Systematic mapping of organism-scale gene-regulatory networks in aging using population asynchrony.

In aging, physiologic networks decline in function at rates that differ between individuals, producing a wide distribution of lifespan. Though 70% of human lifespan variance remains unexplained by heritable factors, little is known about the intrinsic sources of physiologic heterogeneity in aging. To understand how complex physiologic networks generate lifespan variation, new methods are needed. Here, we present Asynch-seq, an approach that uses gene-expression heterogeneity within isogenic populations to study the processes generating lifespan variation. By collecting thousands of single-individual transcriptomes, we capture the Caenorhabditis elegans "pan-transcriptome"-a highly resolved atlas of non-genetic variation. We use our atlas to guide a large-scale perturbation screen that identifies the decoupling of total mRNA content between germline and soma as the largest source of physiologic heterogeneity in aging, driven by pleiotropic genes whose knockdown dramatically reduces lifespan variance. Our work demonstrates how systematic mapping of physiologic heterogeneity can be applied to reduce inter-individual disparities in aging.

An amphioxus neurula stage cell atlas supports a complex scenario for the emergence of vertebrate head mesoderm.

The emergence of new structures can often be linked to the evolution of novel cell types that follows the rewiring of developmental gene regulatory subnetworks. Vertebrates are characterized by a complex body plan compared to the other chordate clades and the question remains of whether and how the emergence of vertebrate morphological innovations can be related to the appearance of new embryonic cell populations. We previously proposed, by studying mesoderm development in the cephalochordate amphioxus, a scenario for the evolution of the vertebrate head mesoderm. To further test this scenario at the cell population level, we used scRNA-seq to construct a cell atlas of the amphioxus neurula, stage at which the main mesodermal compartments are specified. Our data allowed us to validate the presence of a prechordal-plate like territory in amphioxus. Additionally, the transcriptomic profile of somite cell populations supports the homology between specific territories of amphioxus somites and vertebrate cranial/pharyngeal and lateral plate mesoderm. Finally, our work provides evidence that the appearance of the specific mesodermal structures of the vertebrate head was associated to both segregation of pre-existing cell populations, and co-option of new genes for the control of myogenesis.

Single cell atlas of Xenoturbella bocki highlights limited cell-type complexity.

Phylogenetic analyses over the last two decades have united a few small, and previously orphan clades, the nematodermatids, acoels and xenoturbelids, into the phylum Xenacoelomorpha. Some phylogenetic analyses support a sister relationship between Xenacoelomorpha and Ambulacraria (Xenambulacraria), while others suggest that Xenacoelomorpha may be sister to the rest of the Bilateria (Nephrozoa). An understanding of the cell type complements of Xenacoelomorphs is essential to assessing these alternatives as well as to our broader understanding of bilaterian cell type evolution. Employing whole organism single-cell RNA-seq in the marine xenacoelomorph worm Xenoturbella bocki, we show that Xenambulacrarian nerve nets share regulatory features and a peptidergic identity with those found in cnidarians and protostomes and more broadly share muscle and gland cell similarities with other metazoans. Taken together, these data are consistent with broad homologies of animal gland, muscle, and neurons as well as more specific affinities between Xenoturbella and acoel gut and epidermal tissues, consistent with the monophyly of Xenacoelomorpha.

Stepwise emergence of the neuronal gene expression program in early animal evolution.

The assembly of the neuronal and other major cell type programs occurred early in animal evolution. We can reconstruct this process by studying non-bilaterians like placozoans. These small disc-shaped animals not only have nine morphologically described cell types and no neurons but also show coordinated behaviors triggered by peptide-secreting cells. We investigated possible neuronal affinities of these peptidergic cells using phylogenetics, chromatin profiling, and comparative single-cell genomics in four placozoans. We found conserved cell type expression programs across placozoans, including populations of transdifferentiating and cycling cells, suggestive of active cell type homeostasis. We also uncovered fourteen peptidergic cell types expressing neuronal-associated components like the pre-synaptic scaffold that derive from progenitor cells with neurogenesis signatures. In contrast, earlier-branching animals like sponges and ctenophores lacked this conserved expression. Our findings indicate that key neuronal developmental and effector gene modules evolved before the advent of cnidarian/bilaterian neurons in the context of paracrine cell signaling.

Systematic benchmarking of single-cell ATAC-sequencing protocols.

Single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq) has emerged as a powerful tool for dissecting regulatory landscapes and cellular heterogeneity. However, an exploration of systemic biases among scATAC-seq technologies has remained absent. In this study, we benchmark the performance of eight scATAC-seq methods across 47 experiments using human peripheral blood mononuclear cells (PBMCs) as a reference sample and develop PUMATAC, a universal preprocessing pipeline, to handle the various sequencing data formats. Our analyses reveal significant differences in sequencing library complexity and tagmentation specificity, which impact cell-type annotation, genotype demultiplexing, peak calling, differential region accessibility and transcription factor motif enrichment. Our findings underscore the importance of sample extraction, method selection, data processing and total cost of experiments, offering valuable guidance for future research. Finally, our data and analysis pipeline encompasses 169,000 PBMC scATAC-seq profiles and a best practices code repository for scATAC-seq data analysis, which are freely available to extend this benchmarking effort to future protocols.

cGLRs are a diverse family of pattern recognition receptors in innate immunity.

Cyclic GMP-AMP synthase (cGAS) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2'3'-cGAMP that activates STING-dependent downstream immunity. Here, we discover that cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in innate immunity. Building on recent analysis in Drosophila, we identify >3,000 cGLRs present in nearly all metazoan phyla. A forward biochemical screening of 150 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of isomers of the nucleotide signals cGAMP, c-UMP-AMP, and c-di-AMP. Combining structural biology and in vivo analysis in coral and oyster animals, we explain how synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways. Our results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity.

Pancreas agenesis mutations disrupt a lead enhancer controlling a developmental enhancer cluster.

Sequence variants in cis-acting enhancers are important for polygenic disease, but their role in Mendelian disease is poorly understood. Redundancy between enhancers that regulate the same gene is thought to mitigate the pathogenic impact of enhancer mutations. Recent findings, however, have shown that loss-of-function mutations in a single enhancer near PTF1A cause pancreas agenesis and neonatal diabetes. Using mouse and human genetic models, we show that this enhancer activates an entire PTF1A enhancer cluster in early pancreatic multipotent progenitors. This leading role, therefore, precludes functional redundancy. We further demonstrate that transient expression of PTF1A in multipotent progenitors sets in motion an epigenetic cascade that is required for duct and endocrine differentiation. These findings shed insights into the genome regulatory mechanisms that drive pancreas differentiation. Furthermore, they reveal an enhancer that acts as a regulatory master key and is thus vulnerable to pathogenic loss-of-function mutations.

A phylogenetic and proteomic reconstruction of eukaryotic chromatin evolution.

Histones and associated chromatin proteins have essential functions in eukaryotic genome organization and regulation. Despite this fundamental role in eukaryotic cell biology, we lack a phylogenetically comprehensive understanding of chromatin evolution. Here, we combine comparative proteomics and genomics analysis of chromatin in eukaryotes and archaea. Proteomics uncovers the existence of histone post-translational modifications in archaea. However, archaeal histone modifications are scarce, in contrast with the highly conserved and abundant marks we identify across eukaryotes. Phylogenetic analysis reveals that chromatin-associated catalytic functions (for example, methyltransferases) have pre-eukaryotic origins, whereas histone mark readers and chaperones are eukaryotic innovations. We show that further chromatin evolution is characterized by expansion of readers, including capture by transposable elements and viruses. Overall, our study infers detailed evolutionary history of eukaryotic chromatin: from its archaeal roots, through the emergence of nucleosome-based regulation in the eukaryotic ancestor, to the diversification of chromatin regulators and their hijacking by genomic parasites.

Tracing the evolutionary history of Ca2+-signaling modulation by human Bcl-2: Insights from the Capsaspora owczarzaki IP3 receptor ortholog.

Recently, a functional IP3R ortholog (CO.IP3R-A) capable of IP3-induced Ca2+ release has been discovered in Capsaspora owczarzaki, a close unicellular relative to Metazoa. In contrast to mammalian IP3Rs, CO.IP3R-A is not modulated by Ca2+, ATP or PKA. Protein-sequence analysis revealed that CO.IP3R-A contained a putative binding site for anti-apoptotic Bcl-2, although Bcl-2 was not detected in Capsaspora owczarzaki and only appeared in Metazoa. Here, we examined whether human Bcl-2 could form a complex with CO.IP3R-A channels and modulate their Ca2+-flux properties using ectopic expression approaches in a HEK293 cell model in which all three IP3R isoforms were knocked out. We demonstrate that human Bcl-2 via its BH4 domain could functionally interact with CO.IP3R-A, thereby suppressing Ca2+ flux through CO.IP3R-A channels. The BH4 domain of Bcl-2 was sufficient for interaction with CO.IP3R-A channels. Moreover, mutating the Lys17 of Bcl-2's BH4 domain, the residue critical for Bcl-2-dependent modulation of mammalian IP3Rs, abrogated Bcl-2's ability to bind and inhibit CO.IP3R-A channels. Hence, this raises the possibility that a unicellular ancestor of animals already had an IP3R that harbored a Bcl-2-binding site. Bcl-2 proteins may have evolved as controllers of IP3R function by exploiting this pre-existing site, thereby counteracting Ca2+-dependent apoptosis.

Orthology Clusters from Gene Trees with Possvm.

Possvm (Phylogenetic Ortholog Sorting with Species oVerlap and MCL [Markov clustering algorithm]) is a tool that automates the process of identifying clusters of orthologous genes from precomputed phylogenetic trees and classifying gene families. It identifies orthology relationships between genes using the species overlap algorithm to infer taxonomic information from the gene tree topology, and then uses the MCL to identify orthology clusters and provide annotated gene families. Our benchmarking shows that this approach, when provided with accurate phylogenies, is able to identify manually curated orthogroups with very high precision and recall. Overall, Possvm automates the routine process of gene tree inspection and annotation in a highly interpretable manner, and provides reusable outputs and phylogeny-aware gene annotations that can be used to inform comparative genomics and gene family evolution analyses.

Evolutionary cell type mapping with single-cell genomics.

A fundamental characteristic of animal multicellularity is the spatial coexistence of functionally specialized cell types that are all encoded by a single genome sequence. Cell type transcriptional programs are deployed and maintained by regulatory mechanisms that control the asymmetric, differential access to genomic information in each cell. This genome regulation ultimately results in specific cellular phenotypes. However, the emergence, diversity, and evolutionary dynamics of animal cell types remain almost completely unexplored beyond a few species. Single-cell genomics is emerging as a powerful tool to build comprehensive catalogs of cell types and their associated gene regulatory programs in non-traditional model species. We review the current state of sampling efforts across the animal tree of life and challenges ahead for the comparative study of cell type programs. We also discuss how the phylogenetic integration of cell atlases can lead to the development of models of cell type evolution and a phylogenetic taxonomy of cells.

A stony coral cell atlas illuminates the molecular and cellular basis of coral symbiosis, calcification, and immunity.

Stony corals are colonial cnidarians that sustain the most biodiverse marine ecosystems on Earth: coral reefs. Despite their ecological importance, little is known about the cell types and molecular pathways that underpin the biology of reef-building corals. Using single-cell RNA sequencing, we define over 40 cell types across the life cycle of Stylophora pistillata. We discover specialized immune cells, and we uncover the developmental gene expression dynamics of calcium-carbonate skeleton formation. By simultaneously measuring the transcriptomes of coral cells and the algae within them, we characterize the metabolic programs involved in symbiosis in both partners. We also trace the evolution of these coral cell specializations by phylogenetic integration of multiple cnidarian cell type atlases. Overall, this study reveals the molecular and cellular basis of stony coral biology.

ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics.

Single-cell sequencing technologies are revolutionizing biology, but they are limited by the need to dissociate live samples. Here, we present ACME (ACetic-MEthanol), a dissociation approach for single-cell transcriptomics that simultaneously fixes cells. ACME-dissociated cells have high RNA integrity, can be cryopreserved multiple times, and are sortable and permeable. As a proof of principle, we provide single-cell transcriptomic data of different species, using both droplet-based and combinatorial barcoding single-cell methods. ACME uses affordable reagents, can be done in most laboratories and even in the field, and thus will accelerate our knowledge of cell types across the tree of life.

Ectopic activation of GABAB receptors inhibits neurogenesis and metamorphosis in the cnidarian Nematostella vectensis.

The metabotropic gamma-aminobutyric acid B receptor (GABABR) is a G protein-coupled receptor that mediates neuronal inhibition by the neurotransmitter GABA. While GABABR-mediated signalling has been suggested to play central roles in neuronal differentiation and proliferation across evolution, it has mostly been studied in the mammalian brain. Here, we demonstrate that ectopic activation of GABABR signalling affects neurogenic functions in the sea anemone Nematostella vectensis. We identified four putative Nematostella GABABR homologues presenting conserved three-dimensional extracellular domains and residues needed for binding GABA and the GABABR agonist baclofen. Moreover, sustained activation of GABABR signalling reversibly arrests the critical metamorphosis transition from planktonic larva to sessile polyp life stage. To understand the processes that underlie the developmental arrest, we combined transcriptomic and spatial analyses of control and baclofen-treated larvae. Our findings reveal that the cnidarian neurogenic programme is arrested following the addition of baclofen to developing larvae. Specifically, neuron development and neurite extension were inhibited, resulting in an underdeveloped and less organized nervous system and downregulation of proneural factors including NvSoxB(2), NvNeuroD1 and NvElav1. Our results thus point to an evolutionarily conserved function of GABABR in neurogenesis regulation and shed light on early cnidarian development.

IL18 signaling promotes homing of mature Tregs into the thymus.

Foxp3+ regulatory T cells (Tregs) are potent suppressor cells, essential for the maintenance of immune homeostasis. Most Tregs develop in the thymus and are then released into the immune periphery. However, some Tregs populate the thymus and constitute a major subset of yet poorly understood cells. Here we describe a subset of thymus recirculating IL18R+ Tregs with molecular characteristics highly reminiscent of tissue-resident effector Tregs. Moreover, we show that IL18R+ Tregs are endowed with higher capacity to populate the thymus than their IL18R- or IL18R-/- counterparts, highlighting the key role of IL18R in this process. Finally, we demonstrate that IL18 signaling is critical for the induction of the key thymus-homing chemokine receptor - CCR6 on Tregs. Collectively, this study provides a detailed characterization of the mature Treg subsets in the mouse thymus and identifies a key role of IL18 signaling in controlling the CCR6-CCL20-dependent migration of Tregs into the thymus.

A single-cell view on alga-virus interactions reveals sequential transcriptional programs and infection states.

The discovery of giant viruses infecting eukaryotes from diverse ecosystems has revolutionized our understanding of the evolution of viruses and their impact on protist biology, yet knowledge on their replication strategies and transcriptome regulation remains limited. Here, we profile single-cell transcriptomes of the globally distributed microalga Emiliania huxleyi and its specific giant virus during infection. We detected profound heterogeneity in viral transcript levels among individual cells. Clustering single cells based on viral expression profiles enabled reconstruction of the viral transcriptional trajectory. Reordering cells along this path unfolded highly resolved viral genetic programs composed of genes with distinct promoter elements that orchestrate sequential expression. Exploring host transcriptome dynamics across the viral infection states revealed rapid and selective shutdown of protein-encoding nuclear transcripts, while the plastid and mitochondrial transcriptomes persisted into later stages. Single-cell RNA-seq opens a new avenue to unravel the life cycle of giant viruses and their unique hijacking strategies.

MetaCell: analysis of single-cell RNA-seq data using K-nn graph partitions.

scRNA-seq profiles each represent a highly partial sample of mRNA molecules from a unique cell that can never be resampled, and robust analysis must separate the sampling effect from biological variance. We describe a methodology for partitioning scRNA-seq datasets into metacells: disjoint and homogenous groups of profiles that could have been resampled from the same cell. Unlike clustering analysis, our algorithm specializes at obtaining granular as opposed to maximal groups. We show how to use metacells as building blocks for complex quantitative transcriptional maps while avoiding data smoothing. Our algorithms are implemented in the MetaCell R/C++ software package.

Using single-cell transcriptomics to understand functional states and interactions in microbial eukaryotes.

Understanding the diversity and evolution of eukaryotic microorganisms remains one of the major challenges of modern biology. In recent years, we have advanced in the discovery and phylogenetic placement of new eukaryotic species and lineages, which in turn completely transformed our view on the eukaryotic tree of life. But we remain ignorant of the life cycles, physiology and cellular states of most of these microbial eukaryotes, as well as of their interactions with other organisms. Here, we discuss how high-throughput genome-wide gene expression analysis of eukaryotic single cells can shed light on protist biology. First, we review different single-cell transcriptomics methodologies with particular focus on microbial eukaryote applications. Then, we discuss single-cell gene expression analysis of protists in culture and what can be learnt from these approaches. Finally, we envision the application of single-cell transcriptomics to protist communities to interrogate not only community components, but also the gene expression signatures of distinct cellular and physiological states, as well as the transcriptional dynamics of interspecific interactions. Overall, we argue that single-cell transcriptomics can significantly contribute to our understanding of the biology of microbial eukaryotes. This article is part of a discussion meeting issue 'Single cell ecology'.

Origin and evolution of eukaryotic transcription factors.

Transcription factors (TFs) have a central role in genome regulation directing gene transcription through binding specific DNA sequences. Eukaryotic genomes encode a large diversity of TF classes, each defined by unique DNA-interaction domains. Recent advances in genome sequencing and phylogenetic placement of diverse eukaryotic and archaeal species are re-defining the evolutionary history of eukaryotic TFs. The emerging view from a comparative genomics perspective is that the Last Eukaryotic Common Ancestor (LECA) had an extensive repertoire of TFs, most of which represent eukaryotic evolutionary novelties. This burst of TF innovation coincides with the emergence of genomic nuclear segregation and complex chromatin organization.

Global transcriptome analysis of the aphelid Paraphelidium tribonemae supports the phagotrophic origin of fungi.

Aphelids are little-known phagotrophic parasites of algae whose life cycle and morphology resemble those of the parasitic rozellids (Cryptomycota, Rozellomycota). In previous phylogenetic analyses of RNA polymerase and rRNA genes, aphelids, rozellids and Microsporidia (parasites of animals) formed a clade, named Opisthosporidia, which appeared as the sister group to Fungi. However, the statistical support for the Opisthosporidia was always moderate. Here, we generated full life-cycle transcriptome data for the aphelid species Paraphelidium tribonemae. In-depth multi-gene phylogenomic analyses using several protein datasets place this aphelid as the closest relative of fungi to the exclusion of rozellids and Microsporidia. In contrast with the comparatively reduced Rozella allomycis genome, we infer a rich, free-living-like aphelid proteome, with a metabolism similar to fungi, including cellulases likely involved in algal cell-wall penetration and enzymes involved in chitin biosynthesis. Our results suggest that fungi evolved from complex aphelid-like ancestors that lost phagotrophy and became osmotrophic.