Green Fluorescent Protein in the sea urchin: new experimental approaches to transcriptional regulatory analysis in embryos and larvae

The use of Green Fluorescent Protein (GFP) as a reporter for expression transgenes opens the way to several new experimental strategies for the study of gene regulation in sea urchin development. A GFP coding sequence was associated with three different previously studied cis-regulatory systems, viz those of the SM50 gene, expressed in skeletogenic mesenchyme, the CyIIa gene, expressed in archenteron, skeletogenic and secondary mesenchyme, and the Endo16 gene, expressed in vegetal plate, archenteron and midgut. We demonstrate that the sensitivity with which expression can be detected is equal to or greater than that of whole-mount in situ hybridization applied to detection of CAT mRNA synthesized under the control of the same cis-regulatory systems. However, in addition to the important feature that it can be visualized nondestructively in living embryos, GFP has other advantages. First, it freely diffuses even within fine cytoplasmic cables, and thus reveals connections between cells, which in sea urchin embryos is particularly useful for observations on regulatory systems that operate in the syncytial skeletogenic mesenchyme. Second, GFP expression can be dramatically visualized in postembryonic larval tissues. This brings postembryonic larval developmental processes for the first time within the easy range of gene transfer analyses. Third, GFP permits identification and segregation of embryos in which the clonal incorporation of injected DNA has occurred in any particular desired region of the embryo. Thus, we show explicitly that, as expected, GFP transgenes are incorporated in the same nuclei together with other transgenes with which they are co-injected

Genomic characterization of Gli-activator targets in sonic hedgehog-mediated neural patterning

Sonic hedgehog (Shh) acts as a morphogen to mediate the specification of distinct cell identities in the ventral neural tube through a Gli-mediated (Gli1-3) transcriptional network. Identifying Gli targets in a systematic fashion is central to the understanding of the action of Shh. We examined this issue in differentiating neural progenitors in mouse. An epitope-tagged Gli-activator protein was used to directly isolate cis-regulatory sequences by chromatin immunoprecipitation (ChIP). ChIP products were then used to screen custom genomic tiling arrays of putative Hedgehog (Hh) targets predicted from transcriptional profiling studies, surveying 50-150 kb of non-transcribed sequence for each candidate. In addition to identifying expected Gli-target sites, the data predicted a number of unreported direct targets of Shh action. Transgenic analysis of binding regions in Nkx2.2, Nkx2.1 (Titf1) and Rab34 established these as direct Hh targets. These data also facilitated the generation of an algorithm that improved in silico predictions of Hh target genes. Together, these approaches provide significant new insights into both tissue-specific and general transcriptional targets in a crucial Shh-mediated patterning process

Spectrum of turbulent Kelvin-waves cascade in superfluid helium

To explain the observed decay of superfluid turbulence at very low temperature, it has been proposed that a cascade of Kelvin waves (analogous to the classical Kolmogorov cascade) transfers kinetic energy to length scales which are small enough that sound can be radiated away. We report results of numerical simulations of the interaction of quantized vortex filaments. We observe the development of the Kelvin-waves cascade, and compute the statistics of the curvature, the amplitude spectrum (which we compare with competing theories) and the fractal dimension.Comment: 32 pages, 22 figure

Posterior interosseous nerve localization within the proximal forearm - a patient normalized parameter

AIM To provide a “patient-normalized” parameter in the proximal forearm. METHODS Sixty-three cadaveric upper extremities from thirty-five cadavers were studied. A muscle splitting approach was utilized to locate the posterior interosseous nerve (PIN) at the point where it emerges from beneath the supinator. The supinator was carefully incised to expose the midpoint length of the nerve as it passes into the forearm while preserving the associated fascial connections, thereby preserving the relationship of the nerve with the muscle. We measured the transepicondylar distance (TED), PIN distance in the forearm’s neutral rotation position, pronation position, supination position, and the nerve width. Two individuals performed measurements using a digital caliper with inter-observer and intra-observer blinding. The results were analyzed with the Wilcoxon-Mann-Whitney test for paired samples. RESULTS In pronation, the PIN was within two confidence intervals of 1.0 TED in 95% of cases (range 0.7-1.3 TED); in neutral, within two confidence intervals of 0.84 TED in 95% of cases (range 0.5-1.1 TED); in supination, within two confidence intervals of 0.72 TED in 95% of cases (range 0.5-0.9 TED). The mean PIN distance from the lateral epicondyle was 100% of TED in a pronated forearm, 84% in neutral, and 72% in supination. Predictive accuracy was highest in supination; in all cases the majority of specimens (90.47%-95.23%) are within 2 cm of the forearm position-specific percentage of TED. When comparing right to left sides for TEDs with the signed Wilcoxon-Mann-Whitney test for paired samples as well as a significance test (with normal distribution), the P-value was 0.0357 (significance - 0.05) indicating a significant difference between the two sides. CONCLUSION This “patient normalized” parameter localizes the PIN crossing a line drawn between the lateral epicondyle and the radial styloid. Accurate PIN localization will aid in diagnosis, injections, and surgical approaches

Messenger RNA coding for only the alpha subunit of the rat brain Na channel is sufficient for expression of functional channels in Xenopus oocytes

Several cDNA clones coding for the high molecular weight (alpha) subunit of the voltage-sensitive Na channel have been selected by immunoscreening a rat brain cDNA library constructed in the expression vector lambda gt11. As will be reported elsewhere, the amino acid sequence translated from the DNA sequence shows considerable homology to that reported for the Electrophorus electricus electroplax Na channel. Several of the cDNA inserts hybridized with a low-abundance 9-kilobase RNA species from rat brain, muscle, and heart. Sucrose-gradient fractionation of rat brain poly(A) RNA yielded a high molecular weight fraction containing this mRNA, which resulted in functional Na channels when injected into oocytes. This fraction contained undetectable amounts of low molecular weight RNA. The high molecular weight Na channel RNA was selected from rat brain poly(A) RNA by hybridization to a single-strand antisense cDNA clone. Translation of this RNA in Xenopus oocytes resulted in the appearance of tetrodotoxin-sensitive voltage-sensitive Na channels in the oocyte membrane. These results demonstrate that mRNA encoding the alpha subunit of the rat brain Na channel, in the absence of any beta-subunit mRNA, is sufficient for translation to give functional channels in oocytes

Experimental observation and characterization of the magnetorotational instability

Differential rotation occurs in conducting flows in accretion disks and planetary cores. In such systems, the magnetorotational instability can arise from coupling Lorentz and centrifugal forces to cause large radial angular momentum fluxes. We present the first experimental observation of the magnetorotational instability. Our system consists of liquid sodium between differentially rotating spheres, with an imposed coaxial magnetic field. We characterize the observed patterns, dynamics and torque increases, and establish that this instability can occur from a hydrodynamic turbulent background.Comment: 4 pages, 5 figures. Final version, accepted for publication in Physical Review Letter

Seasonal variability of the warm Atlantic Water layer in the vicinity of the Greenland shelf break

The warmest water reaching the east and west coast of Greenland is found between 200?m and 600?m. Whilst important for melting Greenland's outlet glaciers, limited winter observations of this layer prohibit determination of its seasonality. To address this, temperature data from Argo profiling floats, a range of sources within the World Ocean Database and unprecedented coverage from marine-mammal borne sensors have been analysed for the period 2002-2011. A significant seasonal range in temperature (~1-2?°C) is found in the warm layer, in contrast to most of the surrounding ocean. The phase of the seasonal cycle exhibits considerable spatial variability, with the warmest water found near the eastern and southwestern shelf-break towards the end of the calendar year. High-resolution ocean model trajectory analysis suggest the timing of the arrival of the year's warmest water is a function of advection time from the subduction site in the Irminger Basin

The XMM Cluster Survey: The Dynamical State of XMMXCS J2215.9-1738 at z=1.457

We present new spectroscopic observations of the most distant X-ray selected galaxy cluster currently known, XMMXCS J2215.9-1738 at z=1.457, obtained with the DEIMOS instrument at the W. M. Keck Observatory, and the FORS2 instrument on the ESO Very Large Telescope. Within the cluster virial radius, as estimated from the cluster X-ray properties, we increase the number of known spectroscopic cluster members to 17 objects, and calculate the line of sight velocity dispersion of the cluster to be 580+/-140 km/s. We find mild evidence that the velocity distribution of galaxies within the virial radius deviates from a single Gaussian. We show that the properties of J2215.9-1738 are inconsistent with self-similar evolution of local X-ray scaling relations, finding that the cluster is underluminous given its X-ray temperature, and that the intracluster medium contains ~2-3 times the kinetic energy per unit mass of the cluster galaxies. These results can perhaps be explained if the cluster is observed in the aftermath of an off-axis merger. Alternatively, heating of the intracluster medium through supernovae and/or Active Galactic Nuclei activity, as is required to explain the observed slope of the local X-ray luminosity-temperature relation, may be responsible.Comment: 13 pages, 6 figures, accepted for publication in Ap