Wnt dose escalation during the exit from pluripotency identifies tranilast as a regulator of cardiac mesoderm.

Wnt signaling is a critical determinant of cell lineage development. This study used Wnt dose-dependent induction programs to gain insights into molecular regulation of stem cell differentiation. We performed single-cell RNA sequencing of hiPSCs responding to a dose escalation protocol with Wnt agonist CHIR-99021 during the exit from pluripotency to identify cell types and genetic activity driven by Wnt stimulation. Results of activated gene sets and cell types were used to build a multiple regression model that predicts the efficiency of cardiomyocyte differentiation. Cross-referencing Wnt-associated gene expression profiles to the Connectivity Map database, we identified the small-molecule drug, tranilast. We found that tranilast synergistically activates Wnt signaling to promote cardiac lineage differentiation, which we validate by in vitro analysis of hiPSC differentiation and in vivo analysis of developing quail embryos. Our study provides an integrated workflow that links experimental datasets, prediction models, and small-molecule databases to identify drug-like compounds that control cell differentiation.

Inferring cell diversity in single cell data using consortium-scale epigenetic data as a biological anchor for cell identity.

Methods for cell clustering and gene expression from single-cell RNA sequencing (scRNA-seq) data are essential for biological interpretation of cell processes. Here, we present TRIAGE-Cluster which uses genome-wide epigenetic data from diverse bio-samples to identify genes demarcating cell diversity in scRNA-seq data. By integrating patterns of repressive chromatin deposited across diverse cell types with weighted density estimation, TRIAGE-Cluster determines cell type clusters in a 2D UMAP space. We then present TRIAGE-ParseR, a machine learning method which evaluates gene expression rank lists to define gene groups governing the identity and function of cell types. We demonstrate the utility of this two-step approach using atlases of in vivo and in vitro cell diversification and organogenesis. We also provide a web accessible dashboard for analysis and download of data and software. Collectively, genome-wide epigenetic repression provides a versatile strategy to define cell diversity and study gene regulation of scRNA-seq data.

The cellular dynamics of neural tube formation.

The vertebrate brain and spinal cord arise from a common precursor, the neural tube, which forms very early during embryonic development. To shape the forming neural tube, changes in cellular architecture must be tightly co-ordinated in space and time. Live imaging of different animal models has provided valuable insights into the cellular dynamics driving neural tube formation. The most well-characterised morphogenetic processes underlying this transformation are convergent extension and apical constriction, which elongate and bend the neural plate. Recent work has focused on understanding how these two processes are spatiotemporally integrated from the tissue- to the subcellular scale. Various mechanisms of neural tube closure have also been visualised, yielding a growing understanding of how cellular movements, junctional remodelling and interactions with the extracellular matrix promote fusion and zippering of the neural tube. Additionally, live imaging has also now revealed a mechanical role for apoptosis in neural plate bending, and how cell intercalation forms the lumen of the secondary neural tube. Here, we highlight the latest research on the cellular dynamics underlying neural tube formation and provide some perspectives for the future.

Mechanical forces in avian embryo development.

Research using avian embryos has led to major conceptual advances in developmental biology, virology, immunology, genetics and cell biology. The avian embryo has several significant advantages, including ready availability and ease of accessibility, rapid development with marked similarities to mammals and a high amenability to manipulation. As mechanical forces are increasingly recognised as key drivers of morphogenesis, this powerful model system is shedding new light on the mechanobiology of embryonic development. Here, we highlight progress in understanding how mechanical forces direct key morphogenetic processes in the early avian embryo. Recent advances in quantitative live imaging and modelling are elaborating upon traditional work using physical models and embryo manipulations to reveal cell dynamics and tissue forces in ever greater detail. The recent application of transgenic technologies further increases the strength of the avian model and is providing important insights about previously intractable developmental processes.

Specification of the First Mammalian Cell Lineages In Vivo and In Vitro.

Our understanding of how the first mammalian cell lineages arise has been shaped largely by studies of the preimplantation mouse embryo. Painstaking work over many decades has begun to reveal how a single totipotent cell is transformed into a multilayered structure representing the foundations of the body plan. Here, we review how the first lineage decision is initiated by epigenetic regulation but consolidated by the integration of morphological features and transcription factor activity. The establishment of pluripotent and multipotent stem cell lines has enabled deeper analysis of molecular and epigenetic regulation of cell fate decisions. The capability to assemble these stem cells into artificial embryos is an exciting new avenue of research that offers a long-awaited window into cell fate specification in the human embryo. Together, these approaches are poised to profoundly increase our understanding of how the first lineage decisions are made during mammalian embryonic development.

Instructions for Assembling the Early Mammalian Embryo.

The preimplantation mouse embryo is a simple self-contained system, making it an excellent model to discover how mammalian cells function in real time and in vivo. Work over the last decade has revealed some key morphogenetic mechanisms that drive early development, yielding rudimentary instructions for the generation of a mammalian embryo. Here, we review the instructions revealed thus far, and then discuss remaining challenges to discover upstream factors controlling cell fate determination, test the role of mechanisms based on biological noise, and take advantage of recent technological developments to advance the spatial and temporal resolution of our current understanding.

Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation.

Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation.

In Vivo Imaging of Single Mammalian Cells in Development and Disease.

Live imaging has transformed biomedical sciences by enabling visualization and analysis of dynamic cellular processes as they occur in their native contexts. Here, we review key recent efforts applying in vivo optical imaging with single-cell resolution to mammalian systems ranging from embryos to adult tissues and organs. We highlight insights into active processes regulating cell fate and morphogenesis during embryonic development, how neuronal circuitry and non-neuronal cell types contribute to neurological functions, and how novel imaging-based approaches enable the dissection of neurological disorders and cancer with high spatio-temporal resolution. The convergence of technical advancements in accessing, visualizing, and manipulating individual cells provides an unprecedented lens to probe mammalian cellular dynamics in vivo in both physiological and pathological states.

Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy.

Probing transcription factor (TF)-DNA interactions remains challenging in complex in vivo systems such as mammalian embryos, especially when TF copy numbers and fluorescence background are high. To address this difficulty, fluorescence correlation spectroscopy (FCS) can be combined with the use of photoactivatable fluorescent proteins to achieve selective photoactivation of a subset of tagged TF molecules. This approach, termed paFCS, enables FCS measurements within single cell nuclei inside live embryos, and obtains autocorrelation data of a quality previously only attainable in simpler in vitro cell culture systems. Here, we present a protocol demonstrating the applicability of paFCS in developing mouse embryos by outlining its implementation on a commercial laser-scanning microscope. We also provide procedures for optimizing the photoactivation and acquisition parameters and determining key parameters describing TF-DNA binding. The entire procedure can be performed within ∼2 d (excluding embryo culture time), although the acquisition of each paFCS data set takes only ∼10 min. This protocol can be used to noninvasively reveal cell-to-cell variation in TF dynamics, as well as critical, fate-predicting changes over the course of early embryonic development.

How cells change shape and position in the early mammalian embryo.

During preimplantation development, cells of the mammalian embryo must resolve their shape and position to ensure the future viability of the fetus. These initial changes are established as the embryo expands from one to thirty-two cells, and a group of originally spherical cells is transformed into a more polarized structure with distinct cell geometries and lineages. Recent advances in the application of non-invasive imaging technologies have enabled the discovery of mechanisms regulating patterning of the early mammalian embryo. Here, we review recent findings revealing cell protrusions that trigger early changes in cell shape and embryo compaction, and how anisotropies in mechanical forces drive the first spatial segregation of cells in the embryo to form the pluripotent inner mass.

Quantitative imaging of mammalian transcriptional dynamics: from single cells to whole embryos.

Probing dynamic processes occurring within the cell nucleus at the quantitative level has long been a challenge in mammalian biology. Advances in bio-imaging techniques over the past decade have enabled us to directly visualize nuclear processes in situ with unprecedented spatial and temporal resolution and single-molecule sensitivity. Here, using transcription as our primary focus, we survey recent imaging studies that specifically emphasize the quantitative understanding of nuclear dynamics in both time and space. These analyses not only inform on previously hidden physical parameters and mechanistic details, but also reveal a hierarchical organizational landscape for coordinating a wide range of transcriptional processes shared by mammalian systems of varying complexity, from single cells to whole embryos.

Long-Lived Binding of Sox2 to DNA Predicts Cell Fate in the Four-Cell Mouse Embryo.

Transcription factor (TF) binding to DNA is fundamental for gene regulation. However, it remains unknown how the dynamics of TF-DNA interactions change during cell-fate determination in vivo. Here, we use photo-activatable FCS to quantify TF-DNA binding in single cells of developing mouse embryos. In blastocysts, the TFs Oct4 and Sox2, which control pluripotency, bind DNA more stably in pluripotent than in extraembryonic cells. By contrast, in the four-cell embryo, Sox2 engages in more long-lived interactions than does Oct4. Sox2 long-lived binding varies between blastomeres and is regulated by H3R26 methylation. Live-cell tracking demonstrates that those blastomeres with more long-lived binding contribute more pluripotent progeny, and reducing H3R26 methylation decreases long-lived binding, Sox2 target expression, and pluripotent cell numbers. Therefore, Sox2-DNA binding predicts mammalian cell fate as early as the four-cell stage. More generally, we reveal the dynamic repartitioning of TFs between DNA sites driven by physiological epigenetic changes. VIDEO ABSTRACT.

Cortical Tension Allocates the First Inner Cells of the Mammalian Embryo.

Every cell in our body originates from the pluripotent inner mass of the embryo, yet it is unknown how biomechanical forces allocate inner cells in vivo. Here we discover subcellular heterogeneities in tensile forces, generated by actomyosin cortical networks, which drive apical constriction to position the first inner cells of living mouse embryos. Myosin II accumulates specifically around constricting cells, and its disruption dysregulates constriction and cell fate. Laser ablations of actomyosin networks reveal that constricting cells have higher cortical tension, generate tension anisotropies and morphological changes in adjacent regions of neighboring cells, and require their neighbors to coordinate their own changes in shape. Thus, tensile forces determine the first spatial segregation of cells during mammalian development. We propose that, unlike more cohesive tissues, the early embryo dissipates tensile forces required by constricting cells via their neighbors, thereby allowing confined cell repositioning without jeopardizing global architecture.

How adhesion forms the early mammalian embryo.

The early mouse embryo is an excellent system to study how a small group of initially rounded cells start to change shape and establish the first forms of adhesion-based cell-cell interactions in mammals in vivo. In addition to its critical role in the structural integrity of the embryo, we discuss here how adhesion is important to regulate cell polarity and cell fate. Recent evidence suggests that adherens junctions participate in signaling pathways by localizing key proteins to subcellular microdomains. E-cadherin has been identified as the main player required for the establishment of adhesion but other mechanisms involving additional proteins or physical forces acting in the embryo may also contribute. Application of new technologies that enable high-resolution quantitative imaging of adhesion protein dynamics and measurements of biomechanical forces will provide a greater understanding of how adhesion patterns the early mammalian embryo.

Cadherin-dependent filopodia control preimplantation embryo compaction.

Compaction of the preimplantation embryo is the earliest morphogenetic process essential for mammalian development, yet it remains unclear how round cells elongate to form a compacted embryo. Here, using live mouse embryo imaging, we demonstrate that cells extend long E-cadherin-dependent filopodia on to neighbouring cells, which control the cell shape changes necessary for compaction. We found that filopodia extension is tightly coordinated with cell elongation, whereas retraction occurs before cells become round again before dividing. Laser-based ablations revealed that filopodia are required to maintain elongated cell shapes. Moreover, molecular disruption of the filopodia components E-cadherin, α- and ß-catenin, F-actin and myosin-X prevents cells from elongating and compacting the embryo. Finally, we show that early filopodia formation triggered by overexpressing myosin-X is sufficient to induce premature compaction. Our findings establish a role for filopodia during preimplantation embryonic development and provide an in vivo context to investigate the biological functions of filopodia in mammals.

A Molecular Toolbox for Rapid Generation of Viral Vectors to Up- or Down-Regulate Neuronal Gene Expression in vivo.

We introduce a molecular toolbox for manipulation of neuronal gene expression in vivo. The toolbox includes promoters, ion channels, optogenetic tools, fluorescent proteins, and intronic artificial microRNAs. The components are easily assembled into adeno-associated virus (AAV) or lentivirus vectors using recombination cloning. We demonstrate assembly of toolbox components into lentivirus and AAV vectors and use these vectors for in vivo expression of inwardly rectifying potassium channels (Kir2.1, Kir3.1, and Kir3.2) and an artificial microRNA targeted against the ion channel HCN1 (HCN1 miRNA). We show that AAV assembled to express HCN1 miRNA produces efficacious and specific in vivo knockdown of HCN1 channels. Comparison of in vivo viral transduction using HCN1 miRNA with mice containing a germ line deletion of HCN1 reveals similar physiological phenotypes in cerebellar Purkinje cells. The easy assembly and re-usability of the toolbox components, together with the ability to up- or down-regulate neuronal gene expression in vivo, may be useful for applications in many areas of neuroscience.

RNAi for the treatment of prion disease: a window for intervention in neurodegeneration?

Effective treatment of neurodegenerative disease is one of the major challenges facing biomedical research. These disorders, which include Alzheimer's, Huntington's and Parkinson's diseases - as well as the rarer prion diseases - constitute an ever-increasing burden in the developed world, socially, medically and economically. The key barrier to effective therapy is that they present clinically when neuronal loss is advanced, and irreversible. Current treatments are almost all directed at modifying symptoms; few address underlying pathogenic mechanisms and are inevitably delivered too late to rescue dying neurons. In the field of prion diseases, however, insights into the molecular basis and the temporal evolution of prion neurotoxicity are increasing. Recent work in mice leads to new hope for the treatment of these disorders, and potentially for rescuing neurodegeneration more broadly. Using lentivirally mediated RNA interference (RNAi) against native prion protein (PrP), White et al report the first intervention resulting in neuronal rescue, prevention of symptoms and increased survival in mice with established prion disease. Central to the effectiveness of this strategy are both the target and the timing of the intervention: the treatment prevents the formation of the neurotoxic prion agent at a point when diseased neurons can still be saved from death. This review introduces the basic concepts of prion pathogenesis, emerging insights into mechanisms of prion neurotoxicity and the rationale for targeting endogenous prion protein (PrP) in prion therapeutics. It describes the discovery of a window of reversibility of early neuronal damage in prion disease and how together these advances led to the subsequent development of the strategy using RNAi based therapy for these disorders. It discusses the use and relevance of this approach more broadly in neurodegeneration.

Therapy for prion diseases: Insights from the use of RNA interference.

Insights into the molecular basis and the temporal evolution of neurotoxicity in prion disease are increasing, and recent work in mice leads to new avenues for targeting treatment of these disorders. Using lentivirally mediated RNA interference (RNAi) against native prion protein (PrP), White et al. report the first therapeutic intervention that results in neuronal rescue, prevents symptoms and increases survival in mice with established prion disease.(1) Both the target and the timing of treatment here are crucial to the effectiveness of this strategy: the formation of the neurotoxic prion agent is prevented at a point when diseased neurons can still be saved from death. But the data also give new insights into the timing of treatment in the context of the pattern of spread of prion infection throughout the brain, with implications for developing the most effective treatments.

Tuning of synaptic integration in the medial entorhinal cortex to the organization of grid cell firing fields.

Neurons important for cognitive function are often classified by their morphology and integrative properties. However, it is unclear if within a single class of neuron these properties tune synaptic responses to the salient features of the information that each neuron represents. We demonstrate that for stellate neurons in layer II of the medial entorhinal cortex, the waveform of postsynaptic potentials, the time window for detection of coincident inputs, and responsiveness to gamma frequency inputs follow a dorsal-ventral gradient similar to the topographical organization of grid-like spatial firing fields of neurons in this area. We provide evidence that these differences are due to a membrane conductance gradient mediated by HCN and leak potassium channels. These findings suggest key roles for synaptic integration in computations carried out within the medial entorhinal cortex and imply that tuning of neural information processing by membrane ion channels is important for normal cognitive function.

Single treatment with RNAi against prion protein rescues early neuronal dysfunction and prolongs survival in mice with prion disease.

Prion diseases are fatal neurodegenerative conditions for which there is no effective treatment. Prion propagation involves the conversion of cellular prion protein, PrP(C), to its conformational isomer, PrP(Sc), which accumulates in disease. Here, we show effective therapeutic knockdown of PrP(C) expression using RNAi in mice with established prion disease. A single administration of lentivirus expressing a shRNA targeting PrP into each hippocampus of mice with established prion disease significantly prolonged survival time. Treated animals lived 19% and 24% longer than mice given an "empty" lentivirus, or not treated, respectively. Lentivirally mediated RNAi of PrP also prevented the onset of behavioral deficits associated with early prion disease, reduced spongiform degeneration, and protected against neuronal loss. In contrast, mice receiving empty virus or no treatment developed early cognitive impairment and showed severe spongiosis and neuronal loss. The focal use of RNAi therapeutically in prion disease further supports strategies depleting PrP(C), which we previously established to be a valid target for prion-based treatments. This approach can now be used to define the temporal, quantitative, and regional requirements for PrP knockdown for effective treatment of prion disease and to explore mechanisms involved in predegenerative neuronal dysfunction and its rescue.