Visualizing Engrafted Human Cancer and Therapy Responses in Immunodeficient Zebrafish.

Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc-/-, il2rga-/- zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.

Dependence of the evolution of carbon dynamics in the northern permafrost region on the trajectory of climate change.

We conducted a model-based assessment of changes in permafrost area and carbon storage for simulations driven by RCP4.5 and RCP8.5 projections between 2010 and 2299 for the northern permafrost region. All models simulating carbon represented soil with depth, a critical structural feature needed to represent the permafrost carbon-climate feedback, but that is not a universal feature of all climate models. Between 2010 and 2299, simulations indicated losses of permafrost between 3 and 5 million km2 for the RCP4.5 climate and between 6 and 16 million km2 for the RCP8.5 climate. For the RCP4.5 projection, cumulative change in soil carbon varied between 66-Pg C (1015-g carbon) loss to 70-Pg C gain. For the RCP8.5 projection, losses in soil carbon varied between 74 and 652 Pg C (mean loss, 341 Pg C). For the RCP4.5 projection, gains in vegetation carbon were largely responsible for the overall projected net gains in ecosystem carbon by 2299 (8- to 244-Pg C gains). In contrast, for the RCP8.5 projection, gains in vegetation carbon were not great enough to compensate for the losses of carbon projected by four of the five models; changes in ecosystem carbon ranged from a 641-Pg C loss to a 167-Pg C gain (mean, 208-Pg C loss). The models indicate that substantial net losses of ecosystem carbon would not occur until after 2100. This assessment suggests that effective mitigation efforts during the remainder of this century could attenuate the negative consequences of the permafrost carbon-climate feedback.

Long-term warming restructures Arctic tundra without changing net soil carbon storage.

High latitudes contain nearly half of global soil carbon, prompting interest in understanding how the Arctic terrestrial carbon balance will respond to rising temperatures. Low temperatures suppress the activity of soil biota, retarding decomposition and nitrogen release, which limits plant and microbial growth. Warming initially accelerates decomposition, increasing nitrogen availability, productivity and woody-plant dominance. However, these responses may be transitory, because coupled abiotic-biotic feedback loops that alter soil-temperature dynamics and change the structure and activity of soil communities, can develop. Here we report the results of a two-decade summer warming experiment in an Alaskan tundra ecosystem. Warming increased plant biomass and woody dominance, indirectly increased winter soil temperature, homogenized the soil trophic structure across horizons and suppressed surface-soil-decomposer activity, but did not change total soil carbon or nitrogen stocks, thereby increasing net ecosystem carbon storage. Notably, the strongest effects were in the mineral horizon, where warming increased decomposer activity and carbon stock: a 'biotic awakening' at depth.

Coastal sea level rise with warming above 2 °C.

Two degrees of global warming above the preindustrial level is widely suggested as an appropriate threshold beyond which climate change risks become unacceptably high. This "2 °C" threshold is likely to be reached between 2040 and 2050 for both Representative Concentration Pathway (RCP) 8.5 and 4.5. Resulting sea level rises will not be globally uniform, due to ocean dynamical processes and changes in gravity associated with water mass redistribution. Here we provide probabilistic sea level rise projections for the global coastline with warming above the 2 °C goal. By 2040, with a 2 °C warming under the RCP8.5 scenario, more than 90% of coastal areas will experience sea level rise exceeding the global estimate of 0.2 m, with up to 0.4 m expected along the Atlantic coast of North America and Norway. With a 5 °C rise by 2100, sea level will rise rapidly, reaching 0.9 m (median), and 80% of the coastline will exceed the global sea level rise at the 95th percentile upper limit of 1.8 m. Under RCP8.5, by 2100, New York may expect rises of 1.09 m, Guangzhou may expect rises of 0.91 m, and Lagos may expect rises of 0.90 m, with the 95th percentile upper limit of 2.24 m, 1.93 m, and 1.92 m, respectively. The coastal communities of rapidly expanding cities in the developing world, and vulnerable tropical coastal ecosystems, will have a very limited time after midcentury to adapt to sea level rises unprecedented since the dawn of the Bronze Age.

Clusters of circulating tumor cells traverse capillary-sized vessels.

Multicellular aggregates of circulating tumor cells (CTC clusters) are potent initiators of distant organ metastasis. However, it is currently assumed that CTC clusters are too large to pass through narrow vessels to reach these organs. Here, we present evidence that challenges this assumption through the use of microfluidic devices designed to mimic human capillary constrictions and CTC clusters obtained from patient and cancer cell origins. Over 90% of clusters containing up to 20 cells successfully traversed 5- to 10-µm constrictions even in whole blood. Clusters rapidly and reversibly reorganized into single-file chain-like geometries that substantially reduced their hydrodynamic resistances. Xenotransplantation of human CTC clusters into zebrafish showed similar reorganization and transit through capillary-sized vessels in vivo. Preliminary experiments demonstrated that clusters could be disrupted during transit using drugs that affected cellular interaction energies. These findings suggest that CTC clusters may contribute a greater role to tumor dissemination than previously believed and may point to strategies for combating CTC cluster-initiated metastasis.

Ocean-driven thinning enhances iceberg calving and retreat of Antarctic ice shelves.

Iceberg calving from all Antarctic ice shelves has never been directly measured, despite playing a crucial role in ice sheet mass balance. Rapid changes to iceberg calving naturally arise from the sporadic detachment of large tabular bergs but can also be triggered by climate forcing. Here we provide a direct empirical estimate of mass loss due to iceberg calving and melting from Antarctic ice shelves. We find that between 2005 and 2011, the total mass loss due to iceberg calving of 755 ± 24 gigatonnes per year (Gt/y) is only half the total loss due to basal melt of 1516 ± 106 Gt/y. However, we observe widespread retreat of ice shelves that are currently thinning. Net mass loss due to iceberg calving for these ice shelves (302 ± 27 Gt/y) is comparable in magnitude to net mass loss due to basal melt (312 ± 14 Gt/y). Moreover, we find that iceberg calving from these decaying ice shelves is dominated by frequent calving events, which are distinct from the less frequent detachment of isolated tabular icebergs associated with ice shelves in neutral or positive mass balance regimes. Our results suggest that thinning associated with ocean-driven increased basal melt can trigger increased iceberg calving, implying that iceberg calving may play an overlooked role in the demise of shrinking ice shelves, and is more sensitive to ocean forcing than expected from steady state calving estimates.

Atlantic hurricane surge response to geoengineering.

Devastating floods due to Atlantic hurricanes are relatively rare events. However, the frequency of the most intense storms is likely to increase with rises in sea surface temperatures. Geoengineering by stratospheric sulfate aerosol injection cools the tropics relative to the polar regions, including the hurricane Main Development Region in the Atlantic, suggesting that geoengineering may mitigate hurricanes. We examine this hypothesis using eight earth system model simulations of climate under the Geoengineering Model Intercomparison Project (GeoMIP) G3 and G4 schemes that use stratospheric aerosols to reduce the radiative forcing under the Representative Concentration Pathway (RCP) 4.5 scenario. Global mean temperature increases are greatly ameliorated by geoengineering, and tropical temperature increases are at most half of those temperature increases in the RCP4.5. However, sulfate injection would have to double (to nearly 10 teragrams of SO2 per year) between 2020 and 2070 to balance the RCP4.5, approximately the equivalent of a 1991 Pinatubo eruption every 2 y, with consequent implications for stratospheric ozone. We project changes in storm frequencies using a temperature-dependent generalized extreme value statistical model calibrated by historical storm surges and observed temperatures since 1923. The number of storm surge events as big as the one caused by the 2005 Katrina hurricane are reduced by about 50% compared with no geoengineering, but this reduction is only marginally statistically significant. Nevertheless, when sea level rise differences in 2070 between the RCP4.5 and geoengineering are factored into coastal flood risk, we find that expected flood levels are reduced by about 40 cm for 5-y events and about halved for 50-y surges.

Distinct Notch signaling outputs pattern the developing arterial system.

Differentiation of arteries and veins is essential for the development of a functional circulatory system. In vertebrate embryos, genetic manipulation of Notch signaling has demonstrated the importance of this pathway in driving artery endothelial cell differentiation. However, when and where Notch activation occurs to affect endothelial cell fate is less clear. Using transgenic zebrafish bearing a Notch-responsive reporter, we demonstrate that Notch is activated in endothelial progenitors during vasculogenesis prior to blood vessel morphogenesis and is maintained in arterial endothelial cells throughout larval stages. Furthermore, we find that endothelial progenitors in which Notch is activated are committed to a dorsal aorta fate. Interestingly, some arterial endothelial cells subsequently downregulate Notch signaling and then contribute to veins during vascular remodeling. Lineage analysis, together with perturbation of both Notch receptor and ligand function, further suggests several distinct developmental windows in which Notch signaling acts to promote artery commitment and maintenance. Together, these findings demonstrate that Notch acts in distinct contexts to initiate and maintain artery identity during embryogenesis.

Optimized cell transplantation using adult rag2 mutant zebrafish.

Cell transplantation into adult zebrafish has lagged behind mouse models owing to the lack of immunocompromised strains. Here we have created rag2(E450fs) mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft muscle, blood stem cells and various cancers. rag2(E450fs) mutant zebrafish are the first immunocompromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer.

A somitic Wnt16/Notch pathway specifies haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are a self-renewing population of cells that continuously replenish all blood and immune cells during the lifetime of an individual. HSCs are used clinically to treat a wide array of diseases, including acute leukaemias and congenital blood disorders, but obtaining suitable numbers of cells and finding immune-compatible donors remain serious problems. These difficulties have led to an interest in the conversion of embryonic stem cells or induced pluripotent stem cells into HSCs, which is not possible using current methodologies. To accomplish this goal, it is critical to understand the native mechanisms involved in the specification of HSCs during embryonic development. Here we demonstrate in zebrafish that Wnt16 controls a novel genetic regulatory network required for HSC specification. Non-canonical signalling by Wnt16 is required for somitic expression of the Notch ligands deltaC (dlc) and deltaD (dld), and these ligands are, in turn, required for the establishment of definitive haematopoiesis. Notch signalling downstream of Dlc and Dld is earlier than, and distinct from, known cell-autonomous requirements for Notch, strongly suggesting that novel Notch-dependent relay signal(s) induce the first HSCs in parallel to other established pathways. Our results demonstrate that somite-specific gene expression is required for the production of haemogenic endothelium.

Weighting and indirect effects identify keystone species in food webs.

Species extinctions are accelerating globally, yet the mechanisms that maintain local biodiversity remain poorly understood. The extinction of species that feed on or are fed on by many others (i.e. 'hubs') has traditionally been thought to cause the greatest threat of further biodiversity loss. Very little attention has been paid to the strength of those feeding links (i.e. link weight) and the prevalence of indirect interactions. Here, we used a dynamical model based on empirical energy budget data to assess changes in ecosystem stability after simulating the loss of species according to various extinction scenarios. Link weight and/or indirect effects had stronger effects on food-web stability than the simple removal of 'hubs', demonstrating that both quantitative fluxes and species dissipating their effects across many links should be of great concern in biodiversity conservation, and the potential for 'hubs' to act as keystone species may have been exaggerated to date.

A truncation allele in vascular endothelial growth factor c reveals distinct modes of signaling during lymphatic and vascular development.

Vascular endothelial growth factor C (Vegfc) is a secreted protein that guides lymphatic development in vertebrate embryos. However, its role during developmental angiogenesis is not well characterized. Here, we identify a mutation in zebrafish vegfc that severely affects lymphatic development and leads to angiogenesis defects on sensitized genetic backgrounds. The um18 mutation prematurely truncated Vegfc, blocking its secretion and paracrine activity but not its ability to activate its receptor Flt4. When expressed in endothelial cells, vegfc(um18) could not rescue lymphatic defects in mutant embryos, but induced ectopic blood vessel branching. Furthermore, vegfc-deficient endothelial cells did not efficiently contribute to tip cell positions in developing sprouts. Computational modeling together with assessment of endothelial cell dynamics by time-lapse analysis suggested that an autocrine Vegfc/Flt4 loop plays an important role in migratory persistence and filopodia stability during sprouting. Our results suggest that Vegfc acts in two distinct modes during development: as a paracrine factor secreted from arteries to guide closely associated lymphatic vasculature and as an autocrine factor to drive migratory persistence during angiogenesis.

Allograft Cancer Cell Transplantation in Zebrafish.

Allogeneic cell transplantation is the transfer of cells from one individual into another of the same species and has become an indispensable technique for studying development, immunology, regeneration and cancer biology. In experimental settings, tumor cell engraftment into immunologically competent recipients has greatly increased our understanding of the mechanisms that drive self-renewal, progression and metastasis in vivo. Zebrafish have quickly emerged as a powerful genetic model of cancer that has benefited greatly from allogeneic transplantation. Efficient engraftment can be achieved by transplanting cells into either early larval stage zebrafish that have not yet developed a functional acquired immune system or adult zebrafish following radiation or chemical ablation of the immune system. Alternatively, transplantation can be completed in adult fish using either clonal syngeneic strains or newly-generated immune compromised zebrafish models that have mutations in genes required for proper immune cell function. Here, we discuss the current state of cell transplantation as it pertains to zebrafish cancer and the available models used for dissecting important processes underlying cancer. We will also use the zebrafish model to highlight the power of cell transplantation, including its capacity to dynamically assess functional heterogeneity within individual cancer cells, visualize cancer progression and evolution, assess tumor-propagating potential and self-renewal, image cancer cell invasion and dissemination and identify novel therapies for treating cancer.

Projected Atlantic hurricane surge threat from rising temperatures.

Detection and attribution of past changes in cyclone activity are hampered by biased cyclone records due to changes in observational capabilities. Here, we relate a homogeneous record of Atlantic tropical cyclone activity based on storm surge statistics from tide gauges to changes in global temperature patterns. We examine 10 competing hypotheses using nonstationary generalized extreme value analysis with different predictors (North Atlantic Oscillation, Southern Oscillation, Pacific Decadal Oscillation, Sahel rainfall, Quasi-Biennial Oscillation, radiative forcing, Main Development Region temperatures and its anomaly, global temperatures, and gridded temperatures). We find that gridded temperatures, Main Development Region, and global average temperature explain the observations best. The most extreme events are especially sensitive to temperature changes, and we estimate a doubling of Katrina magnitude events associated with the warming over the 20th century. The increased risk depends on the spatial distribution of the temperature rise with highest sensitivity from tropical Atlantic, Central America, and the Indian Ocean. Statistically downscaling 21st century warming patterns from six climate models results in a twofold to sevenfold increase in the frequency of Katrina magnitude events for a 1 °C rise in global temperature (using BNU-ESM, BCC-CSM-1.1, CanESM2, HadGEM2-ES, INM-CM4, and NorESM1-M).

Homogeneous record of Atlantic hurricane surge threat since 1923.

Detection and attribution of past changes in cyclone activity are hampered by biased cyclone records due to changes in observational capabilities. Here we construct an independent record of Atlantic tropical cyclone activity on the basis of storm surge statistics from tide gauges. We demonstrate that the major events in our surge index record can be attributed to landfalling tropical cyclones; these events also correspond with the most economically damaging Atlantic cyclones. We find that warm years in general were more active in all cyclone size ranges than cold years. The largest cyclones are most affected by warmer conditions and we detect a statistically significant trend in the frequency of large surge events (roughly corresponding to tropical storm size) since 1923. In particular, we estimate that Katrina-magnitude events have been twice as frequent in warm years compared with cold years (P < 0.02).

Developed and developing world responsibilities for historical climate change and CO2 mitigation.

At the United Nations Framework Convention on Climate Change Conference in Cancun, in November 2010, the Heads of State reached an agreement on the aim of limiting the global temperature rise to 2 °C relative to preindustrial levels. They recognized that long-term future warming is primarily constrained by cumulative anthropogenic greenhouse gas emissions, that deep cuts in global emissions are required, and that action based on equity must be taken to meet this objective. However, negotiations on emission reduction among countries are increasingly fraught with difficulty, partly because of arguments about the responsibility for the ongoing temperature rise. Simulations with two earth-system models (NCAR/CESM and BNU-ESM) demonstrate that developed countries had contributed about 60-80%, developing countries about 20-40%, to the global temperature rise, upper ocean warming, and sea-ice reduction by 2005. Enacting pledges made at Cancun with continuation to 2100 leads to a reduction in global temperature rise relative to business as usual with a 1/3-2/3 (CESM 33-67%, BNU-ESM 35-65%) contribution from developed and developing countries, respectively. To prevent a temperature rise by 2 °C or more in 2100, it is necessary to fill the gap with more ambitious mitigation efforts.

Post-transcriptional mechanisms contribute to Etv2 repression during vascular development.

etv2 is an endothelial-specific ETS transcription factor that is essential for vascular differentiation and morphogenesis in vertebrates. While recent data suggest that Etv2 is dynamically regulated during vascular development, little is known about the mechanisms involved in this process. Here, we find that etv2 transcript and protein expression are highly dynamic during zebrafish vascular development, with both apparent during early somitogenesis and subsequently down-regulated as development proceeds. Inducible knockdown of Etv2 in zebrafish embryos prior to mid-somitogenesis stages, but not later, caused severe vascular defects, suggesting a specific role in early commitment of lateral mesoderm to the endothelial linage. Accordingly, Etv2-overexpressing cells showed an enhanced ability to commit to endothelial lineages in mosaic embryos. We further find that the etv2 3' untranslated region (UTR) is capable of repressing an endothelial autonomous transgene and contains binding sites for members of the let-7 family of microRNAs. Ectopic expression of let-7a could repress the etv2 3'UTR in sensor assays and was also able to block endogenous Etv2 protein expression, leading to concomitant reduction of endothelial genes. Finally, we observed that Etv2 protein levels persisted in maternal-zygotic dicer1 mutant embryos, suggesting that microRNAs contribute to its repression during vascular development. Taken together, our results suggest that etv2 acts during early development to specify endothelial lineages and is then down-regulated, in part through post-transcriptional repression by microRNAs, to allow normal vascular development.

BMP signaling orchestrates photoreceptor specification in the zebrafish pineal gland in collaboration with Notch.

A variety of signaling pathways have been shown to regulate specification of neuronal subtype identity. However, the mechanisms by which future neurons simultaneously process information from multiple pathways to establish their identity remain poorly understood. The zebrafish pineal gland offers a simple system with which to address questions concerning the integration of signaling pathways during neural specification as it contains only two types of neurons - photoreceptors and projection neurons. We have previously shown that Notch signaling inhibits the projection neuron fate. Here, we show that BMP signaling is both necessary and sufficient to promote the photoreceptor fate. We also demonstrate that crosstalk between BMP and Notch signaling is required for the inhibition of a projection neuron fate in future photoreceptors. In this case, BMP signaling is required as a competence factor for the efficient activation of Notch targets. Our results indicate that both the induction of a photoreceptor fate and the interaction with Notch relies on a canonical BMP/Smad5 pathway. However, the activation of Notch-dependent transcription does not require a canonical Smad5-DNA interaction. Our results provide new insights into how multiple signaling influences are integrated during cell fate specification in the vertebrate CNS.