Zebrafish have become a popular organism for the study of vertebrate gene function1,2. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease3–5. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes6, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.

Aminoglycoside antibiotics are widely used for the treatment of life-threatening bacterial infections, but cause permanent hearing loss in a substantial proportion of treated patients. The sensory hair cells of the inner ear are damaged following entry of these antibiotics via the mechano-electrical transducer (MET) channels located at the tips of the hair cell's stereocilia. d-Tubocurarine (dTC) is a MET channel blocker that reduces the loading of gentamicin-Texas Red (GTTR) into rat cochlear hair cells and protects them from gentamicin treatment. Berbamine is a structurally related alkaloid that reduces GTTR labeling of zebrafish lateral-line hair cells and protects them from aminoglycoside-induced cell death. Both compounds are thought to reduce aminoglycoside entry into hair cells through the MET channels. Here we show that dTC (≥6.25 μM) or berbamine (≥1.55 μM) protect zebrafish hair cells in vivo from neomycin (6.25 μM, 1 h). Protection of zebrafish hair cells against gentamicin (10 μM, 6 h) was provided by ≥25 μM dTC or ≥12.5 μM berbamine. Hair cells in mouse cochlear cultures are protected from longer-term exposure to gentamicin (5 μM, 48 h) by 20 μM berbamine or 25 μM dTC. Berbamine is, however, highly toxic to mouse cochlear hair cells at higher concentrations (≥30 μM) whilst dTC is not. The absence of toxicity in the zebrafish assays prompts caution in extrapolating results from zebrafish neuromasts to mammalian cochlear hair cells. MET current recordings from mouse outer hair cells (OHCs) show that both compounds are permeant open-channel blockers, rapidly and reversibly blocking the MET channel with half-blocking concentrations of 2.2 μM (dTC) and 2.8 μM (berbamine) in the presence of 1.3 mM Ca2+ at −104 mV. Berbamine, but not dTC, also blocks the hair cell's basolateral K+ current, IK,neo, and modeling studies indicate that berbamine permeates the MET channel more readily than dTC. These studies reveal key properties of MET-channel blockers required for the future design of successful otoprotectants.

BackgroundMutations in the SHOX gene are responsible for Leri-Weill Dyschondrosteosis, a disorder characterised by mesomelic limb shortening. Recent investigations into regulatory elements surrounding SHOX have shown that deletions of conserved non-coding elements (CNEs) downstream of the SHOX gene produce a phenotype indistinguishable from Leri-Weill Dyschondrosteosis. As this gene is not found in rodents, we used zebrafish as a model to characterise the expression pattern of the shox gene across the whole embryo and characterise the enhancer domains of different CNEs associated with this gene.Methodology/Principal FindingsExpression of the shox gene in zebrafish was identified using in situ hybridization, with embryos showing expression in the blood, putative heart, hatching gland, brain pharyngeal arch, olfactory epithelium, and fin bud apical ectodermal ridge. By identifying sequences showing 65% identity over at least 40 nucleotides between Fugu, human, dog and opossum we uncovered 35 CNEs around the shox gene. These CNEs were compared with CNEs previously discovered by Sabherwal et al., resulting in the identification of smaller more deeply conserved sub-sequence. Sabherwal et al.'s CNEs were assayed for regulatory function in whole zebrafish embryos resulting in the identification of additional tissues under the regulatory control of these CNEs.Conclusion/SignificanceOur results using whole zebrafish embryos have provided a more comprehensive picture of the expression pattern of the shox gene, and a better understanding of its regulation via deeply conserved noncoding elements. In particular, we identify additional tissues under the regulatory control of previously identified SHOX CNEs. We also demonstrate the importance of these CNEs in evolution by identifying duplicated shox CNEs and more deeply conserved sub-sequences within already identified CNEs.

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The role of disease in the long-term dynamics of threatened species is poorly quantified, as well as being under-represented in ecology and conservation management. To understand persistent host-pathogen interaction operating in a vulnerable habitat, we quantified dynamics driving patterns of seagrass density using a longitudinal study in a relatively pristine site (Isles of Scilly, UK). Replicated samples of eelgrass (Zostera marina) density and wasting disease prevalence, presumably caused by Labyrinthula zosterae, were taken from five meadows at the height of the growing season, over the years 1997-2010. Data were used to parameterise a population dynamic model, incorporating density-dependent factors and sea temperature records. We found that direct density and disease-mediated feedback operate within a network of local populations. Furthermore, our results indicate that the strength of limitation to seagrass growth by disease was increased at higher temperatures. This modification of the coupled host-pathogen dynamics forms a novel hypothesis to account for dramatic die-backs of Z. marina widely reported elsewhere. Our findings highlight the importance of disease in structuring distributions of vulnerable species, as well as the application of population modelling in order to reveal ecological processes and prioritize future mechanistic investigation.

To identify small molecules that shield mammalian sensory hair cells from the ototoxic side effects of aminoglycoside antibiotics, 10,240 compounds were initially screened in zebrafish larvae selecting those that protected lateral-line hair cells against neomycin and gentamicin. When the 64 hits from this screen were retested in mouse cochlear cultures, 8 protected outer hair cells (OHCs) from gentamicin in vitro without causing hair-bundle damage. These 8 hits share structural features and all block, to varying degrees, the OHC's mechano-electrical transducer (MET) channel, a known route of aminoglycoside entry into hair cells. Further characterisation of one of the strongest MET-channel blockers, UoS-7692, revealed it additionally protects against kanamycin and tobramycin, and does not abrogate the bactericidal activity of gentamicin. UoS-7692 behaves, like the aminoglycosides, as a permeant blocker of the MET channel, significantly reduces gentamicin-Texas Red loading into OHCs, and preserves lateral-line function in neomycin-treated zebrafish. Trans-tympanic injection of UoS-7692 protects mouse OHCs from furosemide/kanamycin exposure in vivo and partially preserves hearing. The results confirm the hair-cell MET channel as a viable target for the identification of compounds that protect the cochlea from aminoglycosides, and provide a series of hit compounds that will inform the design of future otoprotectants.

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It is well known that ecological processes such as population regulation and natural enemy interactions potentially occur over a range of spatial scales, and there is a substantial body of literature developing theoretical understanding of the interplay between these processes. However, there are comparatively few studies quantifying the long-term effects of spatial scaling in natural ecosystems. A key challenge is that trophic complexity in real-world biological communities quickly obscures the signal from a focal process. Seagrass meadows provide an excellent opportunity in this respect: in many instances, seagrasses effectively form extensive natural monocultures, in which hypotheses about endogenous dynamics can be formulated and tested. We present amongst the longest unbroken, spatially explict time series of seagrass abundance published to date. Data include annual measures of shoot density, total above-ground abundance, and associated epiphyte cover from five Zostera marina meadows distributed around the Isles of Scilly, UK, from 1996 to 2011. We explore empirical patterns at the local and metapopulation scale using standard time series analysis and develop a simple population dynamic model, testing the hypothesis that both local and metapopulation scale feedback processes are important. We find little evidence of an interaction between scales in seagrass dynamics but that both scales contribute approximately equally to observed local epiphyte abundance. By quantifying the long-term dynamics of seagrass-epiphyte interactions we show how measures of density and extent are both important in establishing baseline information relevant to predicting responses to environmental change and developing management plans. We hope that this study complements existing mechanistic studies of physiology, genetics and productivity in seagrass, whilst highlighting the potential of seagrass as a model ecosystem. More generally, this study provides a rare opportunity to test some of the predictions of ecological theory in a natural ecosystem of global conservation and economic value.