Method of moments framework for differential expression analysis of single-cell RNA sequencing data.

Differential expression analysis of single-cell RNA sequencing (scRNA-seq) data is central for characterizing how experimental factors affect the distribution of gene expression. However, distinguishing between biological and technical sources of cell-cell variability and assessing the statistical significance of quantitative comparisons between cell groups remain challenging. We introduce Memento, a tool for robust and efficient differential analysis of mean expression, variability, and gene correlation from scRNA-seq data, scalable to millions of cells and thousands of samples. We applied Memento to 70,000 tracheal epithelial cells to identify interferon-responsive genes, 160,000 CRISPR-Cas9 perturbed T cells to reconstruct gene-regulatory networks, 1.2 million peripheral blood mononuclear cells (PBMCs) to map cell-type-specific quantitative trait loci (QTLs), and the 50-million-cell CELLxGENE Discover corpus to compare arbitrary cell groups. In all cases, Memento identified more significant and reproducible differences in mean expression compared with existing methods. It also identified differences in variability and gene correlation that suggest distinct transcriptional regulation mechanisms imparted by perturbations.

Designing Biological Circuits: Synthetic Biology Within the Operon Model and Beyond.

In 1961, Jacob and Monod proposed the operon model of gene regulation. At the model's core was the modular assembly of regulators, operators, and structural genes. To illustrate the composability of these elements, Jacob and Monod linked phenotypic diversity to the architectures of regulatory circuits. In this review, we examine how the circuit blueprints imagined by Jacob and Monod laid the foundation for the first synthetic gene networks that launched the field of synthetic biology in 2000. We discuss the influences of the operon model and its broader theoretical framework on the first generation of synthetic biological circuits, which were predominantly transcriptional and posttranscriptional circuits. We also describe how recent advances in molecular biology beyond the operon model-namely, programmable DNA- and RNA-binding molecules as well as models of epigenetic and posttranslational regulation-are expanding the synthetic biology toolkit and enabling the design of more complex biological circuits.

Single-cell protein activity analysis identifies recurrence-associated renal tumor macrophages.

Clear cell renal carcinoma (ccRCC) is a heterogeneous disease with a variable post-surgical course. To assemble a comprehensive ccRCC tumor microenvironment (TME) atlas, we performed single-cell RNA sequencing (scRNA-seq) of hematopoietic and non-hematopoietic subpopulations from tumor and tumor-adjacent tissue of treatment-naive ccRCC resections. We leveraged the VIPER algorithm to quantitate single-cell protein activity and validated this approach by comparison to flow cytometry. The analysis identified key TME subpopulations, as well as their master regulators and candidate cell-cell interactions, revealing clinically relevant populations, undetectable by gene-expression analysis. Specifically, we uncovered a tumor-specific macrophage subpopulation characterized by upregulation of TREM2/APOE/C1Q, validated by spatially resolved, quantitative multispectral immunofluorescence. In a large clinical validation cohort, these markers were significantly enriched in tumors from patients who recurred following surgery. The study thus identifies TREM2/APOE/C1Q-positive macrophage infiltration as a potential prognostic biomarker for ccRCC recurrence, as well as a candidate therapeutic target.

Recovering Gene Interactions from Single-Cell Data Using Data Diffusion.

Single-cell RNA sequencing technologies suffer from many sources of technical noise, including under-sampling of mRNA molecules, often termed "dropout," which can severely obscure important gene-gene relationships. To address this, we developed MAGIC (Markov affinity-based graph imputation of cells), a method that shares information across similar cells, via data diffusion, to denoise the cell count matrix and fill in missing transcripts. We validate MAGIC on several biological systems and find it effective at recovering gene-gene relationships and additional structures. Applied to the epithilial to mesenchymal transition, MAGIC reveals a phenotypic continuum, with the majority of cells residing in intermediate states that display stem-like signatures, and infers known and previously uncharacterized regulatory interactions, demonstrating that our approach can successfully uncover regulatory relations without perturbations.

Toward Minimal Residual Disease-Directed Therapy in Melanoma.

Many patients with advanced cancers achieve dramatic responses to a panoply of therapeutics yet retain minimal residual disease (MRD), which ultimately results in relapse. To gain insights into the biology of MRD, we applied single-cell RNA sequencing to malignant cells isolated from BRAF mutant patient-derived xenograft melanoma cohorts exposed to concurrent RAF/MEK-inhibition. We identified distinct drug-tolerant transcriptional states, varying combinations of which co-occurred within MRDs from PDXs and biopsies of patients on treatment. One of these exhibited a neural crest stem cell (NCSC) transcriptional program largely driven by the nuclear receptor RXRG. An RXR antagonist mitigated accumulation of NCSCs in MRD and delayed the development of resistance. These data identify NCSCs as key drivers of resistance and illustrate the therapeutic potential of MRD-directed therapy. They also highlight how gene regulatory network architecture reprogramming may be therapeutically exploited to limit cellular heterogeneity, a key driver of disease progression and therapy resistance.

Co-optation of Tandem DNA Repeats for the Maintenance of Mesenchymal Identity.

Tandem repeats (TRs) are generated by DNA replication errors and retain a high level of instability, which in principle would make them unsuitable for integration into gene regulatory networks. However, the appearance of DNA sequence motifs recognized by transcription factors may turn TRs into functional cis-regulatory elements, thus favoring their stabilization in genomes. Here, we show that, in human cells, the transcriptional repressor ZEB1, which promotes the maintenance of mesenchymal features largely by suppressing epithelial genes and microRNAs, occupies TRs harboring dozens of copies of its DNA-binding motif within genomic loci relevant for maintenance of epithelial identity. The deletion of one such TR caused quasi-mesenchymal cancer cells to reacquire epithelial features, partially recapitulating the effects of ZEB1 gene deletion. These data demonstrate that the high density of identical motifs in TRs can make them suitable platforms for recruitment of transcriptional repressors, thus promoting their exaptation into pre-existing cis-regulatory networks.

A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain.

The diversity of cell types and regulatory states in the brain, and how these change during aging, remains largely unknown. We present a single-cell transcriptome atlas of the entire adult Drosophila melanogaster brain sampled across its lifespan. Cell clustering identified 87 initial cell clusters that are further subclustered and validated by targeted cell-sorting. Our data show high granularity and identify a wide range of cell types. Gene network analyses using SCENIC revealed regulatory heterogeneity linked to energy consumption. During aging, RNA content declines exponentially without affecting neuronal identity in old brains. This single-cell brain atlas covers nearly all cells in the normal brain and provides the tools to study cellular diversity alongside other Drosophila and mammalian single-cell datasets in our unique single-cell analysis platform: SCope (http://scope.aertslab.org). These results, together with SCope, allow comprehensive exploration of all transcriptional states of an entire aging brain.

An Integrated Epigenomic and Transcriptomic Map of Mouse and Human αß T Cell Development.

αß lineage T cells, most of which are CD4+ or CD8+ and recognize MHC I- or MHC II-presented antigens, are essential for immune responses and develop from CD4+CD8+ thymocytes. The absence of in vitro models and the heterogeneity of αß thymocytes have hampered analyses of their intrathymic differentiation. Here, combining single-cell RNA and ATAC (chromatin accessibility) sequencing, we identified mouse and human αß thymocyte developmental trajectories. We demonstrated asymmetric emergence of CD4+ and CD8+ lineages, matched differentiation programs of agonist-signaled cells to their MHC specificity, and identified correspondences between mouse and human transcriptomic and epigenomic patterns. Through computational analysis of single-cell data and binding sites for the CD4+-lineage transcription factor Thpok, we inferred transcriptional networks associated with CD4+- or CD8+-lineage differentiation, and with expression of Thpok or of the CD8+-lineage factor Runx3. Our findings provide insight into the mechanisms of CD4+ and CD8+ T cell differentiation and a foundation for mechanistic investigations of αß T cell development.

Studying lineage decision-making in vitro: emerging concepts and novel tools.

Correct and timely lineage decisions are critical for normal embryonic development and homeostasis of adult tissues. Therefore, the search for fundamental principles that underlie lineage decision-making lies at the heart of developmental biology. Here, we review attempts to understand lineage decision-making as the interplay of single-cell heterogeneity and gene regulation. Fluctuations at the single-cell level are an important driving force behind cell-state transitions and the creation of cell-type diversity. Gene regulatory networks amplify such fluctuations and define stable cell types. They also mediate the influence of signaling inputs on the lineage decision. In this review, we focus on insights gleaned from in vitro differentiation of embryonic stem cells. We discuss emerging concepts, with an emphasis on transcriptional regulation, dynamical aspects of differentiation, and functional single-cell heterogeneity. We also highlight some novel tools to study lineage decision-making in vitro.

Comparative analysis of gene regulatory networks: from network reconstruction to evolution.

Regulation of gene expression is central to many biological processes. Although reconstruction of regulatory circuits from genomic data alone is therefore desirable, this remains a major computational challenge. Comparative approaches that examine the conservation and divergence of circuits and their components across strains and species can help reconstruct circuits as well as provide insights into the evolution of gene regulatory processes and their adaptive contribution. In recent years, advances in genomic and computational tools have led to a wealth of methods for such analysis at the sequence, expression, pathway, module, and entire network level. Here, we review computational methods developed to study transcriptional regulatory networks using comparative genomics, from sequence to functional data. We highlight how these methods use evolutionary conservation and divergence to reliably detect regulatory components as well as estimate the extent and rate of divergence. Finally, we discuss the promise and open challenges in linking regulatory divergence to phenotypic divergence and adaptation.

A Repeat-Associated Small RNA Controls the Major Virulence Factors of Helicobacter pylori.

Many bacterial pathogens regulate their virulence genes via phase variation, whereby length-variable simple sequence repeats control the transcription or coding potential of those genes. Here, we have exploited this relationship between DNA structure and physiological function to discover a globally acting small RNA (sRNA) regulator of virulence in the gastric pathogen Helicobacter pylori. Our study reports the first sRNA whose expression is affected by a variable thymine (T) stretch in its promoter. We show the sRNA post-transcriptionally represses multiple major pathogenicity factors of H. pylori, including CagA and VacA, by base pairing to their mRNAs. We further demonstrate transcription of the sRNA is regulated by the nickel-responsive transcriptional regulator NikR (thus named NikS for nickel-regulated sRNA), thereby linking virulence factor regulation to nickel concentrations. Using in-vitro infection experiments, we demonstrate NikS affects host cell internalization and epithelial barrier disruption. Together, our results show NikS is a phase-variable, post-transcriptional global regulator of virulence properties in H. pylori.

Genes Involved in the Development and Physiology of Both the Peripheral and Central Auditory Systems.

The genetic approach, based on the study of inherited forms of deafness, has proven to be particularly effective for deciphering the molecular mechanisms underlying the development of the peripheral auditory system, the cochlea and its afferent auditory neurons, and how this system extracts the physical parameters of sound. Although this genetic dissection has provided little information about the central auditory system, scattered data suggest that some genes may have a critical role in both the peripheral and central auditory systems. Here, we review the genes controlling the development and function of the peripheral and central auditory systems, focusing on those with demonstrated intrinsic roles in both systems and highlighting the current underappreciation of these genes. Their encoded products are diverse, from transcription factors to ion channels, as are their roles in the central auditory system, mostly evaluated in brainstem nuclei. We examine the ontogenetic and evolutionary mechanisms that may underlie their expression at different sites.

Understanding developmental system drift.

Developmental system drift (DSD) occurs when the genetic basis for homologous traits diverges over time despite conservation of the phenotype. In this Review, we examine the key ideas, evidence and open problems arising from studies of DSD. Recent work suggests that DSD may be pervasive, having been detected across a range of different organisms and developmental processes. Although developmental research remains heavily reliant on model organisms, extrapolation of findings to non-model organisms can be error-prone if the lineages have undergone DSD. We suggest how existing data and modelling approaches may be used to detect DSD and estimate its frequency. More direct study of DSD, we propose, can inform null hypotheses for how much genetic divergence to expect on the basis of phylogenetic distance, while also contributing to principles of gene regulatory evolution.

Contrasting the development of larval and adult body plans during the evolution of biphasic lifecycles in sea urchins.

Biphasic lifecycles are widespread among animals, but little is known about how the developmental transition between larvae and adults is regulated. Sea urchins are a unique system for studying this phenomenon because of the stark differences between their bilateral larval and pentaradial adult body plans. Here, we use single-cell RNA sequencing to analyze the development of Heliocidaris erythrogramma (He), a sea urchin species with an accelerated, non-feeding mode of larval development. The sequencing time course extends from embryogenesis to roughly a day before the onset of metamorphosis in He larvae, which is a period that has not been covered by previous datasets. We find that the non-feeding developmental strategy of He is associated with several changes in the specification of larval cell types compared to sea urchins with feeding larvae, such as the loss of a larva-specific skeletal cell population. Furthermore, the development of the larval and adult body plans in sea urchins may utilize largely different sets of regulatory genes. These findings lay the groundwork for extending existing developmental gene regulatory networks to cover additional stages of biphasic lifecycles.

Integrative multi-omics increase resolution of the sea urchin posterior gut gene regulatory network at single-cell level.

Drafting gene regulatory networks (GRNs) requires embryological knowledge pertaining to the cell type families, information on the regulatory genes, causal data from gene knockdown experiments and validations of the identified interactions by cis-regulatory analysis. We use multi-omics involving next-generation sequencing to obtain the necessary information for drafting the Strongylocentrotus purpuratus (Sp) posterior gut GRN. Here, we present an update to the GRN using: (1) a single-cell RNA-sequencing-derived cell atlas highlighting the 2 day-post-fertilization (dpf) sea urchin gastrula cell type families, as well as the genes expressed at the single-cell level; (2) a set of putative cis-regulatory modules and transcription factor-binding sites obtained from chromatin accessibility ATAC-seq data; and (3) interactions directionality obtained from differential bulk RNA sequencing following knockdown of the transcription factor Sp-Pdx1, a key regulator of gut patterning in sea urchins. Combining these datasets, we draft the GRN for the hindgut Sp-Pdx1-positive cells in the 2 dpf gastrula embryo. Overall, our data suggest the complex connectivity of the posterior gut GRN and increase the resolution of gene regulatory cascades operating within it.

Bioenergetic costs and the evolution of noise regulation by microRNAs.

Noise control, together with other regulatory functions facilitated by microRNAs (miRNAs), is believed to have played important roles in the evolution of multicellular eukaryotic organisms. miRNAs can dampen protein fluctuations via enhanced degradation of messenger RNA (mRNA), but this requires compensation by increased mRNA transcription to maintain the same expression levels. The overall mechanism is metabolically expensive, leading to questions about how it might have evolved in the first place. We develop a stochastic model of miRNA noise regulation, coupled with a detailed analysis of the associated metabolic costs. Additionally, we calculate binding free energies for a range of miRNA seeds, the short sequences which govern target recognition. We argue that natural selection may have fine-tuned the Michaelis-Menten constant [Formula: see text] describing miRNA-mRNA affinity and show supporting evidence from analysis of experimental data. [Formula: see text] is constrained by seed length, and optimal noise control (minimum protein variance at a given energy cost) is achievable for seeds of 6 to 7 nucleotides in length, the most commonly observed types. Moreover, at optimality, the degree of noise reduction approaches the theoretical bound set by the Wiener-Kolmogorov linear filter. The results illustrate how selective pressure toward energy efficiency has potentially shaped a crucial regulatory pathway in eukaryotes.

A time-resolved single-cell roadmap of the logic driving anterior neural crest diversification from neural border to migration stages.

Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of early molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification toward definitive neural crest. Here, we combine ultradense single-cell transcriptomes with machine-learning and large-scale transcriptomic and epigenomic experimental validation of selected trajectories, to provide the general principles and highlight specific features of the GRN underlying neural crest fate diversification from induction to early migration stages using Xenopus frog embryos as a model. During gastrulation, a transient neural border zone state precedes the choice between neural crest and placodes which includes multiple converging gene programs. During neurulation, transcription factor connectome, and bifurcation analyses demonstrate the early emergence of neural crest fates at the neural plate stage, alongside an unbiased multipotent-like lineage persisting until epithelial-mesenchymal transition stage. We also decipher circuits driving cranial and vagal neural crest formation and provide a broadly applicable high-throughput validation strategy for investigating single-cell transcriptomes in vertebrate GRNs in development, evolution, and disease.

'Building a perfect body': control of vertebrate organogenesis by PBX-dependent regulatory networks.

Pbx genes encode transcription factors that belong to the TALE (three-amino-acid loop extension) superclass of homeodomain proteins. We have witnessed a surge in information about the roles of this gene family as leading actors in the transcriptional control of development. PBX proteins represent a clear example of how transcription factors can regulate developmental processes by combinatorial properties, acting within multimeric complexes to implement activation or repression of transcription depending on their interaction partners. Here, we revisit long-emphasized functions of PBX transcription factors as cofactors for HOX proteins, major architects of the body plan. We further discuss new knowledge on roles of PBX proteins in different developmental contexts as upstream regulators of Hox genes-as factors that interact with non-HOX proteins and can work independently of HOX-as well as potential pioneer factors. Committed to building a perfect body, PBX proteins govern regulatory networks that direct essential morphogenetic processes and organogenesis in vertebrate development. Perturbations of PBX-dependent networks can cause human congenital disease and cancer.

Enhancement of LIN28B-induced hematopoietic reprogramming by IGF2BP3.

Fetal hematopoietic stem and progenitor cells (HSPCs) hold promise to cure a wide array of hematological diseases, and we previously found a role for the RNA-binding protein (RBP) Lin28b in respecifying adult HSPCs to resemble their fetal counterparts. Here we show by single-cell RNA sequencing that Lin28b alone was insufficient for complete reprogramming of gene expression from the adult toward the fetal pattern. Using proteomics and in situ analyses, we found that Lin28b (and its closely related paralog, Lin28a) directly interacted with Igf2bp3, another RBP, and their enforced co-expression in adult HSPCs reactivated fetal-like B-cell development in vivo more efficiently than either factor alone. In B-cell progenitors, Lin28b and Igf2bp3 jointly stabilized thousands of mRNAs by binding at the same sites, including those of the B-cell regulators Pax5 and Arid3a as well as Igf2bp3 mRNA itself, forming an autoregulatory loop. Our results suggest that Lin28b and Igf2bp3 are at the center of a gene regulatory network that mediates the fetal-adult hematopoietic switch. A method to efficiently generate induced fetal-like hematopoietic stem cells (ifHSCs) will facilitate basic studies of their biology and possibly pave a path toward their clinical application.

SWI/SNF complexes are required for retinal pigmented epithelium differentiation and for the inhibition of cell proliferation and neural differentiation programs.

During embryonic development, tissue-specific transcription factors and chromatin remodelers function together to ensure gradual, coordinated differentiation of multiple lineages. Here, we define this regulatory interplay in the developing retinal pigmented epithelium (RPE), a neuroectodermal lineage essential for the development, function and maintenance of the adjacent retina. We present a high-resolution spatial transcriptomic atlas of the developing mouse RPE and the adjacent ocular mesenchyme obtained by geographical position sequencing (Geo-seq) of a single developmental stage of the eye that encompasses young and more mature ocular progenitors. These transcriptomic data, available online, reveal the key transcription factors and their gene regulatory networks during RPE and ocular mesenchyme differentiation. Moreover, conditional inactivation followed by Geo-seq revealed that this differentiation program is dependent on the activity of SWI/SNF complexes, shown here to control the expression and activity of RPE transcription factors and, at the same time, inhibit neural progenitor and cell proliferation genes. The findings reveal the roles of the SWI/SNF complexes in controlling the intersection between RPE and neural cell fates and the coupling of cell-cycle exit and differentiation.