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Background  PacBio high fidelity (HiFi) sequencing reads are both long (15–20 kb) and highly accurate (> Q20). Because of these properties, they have revolutionised genome assembly leading to more accurate and contiguous genomes. In eukaryotes the mitochondrial genome is sequenced alongside the nuclear genome often at very high coverage. A dedicated tool for mitochondrial genome assembly using HiFi reads is still missing. Results  MitoHiFi was developed within the Darwin Tree of Life Project to assemble mitochondrial genomes from the HiFi reads generated for target species. The input for MitoHiFi is either the raw reads or the assembled contigs, and the tool outputs a mitochondrial genome sequence fasta file along with annotation of protein and RNA genes. Variants arising from heteroplasmy are assembled independently, and nuclear insertions of mitochondrial sequences are identified and not used in organellar genome assembly. MitoHiFi has been used to assemble 374 mitochondrial genomes (368 Metazoa and 6 Fungi species) for the Darwin Tree of Life Project, the Vertebrate Genomes Project and the Aquatic Symbiosis Genome Project. Inspection of 60 mitochondrial genomes assembled with MitoHiFi for species that already have reference sequences in public databases showed the widespread presence of previously unreported repeats. Conclusions  MitoHiFi is able to assemble mitochondrial genomes from a wide phylogenetic range of taxa from Pacbio HiFi data. MitoHiFi is written in python and is freely available on GitHub ( https://github.com/marcelauliano/MitoHiFi ). MitoHiFi is available with its dependencies as a Docker container on GitHub (ghcr.io/marcelauliano/mitohifi:master).

Fluorescence microscopy is a key driver of discoveries in the life sciences, with observable phenomena being limited by the optics of the microscope, the chemistry of the fluorophores, and the maximum photon exposure tolerated by the sample. These limits necessitate trade-offs between imaging speed, spatial resolution, light exposure, and imaging depth. In this work we show how content-aware image restoration based on deep learning extends the range of biological phenomena observable by microscopy. We demonstrate on eight concrete examples how microscopy images can be restored even if 60-fold fewer photons are used during acquisition, how near isotropic resolution can be achieved with up to tenfold under-sampling along the axial direction, and how tubular and granular structures smaller than the diffraction limit can be resolved at 20-times-higher frame rates compared to state-of-the-art methods. All developed image restoration methods are freely available as open source software in Python, FIJI, and KNIME.

The transition from ‘well-marked varieties’ of a single species into ‘well-defined species’—especially in the absence of geographic barriers to gene flow (sympatric speciation)—has puzzled evolutionary biologists ever since Darwin 1 , 2 . Gene flow counteracts the buildup of genome-wide differentiation, which is a hallmark of speciation and increases the likelihood of the evolution of irreversible reproductive barriers (incompatibilities) that complete the speciation process 3 . Theory predicts that the genetic architecture of divergently selected traits can influence whether sympatric speciation occurs 4 , but empirical tests of this theory are scant because comprehensive data are difficult to collect and synthesize across species, owing to their unique biologies and evolutionary histories 5 . Here, within a young species complex of neotropical cichlid fishes ( Amphilophus spp.), we analysed genomic divergence among populations and species. By generating a new genome assembly and re-sequencing 453 genomes, we uncovered the genetic architecture of traits that have been suggested to be important for divergence. Species that differ in monogenic or oligogenic traits that affect ecological performance and/or mate choice show remarkably localized genomic differentiation. By contrast, differentiation among species that have diverged in polygenic traits is genomically widespread and much higher overall, consistent with the evolution of effective and stable genome-wide barriers to gene flow. Thus, we conclude that simple trait architectures are not always as conducive to speciation with gene flow as previously suggested, whereas polygenic architectures can promote rapid and stable speciation in sympatry. Population genomic analyses of Midas cichlid fishes in young Nicaraguan crater lakes suggest that sympatric speciation is promoted by polygenic architectures.

Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl ( Ambystoma mexicanum ) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3 . However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3 −/− and Pax7 −/− mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies. Sequencing and assembly of the 32-Gb genome of the Mexican axolotl reveals that it lacks the developmental gene Pax3 , which is essential in other vertebrates; the genome sequence could improve our understanding of the evolution of the axolotl’s remarkable regenerative capabilities. Axolotl genome sequence Elly Tanaka, Eugene Myers and colleagues report the genome sequence of the axolotl, a model organism for developmental, regeneration and evolutionary studies. To sequence and assemble this large and complex genome, the authors used a combination of long- and short-read sequencing, optical mapping and a new genome assembly pipeline, MARVEL, optimized for long-read sequencing of complex genomes. The genome assembly shows an expansion of long terminal repeat retroelements and the presence of a large HoxA cluster, but also a reduction in the number of Pax-family genes in the genome of this popular salamander.

Adaptive light-sheet microscopy improves imaging of live organisms by correcting for optical aberrations in real time. Optimal image quality in light-sheet microscopy requires a perfect overlap between the illuminating light sheet and the focal plane of the detection objective. However, mismatches between the light-sheet and detection planes are common owing to the spatiotemporally varying optical properties of living specimens. Here we present the AutoPilot framework, an automated method for spatiotemporally adaptive imaging that integrates (i) a multi-view light-sheet microscope capable of digitally translating and rotating light-sheet and detection planes in three dimensions and (ii) a computational method that continuously optimizes spatial resolution across the specimen volume in real time. We demonstrate long-term adaptive imaging of entire developing zebrafish ( Danio rerio ) and Drosophila melanogaster embryos and perform adaptive whole-brain functional imaging in larval zebrafish. Our method improves spatial resolution and signal strength two to five-fold, recovers cellular and sub-cellular structures in many regions that are not resolved by non-adaptive imaging, adapts to spatiotemporal dynamics of genetically encoded fluorescent markers and robustly optimizes imaging performance during large-scale morphogenetic changes in living organisms.

Epithelial folding transforms simple sheets of cells into complex three-dimensional tissues and organs during animal development. Epithelial folding has mainly been attributed to mechanical forces generated by an apically localized actomyosin network, however, contributions of forces generated at basal and lateral cell surfaces remain largely unknown. Here we show that a local decrease of basal tension and an increased lateral tension, but not apical constriction, drive the formation of two neighboring folds in developing Drosophila wing imaginal discs. Spatially defined reduction of extracellular matrix density results in local decrease of basal tension in the first fold; fluctuations in F-actin lead to increased lateral tension in the second fold. Simulations using a 3D vertex model show that the two distinct mechanisms can drive epithelial folding. Our combination of lateral and basal tension measurements with a mechanical tissue model reveals how simple modulations of surface and edge tension drive complex three-dimensional morphological changes.

High-throughput imaging generates massive data sets that are difficult to quantitatively analyze by hand. Peng et al . describe customizable software for visualizing and working with multi-gigabyte three-dimensional images in real time. The V3D system provides three-dimensional (3D) visualization of gigabyte-sized microscopy image stacks in real time on current laptops and desktops. V3D streamlines the online analysis, measurement and proofreading of complicated image patterns by combining ergonomic functions for selecting a location in an image directly in 3D space and for displaying biological measurements, such as from fluorescent probes, using the overlaid surface objects. V3D runs on all major computer platforms and can be enhanced by software plug-ins to address specific biological problems. To demonstrate this extensibility, we built a V3D-based application, V3D-Neuron, to reconstruct complex 3D neuronal structures from high-resolution brain images. V3D-Neuron can precisely digitize the morphology of a single neuron in a fruitfly brain in minutes, with about a 17-fold improvement in reliability and tenfold savings in time compared with other neuron reconstruction tools. Using V3D-Neuron, we demonstrate the feasibility of building a 3D digital atlas of neurite tracts in the fruitfly brain.

The structure of axonal arbors controls how signals from individual neurons are routed within the mammalian brain. However, the arbors of very few long-range projection neurons have been reconstructed in their entirety, as axons with diameters as small as 100 nm arborize in target regions dispersed over many millimeters of tissue. We introduce a platform for high-resolution, three-dimensional fluorescence imaging of complete tissue volumes that enables the visualization and reconstruction of long-range axonal arbors. This platform relies on a high-speed two-photon microscope integrated with a tissue vibratome and a suite of computational tools for large-scale image data. We demonstrate the power of this approach by reconstructing the axonal arbors of multiple neurons in the motor cortex across a single mouse brain. Nerve cells or neurons transmit electrical impulses to each other over long distances. These signals travel through highly branching nerve fibers called axons, which are about one hundred times thinner than a human hair, and can extend across the entire brain. Tracing the axon of a neuron from start to end can help to explain how individual neurons and brain areas communicate signals over long distances. A mouse brain contains approximately 70 million neurons, and tracing the axons of many neurons within a brain is a challenging problem. Tackling this problem requires a method for imaging entire brains in high enough detail to unambiguously resolve and follow axons from individual neurons across the brain. Economo, Clack et al. now demonstrate such a method for three-dimensional imaging of tissue samples as large as the whole mouse brain. This system is fully automated and works by first imaging a layer of tissue near the exposed surface of a sample, and then cutting off a slice of tissue that corresponds to the volume that has been imaged. These steps then repeat until the entire sample has been imaged; this takes about a week for a whole mouse brain and produces about 30 terabytes of images. Economo, Clack et al.’s advance can uncover how neurons communicate over long distances with an unprecedented level of precision. The method can now be used to generate a comprehensive database of neurons and their long distance connections. Such a database would aid efforts to model the roles of neural circuits in the brain, and inform the design of experiments to study brain activity during particular behaviors.

This paper describes automated methods for the accurate segmentation and tracking of tens of thousands of nuclei in time-lapse imaging data of developing embryos. The comprehensive reconstruction of cell lineages in complex multicellular organisms is a central goal of developmental biology. We present an open-source computational framework for the segmentation and tracking of cell nuclei with high accuracy and speed. We demonstrate its (i) generality by reconstructing cell lineages in four-dimensional, terabyte-sized image data sets of fruit fly, zebrafish and mouse embryos acquired with three types of fluorescence microscopes, (ii) scalability by analyzing advanced stages of development with up to 20,000 cells per time point at 26,000 cells min −1 on a single computer workstation and (iii) ease of use by adjusting only two parameters across all data sets and providing visualization and editing tools for efficient data curation. Our approach achieves on average 97.0% linkage accuracy across all species and imaging modalities. Using our system, we performed the first cell lineage reconstruction of early Drosophila melanogaster nervous system development, revealing neuroblast dynamics throughout an entire embryo.

In light microscopy, refractive index mismatches between media and sample cause spherical aberrations that often limit penetration depth and resolution. Optical clearing techniques can alleviate these mismatches, but they are so far limited to fixed samples. We present Iodixanol as a non-toxic medium supplement that allows refractive index matching in live specimens and thus substantially improves image quality in live-imaged primary cell cultures, planarians, zebrafish and human cerebral organoids. Light microscopy is a key tool in biomedical research. For perfect images, light needs to be able to pass through the sample, the material (or “mounting medium”) that holds the sample in place, and finally the image-detecting equipment in a straight line. However, in practice, light rays often deviate away from this line because they move at different speeds in different materials; how much the speed of light changes is related to a property called the refractive index of the material. This is exactly the effect that causes a stick stuck into water to look bent at the water’s surface. In light microscopy, mismatches in refractive index significantly reduce quality of the images that can be obtained. Live specimens are particularly challenging to image because different specimens have very different refractive indices compared to the mounting medium, which holds specimens in place but must also keep them alive. Although the addition of chemical compounds can theoretically match the refractive index of the mounting medium to that of the specimen, this approach has so far not been practical because such manipulations tend to kill the specimen. An important challenge has therefore been to identify a compound that can adjust, or “tune”, the refractive index of mounting media over a wide range, yet without harming the specimens. Now, Boothe et al. have identified a chemical called Iodixanol as an ideal and easy to use supplement for tuning the refractive index of water-based live imaging media. Adding Iodixanol to the mounting media did not appear to have any toxic effects on cell cultures, developing zebrafish embryos or regenerating planarian flatworms. Importantly, Boothe et al. found that Iodixanol significantly improved the quality of the images collected from all of these different specimens. It is important to stress that Iodixanol does not change the refractive index of the sample or cancel out refractive index differences within the sample – so it cannot render opaque specimens transparent. Nevertheless, Iodixanol supplementation is a simple and affordable technique to improve image quality in any live imaging application without having to resort to more expensive and highly specialized microscopes.