The intrinsic and extrinsic effects of TET proteins during gastrulation.

Mice deficient for all ten-eleven translocation (TET) genes exhibit early gastrulation lethality. However, separating cause and effect in such embryonic failure is challenging. To isolate cell-autonomous effects of TET loss, we used temporal single-cell atlases from embryos with partial or complete mutant contributions. Strikingly, when developing within a wild-type embryo, Tet-mutant cells retain near-complete differentiation potential, whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential. We map de-repressions of early epiblast factors (e.g., Dppa4 and Gdf3) and failure to activate multiple signaling from nascent mesoderm (Lefty, FGF, and Notch) as likely cell-intrinsic drivers of TET loss phenotypes. We further suggest loss of enhancer demethylation as the underlying mechanism. Collectively, our work demonstrates an unbiased approach for defining intrinsic and extrinsic embryonic gene function based on temporal differentiation atlases and disentangles the intracellular effects of the demethylation machinery from its broader tissue-level ramifications.

A single-embryo, single-cell time-resolved model for mouse gastrulation.

Mouse embryonic development is a canonical model system for studying mammalian cell fate acquisition. Recently, single-cell atlases comprehensively charted embryonic transcriptional landscapes, yet inference of the coordinated dynamics of cells over such atlases remains challenging. Here, we introduce a temporal model for mouse gastrulation, consisting of data from 153 individually sampled embryos spanning 36 h of molecular diversification. Using algorithms and precise timing, we infer differentiation flows and lineage specification dynamics over the embryonic transcriptional manifold. Rapid transcriptional bifurcations characterize the commitment of early specialized node and blood cells. However, for most lineages, we observe combinatorial multi-furcation dynamics rather than hierarchical transcriptional transitions. In the mesoderm, dozens of transcription factors combinatorially regulate multifurcations, as we exemplify using time-matched chimeric embryos of Foxc1/Foxc2 mutants. Our study rejects the notion of differentiation being governed by a series of binary choices, providing an alternative quantitative model for cell fate acquisition.

Reversible, tunable epigenetic silencing of TCF1 generates flexibility in the T cell memory decision.

The immune system encodes information about the severity of a pathogenic threat in the quantity and type of memory cells it forms. This encoding emerges from lymphocyte decisions to maintain or lose self-renewal and memory potential during a challenge. By tracking CD8+ T cells at the single-cell and clonal lineage level using time-resolved transcriptomics, quantitative live imaging, and an acute infection model, we find that T cells will maintain or lose memory potential early after antigen recognition. However, following pathogen clearance, T cells may regain memory potential if initially lost. Mechanistically, this flexibility is implemented by a stochastic cis-epigenetic switch that tunably and reversibly silences the memory regulator, TCF1, in response to stimulation. Mathematical modeling shows how this flexibility allows memory T cell numbers to scale robustly with pathogen virulence and immune response magnitudes. We propose that flexibility and stochasticity in cellular decisions ensure optimal immune responses against diverse threats.

Limited access to antigen drives generation of early B cell memory while restraining the plasmablast response.

Cell fate decisions during early B cell activation determine the outcome of responses to pathogens and vaccines. We examined the early B cell response to T-dependent antigen in mice by single-cell RNA sequencing. Early after immunization, a homogeneous population of activated precursors (APs) gave rise to a transient wave of plasmablasts (PBs), followed a day later by the emergence of germinal center B cells (GCBCs). Most APs rapidly exited the cell cycle, giving rise to non-GC-derived early memory B cells (eMBCs) that retained an AP-like transcriptional profile. Rapid decline of antigen availability controlled these events; provision of excess antigen precluded cell cycle exit and induced a new wave of PBs. Fate mapping revealed a prominent contribution of eMBCs to the MBC pool. Quiescent cells with an MBC phenotype dominated the early response to immunization in primates. A reservoir of APs/eMBCs may enable rapid readjustment of the immune response when failure to contain a threat is manifested by increased antigen availability.

Deterministic and probabilistic fate decisions co-exist in a single retinal lineage.

Correct nervous system development depends on the timely differentiation of progenitor cells into neurons. While the output of progenitor differentiation is well investigated at the population and clonal level, how stereotypic or variable fate decisions are during development is still more elusive. To fill this gap, we here follow the fate outcome of single neurogenic progenitors in the zebrafish retina over time using live imaging. We find that neurogenic progenitor divisions produce two daughter cells, one of deterministic and one of probabilistic fate. Interference with the deterministic branch of the lineage affects lineage progression. In contrast, interference with fate probabilities of the probabilistic branch results in a broader range of fate possibilities than in wild-type and involves the production of any neuronal cell type even at non-canonical developmental stages. Combining the interference data with stochastic modelling of fate probabilities revealed that a simple gene regulatory network is able to predict the observed fate decision probabilities during wild-type development. These findings unveil unexpected lineage flexibility that could ensure robust development of the retina and other tissues.

Self-organized BMP signaling dynamics underlie the development and evolution of digit segmentation patterns in birds and mammals.

Repeating patterns of synovial joints are a highly conserved feature of articulated digits, with variations in joint number and location resulting in diverse digit morphologies and limb functions across the tetrapod clade. During the development of the amniote limb, joints form iteratively within the growing digit ray, as a population of distal progenitors alternately specifies joint and phalanx cell fates to segment the digit into distinct elements. While numerous molecular pathways have been implicated in this fate choice, it remains unclear how they give rise to a repeating pattern. Here, using single-cell RNA sequencing and spatial gene expression profiling, we investigate the transcriptional dynamics of interphalangeal joint specification in vivo. Combined with mathematical modeling, we predict that interactions within the BMP signaling pathway-between the ligand GDF5, the inhibitor NOGGIN, and the intracellular effector pSMAD-result in a self-organizing Turing system that forms periodic joint patterns. Our model is able to recapitulate the spatiotemporal gene expression dynamics observed in vivo, as well as phenocopy digit malformations caused by BMP pathway perturbations. By contrasting in silico simulations with in vivo morphometrics of two morphologically distinct digits, we show how changes in signaling parameters and growth dynamics can result in variations in the size and number of phalanges. Together, our results reveal a self-organizing mechanism that underpins amniote digit segmentation and its evolvability and, more broadly, illustrate how Turing systems based on a single molecular pathway may generate complex repetitive patterns in a wide variety of organisms.

The transcriptional portraits of the neural crest at the individual cell level.

Since the discovery of this cell population by His in 1850, the neural crest has been under intense study for its important role during vertebrate development. Much has been learned about the function and regulation of neural crest cell differentiation, and as a result, the neural crest has become a key model system for stem cell biology in general. The experiments performed in embryology, genetics, and cell biology in the last 150 years in the neural crest field has given rise to several big questions that have been debated intensely for many years: "How does positional information impact developmental potential? Are neural crest cells individually multipotent or a mixed population of committed progenitors? What are the key factors that regulate the acquisition of stem cell identity, and how does a stem cell decide to differentiate towards one cell fate versus another?" Recently, a marriage between single cell multi-omics, statistical modeling, and developmental biology has shed a substantial amount of light on these questions, and has paved a clear path for future researchers in the field.

Cardiac specification during gastrulation - The Yellow Brick Road leading to Tinman.

The question of how the heart develops, and the genetic networks governing this process have become intense areas of research over the past several decades. This research is propelled by classical developmental studies and potential clinical applications to understand and treat congenital conditions in which cardiac development is disrupted. Discovery of the tinman gene in Drosophila, and examination of its vertebrate homolog Nkx2.5, along with other core cardiac transcription factors has revealed how cardiac progenitor differentiation and maturation drives heart development. Careful observation of cardiac morphogenesis along with lineage tracing approaches indicated that cardiac progenitors can be divided into two broad classes of cells, namely the first and second heart fields, that contribute to the heart in two distinct waves of differentiation. Ample evidence suggests that the fate of individual cardiac progenitors is restricted to distinct cardiac structures quite early in development, well before the expression of canonical cardiac progenitor markers like Nkx2.5. Here we review the initial specification of cardiac progenitors, discuss evidence for the early patterning of cardiac progenitors during gastrulation, and consider how early gene expression programs and epigenetic patterns can direct their development. A complete understanding of when and how the developmental potential of cardiac progenitors is determined, and their potential plasticity, is of great interest developmentally and also has important implications for both the study of congenital heart disease and therapeutic approaches based on cardiac stem cell programming.

Dynamics of Chromatin Accessibility During Hematopoietic Stem Cell Differentiation Into Progressively Lineage-Committed Progeny.

Epigenetic mechanisms regulate the multilineage differentiation capacity of hematopoietic stem cells (HSCs) into a variety of blood and immune cells. Mapping the chromatin dynamics of functionally defined cell populations will shed mechanistic insight into 2 major, unanswered questions in stem cell biology: how does epigenetic identity contribute to a cell type's lineage potential, and how do cascades of chromatin remodeling dictate ensuing fate decisions? Our recent work revealed evidence of multilineage gene priming in HSCs, where open cis-regulatory elements (CREs) exclusively shared between HSCs and unipotent lineage cells were enriched for DNA binding motifs of known lineage-specific transcription factors. Oligopotent progenitor populations operating between the HSCs and unipotent cells play essential roles in effecting hematopoietic homeostasis. To test the hypothesis that selective HSC-primed lineage-specific CREs remain accessible throughout differentiation, we used ATAC-seq to map the temporal dynamics of chromatin remodeling during progenitor differentiation. We observed epigenetic-driven clustering of oligopotent and unipotent progenitors into distinct erythromyeloid and lymphoid branches, with multipotent HSCs and MPPs associating with the erythromyeloid lineage. We mapped the dynamics of lineage-primed CREs throughout hematopoiesis and identified both unique and shared CREs as potential lineage reinforcement mechanisms at fate branch points. Additionally, quantification of genome-wide peak count and size revealed overall greater chromatin accessibility in HSCs, allowing us to identify HSC-unique peaks as putative regulators of self-renewal and multilineage potential. Finally, CRISPRi-mediated targeting of ATACseq-identified putative CREs in HSCs allowed us to demonstrate the functional role of selective CREs in lineage-specific gene expression. These findings provide insight into the regulation of stem cell multipotency and lineage commitment throughout hematopoiesis and serve as a resource to test functional drivers of hematopoietic lineage fate.

Stochasticity and determinism in cell fate decisions.

During development, cells need to make decisions about their fate in order to ensure that the correct numbers and types of cells are established at the correct time and place in the embryo. Such cell fate decisions are often classified as deterministic or stochastic. However, although these terms are clearly defined in a mathematical sense, they are sometimes used ambiguously in biological contexts. Here, we provide some suggestions on how to clarify the definitions and usage of the terms stochastic and deterministic in biological experiments. We discuss the frameworks within which such clear definitions make sense and highlight when certain ambiguity prevails. As an example, we examine how these terms are used in studies of neuronal cell fate decisions and point out areas in which definitions and interpretations have changed and matured over time. We hope that this Review will provide some clarification and inspire discussion on the use of terminology in relation to fate decisions.

Principles and mechanisms of asymmetric cell division.

Asymmetric cell division (ACD) is an evolutionarily conserved mechanism used by prokaryotes and eukaryotes alike to control cell fate and generate cell diversity. A detailed mechanistic understanding of ACD is therefore necessary to understand cell fate decisions in health and disease. ACD can be manifested in the biased segregation of macromolecules, the differential partitioning of cell organelles, or differences in sibling cell size or shape. These events are usually preceded by and influenced by symmetry breaking events and cell polarization. In this Review, we focus predominantly on cell intrinsic mechanisms and their contribution to cell polarization, ACD and binary cell fate decisions. We discuss examples of polarized systems and detail how polarization is established and, whenever possible, how it contributes to ACD. Established and emerging model organisms will be considered alike, illuminating both well-documented and underexplored forms of polarization and ACD.

A geometrical perspective on development.

Cell fate decisions emerge as a consequence of a complex set of gene regulatory networks. Models of these networks are known to have more parameters than data can determine. Recent work, inspired by Waddington's metaphor of a landscape, has instead tried to understand the geometry of gene regulatory networks. Here, we describe recent results on the appropriate mathematical framework for constructing these landscapes. This allows the construction of minimally parameterized models consistent with cell behavior. We review existing examples where geometrical models have been used to fit experimental data on cell fate and describe how spatial interactions between cells can be understood geometrically.

Computational analysis of synergism in small networks with different logic.

Cell fate decision processes are regulated by networks which contain different molecules and interactions. Different network topologies may exhibit synergistic or antagonistic effects on cellular functions. Here, we analyze six most common small networks with regulatory logic AND or OR, trying to clarify the relationship between network topologies and synergism (or antagonism) related to cell fate decisions. We systematically examine the contribution of both network topologies and regulatory logic to the cell fate synergism by bifurcation and combinatorial perturbation analysis. Initially, under a single set of parameters, the synergism of three types of networks with AND and OR logic is compared. Furthermore, to consider whether these results depend on the choices of parameter values, statistics on the synergism of five hundred parameter sets is performed. It is shown that the results are not sensitive to parameter variations, indicating that the synergy or antagonism mainly depends on the network topologies rather than the choices of parameter values. The results indicate that the topology with "Dual Inhibition" shows good synergism, while the topology with "Dual Promotion" or "Hybrid" shows antagonism. The results presented here may help us to design synergistic networks based on network structure and regulation combinations, which has promising implications for cell fate decisions and drug combinations.

Immunometabolism shapes B cell fate and functions.

B lymphocyte-mediated humoral immune response is essential for protection against infectious diseases. Deeper research in B cell biology, particularly metabolism is required for the better understanding of its properties in homeostasis and in diseases. Emerging immunometabolism, including anabolism and catabolism, has tremendously impacts on immune cells from development to function and markedly advances our view on immunoregulation. Growing evidence suggests that the ultimate effect of intracellular metabolism on immune cell functions is not only influenced by the external stimuli but also by the balance of the different metabolic pathways. However, B cell immunometabolism is not deeply investigated like T cells. The complex development and differentiation processes of B cell subsets have left many untouched, but fundamental aspects in B cell metabolism. Available evidence demonstrated that the intracellular metabolism has the ubiquitous impact on B cell fate and function decisions at the transcriptional regulation and signal transduction processes. In this review, we update the recent development in the immunometabolism of B cells with the latest findings including the immune-metabolic steering on B cell development, differentiation, and function skewing, and emphasis on how immunometabolism landscape may shape B cell functions in metabolic, autoimmune, and inflammatory disorders. The metabolic interaction of B cells with other immune cells in disease context will also be discussed.

Smooth muscle cell fate decisions decipher a high-resolution heterogeneity within atherosclerosis molecular subtypes.

BACKGROUND: Mounting evidence has revealed the dynamic variations in the cellular status and phenotype of the smooth muscle cell (SMC) are vital for shaping the atherosclerotic plaque microenvironment and ultimately mapping onto heterogeneous clinical outcomes in coronary artery disease. Currently, the underlying clinical significance of SMC evolutions remains unexplored in atherosclerosis. METHODS: The dissociated cells from diseased segments within the right coronary artery of four cardiac transplant recipients and 1070 bulk samples with atherosclerosis from six bulk cohorts were retrieved. Following the SMC fate trajectory reconstruction, the MOVICS algorithm integrating the nearest template prediction was used to develop a stable and robust molecular classification. Subsequently, multi-dimensional potential biological implications, molecular features, and cell landscape heterogeneity among distinct clusters were decoded. RESULTS: We proposed an SMC cell fate decision signature (SCFDS)-based atherosclerosis stratification system and identified three SCFDS subtypes (C1-C3) with distinguishing features: (i) C1 (DNA-damage repair type), elevated base excision repair (BER), DNA replication, as well as oxidative phosphorylation status. (ii) C2 (immune-activated type), stronger immune activation, hyper-inflammatory state, the complex as well as varied lesion microenvironment, advanced stage, the most severe degree of coronary stenosis severity. (iii) C3 (stromal-rich type), abundant fibrous content, stronger ECM metabolism, immune-suppressed microenvironment. CONCLUSIONS: This study uncovered atherosclerosis complex cellular heterogeneity and a differentiated hierarchy of cell populations underlying SMC. The novel high-resolution stratification system could improve clinical outcomes and facilitate individualized management.

An incoherent feedforward loop interprets NFκB/RelA dynamics to determine TNF-induced necroptosis decisions.

Balancing cell death is essential to maintain healthy tissue homeostasis and prevent disease. Tumor necrosis factor (TNF) not only activates nuclear factor κB (NFκB), which coordinates the cellular response to inflammation, but may also trigger necroptosis, a pro-inflammatory form of cell death. Whether TNF-induced NFκB affects the fate decision to undergo TNF-induced necroptosis is unclear. Live-cell microscopy and model-aided analysis of death kinetics identified a molecular circuit that interprets TNF-induced NFκB/RelA dynamics to control necroptosis decisions. Inducible expression of TNFAIP3/A20 forms an incoherent feedforward loop to interfere with the RIPK3-containing necrosome complex and protect a fraction of cells from transient, but not long-term TNF exposure. Furthermore, dysregulated NFκB dynamics often associated with disease diminish TNF-induced necroptosis. Our results suggest that TNF's dual roles in either coordinating cellular responses to inflammation, or further amplifying inflammation are determined by a dynamic NFκB-A20-RIPK3 circuit, that could be targeted to treat inflammation and cancer.

Notch Signaling and Tissue Patterning in Embryology: An Introduction.

The attention of science first turned to the gene that later earned the name Notch over a century ago, when the American scientist John S. Dexter discovered in his laboratory at Olivet College the characteristic notched-wing phenotype (a nick or notch in the wingtip) in mutant fruit flies Drosophila melanogaster. At present, it is generally accepted that the Notch pathway governs tissue patterning and many key cell fate decisions and other core processes during embryonic development and in adult tissues. Not surprisingly, a broad variety of independent inherited diseases (including CADASIL, Alagille, Adams-Oliver, and Hajdu-Cheney syndromes) have now convincingly been linked to defective Notch signaling. In the second edition of the book entitled Notch Signaling in Embryology and Cancer, leading researchers provide a comprehensive, highly readable overview on molecular mechanisms of Notch signaling (Volume I), and notch's roles in embryology (Vol. II) and cancer (Vol. III). In these introductory pages of Vol. II, we give a short overview on its individual chapters, which are intended to provide both basic scientists and clinicians who seek today's clearest understanding of the broad role of Notch signaling in embryology with an authoritative day-to-day source.

Notch Signalling: The Multitask Manager of Inner Ear Development and Regeneration.

Notch signalling is a major regulator of cell fate decisions and tissue patterning in metazoans. It is best known for its role in lateral inhibition, whereby Notch mediates competitive interactions between cells to limit adoption of a given developmental fate. However, it can also function by lateral induction, a cooperative mode of action that was originally described during the patterning of the Drosophila wing disc and creates boundaries or domains of cells of the same character. In this chapter, we introduce these two signalling modes and explain how they contribute to distinct aspects of the development and regeneration of the vertebrate inner ear, the organ responsible for the perception of sound and head movements. We discuss some of the factors that could influence the context-specific outcomes of Notch signalling in the inner ear and the ongoing efforts to target this pathway for the treatment of hearing loss and vestibular dysfunction.

TSEE: an elastic embedding method to visualize the dynamic gene expression patterns of time series single-cell RNA sequencing data.

BACKGROUND: Time series single-cell RNA sequencing (scRNA-seq) data are emerging. However, the analysis of time series scRNA-seq data could be compromised by 1) distortion created by assorted sources of data collection and generation across time samples and 2) inheritance of cell-to-cell variations by stochastic dynamic patterns of gene expression. This calls for the development of an algorithm able to visualize time series scRNA-seq data in order to reveal latent structures and uncover dynamic transition processes. RESULTS: In this study, we propose an algorithm, termed time series elastic embedding (TSEE), by incorporating experimental temporal information into the elastic embedding (EE) method, in order to visualize time series scRNA-seq data. TSEE extends the EE algorithm by penalizing the proximal placement of latent points that correspond to data points otherwise separated by experimental time intervals. TSEE is herein used to visualize time series scRNA-seq datasets of embryonic developmental processed in human and zebrafish. We demonstrate that TSEE outperforms existing methods (e.g. PCA, tSNE and EE) in preserving local and global structures as well as enhancing the temporal resolution of samples. Meanwhile, TSEE reveals the dynamic oscillation patterns of gene expression waves during zebrafish embryogenesis. CONCLUSIONS: TSEE can efficiently visualize time series scRNA-seq data by diluting the distortions of assorted sources of data variation across time stages and achieve the temporal resolution enhancement by preserving temporal order and structure. TSEE uncovers the subtle dynamic structures of gene expression patterns, facilitating further downstream dynamic modeling and analysis of gene expression processes. The computational framework of TSEE is generalizable by allowing the incorporation of other sources of information.

Mitochondrial dynamics in the regulation of neurogenesis: From development to the adult brain.

Mitochondria are classically known to be the cellular energy producers, but a renewed appreciation for these organelles has developed with the accumulating discoveries of additional functions. The importance of mitochondria within the brain has been long known, particularly given the high-energy demanding nature of neurons. The energy demands imposed by neurons require the well-orchestrated morphological adaptation and distribution of mitochondria. Recent studies now reveal the importance of mitochondrial dynamics not only in mature neurons but also during neural development, particularly during the process of neurogenesis and neural stem cell fate decisions. In this review, we will highlight the recent findings that illustrate the importance of mitochondrial dynamics in neurodevelopment and neural stem cell function. Developmental Dynamics 247:47-53, 2018. © 2017 Wiley Periodicals, Inc.