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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., Summary: Introducing an easy-to-use automated anatomical phenotyping pipeline for mouse embryos along with a highly-detailed anatomical E14.5 atlas.

Extended Berkeley Packet Filter (BPF) has emerged as a powerful method to extend packet-processing functionality in the Linux operating system. BPF allows users to write code in high-level languages (like C or Rust) and execute them at specific hooks in the kernel, such as the network device driver. To ensure safe execution of a user-developed BPF program in kernel context, Linux uses an in-kernel static checker. The checker allows a program to execute only if it can prove that the program is crash-free, always accesses memory within safe bounds, and avoids leaking kernel data. BPF programming is not easy. One, even modest-sized BPF programs are deemed too large to analyze and rejected by the kernel checker. Two, the kernel checker may incorrectly determine that a BPF program exhibits unsafe behaviors. Three, even small performance optimizations to BPF code (e.g., 5% gains) must be meticulously hand-crafted by expert developers. Traditional optimizing compilers for BPF are often inadequate since the kernel checker's safety constraints are incompatible with rule-based optimizations. We present K2, a program-synthesis-based compiler that automatically optimizes BPF bytecode with formal correctness and safety guarantees. K2 produces code with 6--26% reduced size, 1.36%--55.03% lower average packet-processing latency, and 0--4.75% higher throughput (packets per second per core) relative to the best clang-compiled program, across benchmarks drawn from Cilium, Facebook, and the Linux kernel. K2 incorporates several domain-specific techniques to make synthesis practical by accelerating equivalence-checking of BPF programs by 6 orders of magnitude.

Programmable switches have emerged as powerful and flexible alternatives to fixed-function forwarding devices. But because of the unique hardware constraints of network switches, the design and implementation of compilers targeting these devices is tedious and error prone. Despite the important role that compilers play in software development, there is a dearth of tools for testing compilers for programmable network devices. We present Druzhba, a programmable switch simulator used for testing compilers targeting programmable packet-processing substrates. We show that we can model the low-level behavior of a switch's programmable hardware. We further show how our machine model can be used by compiler developers to target Druzhba as a compiler backend. Generated machine code programs are fed into Druzhba and tested using a fuzzing-based approach that allows compiler developers to test the correctness of their compilers. Using a program-synthesis-based compiler as a case study, we demonstrate how Druzhba has been successful in testing compiler-generated machine code for our simulated switch pipeline instruction set., 7 pages, 4 figures

Ex vivo magnetic resonance imaging (MRI) requires chemical fixation to preserve tissue during storage or extended imaging sessions. Although it is commonly understood that fixation may alter tissue volume and shape, the potential confounding effects of fixation and storage on morphometric analyses have not been well characterized. With increasing use of ex vivo MRI for mouse brain phenotying and opportunities for inter-study comparisons, we sought to characterize how changes in fixation and/or storage times affected tissue volume, and how this might impact phenotyping results. Mouse brain samples that had been perfusion fixed, within the skull as per our standard protocol, were immersed in formaldehyde-based fixative for 1 to 5days before being stored in saline or water. Throughout fixation and storage, samples were repeatedly scanned using magnetic resonance imaging, and analyzed for volume expansion or shrinkage. We found that most of the brain continued to shrink post fixation, with the rate of shrinkage dependent on the solution in which the samples were submerged. Maximum changes in volume of 3.5% per day and 3% per month were detected during fixation and storage (in PBS), respectively. Most notably, changes were non-uniform, with some structures shrinking slower, or even expanding, when compared to other structures in the brain. Our results highlight that caution is necessary when interpreting results from experiments with inconsistent fixation and storage protocols, so as not to mistake these changes for phenotypic differences.

Despite a massive recent proliferation of Special Economic Zones (SEZs), there is virtually no quantitative research on what drives their dynamism. The aim of this paper is to address this gap and analyse the factors influencing SEZ performance – proxied by economic growth – in emerging countries. The paper relies on two novel datasets, using night-lights data to proxy for SEZ performance and containing a wide range of SEZ policy variables and characteristics across a large number of countries. The main results of the analysis indicate that a) zone growth is difficult to sustain over time; that b) trying to upgrade the technological component or value-added of the economy through SEZ policies is often challenging; and that c) zone size matters: larger zones have an advantage in terms of growth potential. Furthermore, country context significantly determines SEZ performance. Firms look for low cost locations, but in close proximity to large cities. Proximity to large markets as well as pre-existing industrialization also increase SEZ performance. In contrast, incentives and other program specific variables are highly context-specific and not structurally correlated with SEZ performance.

Approximately one third of all mammalian genes are essential for life. Phenotypes resulting from mouse knockouts of these genes have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5000 knockout mouse lines, we have identified 410 lethal genes during the production of the first 1751 unique gene knockouts. Using a standardised phenotyping platform that incorporates high-resolution 3D imaging, we identified novel 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 identified in our screen, thus providing a novel dataset that facilitates prioritization and validation of mutations identified in clinical sequencing efforts.

A missense mutation in mouse Nek8 , which encodes a ciliary kinase, produces the juvenile cystic kidneys ( jck ) model of polycystic kidney disease, but the functions of Nek8 are incompletely understood. Here, we generated a Nek8- null allele and found that homozygous mutant mice die at birth and exhibit randomization of left-right asymmetry, cardiac anomalies, and glomerular kidney cysts. The requirement for Nek8 in left-right patterning is conserved, as knockdown of the zebrafish ortholog caused randomized heart looping. Ciliogenesis was intact in Nek8- deficient embryos and cells, but we observed misexpression of left-sided marker genes early in development, suggesting that nodal ciliary signaling was perturbed. We also generated jck/Nek8 compound heterozygotes; these mutants developed less severe cystic disease than jck homozygotes and provided genetic evidence that the jck allele may encode a gain-of-function protein. Notably, NEK8 and polycystin-2 (PC2) proteins interact, and we found that Nek8 −/− and Pkd2 −/− embryonic phenotypes are strikingly similar. Nek8 -deficient embryos and cells did express PC2 normally, which localized properly to the cilia. However, similar to cells lacking PC2, NEK8-depleted inner medullary collecting duct cells exhibited a defective response to fluid shear, suggesting that NEK8 may play a role in mediating PC2-dependent signaling.

The structural organization of the brain is important for normal brain function and is critical to understand in order to evaluate changes that occur during disease processes. Three-dimensional (3D) imaging of the mouse brain is necessary to appreciate the spatial context of structures within the brain. In addition, the small scale of many brain structures necessitates resolution at the ∼10 μm scale. 3D optical imaging techniques, such as optical projection tomography (OPT), have the ability to image intact large specimens (1 cm3) with ∼5 μm resolution. In this work we assessed the potential of autofluorescence optical imaging methods, and specifically OPT, for phenotyping the mouse brain. We found that both specimen size and fixation methods affected the quality of the OPT image. Based on these findings we developed a specimen preparation method to improve the images. Using this method we assessed the potential of optical imaging for phenotyping. Phenotypic differences between wild-type male and female mice were quantified using computer-automated methods. We found that optical imaging of the endogenous autofluorescence in the mouse brain allows for 3D characterization of neuroanatomy and detailed analysis of brain phenotypes. This will be a powerful tool for understanding mouse models of disease and development and is a technology that fits easily within the workflow of biology and neuroscience labs.

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.

After more than a century of research, the mouse remains the gold-standard model system, for it recapitulates human development and disease and is quickly and highly tractable to genetic manipulations. Fundamental to the power and success of using a mouse model is the ability to stage embryonic mouse development accurately. Past staging systems were limited by the technologies of the day, such that only surface features, visible with a light microscope, could be recognized and used to define stages. With the advent of high-throughput 3D imaging tools that capture embryo morphology in microscopic detail, we now present the first 4D atlas staging system for mouse embryonic development using optical projection tomography and image registration methods. By tracking 3D trajectories of every anatomical point in the mouse embryo from E11.5 to E14.0, we established the first 4D atlas compiled from ex vivo 3D mouse embryo reference images. The resulting 4D atlas comprises 51 interpolated 3D images in this gestational range, resulting in a temporal resolution of 72 min. From this 4D atlas, any mouse embryo image can be subsequently compared and staged at the global, voxel and/or structural level. Assigning an embryonic stage to each point in anatomy allows for unprecedented quantitative analysis of developmental asynchrony among different anatomical structures in the same mouse embryo. This comprehensive developmental data set offers developmental biologists a new, powerful staging system that can identify and compare differences in developmental timing in wild-type embryos and shows promise for localizing deviations in mutant development.

The physical forces that drive morphogenesis are not well characterized in vivo, especially among vertebrates. In the early limb bud, dorsal and ventral ectoderm converge to form the apical ectodermal ridge (AER), although the underlying mechanisms are unclear. By live imaging mouse embryos, we show that prospective AER progenitors intercalate at the dorsoventral boundary and that ectoderm remodels by concomitant cell division and neighbour exchange. Mesodermal expansion and ectodermal tension together generate a dorsoventrally biased stress pattern that orients ectodermal remodelling. Polarized distribution of cortical actin reflects this stress pattern in a β-catenin- and Fgfr2-dependent manner. Intercalation of AER progenitors generates a tensile gradient that reorients resolution of multicellular rosettes on adjacent surfaces, a process facilitated by β-catenin-dependent attachment of cortex to membrane. Therefore, feedback between tissue stress pattern and cell intercalations remodels mammalian ectoderm.

Nature 537, 508–514 (2016); doi:10.1038/nature19356 In this Article, the author Wolfgang Wurst was erroneously omitted from the author list. They are associated with the affiliations: HelmholtzZentrum Munich, Institute of Developmental Genetics, 85764 Munich-Neuherberg, Germany; Technical Universityof Munich, Chair of Developmental Genetics, 85764 Munich-Neuherberg, Germany; German Center for Neurodegenerative Diseases (DZNE) Site Munich, 81377 Munich, Germany; and Munich Cluster for Systems Neurology (SyNergy), 81377 Munich, Germany.

This study used high-frequency ultrasound to evaluate the flow distribution in the mouse fetal circulation at late gestation. We studied 12 fetuses (embryonic day 17.5) from 12 pregnant CD1 mice with 40 MHz ultrasound to assess the flow in 11 vessels based on Doppler measurements of blood velocity and M-mode measurements of diameter. Specifically, the intrahepatic umbilical vein (UVIH), ductus venosus (DV), foramen ovale (FO), ascending aorta (AA), main pulmonary artery (MPA), ductus arteriosus (DA), descending thoracic aorta (DTA), common carotid artery (CCA), inferior vena cava (IVC), and right and left superior vena cavae (RSVC, LSVC) were examined, and anatomically confirmed by micro-CT. The mouse fetal circulatory system was found to be similar to that of the humans in terms of the major circuit and three shunts, but characterized by bilateral superior vena cavae and a single umbilical artery. The combined cardiac output (CCO) was 1.22 ± 0.05 ml/min, with the left ventricle (flow in AA) contributing 47.8 ± 2.3% and the right ventricle (flow in MPA) 52.2 ± 2.3%. Relative to the CCO, the flow percentages were 13.6 ± 1.0% for the UVIH, 10.4 ± 1.1% for the DV, 35.6 ± 2.4% for the DA, 41.9 ± 2.6% for the DTA, 3.8 ± 0.3% for the CCA, 29.5 ± 2.2% for the IVC, 12.7 ± 1.0% for the RSVC, and 9.9 ± 0.9% for the LSVC. The calculated flow percentage was 16.6 ± 3.4% for the pulmonary circulation and 31.2 ± 5.3% for the FO. In conclusion, the flow in mouse fetal circulation can be comprehensively evaluated with ultrasound. The baseline data of the flow distribution in normal mouse fetus serve as the reference range for future studies.

The International Mouse Phenotyping Consortium (IMPC) plans to phenotype 20,000 single-gene knockout mice to gain an insight into gene function. Approximately 30% of these knockout mouse lines will be embryonic or perinatal lethal. The IMPC has selected three-dimensional (3D) imaging to phenotype these mouse lines at relevant stages of embryonic development in an attempt to discover the cause of lethality using detailed anatomical information. Rate of throughput is paramount as IMPC production centers have been given the ambitious task of completing this phenotyping project by 2021. Sifting through the wealth of data within high-resolution 3D mouse embryo data sets by trained human experts is infeasible at this scale. Here, we present a phenotyping pipeline that identifies statistically significant anatomical differences in the knockout, in comparison with the wild type, through a computer-automated image registration algorithm. This phenotyping pipeline consists of three analyses (intensity, deformation, and atlas based) that can detect missing anatomical structures and differences in volume of whole organs as well as on the voxel level. This phenotyping pipeline was applied to micro-CT images of two perinatal lethal mouse lines: a hypomorphic mutation of the Tcf21 gene (Tcf21-hypo) and a knockout of the Satb2 gene. With the proposed pipeline we were able to identify the majority of morphological phenotypes previously published for both the Tcf21-hypo and Satb2 mutant mouse embryos in addition to novel phenotypes. This phenotyping pipeline is an unbiased, automated method that highlights only those structural abnormalities that survive statistical scrutiny and illustrates them in a straightforward fashion.

The International Mouse Phenotyping Consortium has been established to conduct large-scale phenotyping of the approximately 23,000 single-gene knockout mice generated by the International Knockout Mouse Consortium to investigate the role of each gene in the mouse genome. Of the generated mouse lines, 30% are predicted to be embryonic lethal, requiring the implementation of imaging techniques and analysis tools specific to late gestation mouse embryo phenotyping. A well-adopted technique combines the use of iodinated contrast solutions and micro-computed tomography imaging. This simple iodine immersion technique provides superior soft-tissue contrast enhancement, however, the hypertonic nature of iodine promotes dehydration causing moderate to severe tissue deformation. Here, we combine the stabilizing properties of a hydrogel mesh with the enhanced contrast properties of iodine. The protocol promotes cross linking of tissue through formaldehyde fixation and the linking of hydrogel monomers to biomolecules. As a result, the hydrogel supports tissue structure and preserves its conformation taking advantage of iodine-enhanced soft tissue contrast to produce high quality mouse embryo images with minimal tissue distortion. Hydrogel stabilization substantially reduces intersample anatomical variation of mature mouse embryos subjected to iodine preparation protocols. A 20% and 50% reduction in intersample variation of normalized brain and lung volume is achieved through hydrogel stabilization, as well as a 20% reduction in variation in overall embryo anatomy as measured through image registration methods. This increases the sensitivity of computer automated analysis to reveal significant anatomical differences between mutant and wild-type mice.

The goal of the International Mouse Phenotyping Consortium (IMPC) is to phenotype targeted knockout mouse strains throughout the whole mouse genome (23,000 genes) by 2021. A significant percentage of the generated mice will be embryonic lethal; therefore, phenotyping methods tuned to the mouse embryo are needed. Methods that are robust, quantitative, automated and high-throughput are attractive owing to the numbers of mice involved. Three-dimensional (3D) imaging is a useful method for characterizing morphological phenotypes. However, tools to automatically quantify morphological information of mouse embryos from 3D imaging have not been fully developed. We present a representative mouse embryo average 3D atlas comprising micro-CT images of 35 individual C57BL/6J mouse embryos at 15.5 days post-coitum. The 35 micro-CT images were registered into a consensus average image with our automated image registration software and 48 anatomical structures were segmented manually. We report the mean and variation in volumes for each of the 48 segmented structures. Mouse organ volumes vary by 2.6-4.2% on a linear scale when normalized to whole body volume. A power analysis of the volume data reports that a 9-14% volume difference can be detected between two classes of mice with sample sizes of eight. This resource will be crucial in establishing baseline anatomical phenotypic measurements for the assessment of mutant mouse phenotypes, as any future mutant embryo image can be registered to the atlas and subsequent organ volumes calculated automatically.

Mammalian development is a sophisticated program coordinated by a complex set of genetic and physiological factors. Alterations in anatomy or morphology provide intrinsic measures of progress in or deviations from this program. Emerging three-dimensional imaging methods now allow for more sophisticated morphological assessment than ever before, enabling comprehensive phenotyping, visualization of anatomical context and patterns, automated and quantitative morphological analysis, as well as improved understanding of the developmental time course. Furthermore, these imaging tools are becoming increasingly available and will consequently play a prominent role in elucidating the factors that direct and influence mammalian development.

Background The increasing use of the ketogenic diet (KD), particularly by women of child-bearing age, raises a question about its suitability during gestation. To date, no studies have thoroughly investigated the direct implications of a gestational ketogenic diet on embryonic development. Methods To fill this knowledge gap we imaged CD-1 mouse embryos whose mothers were fed either a Standard Diet (SD) or a KD 30 days prior to, as well as during gestation. Images were collected at embryonic days (E) 13.5 using Optical Projection Tomography (OPT) and at E17.5 using Magnetic Resonance Imaging (MRI). Results An anatomical comparison of the SD and KD embryos revealed that at E13.5 the average KD embryo was volumetrically larger, possessed a relatively larger heart but smaller brain, and had a smaller pharynx, cervical spinal cord, hypothalamus, midbrain, and pons, compared with the average SD embryo. At E17.5 the KD embryo was found to be volumetrically smaller with a relatively smaller heart and thymus, but with enlarged cervical spine, thalamus, midbrain and pons. Conclusion A ketogenic diet during gestation results in alterations in embryonic organ growth. Such alterations may be associated with organ dysfunction and potentially behavioral changes in postnatal life.