We describe the development and application of methods for high-throughput neuroanatomy in Drosophila using light microscopy. These tools enable efficient multicolor stochastic labeling of neurons at ...both low and high densities. Expression of multiple membrane-targeted and distinct epitope-tagged proteins is controlled both by a transcriptional driver and by stochastic, recombinase-mediated excision of transcription-terminating cassettes. This MultiColor FlpOut (MCFO) approach can be used to reveal cell shapes and relative cell positions and to track the progeny of precursor cells through development. Using two different recombinases, the number of cells labeled and the number of color combinations observed in those cells can be controlled separately. We demonstrate the utility of MCFO in a detailed study of diversity and variability of Distal medulla (Dm) neurons, multicolumnar local interneurons in the adult visual system. Similar to many brain regions, the medulla has a repetitive columnar structure that supports parallel information processing together with orthogonal layers of cell processes that enable communication between columns. We find that, within a medulla layer, processes of the cells of a given Dm neuron type form distinct patterns that reflect both the morphology of individual cells and the relative positions of their arbors. These stereotyped cell arrangements differ between cell types and can even differ for the processes of the same cell type in different medulla layers. This unexpected diversity of coverage patterns provides multiple independent ways of integrating visual information across the retinotopic columns and implies the existence of multiple developmental mechanisms that generate these distinct patterns.
Significance Nervous systems contain vast numbers of neurons with diverse shapes and complex spatial relationships. We describe new genetic tools for the efficient visualization by light microscopy of individual neurons and their relative positions in Drosophila . The application of these methods to the visual system revealed an unexpected diversity of cell-type–specific arrangements of neuronal processes within a single brain region. This wide range of stereotyped cell arrangements provides distinct circuit elements for processing visual information and implies the existence of a surprisingly large number of genetic programs that produce these arrangements during development.
We present broadband X-ray analyses of a sample of bright ultraluminous X-ray sources (ULX) with the goal of investigating the spectral similarity of this population to the known ULX pulsars, M82 ...X-2, NGC 7793 P13, and NGC 5907 ULX. We perform a phase-resolved analysis of the broadband XMM-Newton+NuSTAR data set of NGC 5907 ULX, finding that the pulsed emission from the accretion column in this source exhibits a similar spectral shape to that seen in both M82 X-2 and NGC 7793 P13, and that this is responsible for the excess emission observed at the highest energies when the spectra are fit with accretion disk models. We then demonstrate that similar "hard" excesses are seen in all ULXs in the broadband sample. Finally, for ULXs where the nature of the accretor is currently unknown, we test whether the hard excesses are all consistent with being produced by an accretion column similar to those present in M82 X-2, NGC 7793 P13, and NGC 5907 ULX. Based on the average shape of the pulsed emission, we find that in all cases a similar accretion column can successfully reproduce the observed data, consistent with the hypothesis that this ULX sample may be dominated by neutron star accretors. Compared to the known pulsar ULXs, our spectral fits for the remaining ULXs suggest that the non-pulsed emission from the accretion flow beyond the magnetosphere makes a stronger relative contribution than the component associated with the accretion column. If these sources do also contain neutron star accretors, this may help to explain the lack of detected pulsations.
The ability to specify the expression levels of exogenous genes inserted in the genomes of transgenic animals is critical for the success of a wide variety of experimental manipulations. Protein ...production can be regulated at the level of transcription, mRNA transport, mRNA half-life, or translation efficiency. In this report, we show that several well-characterized sequence elements derived from plant and insect viruses are able to function in Drosophila to increase the apparent translational efficiency of mRNAs by as much as 20-fold. These increases render expression levels sufficient for genetic constructs previously requiring multiple copies to be effective in single copy, including constructs expressing the temperature-sensitive inactivator of neuronal function Shibirets1, and for the use of cytoplasmic GFP to image the fine processes of neurons.
ABSTRACT We report the detection of coherent pulsations from the ultraluminous X-ray source (ULX) NGC 7793 P13. The 0.42 s nearly sinusoidal pulsations were initially discovered in broadband X-ray ...observations using XMM-Newton and NuSTAR taken in 2016. We subsequently also found pulsations in archival XMM-Newton data taken in 2013 and 2014. The significant (>5 ) detection of coherent pulsations demonstrates that the compact object in P13 is a neutron star, and given the observed peak luminosity of 1040 erg s − 1 (assuming isotropy), it is well above the Eddington limit for a 1.4 M accretor. This makes P13 the second ULX known to be powered by an accreting neutron star. The pulse period varies between epochs, with a slow but persistent spin-up over the 2013-2016 period. This spin-up indicates a magnetic field of B 1.5 × 1012 G, typical of many Galactic accreting pulsars. The most likely explanation for the extreme luminosity is a high degree of beaming; however, this is difficult to reconcile with the sinusoidal pulse profile.
ABSTRACT We present broadband, multi-epoch X-ray spectroscopy of the pulsating ultra-luminous X-ray source (ULX) in NGC 5907. Simultaneous XMM-Newton and NuSTAR data from 2014 are best described by a ...multicolor blackbody model with a temperature gradient as a function of accretion disk radius significantly flatter than expected for a standard thin accretion disk ( , with ). Additionally, we detect a hard power-law tail at energies above 10 keV, which we interpret as being due to Comptonization. We compare this observation to archival XMM-Newton, Chandra, and NuSTAR data from 2003, 2012, and 2013, and investigate possible spectral changes as a function of phase over the 78-day super-orbital period of this source. We find that observations taken around phases 0.3-0.4 show very similar temperature profiles, even though the observed flux varies significantly, while one observation taken around phase 0 has a significantly steeper profile. We discuss these findings in light of the recent discovery that the compact object is a neutron star and show that precession of the accretion disk or the neutron star can self-consistently explain most observed phenomena.
A wide variety of biological experiments rely on the ability to express an exogenous gene in a transgenic animal at a defined level and in a spatially and temporally controlled pattern. We describe ...major improvements of the methods available for achieving this objective in Drosophila melanogaster. We have systematically varied core promoters, UTRs, operator sequences, and transcriptional activating domains used to direct gene expression with the GAL4, LexA, and Split GAL4 transcription factors and the GAL80 transcriptional repressor. The use of site-specific integration allowed us to make quantitative comparisons between different constructs inserted at the same genomic location. We also characterized a set of PhiC31 integration sites for their ability to support transgene expression of both drivers and responders in the nervous system. The increased strength and reliability of these optimized reagents overcome many of the previous limitations of these methods and will facilitate genetic manipulations of greater complexity and sophistication.
Based on phase-resolved broadband spectroscopy using XMM-Newton and NuSTAR, we report on a potential cyclotron resonant scattering feature (CRSF) at E ∼ 13 keV in the pulsed spectrum of the recently ...discovered ultraluminous X-ray source (ULX) pulsar NGC 300 ULX1. If this interpretation is correct, the implied magnetic field of the central neutron star is B ∼ 1012 G (assuming scattering by electrons), similar to that estimated from the observed spin-up of the star, and also similar to known Galactic X-ray pulsars. We discuss the implications of this result for the connection between NGC 300 ULX1 and the other known ULX pulsars, particularly in light of the recent discovery of a likely proton cyclotron line in another ULX, M51 ULX-8.
Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence ...reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.
ABSTRACT We present the detection of an absorption feature at E = 8.77 − 0.06 + 0.05 keV in the combined X-ray spectrum of the ultraluminous X-ray source NGC 1313 X-1 observed with XMM-Newton and ...NuSTAR, significant at the 3 level. If associated with blueshifted ionized iron, the implied outflow velocity is ∼0.2c for Fe xxvi, or ∼0.25c for Fe xxv. These velocities are similar to the ultrafast outflow seen in absorption recently discovered in this source at lower energies by XMM-Newton, and we therefore conclude that this is an iron component to the same outflow. Photoionization modeling marginally prefers the Fe xxv solution, but in either case the outflow properties appear to be extreme, potentially supporting a super-Eddington hypothesis for NGC 1313 X-1.
Site-specific recombinases have been used for two decades to manipulate the structure of animal genomes in highly predictable ways and have become major research tools. However, the small number of ...recombinases demonstrated to have distinct specificities, low toxicity, and sufficient activity to drive reactions to completion in animals has been a limitation. In this report we show that four recombinases derived from yeast—KD, B2, B3, and R—are highly active and nontoxic in Drosophila and that KD, B2, B3, and the widely used FLP recombinase have distinct target specificities. We also show that the KD and B3 recombinases are active in mice.
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