The first two blastomeres contribute unequally to the human embryo.

Retrospective lineage reconstruction of humans predicts that dramatic clonal imbalances in the body can be traced to the 2-cell stage embryo. However, whether and how such clonal asymmetries arise in the embryo is unclear. Here, we performed prospective lineage tracing of human embryos using live imaging, non-invasive cell labeling, and computational predictions to determine the contribution of each 2-cell stage blastomere to the epiblast (body), hypoblast (yolk sac), and trophectoderm (placenta). We show that the majority of epiblast cells originate from only one blastomere of the 2-cell stage embryo. We observe that only one to three cells become internalized at the 8-to-16-cell stage transition. Moreover, these internalized cells are more frequently derived from the first cell to divide at the 2-cell stage. We propose that cell division dynamics and a cell internalization bottleneck in the early embryo establish asymmetry in the clonal composition of the future human body.

Human embryo live imaging reveals nuclear DNA shedding during blastocyst expansion and biopsy.

Proper preimplantation development is essential to assemble a blastocyst capable of implantation. Live imaging has uncovered major events driving early development in mouse embryos; yet, studies in humans have been limited by restrictions on genetic manipulation and lack of imaging approaches. We have overcome this barrier by combining fluorescent dyes with live imaging to reveal the dynamics of chromosome segregation, compaction, polarization, blastocyst formation, and hatching in the human embryo. We also show that blastocyst expansion mechanically constrains trophectoderm cells, causing nuclear budding and DNA shedding into the cytoplasm. Furthermore, cells with lower perinuclear keratin levels are more prone to undergo DNA loss. Moreover, applying trophectoderm biopsy, a mechanical procedure performed clinically for genetic testing, increases DNA shedding. Thus, our work reveals distinct processes underlying human development compared with mouse and suggests that aneuploidies in human embryos may not only originate from chromosome segregation errors during mitosis but also from nuclear DNA shedding.

Temporally multiplexed imaging of dynamic signaling networks in living cells.

Molecular signals interact in networks to mediate biological processes. To analyze these networks, it would be useful to image many signals at once, in the same living cell, using standard microscopes and genetically encoded fluorescent reporters. Here, we report temporally multiplexed imaging (TMI), which uses genetically encoded fluorescent proteins with different clocklike properties-such as reversibly photoswitchable fluorescent proteins with different switching kinetics-to represent different cellular signals. We linearly decompose a brief (few-second-long) trace of the fluorescence fluctuations, at each point in a cell, into a weighted sum of the traces exhibited by each fluorophore expressed in the cell. The weights then represent the signal amplitudes. We use TMI to analyze relationships between different kinase activities in individual cells, as well as between different cell-cycle signals, pointing toward broad utility throughout biology in the analysis of signal transduction cascades in living systems.

Nutrient-regulated control of lysosome function by signaling lipid conversion.

Lysosomes serve dual antagonistic functions in cells by mediating anabolic growth signaling and the catabolic turnover of macromolecules. How these janus-faced activities are regulated in response to cellular nutrient status is poorly understood. We show here that lysosome morphology and function are reversibly controlled by a nutrient-regulated signaling lipid switch that triggers the conversion between peripheral motile mTOR complex 1 (mTORC1) signaling-active and static mTORC1-inactive degradative lysosomes clustered at the cell center. Starvation-triggered relocalization of phosphatidylinositol 4-phosphate (PI(4)P)-metabolizing enzymes reshapes the lysosomal surface proteome to facilitate lysosomal proteolysis and to repress mTORC1 signaling. Concomitantly, lysosomal phosphatidylinositol 3-phosphate (PI(3)P), which marks motile signaling-active lysosomes in the cell periphery, is erased. Interference with this PI(3)P/PI(4)P lipid switch module impairs the adaptive response of cells to altering nutrient supply. Our data unravel a key function for lysosomal phosphoinositide metabolism in rewiring organellar membrane dynamics in response to cellular nutrient status.

Graded mesoderm assembly governs cell fate and morphogenesis of the early mammalian heart.

Using four-dimensional whole-embryo light sheet imaging with improved and accessible computational tools, we longitudinally reconstruct early murine cardiac development at single-cell resolution. Nascent mesoderm progenitors form opposing density and motility gradients, converting the temporal birth sequence of gastrulation into a spatial anterolateral-to-posteromedial arrangement. Migrating precardiac mesoderm does not strictly preserve cellular neighbor relationships, and spatial patterns only become solidified as the cardiac crescent emerges. Progenitors undergo a mesenchymal-to-epithelial transition, with a first heart field (FHF) ridge apposing a motile juxta-cardiac field (JCF). Anchored along the ridge, the FHF epithelium rotates the JCF forward to form the initial heart tube, along with push-pull morphodynamics of the second heart field. In Mesp1 mutants that fail to make a cardiac crescent, mesoderm remains highly motile but directionally incoherent, resulting in density gradient inversion. Our practicable live embryo imaging approach defines spatial origins and behaviors of cardiac progenitors and identifies their unanticipated morphological transitions.

Mechanisms of skin vascular maturation and maintenance captured by longitudinal imaging of live mice.

A functional network of blood vessels is essential for organ growth and homeostasis, yet how the vasculature matures and maintains homeostasis remains elusive in live mice. By longitudinally tracking the same neonatal endothelial cells (ECs) over days to weeks, we found that capillary plexus expansion is driven by vessel regression to optimize network perfusion. Neonatal ECs rearrange positions to evenly distribute throughout the developing plexus and become positionally stable in adulthood. Upon local ablation, adult ECs survive through a plasmalemmal self-repair response, while neonatal ECs are predisposed to die. Furthermore, adult ECs reactivate migration to assist vessel repair. Global ablation reveals coordinated maintenance of the adult vascular architecture that allows for eventual network recovery. Lastly, neonatal remodeling and adult maintenance of the skin vascular plexus are orchestrated by temporally restricted, neonatal VEGFR2 signaling. Our work sheds light on fundamental mechanisms that underlie both vascular maturation and adult homeostasis in vivo.

Intranasal pediatric parainfluenza virus-vectored SARS-CoV-2 vaccine is protective in monkeys.

Pediatric SARS-CoV-2 vaccines are needed that elicit immunity directly in the airways as well as systemically. Building on pediatric parainfluenza virus vaccines in clinical development, we generated a live-attenuated parainfluenza-virus-vectored vaccine candidate expressing SARS-CoV-2 prefusion-stabilized spike (S) protein (B/HPIV3/S-6P) and evaluated its immunogenicity and protective efficacy in rhesus macaques. A single intranasal/intratracheal dose of B/HPIV3/S-6P induced strong S-specific airway mucosal immunoglobulin A (IgA) and IgG responses. High levels of S-specific antibodies were also induced in serum, which efficiently neutralized SARS-CoV-2 variants of concern of alpha, beta, and delta lineages, while their ability to neutralize Omicron sub-lineages was lower. Furthermore, B/HPIV3/S-6P induced robust systemic and pulmonary S-specific CD4+ and CD8+ T cell responses, including tissue-resident memory cells in the lungs. Following challenge, SARS-CoV-2 replication was undetectable in airways and lung tissues of immunized macaques. B/HPIV3/S-6P will be evaluated clinically as pediatric intranasal SARS-CoV-2/parainfluenza virus type 3 vaccine.

Deciphering cell signaling networks with massively multiplexed biosensor barcoding.

Genetically encoded fluorescent biosensors are powerful tools for monitoring biochemical activities in live cells, but their multiplexing capacity is limited by the available spectral space. We overcome this problem by developing a set of barcoding proteins that can generate over 100 barcodes and are spectrally separable from commonly used biosensors. Mixtures of barcoded cells expressing different biosensors are simultaneously imaged and analyzed by deep learning models to achieve massively multiplexed tracking of signaling events. Importantly, different biosensors in cell mixtures show highly coordinated activities, thus facilitating the delineation of their temporal relationship. Simultaneous tracking of multiple biosensors in the receptor tyrosine kinase signaling network reveals distinct mechanisms of effector adaptation, cell autonomous and non-autonomous effects of KRAS mutations, as well as complex interactions in the network. Biosensor barcoding presents a scalable method to expand multiplexing capabilities for deciphering the complexity of signaling networks and their interactions between cells.

Organoid Imaging: Seeing Development and Function.

Organoids are miniaturized and simplified versions of an organ produced in vitro from stem or progenitor cells. They are used as a model system consisting of multiple cell types forming an architecture relevant to the organ and carrying out the function of the organ. They are a useful tool to study development, homeostasis, regeneration, and disease. The imaging of organoids has become a pivotal method to visualize and understand their self-organization, symmetry breaking, growth, differentiation, and function. In this review, we discuss imaging methods, how to analyze these images, and challenges in organoid research.

Phase Separation of a PKA Regulatory Subunit Controls cAMP Compartmentation and Oncogenic Signaling.

The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.

Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics.

In order to analyze how a signal transduction network converts cellular inputs into cellular outputs, ideally one would measure the dynamics of many signals within the network simultaneously. We found that, by fusing a fluorescent reporter to a pair of self-assembling peptides, it could be stably clustered within cells at random points, distant enough to be resolved by a microscope but close enough to spatially sample the relevant biology. Because such clusters, which we call signaling reporter islands (SiRIs), can be modularly designed, they permit a set of fluorescent reporters to be efficiently adapted for simultaneous measurement of multiple nodes of a signal transduction network within single cells. We created SiRIs for indicators of second messengers and kinases and used them, in hippocampal neurons in culture and intact brain slices, to discover relationships between the speed of calcium signaling, and the amplitude of PKA signaling, upon receiving a cAMP-driving stimulus.

Intrinsic Dynamics of a Human Gene Reveal the Basis of Expression Heterogeneity.

Transcriptional regulation in metazoans occurs through long-range genomic contacts between enhancers and promoters, and most genes are transcribed in episodic "bursts" of RNA synthesis. To understand the relationship between these two phenomena and the dynamic regulation of genes in response to upstream signals, we describe the use of live-cell RNA imaging coupled with Hi-C measurements and dissect the endogenous regulation of the estrogen-responsive TFF1 gene. Although TFF1 is highly induced, we observe short active periods and variable inactive periods ranging from minutes to days. The heterogeneity in inactive times gives rise to the widely observed "noise" in human gene expression and explains the distribution of protein levels in human tissue. We derive a mathematical model of regulation that relates transcription, chromosome structure, and the cell's ability to sense changes in estrogen and predicts that hypervariability is largely dynamic and does not reflect a stable biological state.

A Growth-Based Framework for Leaf Shape Development and Diversity.

How do genes modify cellular growth to create morphological diversity? We study this problem in two related plants with differently shaped leaves: Arabidopsis thaliana (simple leaf shape) and Cardamine hirsuta (complex shape with leaflets). We use live imaging, modeling, and genetics to deconstruct these organ-level differences into their cell-level constituents: growth amount, direction, and differentiation. We show that leaf shape depends on the interplay of two growth modes: a conserved organ-wide growth mode that reflects differentiation; and a local, directional mode that involves the patterning of growth foci along the leaf edge. Shape diversity results from the distinct effects of two homeobox genes on these growth modes: SHOOTMERISTEMLESS broadens organ-wide growth relative to edge-patterning, enabling leaflet emergence, while REDUCED COMPLEXITY inhibits growth locally around emerging leaflets, accentuating shape differences created by patterning. We demonstrate the predictivity of our findings by reconstructing key features of C. hirsuta leaf morphology in A. thaliana. VIDEO ABSTRACT.

Human Antibodies that Slow Erythrocyte Invasion Potentiate Malaria-Neutralizing Antibodies.

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the leading target for next-generation vaccines against the disease-causing blood-stage of malaria. However, little is known about how human antibodies confer functional immunity against this antigen. We isolated a panel of human monoclonal antibodies (mAbs) against PfRH5 from peripheral blood B cells from vaccinees in the first clinical trial of a PfRH5-based vaccine. We identified a subset of mAbs with neutralizing activity that bind to three distinct sites and another subset of mAbs that are non-functional, or even antagonistic to neutralizing antibodies. We also identify the epitope of a novel group of non-neutralizing antibodies that significantly reduce the speed of red blood cell invasion by the merozoite, thereby potentiating the effect of all neutralizing PfRH5 antibodies as well as synergizing with antibodies targeting other malaria invasion proteins. Our results provide a roadmap for structure-guided vaccine development to maximize antibody efficacy against blood-stage malaria.

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.

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.

LlamaTags: A Versatile Tool to Image Transcription Factor Dynamics in Live Embryos.

Embryonic cell fates are defined by transcription factors that are rapidly deployed, yet attempts to visualize these factors in vivo often fail because of slow fluorescent protein maturation. Here, we pioneer a protein tag, LlamaTag, which circumvents this maturation limit by binding mature fluorescent proteins, making it possible to visualize transcription factor concentration dynamics in live embryos. Implementing this approach in the fruit fly Drosophila melanogaster, we discovered stochastic bursts in the concentration of transcription factors that are correlated with bursts in transcription. We further used LlamaTags to show that the concentration of protein in a given nucleus heavily depends on transcription of that gene in neighboring nuclei; we speculate that this inter-nuclear signaling is an important mechanism for coordinating gene expression to delineate straight and sharp boundaries of gene expression. Thus, LlamaTags now make it possible to visualize the flow of information along the central dogma in live embryos.

Light-Sheet Microscopy and Its Potential for Understanding Developmental Processes.

The ability to visualize and quantitatively measure dynamic biological processes in vivo and at high spatiotemporal resolution is of fundamental importance to experimental investigations in developmental biology. Light-sheet microscopy is particularly well suited to providing such data, since it offers exceptionally high imaging speed and good spatial resolution while minimizing light-induced damage to the specimen. We review core principles and recent advances in light-sheet microscopy, with a focus on concepts and implementations relevant for applications in developmental biology. We discuss how light-sheet microcopy has helped advance our understanding of developmental processes from single-molecule to whole-organism studies, assess the potential for synergies with other state-of-the-art technologies, and introduce methods for computational image and data analysis. Finally, we explore the future trajectory of light-sheet microscopy, discuss key efforts to disseminate new light-sheet technology, and identify exciting opportunities for further advances.

p300 is an obligate integrator of combinatorial transcription factor inputs.

Transcription coactivators are proteins or protein complexes that mediate transcription factor (TF) function. However, they lack DNA-binding capacity, prompting the question of how they engage target loci. Three non-exclusive hypotheses have been posited: coactivators are recruited by complexing with TFs, by binding histones through epigenetic reader domains, or by partitioning into condensates through their extensive intrinsically disordered regions. Using p300 as a prototypical coactivator, we systematically mutated its annotated domains and show by single-molecule tracking in live U2OS cells that coactivator-chromatin binding depends entirely on combinatorial binding of multiple TF-interaction domains. Furthermore, we demonstrate that acetyltransferase activity opposes p300-chromatin association and that the N-terminal TF-interaction domains regulate that activity. Single TF-interaction domains are insufficient for chromatin binding and regulation of catalytic activity, implying a principle that we speculate could broadly apply to eukaryotic gene regulation: a TF must act in coordination with other TFs to recruit coactivator activity.

Live-cell imaging of RNA Pol II and elongation factors distinguishes competing mechanisms of transcription regulation.

RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.