3D reconstruction of a gastrulating human embryo.

Progress in understanding early human development has been impeded by the scarcity of reference datasets from natural embryos, particularly those with spatial information during crucial stages like gastrulation. We conducted high-resolution spatial transcriptomics profiling on 38,562 spots from 62 transverse sections of an intact Carnegie stage (CS) 8 human embryo. From this spatial transcriptomic dataset, we constructed a 3D model of the CS8 embryo, in which a range of cell subtypes are identified, based on gene expression patterns and positional register, along the anterior-posterior, medial-lateral, and dorsal-ventral axis in the embryo. We further characterized the lineage trajectories of embryonic and extra-embryonic tissues and associated regulons and the regionalization of signaling centers and signaling activities that underpin lineage progression and tissue patterning during gastrulation. Collectively, the findings of this study provide insights into gastrulation and post-gastrulation development of the human embryo.

Time-series reconstruction of the molecular architecture of human centriole assembly.

Centriole biogenesis, as in most organelle assemblies, involves the sequential recruitment of sub-structural elements that will support its function. To uncover this process, we correlated the spatial location of 24 centriolar proteins with structural features using expansion microscopy. A time-series reconstruction of protein distributions throughout human procentriole assembly unveiled the molecular architecture of the centriole biogenesis steps. We found that the process initiates with the formation of a naked cartwheel devoid of microtubules. Next, the bloom phase progresses with microtubule blade assembly, concomitantly with radial separation and rapid cartwheel growth. In the subsequent elongation phase, the tubulin backbone grows linearly with the recruitment of the A-C linker, followed by proteins of the inner scaffold (IS). By following six structural modules, we modeled 4D assembly of the human centriole. Collectively, this work provides a framework to investigate the spatial and temporal assembly of large macromolecules.

Reconstruction of neocortex: Organelles, compartments, cells, circuits, and activity.

We assembled a semi-automated reconstruction of L2/3 mouse primary visual cortex from ∼250 × 140 × 90 µm3 of electron microscopic images, including pyramidal and non-pyramidal neurons, astrocytes, microglia, oligodendrocytes and precursors, pericytes, vasculature, nuclei, mitochondria, and synapses. Visual responses of a subset of pyramidal cells are included. The data are publicly available, along with tools for programmatic and three-dimensional interactive access. Brief vignettes illustrate the breadth of potential applications relating structure to function in cortical circuits and neuronal cell biology. Mitochondria and synapse organization are characterized as a function of path length from the soma. Pyramidal connectivity motif frequencies are predicted accurately using a configuration model of random graphs. Pyramidal cells receiving more connections from nearby cells exhibit stronger and more reliable visual responses. Sample code shows data access and analysis.

Mapping transcriptomic vector fields of single cells.

Single-cell (sc)RNA-seq, together with RNA velocity and metabolic labeling, reveals cellular states and transitions at unprecedented resolution. Fully exploiting these data, however, requires kinetic models capable of unveiling governing regulatory functions. Here, we introduce an analytical framework dynamo (https://github.com/aristoteleo/dynamo-release), which infers absolute RNA velocity, reconstructs continuous vector fields that predict cell fates, employs differential geometry to extract underlying regulations, and ultimately predicts optimal reprogramming paths and perturbation outcomes. We highlight dynamo's power to overcome fundamental limitations of conventional splicing-based RNA velocity analyses to enable accurate velocity estimations on a metabolically labeled human hematopoiesis scRNA-seq dataset. Furthermore, differential geometry analyses reveal mechanisms driving early megakaryocyte appearance and elucidate asymmetrical regulation within the PU.1-GATA1 circuit. Leveraging the least-action-path method, dynamo accurately predicts drivers of numerous hematopoietic transitions. Finally, in silico perturbations predict cell-fate diversions induced by gene perturbations. Dynamo, thus, represents an important step in advancing quantitative and predictive theories of cell-state transitions.

Characterizing genetic intra-tumor heterogeneity across 2,658 human cancer genomes.

Intra-tumor heterogeneity (ITH) is a mechanism of therapeutic resistance and therefore an important clinical challenge. However, the extent, origin, and drivers of ITH across cancer types are poorly understood. To address this, we extensively characterize ITH across whole-genome sequences of 2,658 cancer samples spanning 38 cancer types. Nearly all informative samples (95.1%) contain evidence of distinct subclonal expansions with frequent branching relationships between subclones. We observe positive selection of subclonal driver mutations across most cancer types and identify cancer type-specific subclonal patterns of driver gene mutations, fusions, structural variants, and copy number alterations as well as dynamic changes in mutational processes between subclonal expansions. Our results underline the importance of ITH and its drivers in tumor evolution and provide a pan-cancer resource of comprehensively annotated subclonal events from whole-genome sequencing data.

PspA adopts an ESCRT-III-like fold and remodels bacterial membranes.

PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.

MorphoSeq: Full Single-Cell Transcriptome Dynamics Up to Gastrulation in a Chordate.

Single-cell RNA sequencing (scRNA-seq) provides a leap forward in resolving cellular diversity and developmental trajectories but fails to comprehensively delineate the spatial organization and precise cellular makeup of individual embryos. Here, we reconstruct from scRNA-seq and light sheet imaging data a canonical digital embryo that captures the genome-wide gene expression trajectory of every single cell at every cell division in the 18 lineages up to gastrulation in the ascidian Phallusia mammillata. By using high-coverage scRNA-seq, we devise a computational framework that stratifies single cells of individual embryos into cell types without prior knowledge. Unbiased transcriptome data analysis mapped each cell's physical position and lineage history, yielding the complete history of gene expression at the genome-wide level for every single cell in a developing embryo. A comparison of individual embryos reveals both extensive reproducibility between symmetric embryo sides and a large inter-embryonic variability due to small differences in embryogenesis timing.

Reconstruction of 1,000 Projection Neurons Reveals New Cell Types and Organization of Long-Range Connectivity in the Mouse Brain.

Neuronal cell types are the nodes of neural circuits that determine the flow of information within the brain. Neuronal morphology, especially the shape of the axonal arbor, provides an essential descriptor of cell type and reveals how individual neurons route their output across the brain. Despite the importance of morphology, few projection neurons in the mouse brain have been reconstructed in their entirety. Here we present a robust and efficient platform for imaging and reconstructing complete neuronal morphologies, including axonal arbors that span substantial portions of the brain. We used this platform to reconstruct more than 1,000 projection neurons in the motor cortex, thalamus, subiculum, and hypothalamus. Together, the reconstructed neurons constitute more than 85 meters of axonal length and are available in a searchable online database. Axonal shapes revealed previously unknown subtypes of projection neurons and suggest organizational principles of long-range connectivity.

Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes.

Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryo-EM structures of core and complete vRNAP enzymes from Vaccinia virus at 2.8 Å resolution. The vRNAP core enzyme resembles eukaryotic RNA polymerase II (Pol II) but also reveals many virus-specific features, including the transcription factor Rap94. The complete enzyme additionally contains the transcription factor VETF, the mRNA processing factors VTF/CE and NPH-I, the viral core protein E11, and host tRNAGln. This complex can carry out the entire early transcription cycle. The structures show that Rap94 partially resembles the Pol II initiation factor TFIIB, that the vRNAP subunit Rpo30 resembles the Pol II elongation factor TFIIS, and that NPH-I resembles chromatin remodeling enzymes. Together with the accompanying paper (Hillen et al., 2019), these results provide the basis for unraveling the mechanisms of poxvirus transcription and RNA processing.

The Evolutionary Landscape of Localized Prostate Cancers Drives Clinical Aggression.

The majority of newly diagnosed prostate cancers are slow growing, with a long natural life history. Yet a subset can metastasize with lethal consequences. We reconstructed the phylogenies of 293 localized prostate tumors linked to clinical outcome data. Multiple subclones were detected in 59% of patients, and specific subclonal architectures associate with adverse clinicopathological features. Early tumor development is characterized by point mutations and deletions followed by later subclonal amplifications and changes in trinucleotide mutational signatures. Specific genes are selectively mutated prior to or following subclonal diversification, including MTOR, NKX3-1, and RB1. Patients with low-risk monoclonal tumors rarely relapse after primary therapy (7%), while those with high-risk polyclonal tumors frequently do (61%). The presence of multiple subclones in an index biopsy may be necessary, but not sufficient, for relapse of localized prostate cancer, suggesting that evolution-aware biomarkers should be studied in prospective studies of low-risk tumors suitable for active surveillance.

Digital Museum of Retinal Ganglion Cells with Dense Anatomy and Physiology.

When 3D electron microscopy and calcium imaging are used to investigate the structure and function of neural circuits, the resulting datasets pose new challenges of visualization and interpretation. Here, we present a new kind of digital resource that encompasses almost 400 ganglion cells from a single patch of mouse retina. An online "museum" provides a 3D interactive view of each cell's anatomy, as well as graphs of its visual responses. The resource reveals two aspects of the retina's inner plexiform layer: an arbor segregation principle governing structure along the light axis and a density conservation principle governing structure in the tangential plane. Structure is related to visual function; ganglion cells with arbors near the layer of ganglion cell somas are more sustained in their visual responses on average. Our methods are potentially applicable to dense maps of neuronal anatomy and physiology in other parts of the nervous system.

Structural Basis for Gating and Activation of RyR1.

The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca(2+)) release channel required for skeletal muscle contraction. Here, we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca(2+), ATP, and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca(2+) alone induces conformational changes in the cytoplasmic assembly ("priming"), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement, and deformation of the S4-S5 linker and conformational changes in the pseudo-voltage-sensor domain.

Cryogenic electron microscopy and single-particle analysis.

About 20 years ago, the first three-dimensional (3D) reconstructions at subnanometer (<10-Å) resolution of an icosahedral virus assembly were obtained by cryogenic electron microscopy (cryo-EM) and single-particle analysis. Since then, thousands of structures have been determined to resolutions ranging from 30 Å to near atomic (<4 Å). Almost overnight, the recent development of direct electron detectors and the attendant improvement in analysis software have advanced the technology considerably. Near-atomic-resolution reconstructions can now be obtained, not only for megadalton macromolecular complexes or highly symmetrical assemblies but also for proteins of only a few hundred kilodaltons. We discuss the developments that led to this breakthrough in high-resolution structure determination by cryo-EM and point to challenges that lie ahead.

Ancestral ribonucleases back in motion for evolutionary-dynamics guided protein design.

The dynamics behavior of a protein is essential for its functionality. Here, Doucet et al. demonstrate how the evolutionary analysis of conformational pathways within a protein family serves to identify common core scaffolds that accommodate branch-specific functional regions controlled by flexibility switches, offering a model for evolutionary-dynamics based protein design.

Computationally guided AAV engineering for enhanced gene delivery.

Gene delivery vehicles based on adeno-associated viruses (AAVs) are enabling increasing success in human clinical trials, and they offer the promise of treating a broad spectrum of both genetic and non-genetic disorders. However, delivery efficiency and targeting must be improved to enable safe and effective therapies. In recent years, considerable effort has been invested in creating AAV variants with improved delivery, and computational approaches have been increasingly harnessed for AAV engineering. In this review, we discuss how computationally designed AAV libraries are enabling directed evolution. Specifically, we highlight approaches that harness sequences outputted by next-generation sequencing (NGS) coupled with machine learning (ML) to generate new functional AAV capsids and related regulatory elements, pushing the frontier of what vector engineering and gene therapy may achieve.

Metamorphic protein folding as evolutionary adaptation.

Metamorphic proteins switch reversibly between multiple distinct, stable structures, often with different functions. It was previously hypothesized that metamorphic proteins arose as intermediates in the evolution of a new fold - rare and transient exceptions to the 'one sequence, one fold' paradigm. However, as described herein, mounting evidence suggests that metamorphic folding is an adaptive feature, preserved and optimized over evolutionary time as exemplified by the NusG family and the chemokine XCL1. Analysis of extant protein families and resurrected protein ancestors demonstrates that large regions of sequence space are compatible with metamorphic folding. As a category that enhances biological fitness, metamorphic proteins are likely to employ fold switching to perform important biological functions and may be more common than previously thought.

High-throughput morphometric and transcriptomic profiling uncovers composition of naïve and sensory-deprived cortical cholinergic VIP/CHAT neurons.

Cortical neuronal networks control cognitive output, but their composition and modulation remain elusive. Here, we studied the morphological and transcriptional diversity of cortical cholinergic VIP/ChAT interneurons (VChIs), a sparse population with a largely unknown function. We focused on VChIs from the whole barrel cortex and developed a high-throughput automated reconstruction framework, termed PopRec, to characterize hundreds of VChIs from each mouse in an unbiased manner, while preserving 3D cortical coordinates in multiple cleared mouse brains, accumulating thousands of cells. We identified two fundamentally distinct morphological types of VChIs, bipolar and multipolar that differ in their cortical distribution and general morphological features. Following mild unilateral whisker deprivation on postnatal day seven, we found after three weeks both ipsi- and contralateral dendritic arborization differences and modified cortical depth and distribution patterns in the barrel fields alone. To seek the transcriptomic drivers, we developed NuNeX, a method for isolating nuclei from fixed tissues, to explore sorted VChIs. This highlighted differentially expressed neuronal structural transcripts, altered exitatory innervation pathways and established Elmo1 as a key regulator of morphology following deprivation.

Cardiomyocyte orientation recovery at micrometer scale reveals long-axis fiber continuum in heart walls.

Coordinated cardiomyocyte contraction drives the mammalian heart to beat and circulate blood. No consensus model of cardiomyocyte geometrical arrangement exists, due to the limited spatial resolution of whole heart imaging methods and the piecemeal nature of studies based on histological sections. By combining microscopy and computer vision, we produced the first-ever three-dimensional cardiomyocyte orientation reconstruction across mouse ventricular walls at the micrometer scale, representing a gain of three orders of magnitude in spatial resolution. We recovered a cardiomyocyte arrangement aligned to the long-axis direction of the outer ventricular walls. This cellular network lies in a thin shell and forms a continuum with longitudinally arranged cardiomyocytes in the inner walls, with a complex geometry at the apex. Our reconstruction methods can be applied at fine spatial scales to further understanding of heart wall electrical function and mechanics, and set the stage for the study of micron-scale fiber remodeling in heart disease.

Interface-confined intermediate phase in TiO2 enables efficient photocatalysis.

As a prototypical photocatalyst, TiO[Formula: see text] has been extensively studied. An interesting yet puzzling experimental fact was that P25-a mixture of anatase and rutile TiO[Formula: see text]-outperforms the individual phases; the origin of this mysterious fact, however, remains elusive. Employing rigorous first-principles calculations, here we uncover a metastable intermediate structure (MIS), which is formed due to confinement at the anatase/rutile interface. The MIS has a high conduction-band minimum level and thus substantially enhances the overpotential of the hydrogen evolution reaction. Also, the corresponding band alignment at the interface leads to efficient separation of electrons and holes. The interfacial confinement additionally creates a wide distribution of the band gap in the vicinity of the interface, which in turn improves optical absorption. These factors all contribute to the enhanced photocatalytic efficiency in P25. Our insights provide a rationale to the puzzling superior photocatalytic performance of P25 and enable a strategy to achieve highly efficient photocatalysis via interface engineering.

Isotope and morphometrical evidence reveals the technological package associated with agriculture adoption in western Europe.

This study aimed to reconstruct the environmental conditions and the crop management practices and plant characteristics when agriculture appeared in western Europe. We analyzed oak charcoal and a large number of cereal caryopsides recovered from La Draga (Girona, Spain), an early (5300 to 4800 cal. BC) agricultural site from the Iberian Peninsula. The carbon isotope discrimination (Δ13C) values of oak, the dominant forest species in the region, indicates prevalence of a wet climate at the site. Further, we reconstructed crop management conditions, achievable yield, and crop characteristics through the analysis of Δ13C, nitrogen isotope composition (δ15N), nitrogen content, and the reconstructed weight of wheat and barley caryopsides, following protocols developed by our team [Araus et al., Nat. Commun. 5, 3953 (2014)] and comparison of these parameters with present-day organic agriculture in the region. In parallel, a regional perspective was achieved through the study of wheat and barley grains of seventeen Neolithic sites from the western Mediterranean. The results suggest that rather than small-garden cultivation, a more extensive agriculture was practiced under good water availability and moderate manuring. Moreover, results from La Draga evidence that grain weight and spike morphology were comparable to contemporary cereals. Growing conditions and the prevalence of improved crop traits indicate that agriculture was fairly consolidated at the time it reached the western edge of Europe.