuable model in cancer research. In this chapter, we will summarize, how zebrafish has been used to characterize cells of the tumor microenvironment. We will cover both genetically engineered cancer models and xenograft models in zebrafish. The majority of work has been done on the role of innate immune cells and their role during tumor initiation and metastasis, but we will also cover studies focusing on adipocytes, fibroblasts, and endothelial cells. Taken together, we will highlight the versatile use of the zebrafish model for in vivo tumor microenvironment studies.

Discrete Notch signaling requirements in the specification of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) require multiple molecular inputs for proper specification, including activity of the Notch signaling pathway. A requirement for the Notch1 and dispensability of the Notch2 receptor has been demonstrated in mice, but the role of the remaining Notch receptors has not been investigated. Here, we demonstrate that three of the four Notch receptors are independently required for the specification of HSCs in the zebrafish. The orthologues of the murine Notch1 receptor, Notch1a and Notch1b, are each required intrinsically to fate HSCs, just prior to their emergence from aortic hemogenic endothelium. By contrast, the Notch3 receptor is required earlier within the developing somite to regulate HSC emergence in a non-cell-autonomous manner. Epistatic analyses demonstrate that Notch3 function lies downstream of Wnt16, which is required for HSC specification through its regulation of two Notch ligands, dlc and dld. Collectively, these findings demonstrate for the first time that multiple Notch signaling inputs are required to specify HSCs and that Notch3 performs a novel role within the somite to regulate the neighboring precursors of hemogenic endothelium.

Gata2b is a restricted early regulator of hemogenic endothelium in the zebrafish embryo.

The adult blood system is established by hematopoietic stem cells (HSCs), which arise during development from an endothelial-to-hematopoietic transition of cells comprising the floor of the dorsal aorta. Expression of aortic runx1 has served as an early marker of HSC commitment in the zebrafish embryo, but recent studies have suggested that HSC specification begins during the convergence of posterior lateral plate mesoderm (PLM), well before aorta formation and runx1 transcription. Further understanding of the earliest stages of HSC specification necessitates an earlier marker of hemogenic endothelium. Studies in mice have suggested that GATA2 might function at early stages within hemogenic endothelium. Two orthologs of Gata2 exist in zebrafish: gata2a and gata2b. Here, we report that gata2b expression initiates during the convergence of PLM, becoming restricted to emerging HSCs. We observe Notch-dependent gata2b expression within the hemogenic subcompartment of the dorsal aorta that is in turn required to initiate runx1 expression. Our results indicate that Gata2b functions within hemogenic endothelium from an early stage, whereas Gata2a functions more broadly throughout the vascular system.

Dual modality reflection mode optical coherence and photoacoustic microscopy using an akinetic sensor: publisher's note.

This publisher's note corrects an error in the funding section in Opt. Lett.42, 4319 (2017)OPLEDP0146-959210.1364/OL.42.004319.

Dual modality reflection mode optical coherence and photoacoustic microscopy using an akinetic sensor.

This Letter presents a novel dual modality reflection mode optical coherence and photoacoustic microscopy (OC-PAM) system. The optical coherence microscopy modality features a broadband source to accomplish 5 µm axial resolution. The photoacoustic microscopy modality uses a rigid akinetic Fabry-Perot etalon encapsulated in an optically transparent medium, which forms a 2 mm×11 mm translucent imaging window, permitting reflection mode dual modality imaging. After characterization, the OC-PAM system was applied to image zebrafish larvae in vivo, demonstrating its capability in biomedical imaging with complementary optical scattering and absorption contrasts by revealing morphology in the fish larvae.

A pH-sensitive Macromolecular Prodrug as TLR7/8 Targeting Immune Response Modifier.

The chemical synthesis and biological activity of novel functionalized imidazoquinoline derivatives (ImQ) to generate Toll-like receptor (TLR) 7/8 specific prodrugs are presented. In vivo activity of ImQs to induce inflammation was confirmed in zebrafish larvae. After covalent ligation to fully biodegradable polyphosphazenes (ImQ-polymer), the macromolecular prodrugs were designed to undergo intracellular pH-sensitive release of ImQs to induce inflammation through binding to endosomal TLR7/8 (danger signal). We showed ImQ dissociation from prodrugs at a pH 5 pointing towards endosomal prodrug degradability. ImQ-polymers strongly activated ovalbumin-specific T cells in murine splenocytes as shown by increased proliferation and expression of the IL-2 receptor (CD25) on CD8+ T cells accompanied by strong IFN-γ release. ImQ prodrugs presented here are suggested to form the basis of novel nanovaccines, for example, for intravenous or intratumoral cancer immunotherapeutic applications to trigger physiological antitumor immune responses.

Green-Light-Induced Inactivation of Receptor Signaling Using Cobalamin-Binding Domains.

Optogenetics and photopharmacology provide spatiotemporally precise control over protein interactions and protein function in cells and animals. Optogenetic methods that are sensitive to green light and can be used to break protein complexes are not broadly available but would enable multichromatic experiments with previously inaccessible biological targets. Herein, we repurposed cobalamin (vitamin B12) binding domains of bacterial CarH transcription factors for green-light-induced receptor dissociation. In cultured cells, we observed oligomerization-induced cell signaling for the fibroblast growth factor receptor 1 fused to cobalamin-binding domains in the dark that was rapidly eliminated upon illumination. In zebrafish embryos expressing fusion receptors, green light endowed control over aberrant fibroblast growth factor signaling during development. Green-light-induced domain dissociation and light-inactivated receptors will critically expand the optogenetic toolbox for control of biological processes.

Zebrafish jam-b2 Gal4-enhancer trap line recapitulates endogenous jam-b2 expression in extraocular muscles.

BACKGROUND: Members of the junctional adhesion molecule (JAM) family function as cell adhesion molecules and cell surface receptors. The zebrafish genome contains six different jam genes, and jam-b and jam-c were shown to be essential for myoblast fusion during skeletal muscle development. However, little is known about jam-b2 expression and function. RESULTS: We isolated the cDNA of zebrafish jam-b2. jam-b2 is expressed specifically in extraocular muscles (EOMs), jaw muscles, and pectoral fins in zebrafish larvae, but not in trunk muscles. The identified jam-b2 expression pattern is supported by the analysis of a zebrafish Gal4-enhancer trap line, in which the coding sequence of the transcriptional activator KalTA4 together with a Gal4-dependent UAS-mCherry expression cassette was inserted into the jam-b2 locus. Intercrosses with an UAS:EGFP strain proves the possibility for targeting transgene expression to EOMs, jaw muscles and fins. Finally, we characterized the concerted contraction pattern of EOMs in larvae performing an optokinetic response. CONCLUSIONS: The expression pattern of jam-b2 suggests that it may contribute different properties to EOMs, jaw muscles, and pectoral fins. The jam-b2:KalTA4-UAS-mCherry transgenic strain serves a dual role as both a reporter for these muscles and as a valuable genetic tool for targeting transgene expression to EOMs.

Senescence-associated ß-galactosidase staining over the lifespan differs in a short- and a long-lived fish species.

During the aging process, cells can enter cellular senescence, a state in which cells leave the cell cycle but remain viable. This mechanism is thought to protect tissues from propagation of damaged cells and the number of senescent cells has been shown to increase with age. The speed of aging determines the lifespan of a species and it varies significantly in different species. To assess the progress of cellular senescence during lifetime, we performed a comparative longitudinal study using histochemical detection of the senescence-associated beta-galactosidase as senescence marker to map the staining patterns in organs of the long-lived zebrafish and the short-lived turquoise killifish using light- and electron microscopy. We compared age stages corresponding to human stages of newborn, childhood, adolescence, adult and old age. We found tissue-specific but conserved signal patterns with respect to organ distribution. However, we found dramatic differences in the onset of tissue staining. The stained zebrafish organs show little to no signal at newborn age followed by a gradual increase in signal intensity, whereas the organs of the short-lived killifish show an early onset of staining already at newborn stage, which remains conspicuous at all age stages. The most prominent signal was found in liver, intestine, kidney and heart, with the latter showing the most prominent interspecies divergence in onset of staining and in staining intensity. In addition, we found staining predominantly in epithelial cells, some of which are post-mitotic, such as the intestinal epithelial lining. We hypothesize that the association of the strong and early-onset signal pattern in the short-lived killifish is consistent with a protective mechanism in a fast growing species. Furthermore, we believe that staining in post-mitotic cells may play a role in maintaining tissue integrity, suggesting different roles for cellular senescence during life.

Exploration and optimisation of structure-activity relationships of new triazole-based C-terminal Hsp90 inhibitors towards in vivo anticancer potency.

The development of new anticancer agents is one of the most urgent topics in drug discovery. Inhibition of molecular chaperone Hsp90 stands out as an approach that affects various oncogenic proteins in different types of cancer. These proteins rely on Hsp90 to obtain their functional structure, and thus Hsp90 is indirectly involved in the pathophysiology of cancer. However, the most studied ATP-competitive inhibition of Hsp90 at the N-terminal domain has proven to be largely unsuccessful clinically. Therefore, research has shifted towards Hsp90 C-terminal domain (CTD) inhibitors, which are also the focus of this study. Our recent discovery of compound C has provided us with a starting point for exploring the structure-activity relationship and optimising this new class of triazole-based Hsp90 inhibitors. This investigation has ultimately led to a library of 33 analogues of C that have suitable physicochemical properties and several inhibit the growth of different cancer types in the low micromolar range. Inhibition of Hsp90 was confirmed by biophysical and cellular assays and the binding epitopes of selected inhibitors were studied by STD NMR. Furthermore, the most promising Hsp90 CTD inhibitor 5x was shown to induce apoptosis in breast cancer (MCF-7) and Ewing sarcoma (SK-N-MC) cells while inducing cause cell cycle arrest in MCF-7 cells. In MCF-7 cells, it caused a decrease in the levels of ERα and IGF1R, known Hsp90 client proteins. Finally, 5x was tested in zebrafish larvae xenografted with SK-N-MC tumour cells, where it limited tumour growth with no obvious adverse effects on normal zebrafish development.

Comparative transcriptomics coupled to developmental grading via transgenic zebrafish reporter strains identifies conserved features in neutrophil maturation.

Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defense. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here we show, by generating and analysing transgenic zebrafish strains representing distinct neutrophil differentiation stages, a high-resolution transcriptional profile of neutrophil maturation. We link gene expression at each stage to characteristic transcription factors, including C/ebp-ß, which is important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human samples confirms high molecular similarity of immature stages and discriminates zebrafish-specific from pan-species gene signatures. Applying the pan-species neutrophil maturation signature to RNA-sequencing data from human neuroblastoma patients reveals association between metastatic tumor cell infiltration in the bone marrow and an overall increase in mature neutrophils. Our detailed neutrophil maturation atlas thus provides a valuable resource for studying neutrophil function at different stages across species in health and disease.

A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations.

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

Silencer-delimited transgenesis: NRSE/RE1 sequences promote neural-specific transgene expression in a NRSF/REST-dependent manner.

BACKGROUND: We have investigated a simple strategy for enhancing transgene expression specificity by leveraging genetic silencer elements. The approach serves to restrict transgene expression to a tissue of interest - the nervous system in the example provided here - thereby promoting specific/exclusive targeting of discrete cellular subtypes. Recent innovations are bringing us closer to understanding how the brain is organized, how neural circuits function, and how neurons can be regenerated. Fluorescent proteins enable mapping of the 'connectome', optogenetic tools allow excitable cells to be short-circuited or hyperactivated, and targeted ablation of neuronal subtypes facilitates investigations of circuit function and neuronal regeneration. Optimally, such toolsets need to be expressed solely within the cell types of interest as off-site expression makes establishing causal relationships difficult. To address this, we have exploited a gene 'silencing' system that promotes neuronal specificity by repressing expression in non-neural tissues. This methodology solves non-specific background issues that plague large-scale enhancer trap efforts and may provide a means of leveraging promoters/enhancers that otherwise express too broadly to be of value for in vivo manipulations. RESULTS: We show that a conserved neuron-restrictive silencer element (NRSE) can function to restrict transgene expression to the nervous system. The neuron-restrictive silencing factor/repressor element 1 silencing transcription factor (NRSF/REST) transcriptional repressor binds NRSE/repressor element 1 (RE1) sites and silences gene expression in non-neuronal cells. Inserting NRSE sites into transgenes strongly biased expression to neural tissues. NRSE sequences were effective in restricting expression of bipartite Gal4-based 'driver' transgenes within the context of an enhancer trap and when associated with a defined promoter and enhancer. However, NRSE sequences did not serve to restrict expression of an upstream activating sequence (UAS)-based reporter/effector transgene when associated solely with the UAS element. Morpholino knockdown assays showed that NRSF/REST expression is required for NRSE-based transgene silencing. CONCLUSIONS: Our findings demonstrate that the addition of NRSE sequences to transgenes can provide useful new tools for functional studies of the nervous system. However, the general approach may be more broadly applicable; tissue-specific silencer elements are operable in tissues other than the nervous system, suggesting this approach can be similarly applied to other paradigms. Thus, creating synthetic associations between endogenous regulatory elements and tissue-specific silencers may facilitate targeting of cellular subtypes for which defined promoters/enhancers are lacking.

High-content drug screening in zebrafish xenografts reveals high efficacy of dual MCL-1/BCL-XL inhibition against Ewing sarcoma.

Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.