Tools for neuroanatomy and neurogenetics in Drosophila Pfeiffer, Barret D; Jenett, Arnim; Hammonds, Ann S ...
Proceedings of the National Academy of Sciences - PNAS,
07/2008, Letnik:
105, Številka:
28
Journal Article
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We demonstrate the feasibility of generating thousands of transgenic Drosophila melanogaster lines in which the expression of an exogenous gene is reproducibly directed to distinct small subsets of ...cells in the adult brain. We expect the expression patterns produced by the collection of 5,000 lines that we are currently generating to encompass all neurons in the brain in a variety of intersecting patterns. Overlapping 3-kb DNA fragments from the flanking noncoding and intronic regions of genes thought to have patterned expression in the adult brain were inserted into a defined genomic location by site-specific recombination. These fragments were then assayed for their ability to function as transcriptional enhancers in conjunction with a synthetic core promoter designed to work with a wide variety of enhancer types. An analysis of 44 fragments from four genes found that >80% drive expression patterns in the brain; the observed patterns were, on average, comprised of <100 cells. Our results suggest that the D. melanogaster genome contains >50,000 enhancers and that multiple enhancers drive distinct subsets of expression of a gene in each tissue and developmental stage. We expect that these lines will be valuable tools for neuroanatomy as well as for the elucidation of neuronal circuits and information flow in the fly brain.
Drosophila central neurons arise from neuroblasts that generate neurons in a pair-wise fashion, with the two daughters providing the basis for distinct A and B hemilineage groups. 33 ...postembryonically-born hemilineages contribute over 90% of the neurons in each thoracic hemisegment. We devised genetic approaches to define the anatomy of most of these hemilineages and to assessed their functional roles using the heat-sensitive channel dTRPA1. The simplest hemilineages contained local interneurons and their activation caused tonic or phasic leg movements lacking interlimb coordination. The next level was hemilineages of similar projection cells that drove intersegmentally coordinated behaviors such as walking. The highest level involved hemilineages whose activation elicited complex behaviors such as takeoff. These activation phenotypes indicate that the hemilineages vary in their behavioral roles with some contributing to local networks for sensorimotor processing and others having higher order functions of coordinating these local networks into complex behavior.
Hot gas pervades the Universe: about half of the baryonic content in the Universe is expected to be at T > 105 K, and there are as many baryons at T > 107 trapped in galaxy clusters as there are ...locked into stars. There is an intimate relation between this hot gas, which delineates the large-scale structure of the Universe, and the most energetic phenomena occurring in the immediate vicinity of super-massive black holes, through a poorly known process called Cosmic Feedback. Studying the hot and energetic universe requires X-ray observatories in space, whose capabilities greatly exceed those of the current workhorse observatories: NASA's Chandra and ESA's XMM- Newton. Athena has been selected by ESA as the L2 mission (due for launch in 2028), to address the "Hot and Energetic Universe" science theme. It will be a large X-ray observatory capable of addressing the above topics, and many other fundamental questions in contemporary astrophysics. Here we present the Athena science objectives, the mission concept and its payload, including the X-ray telescope and its two baseline instruments: a Wide Field Imager (WFI) and an X-ray Integral Field Unit (X-IFU).
Diffuse neuromodulatory systems such as norepinephrine (NE) control brain-wide states such as arousal, but whether they control complex social behaviors more specifically is not clear. Octopamine ...(OA), the insect homolog of NE, is known to promote both arousal and aggression. We have performed a systematic, unbiased screen to identify OA receptor-expressing neurons (OARNs) that control aggression in Drosophila. Our results uncover a tiny population of male-specific aSP2 neurons that mediate a specific influence of OA on aggression, independent of any effect on arousal. Unexpectedly, these neurons receive convergent input from OA neurons and P1 neurons, a population of FruM+ neurons that promotes male courtship behavior. Behavioral epistasis experiments suggest that aSP2 neurons may constitute an integration node at which OAergic neuromodulation can bias the output of P1 neurons to favor aggression over inter-male courtship. These results have potential implications for thinking about the role of related neuromodulatory systems in mammals.
•OAR+ aSP2 neurons control aggression independently of arousal in Drosophila•Octopamine receptor Oamb in aSP2 neurons modulates aggression in response to OA•aSP2 neurons receive input from P1 neurons, which promote courtship and aggression•aSP2 neurons integrate input from OANs and P1 neurons to promote aggression
Watanabe et al. describe a circuit mechanism by which the neuromodulator octopamine regulates an output relay from a multifunctional social behavior network to control the balance between aggression and courtship in Drosophila.
In animals, each sequence-specific transcription factor typically binds to thousands of genomic regions in vivo. Our previous studies of 20 transcription factors show that most genomic regions bound ...at high levels in Drosophila blastoderm embryos are known or probable functional targets, but genomic regions occupied only at low levels have characteristics suggesting that most are not involved in the cis -regulation of transcription. Here we use transgenic reporter gene assays to directly test the transcriptional activity of 104 genomic regions bound at different levels by the 20 transcription factors. Fifteen genomic regions were selected based solely on the DNA occupancy level of the transcription factor Kruppel. Five of the six most highly bound regions drive blastoderm patterns of reporter transcription. In contrast, only one of the nine lowly bound regions drives transcription at this stage and four of them are not detectably active at any stage of embryogenesis. A larger set of 89 genomic regions chosen using criteria designed to identify functional cis -regulatory regions supports the same trend: genomic regions occupied at high levels by transcription factors in vivo drive patterned gene expression, whereas those occupied only at lower levels mostly do not. These results support studies that indicate that the high cellular concentrations of sequence-specific transcription factors drive extensive, low-occupancy, nonfunctional interactions within the accessible portions of the genome.
Chemical enrichment of the Universe at all scales is related to stellar winds and explosive supernovae phenomena. Metals produced by stars and later spread throughout the intracluster medium (ICM) at ...the megaparsec scale become a fossil record of the chemical enrichment of the Universe and of the dynamical and feedback mechanisms determining their circulation. As demonstrated by the results of the soft X-ray spectrometer onboard Hitomi, high-resolution X-ray spectroscopy is the path to differentiating among the models that consider different metal-production mechanisms, predict the outcoming yields, and are a function of the nature, mass, and/or initial metallicity of their stellar progenitor. Transformational results shall be achieved through improvements in the energy resolution and effective area of X-ray observatories, allowing them to detect rarer metals (e.g. Na, Al) and constrain yet-uncertain abundances (e.g. C, Ne, Ca, Ni). The X-ray Integral Field Unit (X-IFU) instrument onboard the next-generation European X-ray observatory
Athena
is expected to deliver such breakthroughs. Starting from 100 ks of synthetic observations of 12 abundance ratios in the ICM of four simulated clusters, we demonstrate that the X-IFU will be capable of recovering the input chemical enrichment models at both low (
z
= 0.1) and high (
z
= 1) redshifts, while statistically excluding more than 99.5% of all the other tested combinations of models. By fixing the enrichment models which provide the best fit to the simulated data, we also show that the X-IFU will constrain the slope of the stellar initial mass function within ∼12%. These constraints will be key ingredients in our understanding of the chemical enrichment of the Universe and its evolution.
ABSTRACT
Most ultraluminous X-ray sources (ULXs) are believed to be powered by super-Eddington accreting neutron stars and, perhaps, black holes. Above the Eddington rate the disc is expected to ...thicken and to launch powerful winds through radiation pressure. Winds have been recently discovered in several ULXs. However, it is yet unclear whether the thickening of the disc or the wind variability causes the switch between the classical soft and supersoft states observed in some ULXs. In order to understand such phenomenology and the overall super-Eddington mechanism, we undertook a large (800 ks) observing campaign with XMM-Newton to study NGC 247 ULX-1, which shifts between a supersoft and classical soft ULX state. The new observations show unambiguous evidence of a wind in the form of emission and absorption lines from highly ionized ionic species, with the latter indicating a mildly relativistic outflow (−0.17c) in line with the detections in other ULXs. Strong dipping activity is observed in the light curve and primarily during the brightest observations, which is typical among soft ULXs, and indicates a close relationship between the accretion rate and the appearance of the dips. The latter is likely due to a thickening of the disc scale height and the wind as shown by a progressively increasing blueshift in the spectral lines.
Atoll sources are accreting neutron star (NS) low-mass X-ray binaries. We present a spectral analysis of four persistent atoll sources (GX 3+1, 4U 1702−429, 4U 0614+091, and 4U 1746−371) observed for ...∼20 ks each with NuSTAR to determine the extent of the inner accretion disk. These sources range from an apparent luminosity of 0.006-0.11 of the Eddington limit (assuming the empirical limit of 3.8 × 1038 erg s−1). Broad Fe emission features shaped by Doppler and relativistic effects close to the NS were firmly detected in three of these sources. The position of the disk appears to be close to the innermost stable circular orbit (ISCO) in each case. For GX 3+1, we determine (90% confidence level) and an inclination of 27°-31°. For 4U 1702−429, we find a and inclination of 53°-64°. For 4U 0614+091, the disk has a position of and inclination of 50°-62°. If the disk does not extend to the innermost stable circular orbit, we can place conservative limits on the magnetic field strength in these systems in the event that the disk is truncated at the Alfvén radius. This provides the limit at the poles of B ≤ 6.7 × 108 G, 3.3 × 108 G, and 14.5 × 108 G for GX 3+1, 4U 1702−429, and 4U 0614+091, respectively. For 4U 1746−371, we argue that the most plausible explanation for the lack of reflection features is a combination of source geometry and strong Comptonization. We place these sources among the larger sample of NSs that have been observed with NuSTAR.
ABSTRACT We present results from spectral fitting of the very high state of GX 339-4 with Nuclear Spectroscopic Telescope Array (NuSTAR) and Swift. We use relativistic reflection modeling to measure ...the spin of the black hole and inclination of the inner disk and find a spin of a = 0.95 − 0.08 + 0.02 and inclination of 30° 1° (statistical errors). These values agree well with previous results from reflection modeling. With the exceptional sensitivity of NuSTAR at the high-energy side of the disk spectrum, we are able to constrain multiple physical parameters simultaneously using continuum fitting. By using the constraints from reflection as input for the continuum fitting method, we invert the conventional fitting procedure to estimate the mass and distance of GX 339-4 using just the X-ray spectrum, finding a mass of 9.0 − 1.2 + 1.6 M and distance of 8.4 0.9 kpc (statistical errors).
Conditional expression of hairpin constructs in Drosophila is a powerful method to disrupt the activity of single genes with a spatial and temporal resolution that is impossible, or exceedingly ...difficult, using classical genetic methods. We previously described a method (Ni et al. 2008) whereby RNAi constructs are targeted into the genome by the phiC31-mediated integration approach using Vermilion-AttB-Loxp-Intron-UAS-MCS (VALIUM), a vector that contains vermilion as a selectable marker, an attB sequence to allow for phiC31-targeted integration at genomic attP landing sites, two pentamers of UAS, the hsp70 core promoter, a multiple cloning site, and two introns. As the level of gene activity knockdown associated with transgenic RNAi depends on the level of expression of the hairpin constructs, we generated a number of derivatives of our initial vector, called the "VALIUM" series, to improve the efficiency of the method. Here, we report the results from the systematic analysis of these derivatives and characterize VALIUM10 as the most optimal vector of this series. A critical feature of VALIUM10 is the presence of gypsy insulator sequences that boost dramatically the level of knockdown. We document the efficacy of VALIUM as a vector to analyze the phenotype of genes expressed in the nervous system and have generated a library of 2282 constructs targeting 2043 genes that will be particularly useful for studies of the nervous system as they target, in particular, transcription factors, ion channels, and transporters.
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