A small molecule screen identifies in vivo modulators of peripheral nerve regeneration in zebrafish

Adult vertebrates have retained the ability to regenerate peripheral nerves after injury, although regeneration is frequently incomplete, often leading to functional impairments. Small molecule screens using whole organisms have high potential to identify biologically relevant targets, yet currently available assays for in vivo peripheral nerve regeneration are either very laborious and/or require complex technology. Here we take advantage of the optical transparency of larval zebrafish to develop a simple and fast pectoral fin removal assay that measures peripheral nerve regeneration in vivo. Twenty-four hours after fin amputation we observe robust and stereotyped nerve regrowth at the fin base. Similar to laser mediated nerve transection, nerve regrowth after fin amputation requires Schwann cells and FGF signaling, confirming that the fin amputation assay identifies pathways relevant for peripheral nerve regeneration. From a library of small molecules with known targets, we identified 21 compounds that impair peripheral nerve regeneration. Several of these compounds target known regulators of nerve regeneration, further validating the fin removal assay. Twelve of the identified compounds affect targets not previously known to control peripheral nerve regeneration. Using a laser-mediated nerve transection assay we tested ten of those compounds and confirmed six of these compounds to impair peripheral nerve regeneration: an EGFR inhibitor, a glucocorticoid, prostaglandin D2, a retinoic acid agonist, an inhibitor of calcium channels and a topoisomerase I inhibitor. Thus, we established a technically simple assay to rapidly identify valuable entry points into pathways critical for vertebrate peripheral nerve regeneration

Temporal and Spatial Requirements of unplugged/MuSK Function during Zebrafish Neuromuscular Development

One of the earliest events in neuromuscular junction (NMJ) development is the accumulation of acetylcholine receptor (AChR) at the center of muscle cells. The unplugged/MuSK (muscle specific tyrosine kinase) gene is essential to initiate AChR clustering but also to restrict approaching growth cones to the muscle center, thereby coordinating pre- and postsynaptic development. To determine how unplugged/MuSK signaling coordinates these two processes, we examined the temporal and spatial requirements of unplugged/MuSK in zebrafish embryos using heat-shock inducible transgenes. Here, we show that despite its expression in muscle cells from the time they differentiate, unplugged/MuSK activity is first required just prior to the appearance of AChR clusters to simultaneously induce AChR accumulation and to guide motor axons. Furthermore, we demonstrate that ectopic expression of unplugged/MuSK throughout the muscle membrane results in wildtype-like AChR prepattern and neuromuscular synapses in the central region of muscle cells. We propose that AChR prepatterning and axonal guidance are spatio-temporally coordinated through common unplugged/MuSK signals, and that additional factor(s) restrict unplugged/MuSK signaling to a central muscle zone critical for establishing mid-muscle synaptogenesis

Wnt Signals Organize Synaptic Prepattern and Axon Guidance through the Zebrafish unplugged/MuSK Receptor

SummaryEarly during neuromuscular development, acetylcholine receptors (AChRs) accumulate at the center of muscle fibers, precisely where motor growth cones navigate and synapses eventually form. Here, we show that Wnt11r binds to the zebrafish unplugged/MuSK ectodomain to organize this central muscle zone. In the absence of such a zone, prepatterned AChRs fail to aggregate and, as visualized by live-cell imaging, growth cones stray from their central path. Using inducible unplugged/MuSK transgenes, we show that organization of the central muscle zone is dispensable for the formation of neural synapses, but essential for AChR prepattern and motor growth cone guidance. Finally, we show that blocking noncanonical dishevelled signaling in muscle fibers disrupts AChR prepatterning and growth cone guidance. We propose that Wnt ligands activate unplugged/MuSK signaling in muscle fibers to restrict growth cone guidance and AChR prepatterns to the muscle center through a mechanism reminiscent of the planar cell polarity pathway

Quantifying dust and the ultraviolet radiation-density in the local universe

A sample of local galaxies for which far infrared and uv fluxes are available is used to estimate the characteristic dust extinction in galaxies and to test whether standard dust properties are plausible. Assuming galaxies can be characterized by a single dust optical depth (certainly not valid for galaxies with a dominant starburst component), the infrared excess and ultraviolet colours of local galaxies are found to be consistent with normal Milky Way dust, with a mean value for E(B-V) of 0.16. A significant fraction of the dust heating is due to older, lower mass stars, and this fraction increases towards earlier galaxy types. Analysis of F_fir/F_uv versus uv colour diagrams for starburst galaxies in terms of a simple screen dust model does not support a Calzetti (1999) rather than a Milky Way extinction law, though the absence of the expected strong 2200 A feature in several galaxies with IUE spectra does show that more detailed radiative transfer models are needed, probably with non-spherical geometry. A simple treatment in which the 100/60 mu flux-ratio is used to subtract the optically thick starburst contribution to the far infrared radiation results in lower extinction estimates for the optically thin cirrus component, with a mean E(B-V) of 0.10 The uv luminosity density, corrected for dust extinction, is derived and a value for the local mean star-formation rate inferred. This is consistent with previous estimates from uv surveys and from H-alpha surveys.Comment: Accepted MNRAS, 11 pages, 11 figure

Molecular-Genetic Mapping of Zebrafish Mutants with Variable Phenotypic Penetrance

Forward genetic screens in vertebrates are powerful tools to generate models relevant to human diseases, including neuropsychiatric disorders. Variability in phenotypic penetrance and expressivity is common in these disorders and behavioral mutant models, making their molecular-genetic mapping a formidable task. Using a ‘phenotyping by segregation’ strategy, we molecularly map the hypersensitive zebrafish houdini mutant despite its variable phenotypic penetrance, providing a generally applicable strategy to map zebrafish mutants with subtle phenotypes

CNS Hypomyelination Disrupts Axonal Conduction and Behavior in Larval Zebrafish

Myelination is essential for central nervous system (CNS) formation, health and function. As a model organism, larval zebrafish have been extensively employed to investigate the molecular and cellular basis of CNS myelination, because of their genetic tractability and suitability for non-invasive live cell imaging. However, it has not been assessed to what extent CNS myelination affects neural circuit function in zebrafish larvae, prohibiting the integration of molecular and cellular analyses of myelination with concomitant network maturation. To test whether larval zebrafish might serve as a suitable platform with which to study the effects of CNS myelination and its dysregulation on circuit function, we generated zebrafish myelin regulatory factor (myrf) mutants with CNS-specific hypomyelination and investigated how this affected their axonal conduction properties and behavior. We found that myrf mutant larvae exhibited increased latency to perform startle responses following defined acoustic stimuli. Furthermore, we found that hypomyelinated animals often selected an impaired response to acoustic stimuli, exhibiting a bias toward reorientation behavior instead of the stimulus-appropriate startle response. To begin to study how myelination affected the underlying circuitry, we established electrophysiological protocols to assess various conduction properties along single axons. We found that the hypomyelinated myrf mutants exhibited reduced action potential conduction velocity and an impaired ability to sustain high-frequency action potential firing. This study indicates that larval zebrafish can be used to bridge molecular and cellular investigation of CNS myelination with multiscale assessment of neural circuit function. SIGNIFICANCE STATEMENT Myelination of CNS axons is essential for their health and function, and it is now clear that myelination is a dynamic life-long process subject to modulation by neuronal activity. However, it remains unclear precisely how changes to myelination affects animal behavior and underlying action potential conduction along axons in intact neural circuits. In recent years, zebrafish have been employed to study cellular and molecular mechanisms of myelination, because of their relatively simple, optically transparent, experimentally tractable vertebrate nervous system. Here we find that changes to myelination alter the behavior of young zebrafish and action potential conduction along individual axons, providing a platform to integrate molecular, cellular, and circuit level analyses of myelination using this model

Scattered X-rays in Obscured Active Galactic Nuclei and their Implications for Geometrical Structure and Evolution

We construct a new sample of 32 obscured active galactic nuclei (AGNs) selected from the Second XMM-Newton Serendipitous Source Catalogue to investigate their multiwavelength properties in relation to the "scattering fraction", the ratio of the soft X-ray flux to the absorption-corrected direct emission. The sample covers a broad range of the scattering fraction (0.1%-10%). A quarter of the 32 AGNs have a very low scattering fraction (smaller than 0.5%), which suggests that they are buried in a geometrically thick torus with a very small opening angle. We investigate correlations between the scattering fraction and multiwavelength properties. We find that AGNs with a small scattering fraction tend to have low [O III]lambda5007/X-ray luminosity ratios. This result agrees with the expectation that the extent of the narrow-line region is small because of the small opening angle of the torus. There is no significant correlation between scattering fraction and far-infrared luminosity. This implies that a scale height of the torus is not primarily determined by starburst activity. We also compare scattering fraction with black hole mass or Eddington ratio and find a weak anti-correlation between the Eddington ratio and scattering fraction. This implies that more rapidly growing supermassive black holes tend to have thicker tori.Comment: 13 pages, 10 figures, accepted for publication in Ap

A Spectropolarimetric Test of the Structure of the Intrinsic Absorbers in the Quasar HS1603+3820

We report the results of a spectropolarimetric observation of the C IV mini-BAL in the quasar HS1603+3820. The observations were carried out with the FOCAS instrument on the Subaru telescope and yielded an extremely high polarization sensitivity of 0.1%, at a resolving power of 1500. HS1603+3820 has been the target of a high-resolution spectroscopic monitoring campaign for more than four years, aimed at studying its highly variable C IV mini-BAL profile. Using the monitoring observations, in an earlier paper we were able to narrow down the causes of the variability to the following two scenarios: (1) scattering material of variable optical depth redirecting photons around the absorber, and (2) a variable, highly-ionized screen between the continuum source and the absorber which modulates the UV continuum incident on the absorber. The observations presented here provide a crucial test of the scattering scenario and lead us to disfavor it because (a) the polarization level is very small (p~0.6%) throughout the spectrum, and (b) the polarization level does not increase across the mini-BAL trough. Thus, the variable screen scenario emerges as our favored explanation of the C IV mini-BAL variability. Our conclusion is bolstered by recent X-ray observations of nearby mini-BAL quasars, which show a rapidly variable soft X-ray continuum that appears to be the result of transmission through an ionized absorber of variable ionization parameter and optical depth.Comment: To appear in the Astrophysical Journal, vol. 719, August 201

Modelling the Spoon IRS diagnostic diagram

We explore whether our models for starbursts, quiescent star-forming galaxies and for AGN dust tori are able to model the full range of IRS spectra measured with Spitzer. The diagnostic plot of 9.7 mu silicate optical depth versus 6.2 mu PAH equivalent width, introduced by Spoon and coworkers in 2007, gives a good indication of the age and optical depth of a starburst, and of the contribution of an AGN dust torus. However there is aliasing between age and optical depth at later times in the evolution of a starburst, and between age and the presence of an AGN dust torus. Modeling the full IRS spectra and using broad-band 25-850 mu fluxes can help to resolve these aliases. The observed spectral energy distributions require starbursts of a range of ages with initial dust optical depth ranging from 50-200, optically thin dust emission ('cirrus') illuminated by a range of surface brightnesses of the interstellar radiation field, and AGN dust tori with a range of viewing angles.Comment: Accepted for publication by MNRAS. 8 pages, 10 figure

Crossover and scaling in a two-dimensional field-tuned superconductor

Using an analysis similar to that of Imry and Wortis, it is shown that the apparent first order superconductor to metal transition, which has been claimed to exist at low values of the magnetic field in a two-dimensional field-tuned system at zero temperature,can be consistentlyinterpreted as a sharp crossover from a strong superconductor to an inhomogeneous state, which is a weak superconductor. The true zero-temperature superconductor to insulator transition within the inhomogenous state is conjectured to be that of randomly diluted XY model. An explaination of the observed finite temperature approximate scaling of resistivity close to the critical point is speculated within this model.Comment: 5 pages, 2 figures, corrected and modified according to referee Report