Imaging plant germline differentiation within Arabidopsis flower by light sheet microscopy, bioRxiv, 2019-09-20
AbstractIn higher plants, germline differentiation occurs during a relatively short period within developing flowers. Understanding of the mechanisms that govern germline differentiation lags behind other plant developmental processes. This is largely because the germline is restricted to relatively few cells buried deep within floral tissues, which makes them difficult to study. To overcome this limitation, we have developed a methodology for live imaging of the germ cell lineage within floral organs of Arabidopsis using light sheet fluorescence microscopy. We have established reporter lines, cultivation conditions, and imaging protocols for high-resolution microscopy of developing flowers continuously for up to several days. We used multiview imagining to reconstruct a three-dimensional model of a flower at subcellular resolution. We demonstrate the power of this approach by capturing male and female meiosis, asymmetric pollen division, movement of meiotic chromosomes, and unusual restitution mitosis in tapetum cells. This method will enable new avenues of research into plant sexual reproduction.
biorxiv plant-biology 200-500-users 2019Multiple testing correction over contrasts for brain imaging, bioRxiv, 2019-09-20
AbstractThe multiple testing problem arises not only when there are many voxels or vertices in an image representation of the brain, but also when multiple contrasts of parameter estimates (that is, hypotheses) are tested in the same general linear model. Here we argue that a correction for this multiplicity must be performed to avoid excess of false positives. Various methods have been proposed in the literature, but few have been applied to brain imaging. Here we discuss and compare different methods to make such correction in different scenarios, showing that one classical and well known method is invalid, and argue that permutation is the best option to perform such correction due to its exactness and flexibility to handle a variety of common imaging situations.
biorxiv neuroscience 100-200-users 2019The genome of Caenorhabditis bovis, bioRxiv, 2019-09-20
AbstractThe free-living nematode Caenorhabditis elegans is a key laboratory model for metazoan biology. C. elegans is also used as a model for parasitic nematodes despite being only distantly related to most parasitic species. All ∼65 Caenorhabditis species currently in culture are free-living with most having been isolated from decaying plant or fungal matter. Caenorhabditis bovis is a particularly unusual species, having been isolated several times from the inflamed ears of Zebu cattle in Eastern Africa where it is believed to be the cause of bovine parasitic otitis. C. bovis is therefore of particular interest to researchers interested in the evolution of nematode parasitism and in Caenorhabditis diversity. However, as C. bovis is not in laboratory culture, it remains little studied and details of its prevalence, role in bovine parasitic otitis and relationships to other Caenorhabditis species are scarce. Here, by sampling livestock markets and slaughterhouses in Western Kenya, we successfully reisolate C. bovis from the ear of adult female Zebu. We sequence the genome of C. bovis using the Oxford Nanopore MinION platform in a nearby field laboratory and use the data to generate a chromosome-scale draft genome sequence. We exploit this draft genome to reconstruct the phylogenetic relationships of C. bovis to other Caenorhabditis species and reveal the changes in genome size and content that have occurred during its evolution. We also identify expansions in several gene families that have been implicated in parasitism in other nematode species, including those associated with resistance to antihelminthic drugs. The high-quality draft genome and our analyses thereof represent a significant advancement in our understanding of this unusual Caenorhabditis species.
biorxiv genomics 0-100-users 2019Selection of representative genomes for 24,706 bacterial and archaeal species clusters provide a complete genome-based taxonomy, bioRxiv, 2019-09-19
AbstractWe recently introduced the Genome Taxonomy Database (GTDB), a phylogenetically consistent, genome-based taxonomy providing rank normalized classifications for nearly 150,000 genomes from domain to genus. However, nearly 40% of the genomes used to infer the GTDB reference tree lack a species name, reflecting the large number of genomes in public repositories without complete taxonomic assignments. Here we address this limitation by proposing 24,706 species clusters which encompass all publicly available bacterial and archaeal genomes when using commonly accepted average nucleotide identity (ANI) criteria for circumscribing species. In contrast to previous ANI studies, we selected a single representative genome to serve as the nomenclatural type for circumscribing each species with type strains used where available. We complemented the 8,792 species clusters with validly or effectively published names with 15,914de novospecies clusters in order to assign placeholder names to the growing number of genomes from uncultivated species. This provides the first complete domain to species taxonomic framework which will improve communication of scientific results.
biorxiv microbiology 200-500-users 2019Precision calcium imaging of dense neural populations via a cell body-targeted calcium indicator, bioRxiv, 2019-09-18
AbstractMethods for one-photon fluorescent imaging of calcium dynamics in vivo are popular due to their ability to simultaneously capture the dynamics of hundreds of neurons across large fields of view, at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk between cell bodies and the surrounding neuropil, resulting in decreased signal-to-noise and artifactual correlations of neural activity. Here, we address this problem by engineering cell body-targeted variants of the fluorescent calcium indicator GCaMP6f. We screened fusions of GCaMP6f to both natural as well as engineered peptides, and identified fusions that localized GCaMP6f to within approximately 50 microns of the cell body of neurons in live mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP6f in dense neural circuits reported fewer artifactual spikes from neuropil, increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators increases neuronal signal fidelity and may facilitate even greater usage of simple, powerful, one-photon methods of population imaging of neural calcium dynamics.
biorxiv neuroscience 0-100-users 2019centroFlye Assembling Centromeres with Long Error-Prone Reads, bioRxiv, 2019-09-17
AbstractAlthough variations in centromeres have been linked to cancer and infertility, centromeres still represent the “dark matter of the human genome” and remain an enigma for both biomedical and evolutionary studies. Since centromeres have withstood all previous attempts to develop an automated tool for their assembly and since their assembly using short reads is viewed as intractable, recent efforts attempted to manually assemble centromeres using long error-prone reads. We describe the centroFlye algorithm for centromere assembly using long error-prone reads, apply it for assembling the human X centromere, and use the constructed assembly to gain insights into centromere evolution. Our analysis reveals putative breakpoints in the previous manual reconstruction of the human X centromere and opens a possibility to automatically close the remaining multi-megabase gaps in the reference human genome.
biorxiv bioinformatics 100-200-users 2019