RESUMEN
Synchronization of coupled oscillators is a universal phenomenon encountered across different scales and contexts, e.g., chemical wave patterns, superconductors, and the unison applause we witness in concert halls. The existence of common underlying coupling rules defines universality classes, revealing a fundamental sameness between seemingly distinct systems. Identifying rules of synchronization in any particular setting is hence of paramount relevance. Here, we address the coupling rules within an embryonic oscillator ensemble linked to vertebrate embryo body axis segmentation. In vertebrates, the periodic segmentation of the body axis involves synchronized signaling oscillations in cells within the presomitic mesoderm (PSM), from which somites, the prevertebrae, form. At the molecular level, it is known that intact Notch-signaling and cell-to-cell contact are required for synchronization between PSM cells. However, an understanding of the coupling rules is still lacking. To identify these, we develop an experimental assay that enables direct quantification of synchronization dynamics within mixtures of oscillating cell ensembles, for which the initial input frequency and phase distribution are known. Our results reveal a "winner-takes-it-all" synchronization outcome, i.e., the emerging collective rhythm matches one of the input rhythms. Using a combination of theory and experimental validation, we develop a coupling model, the "Rectified Kuramoto" (ReKu) model, characterized by a phase-dependent, nonreciprocal interaction in the coupling of oscillatory cells. Such nonreciprocal synchronization rules reveal fundamental similarities between embryonic oscillators and a class of collective behaviors seen in neurons and fireflies, where higher-level computations are performed and linked to nonreciprocal synchronization.
Asunto(s)
Tipificación del Cuerpo , Animales , Tipificación del Cuerpo/fisiología , Relojes Biológicos/fisiología , Embrión no Mamífero/fisiología , Transducción de Señal/fisiología , Somitos/embriología , Mesodermo/embriología , Modelos BiológicosRESUMEN
A fundamental requirement for embryonic development is the coordination of signaling activities in space and time. A notable example in vertebrate embryos is found during somitogenesis, where gene expression oscillations linked to the segmentation clock are synchronized across cells in the presomitic mesoderm (PSM) and result in tissue-level wave patterns. To examine their onset during mouse embryo development, we studied the dynamics of the segmentation clock gene Lfng during gastrulation. To this end, we established an imaging setup using selective plane illumination microscopy (SPIM) that enables culture and simultaneous imaging of up to four embryos ('SPIM- for-4'). Using SPIM-for-4, combined with genetically encoded signaling reporters, we detected the onset of Lfng oscillations within newly formed mesoderm at presomite stages. Functionally, we found that initial synchrony and the first â¼6-8 oscillation cycles occurred even when Notch signaling was impaired, revealing similarities to previous findings made in zebrafish embryos. Finally, we show that a spatial period gradient is present at the onset of oscillatory activity, providing a potential mechanism accounting for our observation that wave patterns build up gradually over the first oscillation cycles.
Asunto(s)
Gastrulación , Somitos , Animales , Regulación del Desarrollo de la Expresión Génica , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Mesodermo/metabolismo , Ratones , Receptores Notch/genética , Receptores Notch/metabolismo , Somitos/metabolismo , Pez Cebra/genéticaRESUMEN
Size control is fundamental in tissue development and homeostasis1,2. Although the role of cell proliferation in these processes has been widely studied, the mechanisms that control embryo size-and how these mechanisms affect cell fate-remain unknown. Here we use the mouse blastocyst as a model to unravel a key role of fluid-filled lumen in the control of embryo size and specification of cell fate. We find that there is a twofold increase in lumenal pressure during blastocyst development, which translates into a concomitant increase in cell cortical tension and tissue stiffness of the trophectoderm that lines the lumen. Increased cortical tension leads to vinculin mechanosensing and maturation of functional tight junctions, which establishes a positive feedback loop to accommodate lumen growth. When the cortical tension reaches a critical threshold, cell-cell adhesion cannot be sustained during mitotic entry, which leads to trophectoderm rupture and blastocyst collapse. A simple theory of hydraulically gated oscillations recapitulates the observed dynamics of size oscillations, and predicts the scaling of embryo size with tissue volume. This theory further predicts that disrupted tight junctions or increased tissue stiffness lead to a smaller embryo size, which we verified by biophysical, embryological, pharmacological and genetic perturbations. Changes in lumenal pressure and size can influence the cell division pattern of the trophectoderm, and thereby affect cell allocation and fate. Our study reveals how lumenal pressure and tissue mechanics control embryo size at the tissue scale, which is coupled to cell position and fate at the cellular scale.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Desarrollo Embrionario , Mecanotransducción Celular/fisiología , Animales , Blastocisto/citología , Adhesión Celular , División Celular , Forma de la Célula , Embrión de Mamíferos/anatomía & histología , Femenino , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Uniones Estrechas , Vinculina/metabolismoRESUMEN
Clonal cell population dynamics play a critical role in both disease and development. Due to high mitochondrial mutation rates under both healthy and diseased conditions, mitochondrial genomic variability is a particularly useful resource in facilitating the identification of clonal population structure. Here we present mitoClone2, an all-inclusive R package allowing for the identification of clonal populations through integration of mitochondrial heteroplasmic variants discovered from single-cell sequencing experiments. Our package streamlines the investigation of this phenomenon by providing: built-in compatibility with commonly used tools for the delineation of clonal structure, the ability to directly use multiplexed BAM files as input, annotations for both human and mouse mitochondrial genomes, and helper functions for calling, filtering, clustering, and visualizing variants.
RESUMEN
Experiments that compare rhythmic properties across different genetic alterations and entrainment conditions underlie some of the most important breakthroughs in circadian biology. A robust estimation of the rhythmic properties of the circadian signals goes hand in hand with these discoveries. Widely applied traditional signal analysis methods such as fitting cosine functions or Fourier transformations rely on the assumption that oscillation periods do not change over time. However, novel high-resolution recording techniques have shown that, most commonly, circadian signals exhibit time-dependent changes of periods and amplitudes which cannot be captured with the traditional approaches. In this chapter we introduce a method to determine time-dependent properties of oscillatory signals, using the novel open-source Python-based Biological Oscillations Analysis Toolkit (pyBOAT). We show with examples how to detect rhythms, compute and interpret high-resolution time-dependent spectral results, analyze the main oscillatory component, and to subsequently determine these main components' time-dependent instantaneous period, amplitude, and phase. We introduce step-by-step how such an analysis can be done by means of the easy-to-use point-and-click graphical user interface (GUI) provided by pyBOAT or executed within a Python programming environment. Concepts are explained using simulated signals as well as experimentally obtained time series.
Asunto(s)
Ritmo Circadiano , Factores de TiempoRESUMEN
Living systems exhibit an unmatched complexity, due to countless, entangled interactions across scales. Here, we aim to understand a complex system, that is, segmentation timing in mouse embryos, without a reference to these detailed interactions. To this end, we develop a coarse-grained approach, in which theory guides the experimental identification of the segmentation clock entrainment responses. We demonstrate period- and phase-locking of the segmentation clock across a wide range of entrainment parameters, including higher-order coupling. These quantifications allow to derive the phase response curve (PRC) and Arnold tongues of the segmentation clock, revealing its essential dynamical properties. Our results indicate that the somite segmentation clock has characteristics reminiscent of a highly non-linear oscillator close to an infinite period bifurcation and suggests the presence of long-term feedbacks. Combined, this coarse-grained theoretical-experimental approach reveals how we can derive simple, essential features of a highly complex dynamical system, providing precise experimental control over the pace and rhythm of the somite segmentation clock.
Asunto(s)
Somitos , Lengua , Animales , RatonesRESUMEN
Development is a highly dynamic process in which organisms often experience changes in both form and behavior, which are typically coupled to each other. However, little is known about how organismal-scale behaviors such as body contractility and motility impact morphogenesis. Here, we use the cnidarian Nematostella vectensis as a developmental model to uncover a mechanistic link between organismal size, shape, and behavior. Using quantitative live imaging in a large population of developing animals, combined with molecular and biophysical experiments, we demonstrate that the muscular-hydraulic machinery that controls body movement also drives larva-polyp morphogenesis. We show that organismal size largely depends on cavity inflation through fluid uptake, whereas body shape is constrained by the organization of the muscular system. The generation of ethograms identifies different trajectories of size and shape development in sessile and motile animals, which display distinct patterns of body contractions. With a simple theoretical model, we conceptualize how pressures generated by muscular hydraulics can act as a global mechanical regulator that coordinates tissue remodeling. Altogether, our findings illustrate how organismal contractility and motility behaviors can influence morphogenesis.
Asunto(s)
Anémonas de Mar , Animales , Larva , MorfogénesisRESUMEN
The intrinsic genetic program of a cell is not sufficient to explain all of the cell's activities. External mechanical stimuli are increasingly recognized as determinants of cell behavior. In the epithelial folding event that constitutes the beginning of gastrulation in Drosophila, the genetic program of the future mesoderm leads to the establishment of a contractile actomyosin network that triggers apical constriction of cells and thereby tissue folding. However, some cells do not constrict but instead stretch, even though they share the same genetic program as their constricting neighbors. We show here that tissue-wide interactions force these cells to expand even when an otherwise sufficient amount of apical, active actomyosin is present. Models based on contractile forces and linear stress-strain responses do not reproduce experimental observations, but simulations in which cells behave as ductile materials with nonlinear mechanical properties do. Our models show that this behavior is a general emergent property of actomyosin networks in a supracellular context, in accordance with our experimental observations of actin reorganization within stretching cells.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Citoesqueleto de Actina/genética , Actinas/genética , Actomiosina/genética , Animales , Forma de la Célula/genética , Citoesqueleto/genética , Gastrulación/genética , Mesodermo/fisiologíaRESUMEN
The migrational propensity of neuroblastoma is affected by cell identity, but the mechanisms behind the divergence remain unknown. Using RNAi and time-lapse imaging, we show that ADRN-type NB cells exhibit RAC1- and kalirin-dependent nucleokinetic (NUC) migration that relies on several integral components of neuronal migration. Inhibition of NUC migration by RAC1 and kalirin-GEF1 inhibitors occurs without hampering cell proliferation and ADRN identity. Using three clinically relevant expression dichotomies, we reveal that most of up-regulated mRNAs in RAC1- and kalirin-GEF1-suppressed ADRN-type NB cells are associated with low-risk characteristics. The computational analysis shows that, in a context of overall gene set poverty, the upregulomes in RAC1- and kalirin-GEF1-suppressed ADRN-type cells are a batch of AU-rich element-containing mRNAs, which suggests a link between NUC migration and mRNA stability. Gene set enrichment analysis-based search for vulnerabilities reveals prospective weak points in RAC1- and kalirin-GEF1-suppressed ADRN-type NB cells, including activities of H3K27- and DNA methyltransferases. Altogether, these data support the introduction of NUC inhibitors into cancer treatment research.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Neuroblastoma/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Neuronas Adrenérgicas/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Células Cultivadas , Preescolar , Bases de Datos Genéticas , Femenino , Factores de Intercambio de Guanina Nucleótido/fisiología , Humanos , Masculino , Neuroblastoma/patología , Estudios Prospectivos , Proteínas Serina-Treonina Quinasas/fisiología , Proteína de Unión al GTP rac1/fisiologíaRESUMEN
Cellular signaling systems precisely transmit information in the presence of molecular noise while retaining flexibility to accommodate the needs of individual cells. To understand design principles underlying such versatile signaling, we analyzed the response of the tumor suppressor p53 to varying levels of DNA damage in hundreds of individual cells and observed a switch between distinct signaling modes characterized by isolated pulses and sustained oscillations of p53 accumulation. Guided by dynamic systems theory we show that this requires an excitable network structure comprising positive feedback and provide experimental evidence for its molecular identity. The resulting data-driven model reproduced all features of measured signaling responses and is sufficient to explain their heterogeneity in individual cells. We present evidence that heterogeneity in the levels of the feedback regulator Wip1 sets cell-specific thresholds for p53 activation, providing means to modulate its response through interacting signaling pathways. Our results demonstrate how excitable signaling networks can provide high specificity, sensitivity and robustness while retaining unique possibilities to adjust their function to the physiology of individual cells.
Asunto(s)
Daño del ADN , Modelos Biológicos , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Células A549 , Humanos , Células MCF-7 , Proteína Fosfatasa 2C/metabolismoRESUMEN
While many tumors initially respond to chemotherapy, regrowth of surviving cells compromises treatment efficacy in the long term. The cell-biological basis of this regrowth is not understood. Here, we characterize the response of individual, patient-derived neuroblastoma cells driven by the prominent oncogene MYC to the first-line chemotherapy, doxorubicin. Combining live-cell imaging, cell-cycle-resolved transcriptomics, and mathematical modeling, we demonstrate that a cell's treatment response is dictated by its expression level of MYC and its cell-cycle position prior to treatment. All low-MYC cells enter therapy-induced senescence. High-MYC cells, by contrast, disable their cell-cycle checkpoints, forcing renewed proliferation despite treatment-induced DNA damage. After treatment, the viability of high-MYC cells depends on their cell-cycle position during treatment: newborn cells promptly halt in G1 phase, repair DNA damage, and form re-growing clones; all other cells show protracted DNA repair and ultimately die. These findings demonstrate that fast-proliferating tumor cells may resist cytotoxic treatment non-genetically, by arresting within a favorable window of the cell cycle.