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.

Brahma safeguards canalization of cardiac mesoderm differentiation.

Differentiation proceeds along a continuum of increasingly fate-restricted intermediates, referred to as canalization1,2. Canalization is essential for stabilizing cell fate, but the mechanisms that underlie robust canalization are unclear. Here we show that the BRG1/BRM-associated factor (BAF) chromatin-remodelling complex ATPase gene Brm safeguards cell identity during directed cardiogenesis of mouse embryonic stem cells. Despite the establishment of a well-differentiated precardiac mesoderm, Brm-/- cells predominantly became neural precursors, violating germ layer assignment. Trajectory inference showed a sudden acquisition of a non-mesodermal identity in Brm-/- cells. Mechanistically, the loss of Brm prevented de novo accessibility of primed cardiac enhancers while increasing the expression of neurogenic factor POU3F1, preventing the binding of the neural suppressor REST and shifting the composition of BRG1 complexes. The identity switch caused by the Brm mutation was overcome by increasing BMP4 levels during mesoderm induction. Mathematical modelling supports these observations and demonstrates that Brm deletion affects cell fate trajectory by modifying saddle-node bifurcations2. In the mouse embryo, Brm deletion exacerbated mesoderm-deleted Brg1-mutant phenotypes, severely compromising cardiogenesis, and reveals an in vivo role for Brm. Our results show that Brm is a compensable safeguard of the fidelity of mesoderm chromatin states, and support a model in which developmental canalization is not a rigid irreversible path, but a highly plastic trajectory.

Tissue mechanics in stem cell fate, development, and cancer.

Cells in tissues experience a plethora of forces that regulate their fate and modulate development and homeostasis. Cells sense mechanical cues through localized mechanoreceptors or by influencing cytoskeletal or plasma membrane organization. Cells translate force and modulate their behavior through a process termed mechanotransduction. Cells tune their tension upon exposure to chronic force by engaging cellular machinery that modulates actin tension, which in turn stimulates matrix remodeling and stiffening and alters cell-cell adhesions until cells achieve a state of tensional homeostasis. Loss of tensional homeostasis can be induced through oncogene activity and/or tissue fibrosis, accompanies tumor progression, and is associated with increased cancer risk. The mechanical stresses that develop in tumors can also foster the mesenchymal-like transdifferentiation of cells to induce a stem-like phenotype that contributes to their aggression, metastatic dissemination, and treatment resistance. Thus, strategies that ameliorate tumor mechanics may comprise an effective strategy to prevent aggressive tumor behavior.

Adhesion-mediated mechanosignaling forces mitohormesis.

Mitochondria control eukaryotic cell fate by producing the energy needed to support life and the signals required to execute programed cell death. The biochemical milieu is known to affect mitochondrial function and contribute to the dysfunctional mitochondrial phenotypes implicated in cancer and the morbidities of aging. However, the physical characteristics of the extracellular matrix are also altered in cancerous and aging tissues. Here, we demonstrate that cells sense the physical properties of the extracellular matrix and activate a mitochondrial stress response that adaptively tunes mitochondrial function via solute carrier family 9 member A1-dependent ion exchange and heat shock factor 1-dependent transcription. Overall, our data indicate that adhesion-mediated mechanosignaling may play an unappreciated role in the altered mitochondrial functions observed in aging and cancer.

EPH/EPHRIN regulates cellular organization by actomyosin contractility effects on cell contacts.

EPH/EPHRIN signaling is essential to many aspects of tissue self-organization and morphogenesis, but little is known about how EPH/EPHRIN signaling regulates cell mechanics during these processes. Here, we use a series of approaches to examine how EPH/EPHRIN signaling drives cellular self-organization. Contact angle measurements reveal that EPH/EPHRIN signaling decreases the stability of heterotypic cell:cell contacts through increased cortical actomyosin contractility. We find that EPH/EPHRIN-driven cell segregation depends on actomyosin contractility but occurs independently of directed cell migration and without changes in cell adhesion. Atomic force microscopy and live cell imaging of myosin localization support that EPH/EPHRIN signaling results in increased cortical tension. Interestingly, actomyosin contractility also nonautonomously drives increased EPHB2:EPHB2 homotypic contacts. Finally, we demonstrate that changes in tissue organization are driven by minimization of heterotypic contacts through actomyosin contractility in cell aggregates and by mouse genetics experiments. These data elucidate the biomechanical mechanisms driving EPH/EPHRIN-based cell segregation wherein differences in interfacial tension, regulated by actomyosin contractility, govern cellular self-organization.

Membrane Tension Locks In Pluripotency.

In this issue of Cell Stem Cell, De Belly et al. (2021) and Bergert et al. (2021) reveal that membrane tension regulates the pluripotent state via endocytosis-mediated ERK signaling. These findings advance our understanding of naive pluripotency and highlight how cell mechanics are intertwined with molecular signaling to drive cell fate decisions.

Mechanical Tension Promotes Formation of Gastrulation-like Nodes and Patterns Mesoderm Specification in Human Embryonic Stem Cells.

Embryogenesis is directed by morphogens that induce differentiation within a defined tissue geometry. Tissue organization is mediated by cell-cell and cell-extracellular matrix (ECM) adhesions and is modulated by cell tension and tissue-level forces. Whether cell tension regulates development by modifying morphogen signaling is less clear. Human embryonic stem cells (hESCs) exhibit an intrinsic capacity for self-organization, which motivates their use as a tractable model of early human embryogenesis. We engineered patterned substrates that recapitulate the biophysical properties of the early embryo and mediate the self-organization of "gastrulation-like" nodes in cultured hESCs. Tissue geometries that generated local nodes of high cell-adhesion tension directed the spatial patterning of the BMP4-dependent "gastrulation-like" phenotype by enhancing phosphorylation and junctional release of ß-catenin to promote Wnt signaling and mesoderm specification. Furthermore, direct force application via mechanical stretching promoted BMP-dependent mesoderm specification, confirming that tissue-level forces can directly regulate cell fate specification in early human development.

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Wnt4 from the Niche Controls the Mechano-Properties and Quiescent State of Muscle Stem Cells.

Satellite cells (SCs) reside in a dormant state during tissue homeostasis. The specific paracrine agents and niche cells that maintain SC quiescence remain unknown. We find that Wnt4 produced by the muscle fiber maintains SC quiescence through RhoA. Using cell-specific inducible genetics, we find that a Wnt4-Rho signaling axis constrains SC numbers and activation during tissue homeostasis in adult mice. Wnt4 activates Rho in quiescent SCs to maintain mechanical strain, restrict movement in the niche, and repress YAP. The induction of YAP upon disruption of RhoA is essential for SC activation under homeostasis. In the context of injury, the loss of Wnt4 from the niche accelerates SC activation and muscle repair, whereas overexpression of Wnt4 transitions SCs into a deeper state of quiescence and delays muscle repair. In conclusion, the SC pool undergoes dynamic transitions during early activation with changes in mechano-properties and cytoskeleton signaling preceding cell-cycle entry.

Controlled modelling of human epiblast and amnion development using stem cells.

Early human embryonic development involves extensive lineage diversification, cell-fate specification and tissue patterning1. Despite its basic and clinical importance, early human embryonic development remains relatively unexplained owing to interspecies divergence2,3 and limited accessibility to human embryo samples. Here we report that human pluripotent stem cells (hPSCs) in a microfluidic device recapitulate, in a highly controllable and scalable fashion, landmarks of the development of the epiblast and amniotic ectoderm parts of the conceptus, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and primitive streak cells. We further show that amniotic ectoderm-like cells function as a signalling centre to trigger the onset of gastrulation-like events in hPSCs. Given its controllability and scalability, the microfluidic model provides a powerful experimental system to advance knowledge of human embryology and reproduction. This model could assist in the rational design of differentiation protocols of hPSCs for disease modelling and cell therapy, and in high-throughput drug and toxicity screens to prevent pregnancy failure and birth defects.

Spatiotemporal mosaic self-patterning of pluripotent stem cells using CRISPR interference.

Morphogenesis involves interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events lack control over cell-type co-emergence and offer little capability to selectively perturb specific cell subpopulations. Our in vitro system interrogates cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined effects of induced mosaic knockdown of molecular regulators of cortical tension (ROCK1) and cell-cell adhesion (CDH1) with CRISPR interference. Mosaic knockdown of ROCK1 or CDH1 resulted in differential patterning within hiPSC colonies due to cellular self-organization, while retaining an epithelial pluripotent phenotype. Knockdown induction stimulates a transient wave of differential gene expression within the mixed populations that stabilized in coordination with observed self-organization. Mosaic patterning enables genetic interrogation of emergent multicellular properties, which can facilitate better understanding of the molecular pathways that regulate symmetry-breaking during morphogenesis.

The Physical and Biochemical Properties of the Extracellular Matrix Regulate Cell Fate.

The extracellular matrix is a complex network of hydrated macromolecular proteins and sugars that, in concert with bound soluble factors, comprise the acellular stromal microenvironment of tissues. Rather than merely providing structural information to cells, the extracellular matrix plays an instructive role in development and is critical for the maintenance of tissue homeostasis. In this chapter, we review the composition of the extracellular matrix and summarize data illustrating its importance in embryogenesis, tissue-specific development, and stem cell differentiation. We discuss how the biophysical and biochemical properties of the extracellular matrix ligate specific transmembrane receptors to activate intracellular signaling that alter cell shape and cytoskeletal dynamics to modulate cell growth and viability, and direct cell migration and cell fate. We present examples describing how the extracellular matrix functions as a highly complex physical and chemical entity that regulates tissue organization and cell behavior through a dynamic and reciprocal dialogue with the cellular constituents of the tissue. We suggest that the extracellular matrix not only transmits cellular and tissue-level force to shape development and tune cellular activities that are key for coordinated tissue behavior, but that it is itself remodeled such that it temporally evolves to maintain the integrated function of the tissue. Accordingly, we argue that perturbations in extracellular matrix composition and structure compromise key developmental events and tissue homeostasis, and promote disease.

Mechanical Control of Epithelial-to-Mesenchymal Transitions in Development and Cancer.

Mechanical force modulates development, influences tissue homeostasis, and contributes to disease. Forces sculpt tissue-level behaviors and direct cell fate by engaging mechanoreceptors and by altering organization of the cytoskeleton and actomyosin contractility to stimulate mechanotransduction mechanisms that alter transcription. Nevertheless, how force specifically leverages mechanotransduction pathways to control transcriptional regulation of cell fate remains unclear. Here we review recent findings specifically in the context of epithelial-to-mesenchymal transitions that have revealed conserved mechanisms whereby extracellular force, mediated through cell-extracellular matrix and cell-cell junctional complexes, induces transcriptional reprogramming to alter cell and tissue fate. We focus on the interplay between tissue mechanics and the epithelial-to-mesenchymal transitions that occur during embryonic development and cancer malignancy. We describe the adhesion-linked cellular machinery that mediates mechano-transduction and elaborate on how these force-linked networks stimulate key transcriptional programs that induce an epithelial-to-mesenchymal phenotypic transition, thereby providing an overview of how mechanical signals can be translated into a change in cell fate.