RESUMEN
The vertebrate anteroposterior axis forms through elongation of multiple tissues during embryogenesis. This process is based on tissue-autonomous mechanisms of force generation and intertissue mechanical coupling whose failure leads to severe developmental anomalies such as body truncation and spina bifida. Similar to other morphogenetic modules, anteroposterior body extension requires both the rearrangement of existing materials-such as cells and extracellular matrix-and the local addition of new materials, i.e., anisotropic growth, through cell proliferation, cell growth, and matrix deposition. Numerous signaling pathways coordinate body axis formation via regulation of cell behavior during tissue rearrangements and/or volumetric growth. From a physical perspective, morphogenesis depends on both cell-generated forces and tissue material properties. As the spatiotemporal variation of these mechanical parameters has recently been explored in the context of vertebrate body elongation, the study of this process is likely to shed light on the cross talk between signaling and mechanics during morphogenesis.
Asunto(s)
Tipificación del Cuerpo , Desarrollo Embrionario , Vertebrados/embriología , Animales , Movimiento Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Humanos , Transducción de Señal , Vertebrados/metabolismoRESUMEN
Epithelial cells acquire functionally important shapes (e.g., squamous, cuboidal, columnar) during development. Here, we combine theory, quantitative imaging, and perturbations to analyze how tissue geometry, cell divisions, and mechanics interact to shape the presumptive enveloping layer (pre-EVL) on the zebrafish embryonic surface. We find that, under geometrical constraints, pre-EVL flattening is regulated by surface cell number changes following differentially oriented cell divisions. The division pattern is, in turn, determined by the cell shape distribution, which forms under geometrical constraints by cell-cell mechanical coupling. An integrated mathematical model of this shape-division feedback loop recapitulates empirical observations. Surprisingly, the model predicts that cell shape is robust to changes of tissue surface area, cell volume, and cell number, which we confirm in vivo. Further simulations and perturbations suggest the parameter linking cell shape and division orientation contributes to epithelial diversity. Together, our work identifies an evolvable design logic that enables robust cell-level regulation of tissue-level development.
Asunto(s)
Células Epiteliales/citología , Modelos Biológicos , Morfogénesis , Pez Cebra/embriología , Animales , Fenómenos Biomecánicos , Recuento de Células , División Celular , Forma de la Célula , Embrión no Mamífero/citologíaRESUMEN
Sharply delineated domains of cell types arise in developing tissues under instruction of inductive signal (morphogen) gradients, which specify distinct cell fates at different signal levels. The translation of a morphogen gradient into discrete spatial domains relies on precise signal responses at stable cell positions. However, cells in developing tissues undergoing morphogenesis and proliferation often experience complex movements, which may affect their morphogen exposure, specification, and positioning. How is a clear pattern achieved with cells moving around? Using in toto imaging of the zebrafish neural tube, we analyzed specification patterns and movement trajectories of neural progenitors. We found that specified progenitors of different fates are spatially mixed following heterogeneous Sonic Hedgehog signaling responses. Cell sorting then rearranges them into sharply bordered domains. Ectopically induced motor neuron progenitors also robustly sort to correct locations. Our results reveal that cell sorting acts to correct imprecision of spatial patterning by noisy inductive signals.
Asunto(s)
Morfogénesis , Células-Madre Neurales/metabolismo , Tubo Neural/citología , Transducción de Señal , Pez Cebra/embriología , Animales , Movimiento Celular , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Proteínas Hedgehog/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Developmental morphogenesis is driven by tissue stresses acting on tissue rheology. Direct measurements of forces in small tissues (100â µm-1â mm) in situ, such as in early embryos, require high spatial precision and minimal invasiveness. Here, we introduce a control-based approach, tissue force microscopy (TiFM), that integrates a mechanical cantilever probe and live imaging with closed-loop feedback control of mechanical loading in early chicken embryos. By testing previously qualitatively characterized force-producing tissues in the elongating body axis, we show that TiFM quantitatively captures stress dynamics with high sensitivity. TiFM also provides the means to apply stable, minimally invasive and physiologically relevant loads to drive tissue deformation and to follow the resulting morphogenetic progression associated with large-scale cell movements. Together, TiFM allows us to control tissue force measurement and manipulation in small developing embryos, and promises to contribute to the quantitative understanding of complex multi-tissue mechanics during development.
Asunto(s)
Pollos , Fenómenos Mecánicos , Animales , Embrión de Pollo , Morfogénesis/fisiologíaRESUMEN
Formation of the body of vertebrate embryos proceeds sequentially by posterior addition of tissues from the tail bud. Cells of the tail bud and the posterior presomitic mesoderm, which control posterior elongation1, exhibit a high level of aerobic glycolysis that is reminiscent of the metabolic status of cancer cells experiencing the Warburg effect2,3. Glycolytic activity downstream of fibroblast growth factor controls WNT signalling in the tail bud3. In the neuromesodermal precursors of the tail bud4, WNT signalling promotes the mesodermal fate that is required for sustained axial elongation, at the expense of the neural fate3,5. How glycolysis regulates WNT signalling in the tail bud is currently unknown. Here we used chicken embryos and human tail bud-like cells differentiated in vitro from induced pluripotent stem cells to show that these cells exhibit an inverted pH gradient, with the extracellular pH lower than the intracellular pH, as observed in cancer cells6. Our data suggest that glycolysis increases extrusion of lactate coupled to protons via the monocarboxylate symporters. This contributes to elevating the intracellular pH in these cells, which creates a favourable chemical environment for non-enzymatic ß-catenin acetylation downstream of WNT signalling. As acetylated ß-catenin promotes mesodermal rather than neural fate7, this ultimately leads to activation of mesodermal transcriptional WNT targets and specification of the paraxial mesoderm in tail bud precursors. Our work supports the notion that some tumour cells reactivate a developmental metabolic programme.
Asunto(s)
Amnios/embriología , Glucólisis , Proteínas Wnt/metabolismo , Acetilación , Animales , Tipificación del Cuerpo , Embrión de Pollo , Humanos , Concentración de Iones de Hidrógeno , Ácido Láctico/metabolismo , Mesodermo/metabolismo , beta Catenina/metabolismoRESUMEN
The neural tube is an important model system of morphogenesis representing the developmental module of out-of-plane epithelial deformation. As the embryonic precursor of the central nervous system, the neural tube also holds keys to many defects and diseases. Recent advances begin to reveal how genetic, cellular and environmental mechanisms work in concert to ensure correct neural tube shape. A physical model is emerging where these factors converge at the regulation of the mechanical forces and properties within and around the tissue that drive tube formation towards completion. Here we review the dynamics and mechanics of neural tube morphogenesis and discuss the underlying cellular behaviours from the viewpoint of tissue mechanics. We will also highlight some of the conceptual and technical next steps.
Asunto(s)
Modelos Biológicos , Tubo Neural , Fenómenos Biomecánicos , Sistema Nervioso Central , Morfogénesis/genéticaRESUMEN
Embryonic morphogenesis is a biological process which depicts shape forming of tissues and organs during development. Unveiling the roles of mechanical forces generated, transmitted, and regulated in cells and tissues through these processes is key to understanding the biophysical mechanisms governing morphogenesis. To this end, it is imperative to measure, simulate, and predict the regulation and control of these mechanical forces during morphogenesis. This article aims to provide a comprehensive review of the recent advances on mechanical properties of cells and tissues, generation of mechanical forces in cells and tissues, the transmission processes of these generated forces during cells and tissues, the tools and methods used to measure and predict these mechanical forces in vivo, in vitro, or in silico, and to better understand the corresponding regulation and control of generated forces. Understanding the biomechanics and mechanobiology of morphogenesis will not only shed light on the fundamental physical mechanisms underlying these concerted biological processes during normal development, but also uncover new information that will benefit biomedical research in preventing and treating congenital defects or tissue engineering and regeneration.
Asunto(s)
Desarrollo Embrionario , Fenómenos Biomecánicos , Biofisica , MorfogénesisRESUMEN
During the development of the spinal cord, proliferative neural progenitors differentiate into postmitotic neurons with distinct fates. How cells switch from progenitor states to differentiated fates is poorly understood. To address this question, we studied the differentiation of progenitors in the zebrafish spinal cord, focusing on the differentiation of Kolmer-Agduhrâ³ (KAâ³) interneurons from lateral floor plate (LFP) progenitors. In vivo cell tracking demonstrates that KAâ³ cells are generated from LFP progenitors by both symmetric and asymmetric cell divisions. A photoconvertible reporter of signaling history (PHRESH) reveals distinct temporal profiles of Hh response: LFP progenitors continuously respond to Hh, while KAâ³ cells lose Hh response upon differentiation. Hh signaling is required in LFP progenitors for KAâ³ fate specification, but prolonged Hh signaling interferes with KAâ³ differentiation. Notch signaling acts permissively to maintain LFP progenitor cells: activation of Notch signaling prevents differentiation, whereas inhibition of Notch signaling results in differentiation of ectopic KAâ³ cells. These results indicate that neural progenitors depend on Notch signaling to maintain Hh responsiveness and rely on Hh signaling to induce fate identity, whereas proper differentiation depends on the attenuation of both Notch and Hh signaling.
Asunto(s)
Proteínas Hedgehog/metabolismo , Receptores de Superficie Celular/genética , Receptores Notch/metabolismo , Médula Espinal/crecimiento & desarrollo , Pez Cebra , Animales , Animales Modificados Genéticamente , Diferenciación Celular , División Celular/genética , Linaje de la Célula , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Interneuronas/citología , Interneuronas/metabolismo , Receptores Patched , Receptores de Superficie Celular/metabolismo , Receptores Notch/genética , Transducción de Señal , Médula Espinal/metabolismo , Células Madre/citología , Células Madre/metabolismo , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Pressure exerted by fluid contained within a lumen plays a crucial role in the growth, morphogenesis, and patterning of epithelial organs. Accurate modulation of lumen pressure in the developing embryo requires sensitive and robust methods that can detect and vary pressure in the range of tens to hundreds of Pascals (Pa). Here we describe a simple, cost-effective protocol for setting up a pressure modulation apparatus combining a high-sensitivity pressure sensor and a water column whose height can be finely tuned. We demonstrate lumen pressure control using the developing brain of early chicken embryos.
Asunto(s)
Presión , Animales , Embrión de Pollo , Encéfalo/embriología , Encéfalo/fisiologíaRESUMEN
The functional and sensory augmentation of living structures, such as human skin and plant epidermis, with electronics can be used to create platforms for health management and environmental monitoring. Ideally, such bioelectronic interfaces should not obstruct the inherent sensations and physiological changes of their hosts. The full life cycle of the interfaces should also be designed to minimize their environmental footprint. Here we report imperceptible augmentation of living systems through in situ tethering of organic bioelectronic fibres. Using an orbital spinning technique, substrate-free and open fibre networks-which are based on poly (3,4-ethylenedioxythiophene):polystyrene sulfonate-can be tethered to biological surfaces, including fingertips, chick embryos and plants. We use customizable fibre networks to create on-skin electrodes that can record electrocardiogram and electromyography signals, skin-gated organic electrochemical transistors and augmented touch and plant interfaces. We also show that the fibres can be used to couple prefabricated microelectronics and electronic textiles, and that the fibres can be repaired, upgraded and recycled.
RESUMEN
The quantification of cell shape, cell migration, and cell rearrangements is important for addressing classical questions in developmental biology such as patterning and tissue morphogenesis. Time-lapse microscopic imaging of transgenic embryos expressing fluorescent reporters is the method of choice for tracking morphogenetic changes and establishing cell lineages and fate maps in vivo. However, the manual steps involved in curating thousands of putative cell segmentations have been a major bottleneck in the application of these technologies especially for cell membranes. Segmentation of cell membranes while more difficult than nuclear segmentation is necessary for quantifying the relations between changes in cell morphology and morphogenesis. We present a novel and fully automated method to first reconstruct membrane signals and then segment out cells from 3D membrane images even in dense tissues. The approach has three stages: 1) detection of local membrane planes, 2) voting to fill structural gaps, and 3) region segmentation. We demonstrate the superior performance of the algorithms quantitatively on time-lapse confocal and two-photon images of zebrafish neuroectoderm and paraxial mesoderm by comparing its results with those derived from human inspection. We also compared with synthetic microscopic images generated by simulating the process of imaging with fluorescent reporters under varying conditions of noise. Both the over-segmentation and under-segmentation percentages of our method are around 5%. The volume overlap of individual cells, compared to expert manual segmentation, is consistently over 84%. By using our software (ACME) to study somite formation, we were able to segment touching cells with high accuracy and reliably quantify changes in morphogenetic parameters such as cell shape and size, and the arrangement of epithelial and mesenchymal cells. Our software has been developed and tested on Windows, Mac, and Linux platforms and is available publicly under an open source BSD license (https://github.com/krm15/ACME).
Asunto(s)
Automatización , Membrana Celular , Forma de la Célula , Algoritmos , Animales , Fluorescencia , Pez CebraRESUMEN
Embryonic tissues undergoing shape change draw mechanical input from extraembryonic substrates. In avian eggs, the early blastoderm disk is under the tension of the vitelline membrane (VM). Here we report that the chicken VM characteristically downregulates tension and stiffness to facilitate stage-specific embryo morphogenesis. Experimental relaxation of the VM early in development impairs blastoderm expansion, while maintaining VM tension in later stages resists the convergence of the posterior body causing stalled elongation, failure of neural tube closure, and axis rupture. Biochemical and structural analysis shows that VM weakening is associated with the reduction of outer-layer glycoprotein fibers, which is caused by an increasing albumen pH due to CO2 release from the egg. Our results identify a previously unrecognized potential cause of body axis defects through mis-regulation of extraembryonic tissue tension.
Asunto(s)
Blastodermo , Pollos , Animales , Regulación hacia Abajo , Blastodermo/fisiología , Desarrollo Embrionario/genéticaRESUMEN
Tissues undergoing morphogenesis impose mechanical effects on one another. How developmental programs adapt to or take advantage of these effects remains poorly explored. Here, using a combination of live imaging, modeling, and microsurgical perturbations, we show that the axial and paraxial tissues in the forming avian embryonic body coordinate their rates of elongation through mechanical interactions. First, a cell motility gradient drives paraxial presomitic mesoderm (PSM) expansion, resulting in compression of the axial neural tube and notochord; second, elongation of axial tissues driven by PSM compression and polarized cell intercalation pushes the caudal progenitor domain posteriorly; finally, the axial push drives the lateral movement of midline PSM cells to maintain PSM growth and cell motility. These interactions form an engine-like positive feedback loop, which sustains a shared elongation rate for coupled tissues. Our results demonstrate a key role of inter-tissue forces in coordinating distinct body axis tissues during their co-elongation.
Asunto(s)
Embrión no Mamífero/anatomía & histología , Organogénesis , Animales , Fenómenos Biomecánicos , Tipificación del Cuerpo , Movimiento Celular , Polaridad Celular , Rastreo Celular , Embrión de Pollo , Simulación por Computador , Mesodermo/embriología , Codorniz/embriologíaRESUMEN
Mammalian embryos transiently exhibit aerobic glycolysis (Warburg effect), a metabolic adaptation also observed in cancer cells. The role of this particular type of metabolism during vertebrate organogenesis is currently unknown. Here, we provide evidence for spatiotemporal regulation of glycolysis in the posterior region of mouse and chicken embryos. We show that a posterior glycolytic gradient is established in response to graded transcription of glycolytic enzymes downstream of fibroblast growth factor (FGF) signaling. We demonstrate that glycolysis controls posterior elongation of the embryonic axis by regulating cell motility in the presomitic mesoderm and by controlling specification of the paraxial mesoderm fate in the tail bud. Our results suggest that glycolysis in the tail bud coordinates Wnt and FGF signaling to promote elongation of the embryonic axis.
Asunto(s)
Amnios/embriología , Amnios/metabolismo , Tipificación del Cuerpo , Factores de Crecimiento de Fibroblastos/metabolismo , Glucólisis , Vertebrados/embriología , Vertebrados/metabolismo , Vía de Señalización Wnt , Animales , Tipificación del Cuerpo/genética , Movimiento Celular , Embrión de Pollo , Espacio Extracelular/metabolismo , Glucólisis/genética , Concentración de Iones de Hidrógeno , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Fenotipo , Cola (estructura animal)/embriología , Transcripción Genética , Vertebrados/genética , Vía de Señalización Wnt/genéticaRESUMEN
Here we look at modern developmental biology with a focus on the relationship between different approaches of investigation. We argue that direct imaging is a powerful approach not only for obtaining descriptive information but also for model generation and testing that lead to mechanistic insights. Modeling, on the other hand, conceptualizes imaging data and provides guidance to perturbations. The inquiry progresses most efficiently when a trinity of approachesquantitative imaging (measurement), modeling (theory) and perturbation (test)are pursued in concert, but not when one approach is dominant. Using recent studies of the zebrafish system, we show how this combination has effectively advanced classic topics in developmental biology compared to a perturbation-centric approach. Finally, we show that interdisciplinary expertise and perhaps specialization are necessary for carrying out a systematic approach, and discuss the technical hurdles.
Asunto(s)
Biología Evolutiva/métodos , Modelos Biológicos , Animales , Tipificación del Cuerpo , Biología Evolutiva/tendencias , Imagen Óptica/métodos , Biología de Sistemas , Pez Cebra/embriologíaRESUMEN
We introduce a multicolor labeling strategy (Multibow) for cell tracing experiments in developmental and regenerative processes. Building on Brainbow-based approaches that produce colors by differential expression levels of different fluorescent proteins, Multibow adds a layer of label diversity by introducing a binary code in which reporters are initially OFF and then probabilistically ON or OFF following Cre recombination. We have developed a library of constructs that contains seven different colors and three different subcellular localizations. Combining constructs from this library in the presence of Cre generates cells labeled with multiple independently expressed colors based on if each construct is ON or OFF following recombination. These labels form a unique "barcode" that allows the tracking of the cell and its clonal progenies in addition to expression level differences of each color. We tested Multibow in zebrafish which validates its design concept and suggests its utility for cell tracing applications in development and regeneration.
Asunto(s)
Células/citología , Biología Evolutiva/métodos , Técnica del Anticuerpo Fluorescente/métodos , Tipificación Molecular/métodos , Manejo de Especímenes/métodos , Animales , Animales Modificados Genéticamente , Clonación Molecular , Procesamiento de Imagen Asistido por Computador , Microscopía Confocal , Coloración y Etiquetado/métodos , Pez CebraRESUMEN
In mammalian embryonic stem cells, the acquisition of pluripotency is dependent on Nanog, but the in vivo analysis of Nanog has been hampered by its requirement for early mouse development. In an effort to examine the role of Nanog in vivo, we identified a zebrafish Nanog ortholog and found that its knockdown impaired endoderm formation. Genome-wide transcription analysis revealed that nanog-like morphants fail to develop the extraembryonic yolk syncytial layer (YSL), which produces Nodal, required for endoderm induction. We examined the genes that were regulated by Nanog-like and identified the homeobox gene mxtx2, which is both necessary and sufficient for YSL induction. Chromatin immunoprecipitation assays and genetic studies indicated that Nanog-like directly activates mxtx2, which, in turn, specifies the YSL lineage by directly activating YSL genes. Our study identifies a Nanog-like-Mxtx2-Nodal pathway and establishes a role for Nanog-like in regulating the formation of the extraembryonic tissue required for endoderm induction.