LAMA: automated image analysis for the developmental phenotyping of mouse embryos.

Advanced 3D imaging modalities, such as micro-computed tomography (micro-CT), have been incorporated into the high-throughput embryo pipeline of the International Mouse Phenotyping Consortium (IMPC). This project generates large volumes of raw data that cannot be immediately exploited without significant resources of personnel and expertise. Thus, rapid automated annotation is crucial to ensure that 3D imaging data can be integrated with other multi-dimensional phenotyping data. We present an automated computational mouse embryo phenotyping pipeline that harnesses the large amount of wild-type control data available in the IMPC embryo pipeline in order to address issues of low mutant sample number as well as incomplete penetrance and variable expressivity. We also investigate the effect of developmental substage on automated phenotyping results. Designed primarily for developmental biologists, our software performs image pre-processing, registration, statistical analysis and segmentation of embryo images. We also present a novel anatomical E14.5 embryo atlas average and, using it with LAMA, show that we can uncover known and novel dysmorphology from two IMPC knockout lines.

Cleft palate and minor metabolic disturbances in a mouse global Arl15 gene knockout.

ARL15, a small GTPase protein, was linked to metabolic traits in association studies. We aimed to test the Arl15 gene as a functional candidate for metabolic traits in the mouse. CRISPR/Cas9 germline knockout (KO) of Arl15 showed that homozygotes were postnatal lethal and exhibited a complete cleft palate (CP). Also, decreased cell migration was observed from Arl15 KO mouse embryonic fibroblasts (MEFs). Metabolic phenotyping of heterozygotes showed that females had reduced fat mass on a chow diet from 14 weeks of age. Mild body composition phenotypes were also observed in heterozygous mice on a high-fat diet (HFD)/low-fat diet (LFD). Females on a HFD showed reduced body weight, gonadal fat depot weight and brown adipose tissue (BAT) weight. In contrast, in the LFD group, females showed increased bone mineral density (BMD), while males showed a trend toward reduced BMD. Clinical biochemistry analysis of plasma on HFD showed transient lower adiponectin at 20 weeks of age in females. Urinary and plasma Mg2+ concentrations were not significantly different. Our phenotyping data showed that Arl15 is essential for craniofacial development. Adult metabolic phenotyping revealed potential roles in brown adipose tissue and bone development.

Corrigendum: High-throughput discovery of novel developmental phenotypes.

This corrects the article DOI: 10.1038/nature19356.

High-throughput discovery of novel developmental phenotypes.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.

A bioimage informatics platform for high-throughput embryo phenotyping.

High-throughput phenotyping is a cornerstone of numerous functional genomics projects. In recent years, imaging screens have become increasingly important in understanding gene-phenotype relationships in studies of cells, tissues and whole organisms. Three-dimensional (3D) imaging has risen to prominence in the field of developmental biology for its ability to capture whole embryo morphology and gene expression, as exemplified by the International Mouse Phenotyping Consortium (IMPC). Large volumes of image data are being acquired by multiple institutions around the world that encompass a range of modalities, proprietary software and metadata. To facilitate robust downstream analysis, images and metadata must be standardized to account for these differences. As an open scientific enterprise, making the data readily accessible is essential so that members of biomedical and clinical research communities can study the images for themselves without the need for highly specialized software or technical expertise. In this article, we present a platform of software tools that facilitate the upload, analysis and dissemination of 3D images for the IMPC. Over 750 reconstructions from 80 embryonic lethal and subviable lines have been captured to date, all of which are openly accessible at mousephenotype.org. Although designed for the IMPC, all software is available under an open-source licence for others to use and develop further. Ongoing developments aim to increase throughput and improve the analysis and dissemination of image data. Furthermore, we aim to ensure that images are searchable so that users can locate relevant images associated with genes, phenotypes or human diseases of interest.

Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica.

Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.

A mouse informatics platform for phenotypic and translational discovery.

The International Mouse Phenotyping Consortium (IMPC) is providing the world's first functional catalogue of a mammalian genome by characterising a knockout mouse strain for every gene. A robust and highly structured informatics platform has been developed to systematically collate, analyse and disseminate the data produced by the IMPC. As the first phase of the project, in which 5000 new knockout strains are being broadly phenotyped, nears completion, the informatics platform is extending and adapting to support the increasing volume and complexity of the data produced as well as addressing a large volume of users and emerging user groups. An intuitive interface helps researchers explore IMPC data by giving overviews and the ability to find and visualise data that support a phenotype assertion. Dedicated disease pages allow researchers to find new mouse models of human diseases, and novel viewers provide high-resolution images of embryonic and adult dysmorphologies. With each monthly release, the informatics platform will continue to evolve to support the increased data volume and to maintain its position as the primary route of access to IMPC data and as an invaluable resource for clinical and non-clinical researchers.

Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans.

Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at approximately 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for approximately 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.

VarB: a variation browsing and analysis tool for variants derived from next-generation sequencing data.

SUMMARY: There is an immediate need for tools to both analyse and visualize in real-time single-nucleotide polymorphisms, insertions and deletions, and other structural variants from new sequence file formats. We have developed VarB software that can be used to visualize variant call format files in real time, as well as identify regions under balancing selection and informative markers to differentiate user-defined groups (e.g. populations). We demonstrate its utility using sequence data from 50 Plasmodium falciparum isolates comprising two different continents and confirm known signals from genomic regions that contain important antigenic and anti-malarial drug-resistance genes. AVAILABILITY AND IMPLEMENTATION: The C++-based software VarB and user manual are available from www.pathogenseq.org/varb. CONTACT: taane.clark@lshtm.ac.uk

Application of a convolutional neural network to the quality control of MRI defacing.

Large-scale neuroimaging datasets present unique challenges for automated processing pipelines. Motivated by a large clinical trials dataset with over 235,000 MRI scans, we consider the challenge of defacing - anonymisation to remove identifying facial features. The defacing process must undergo quality control (QC) checks to ensure that the facial features have been removed and that the brain tissue is left intact. Visual QC checks are time-consuming and can cause delays in preparing data. We have developed a convolutional neural network (CNN) that can assist with the QC of the application of MRI defacing; our CNN is able to distinguish between scans that are correctly defaced and can classify defacing failures into three sub-types to facilitate parameter tuning during remedial re-defacing. Since integrating the CNN into our anonymisation pipeline, over 75,000 scans have been processed. Strict thresholds have been applied so that ambiguous classifications are referred for visual QC checks, however all scans still undergo an efficient verification check before being marked as passed. After applying the thresholds, our network is 92% accurate and can classify nearly half of the scans without the need for protracted manual checks. Our model can generalise across MRI modalities and has comparable performance when tested on an independent dataset. Even with the introduction of the verification checks, incorporation of the CNN has reduced the time spent undertaking QC checks by 42% during initial defacing, and by 35% overall. With the help of the CNN, we have been able to successfully deface 96% of the scans in the project whilst maintaining high QC standards. In a similarly sized new project, we would expect the model to reduce the time spent on manual QC checks by 125 h. Our approach is applicable to other projects with the potential to greatly improve the efficiency of imaging anonymisation pipelines.

Simultaneous Assessment of Circadian Rhythms and Sleep in Mice Using Passive Infrared Sensors: A User's Guide.

The 24-hr cycle of activity and sleep provides perhaps the most familiar example of circadian rhythms. In mammals, circadian activity rhythms are generated by a master biological clock located in the hypothalamic suprachiasmatic nuclei (SCN). This clock is synchronized (entrained) to the external light environment via light input from retinal photoreceptors. However, sleep is not a simple circadian output and also is regulated by a homeostatic process whereby extended wakefulness increases the need for subsequent sleep. As such, the amount and distribution of sleep depends upon the interaction between both circadian and homeostatic processes. Moreover, the study of circadian activity and sleep is not confined only to these specialized fields. Sleep and circadian rhythm disruption is common in many conditions, ranging from neurological and metabolic disorders to aging. Such disruption is associated with a range of negative consequences including cognitive impairment and mood disorders, as well as immune and metabolic dysfunction. As circadian activity and sleep are hallmarks of normal healthy physiology, they also provide valuable welfare indicators. However, traditional methods for the monitoring of circadian rhythms and sleep in mice can require separate specialized resources as well as significant expertise. Here, we outline a low-cost, non-invasive, and open-source method for the simultaneous assessment of circadian activity and sleep in mice. This protocol describes both the assembly of the hardware used and the capture and analysis of data without the need for expertise in electronics or data processing. © 2020 Wiley Periodicals LLC. Basic Protocol: Assembly of a PIR system for basic activity and sleep recordings Alternate Protocol: Data collection using Raspberry Pi Support Protocol: Circadian analysis using PIR sensors.

A putative DEAD-box RNA-helicase is required for normal zoospore development in the late blight pathogen Phytophthora infestans.

The asexual multinucleated sporangia of Phytophthora infestans can germinate directly through a germ tube or indirectly by releasing zoospores. The molecular mechanisms controlling sporangial cytokinesis or sporangial cleavage, and zoospore release are largely unknown. Sporangial cleavage is initiated by a cold shock that eventually compartmentalizes single nuclei within each zoospore. Comparison of EST representation in different cDNA libraries revealed a putative ATP-dependent DEAD-box RNA-helicase gene in P. infestans, Pi-RNH1, which has a 140-fold increased expression level in young zoospores compared to uncleaved sporangia. RNA interference was employed to determine the role of Pi-RNH1 in zoospore development. Silencing efficiencies of up to 99% were achieved in some transiently-silenced lines. These Pi-RNH1-silenced lines produced large aberrant zoospores that had undergone partial cleavage and often had multiple flagella on their surface. Transmission electron microscopy revealed that cytoplasmic vesicles fused in the silenced lines, resulting in the formation of large vesicles. The Pi-RNH1-silenced zoospores were also sensitive to osmotic pressure and often ruptured upon release from the sporangia. These findings indicate that Pi-RNH1 has a major function in zoospore development and its potential role in cytokinesis is discussed.

Crossed Aphasia in a Patient with Anaplastic Astrocytoma of the Non-Dominant Hemisphere.

Aphasia describes a spectrum of speech impairments due to damage in the language centers of the brain. Insult to the inferior frontal gyrus of the dominant cerebral hemisphere results in Broca's aphasia - the inability to produce fluent speech. The left cerebral hemisphere has historically been considered the dominant side, a characteristic long presumed to be related to a person's "handedness". However, recent studies utilizing fMRI have shown that right hemispheric dominance occurs more frequently than previously proposed and despite a person's handedness. Here we present a case of a right-handed patient with Broca's aphasia caused by a right-sided brain tumor. This is significant not only because the occurrence of aphasia in right-handed-individuals with right hemispheric brain damage (so-called "crossed aphasia") is unusual but also because such findings support dissociation between hemispheric linguistic dominance and handedness.

Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium.

Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.

A family of small tyrosine rich proteins is essential for oogonial and oospore cell wall development of the mycoparasitic oomycete Pythium oligandrum.

The mycoparasitic oomycete Pythium oligandrum is homothallic, producing an abundance of thick-walled spiny oospores in culture. After mining a cDNA sequence dataset, we identified a family of genes that code for small tyrosine rich (Pythium oligandrumsmall tyrosine rich (PoStr)) proteins. Sequence analysis identified similarity between the PoStr proteins and putative glycine-rich cell wall proteins from the related plant pathogenic oomycete Pythium ultimum, and mating-induced genes from the oomycete Phytophthora infestans. Expression analysis showed that PoStr transcripts accumulate during oospore production in culture and immunolocalisation indicates the presence of these proteins in oogonial and oospore cell walls. PoStr protein abundance correlated positively with production of oogonia as determined by antibiotic-mediated oogonia suppression. To further characterise the role of PoStr proteins in P. oligandrum oospore production, we silenced this gene family using homology-dependent gene silencing. This represents the first characterisation of genes using gene silencing in a Pythium species. Oospores from silenced strains displayed major ultrastructural changes and were sensitive to degradative enzyme treatment. Oogonia of silenced strains either appeared to be arrested at the mature oosphere stage of development or in around 40 % of the structures, showed a complete suppression of oospore formation. Suppressed oogonia were highly vacuolated and the oogonium wall was thickened by a new inner wall layer. Our data suggest PoStr proteins are probably integral structural components of the normal oospore cell wall and play a key role in oospore formation.

The oomycete Pythium oligandrum expresses putative effectors during mycoparasitism of Phytophthora infestans and is amenable to transformation.

The oomycete Pythium oligandrum is a mycoparasitic biocontrol agent that is able to antagonise several plant pathogens, and can promote plant growth. In order to test the potential usefulness of P. oligandrum as a biocontrol agent against late blight disease caused by the oomycete Phytophthora infestans, we investigated the interaction between P. oligandrum and Ph. infestans using the green fluorescent protein (GFP) as a reporter gene. A CaCl(2) and polyethylene-glycol-based DNA transformation protocol was developed for P. oligandrum and transformants constitutively expressing GFP were produced. Up to 56 % of P. oligandrum transformants showed both antibiotic resistance and fluorescence. Mycoparasitic interactions, including coiling of P. oligandrum hyphae around Ph. infestans hyphae, were observed with fluorescent microscopy. To gain further insights into the nature of P. oligandrum mycoparasitism, we sequenced 2376 clones from cDNA libraries of P. oligandrum mycelium grown in vitro, or on heat-killed Ph. infestans mycelium as the sole nutrient source. 1219 consensus sequences were obtained including transcripts encoding glucanases, proteases, protease inhibitors, putative effectors and elicitors, which may play a role in mycoparasitism. This represents the first published expressed sequence tag (EST) resource for P. oligandrum and provides a platform for further molecular studies and comparative analysis in the Pythiales.

Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire.

BACKGROUND: Pythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species. RESULTS: The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome, including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report of these in a genome outside the metazoans. CONCLUSIONS: Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae.