The Gal4-UAS regulatory system of yeast is widely used to modulate gene expression in Drosophila; however, there are limitations to its usefulness in transgenic zebrafish, owing to progressive methylation and silencing of the CpG-rich multicopy upstream activation sequence. Although a modified, less repetitive UAS construct may overcome this problem, it is highly desirable to have additional transcriptional regulatory systems that can be applied independently or in combination with the Gal4/UAS system for intersectional gene expression. The Q transcriptional regulatory system of Neurospora crassa functions similarly to Gal4/UAS. QF is a transcriptional activator that binds to the QUAS upstream regulatory sequence to drive reporter gene expression. Unlike Gal4, the QF binding site does not contain essential CpG dinucleotide sequences that are subject to DNA methylation. The QS protein is a repressor of QF mediated transcriptional activation akin to Gal80. The functionality of the Q system has been demonstrated in Drosophila and Caenorhabditis elegans and we now report its successful application to a vertebrate model, the zebrafish, Danio redo. Several tissue-specific promoters were used to drive QF expression in stable transgenic lines, as assessed by activation of a QUAS:GFP transgene. The QS repressor was found to dramatically reduce QF activity in injected zebrafish embryos: however, a similar repression has not yet been achieved in transgenic animals expressing QS under the control of ubiquitous promoters. A dual reporter construct containing both QUAS and UAS, each upstream of different fluorescent proteins was also generated and tested in transient assays, demonstrating that the two systems can work in parallel within the same cell. The adoption of the Q system should greatly increase the versatility and power of transgenic approaches for regulating gene expression in zebrafish. (C) 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Hemispheric specializations are well studied at the functional level but less is known about the underlying neural mechanisms. We identified a small cluster of cholinergic neurons in the dorsal habenula (dHb) of zebrafish, defined by their expression of the lecithin retinol acyltransferase domain containing 2 a (lratd2a) gene and their efferent connections with a subregion of the ventral interpeduncular nucleus (vIPN). The lratd2a-expressing neurons in the right dHb are innervated by a subset of mitral cells from both the left and right olfactory bulb and are activated upon exposure to the odorant cadaverine that is repellent to adult zebrafish. Using an intersectional strategy to drive expression of the botulinum neurotoxin specifically in these neurons, we find that adults no longer show aversion to cadaverine. Mutants with left-isomerized dHb that lack these neurons are also less repelled by cadaverine and their behavioral response to alarm substance, a potent aversive cue, is diminished. However, mutants in which both dHb have right identity appear more reactive to alarm substance. The results implicate an asymmetric dHb-vIPN neural circuit in the processing of repulsive olfactory cues and in modulating the resultant behavioral response.

Hepatic cysts are fluid-filled lesions in the liver that are estimated to occur in 5% of the population. They may cause hepatomegaly and abdominal pain. Progression to secondary fibrosis, cirrhosis, or cholangiocarcinoma can lead to morbidity and mortality. Previous studies of patients and rodent models have associated hepatic cyst formation with increased proliferation and fluid secretion in cholangiocytes, which are partially due to impaired primary cilia. Congenital hepatic cysts are thought to originate from faulty bile duct development, but the underlying mechanisms are not fully understood. In a forward genetic screen, we identified a zebrafish mutant that developed hepatic cysts during larval stages. The cyst formation was not due to changes in biliary cell proliferation, bile secretion, or impairment of primary cilia. Instead, time-lapse live imaging data showed that the mutant biliary cells failed to form interconnecting bile ducts because of defects in motility and protrusive activity. Accordingly, immunostaining revealed a disorganized actin and microtubule cytoskeleton in the mutant biliary cells. By whole-genome sequencing, we determined that the cystic phenotype in the mutant was caused by a missense mutation in the furinb gene, which encodes a proprotein convertase. The mutation altered Furinb localization and caused endoplasmic reticulum (ER) stress. The cystic phenotype could be suppressed by treatment with the ER stress inhibitor 4-phenylbutyric acid and exacerbated by treatment with the ER stress inducer tunicamycin. The mutant liver also exhibited increased mammalian target of rapamycin (mTOR) signaling. Treatment with mTOR inhibitors halted cyst formation at least partially through reducing ER stress. Conclusion: Our study has established a vertebrate model for studying hepatic cystogenesis and illustrated the contribution of ER stress in the disease pathogenesis.

The identification of biosignatures in Earth's ancient rock record and detection of extraplanetary life is one of the primary goals in astrobiology. Intrinsic to this goal is the improvement of analytical techniques and protocols used to identify an unambiguous signal of life. Micro Raman spectroscopy is a nondestructive method that allows for in situ identification of a wide range of minerals and compounds. The use of D (similar to 1350 cm(-1)) and G (similar to 1580 cm(-1)) band parameters to infer the biogenicity of carbonaceous materials in fossils has become a commonly used analytical tool, but carbonaceous compounds from different sources often share the same spectroscopic characteristics. Microfossil studies do not always take into consideration a nonbiological source for the carbon in their samples and therefore still rely on morphology as the primary mode of identification. Comprehensive studies that consider all carbon sources are typically done on metasediments, coals, or meteorites, and the results are not clearly applicable to microfossil identification. In this study, microfossils from a suite of sedimentary rock samples of various ages were analyzed with micro Raman spectroscopy to investigate the nature and provenance of carbonaceous material. To further constrain D-and G-band carbon characteristics, micro Raman analyses were also performed on well-characterized meteorite samples as abiological controls. The results appear to show a correlation of precursor carbonaceous material with D-band parameters and thermal history with G-band parameters. This systematic study lays the groundwork for improving the use of the G-and D-band trends as useful indicators of the origin of carbon in microfossils. Before unambiguous biosignatures can be established, further work characterizing the carbonaceous material in microfossils of different ages, thermal histories, and host rock compositions is needed.

Environmental conditions shape community composition. Arctic thermal springs provide an opportunity to study how environmental gradients can impose strong selective pressures on microbial communities and provide a continuum of niche opportunities. We use microscopic and molecular methods to conduct a survey of microbial community composition at Troll Springs on Svalbard, Norway, in the high Arctic. Microorganisms there exist under a wide range of environmental conditions: in warm water as periphyton, in moist granular materials, and in cold, dry rock as endoliths. Troll Springs has two distinct ecosystems, aquatic and terrestrial, together in close proximity, with different underlying environmental factors shaping each microbial community. Periphyton are entrapped during precipitation of calcium carbonate from the spring's waters, providing microbial populations that serve as precursors for the development of endolithic communities. This process differs from most endolith colonization, in which the rock predates the communities that colonize it. Community composition is modulated as environmental conditions change within the springs. At Troll, the aquatic environments show a small number of dominant operational taxonomic units (OTUs) that are specific to each sample. The terrestrial environments show a more even distribution of OTUs common to multiple samples.

A single scoop of the Rocknest aeolian deposit was sieved (<150 mu m), and four separate sample portions, each with a mass of similar to 50mg, were delivered to individual cups inside the Sample Analysis at Mars (SAM) instrument by the Mars Science Laboratory rover's sample acquisition system. The samples were analyzed separately by the SAM pyrolysis evolved gas and gas chromatograph mass spectrometer analysis modes. Several chlorinated hydrocarbons including chloromethane, dichloromethane, trichloromethane, a chloromethylpropene, and chlorobenzene were identified by SAM above background levels with abundances of similar to 0.01 to 2.3nmol. The evolution of the chloromethanes observed during pyrolysis is coincident with the increase in O-2 released from the Rocknest sample and the decomposition of a product of N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA), a chemical whose vapors were released from a derivatization cup inside SAM. The best candidate for the oxychlorine compounds in Rocknest is a hydrated calcium perchlorate (Ca(ClO4)(2)nH(2)O), based on the temperature release of O-2 that correlates with the release of the chlorinated hydrocarbons measured by SAM, although other chlorine-bearing phases are being considered. Laboratory analog experiments suggest that the reaction of Martian chlorine from perchlorate decomposition with terrestrial organic carbon from MTBSTFA during pyrolysis can explain the presence of three chloromethanes and a chloromethylpropene detected by SAM. Chlorobenzene may be attributed to reactions of Martian chlorine released during pyrolysis with terrestrial benzene or toluene derived from 2,6-diphenylphenylene oxide (Tenax) on the SAM hydrocarbon trap. At this time we do not have definitive evidence to support a nonterrestrial carbon source for these chlorinated hydrocarbons, nor do we exclude the possibility that future SAM analyses will reveal the presence of organic compounds native to the Martian regolith.

The Sample Analysis at Mars (SAM) instrument suite on the Mars Science Laboratory (MSL) measured a Mars atmospheric(14)N/N-15 ratio of 173 11 on sol 341 of the mission, agreeing with Viking's measurement of 168 17. The MSL/SAM value was based on Quadrupole Mass Spectrometer measurements of an enriched atmospheric sample, with CO2 and H2O removed. Doubly ionized nitrogen data at m/z 14 and 14.5 had the highest signal/background ratio, with results confirmed by m/z 28 and 29 data. Gases in SNC meteorite glasses have been interpreted as mixtures containing a Martian atmospheric component, based partly on distinctive(14)N/N-15 and(40)Ar/N-14 ratios. Recent MSL/SAM measurements of the(40)Ar/N-14 ratio (0.51 0.01) are incompatible with the Viking ratio (0.35 0.08). The meteorite mixing line is more consistent with the atmospheric composition measured by Viking than by MSL.

The Sample Analysis at Mars (SAM) instrument on the Mars Science Laboratory (MSL) rover Curiosity detected evolved gases during thermal analysis of soil samples from the Rocknest aeolian deposit in Gale Crater. Major species detected (in order of decreasing molar abundance) were H2O, SO2, CO2, and O-2, all at the mu mol level, with HCl, H2S, NH3, NO, and HCN present at the tens to hundreds of nmol level. We compute weight % numbers for the major gases evolved by assuming a likely source and calculate abundances between 0.5 and 3 wt.%. The evolution of these gases implies the presence of both oxidized (perchlorates) and reduced (sulfides or H-bearing) species as well as minerals formed under alkaline (carbonates) and possibly acidic (sulfates) conditions. Possible source phases in the Rocknest material are hydrated amorphous material, minor clay minerals, and hydrated perchlorate salts (all potential H2O sources), carbonates (CO2), perchlorates (O-2 and HCl), and potential N-bearing materials (e.g., Martian nitrates, terrestrial or Martian nitrogenated organics, ammonium salts) that evolve NH3, NO, and/or HCN. We conclude that Rocknest materials are a physical mixture in chemical disequilibrium, consistent with aeolian mixing, and that although weathering is not extensive, it may be ongoing even under current Martian surface conditions.

The Sample Analysis at Mars (SAM) instrument suite detected SO2, H2S, OCS, and CS2 from similar to 450 to 800 degrees C during evolved gas analysis (EGA) of materials from the Rocknest aeolian deposit in Gale Crater, Mars. This was the first detection of evolved sulfur species from a Martian surface sample during in situ EGA. SO2 (similar to 3-22 mu mol) is consistent with the thermal decomposition of Fe sulfates or Ca sulfites, or evolution/desorption from sulfur-bearing amorphous phases. Reactions between reduced sulfur phases such as sulfides and evolved O-2 or H2O in the SAM oven are another candidate SO2 source. H2S (similar to 41-109nmol) is consistent with interactions of H2O, H-2 and/or HCl with reduced sulfur phases and/or SO2 in the SAM oven. OCS (similar to 1-5nmol) and CS2 (similar to 0.2-1nmol) are likely derived from reactions between carbon-bearing compounds and reduced sulfur. Sulfates and sulfites indicate some aqueous interactions, although not necessarily at the Rocknest site; Fe sulfates imply interaction with acid solutions whereas Ca sulfites can form from acidic to near-neutral solutions. Sulfides in the Rocknest materials suggest input from materials originally deposited in a reducing environment or from detrital sulfides from an igneous source. The presence of sulfides also suggests that the materials have not been extensively altered by oxidative aqueous weathering. The possibility of both reduced and oxidized sulfur compounds in the deposit indicates a nonequilibrium assemblage. Understanding the sulfur mineralogy in Rocknest materials, which exhibit chemical similarities to basaltic fines analyzed elsewhere on Mars, can provide insight in to the origin and alteration history of Martian surface materials.