Understanding Of Morphogenesis Research Articles

Transitions in development - an interview with Amy Shyer and Alan Rodrigues.

Amy Shyer and Alan Rodrigues co-lead the Laboratory of Morphogenesis at Rockefeller University. Having first met as graduate students in Cliff Tabin's lab at Harvard University, they then moved to the Bay Area (California), where Amy carried out her postdoctoral work in Richard Harland's lab while Alan pursued independent postdoctoral research based on the work of Pere Alberch. In 2018, they were recruited to Rockefeller, where their lab investigates how morphogenesis regulates patterning across length-scales, with a focus on collective behaviours operating at the supracellular scale. We met Amy and Alan over zoom to discuss their unique approach to understanding morphogenesis, the importance of emotional resilience in becoming a PI, and the benefits and challenges of running a singular research programme through two people.

GPI-anchored FGF directs cytoneme-mediated bidirectional contacts to regulate its tissue-specific dispersion

How signaling proteins generate a multitude of information to organize tissue patterns is critical to understanding morphogenesis. In Drosophila, FGF produced in wing-disc cells regulates the development of the disc-associated air-sac-primordium (ASP). Here, we show that FGF is Glycosylphosphatidylinositol-anchored to the producing cell surface and that this modification both inhibits free FGF secretion and promotes target-specific cytoneme contacts and contact-dependent FGF release. FGF-source and ASP cells extend cytonemes that present FGF and FGFR on their surfaces and reciprocally recognize each other over distance by contacting through cell-adhesion-molecule (CAM)-like FGF-FGFR binding. Contact-mediated FGF-FGFR interactions induce bidirectional responses in ASP and source cells that, in turn, polarize FGF-sending and FGF-receiving cytonemes toward each other to reinforce signaling contacts. Subsequent un-anchoring of FGFR-bound-FGF from the source membrane dissociates cytoneme contacts and delivers FGF target-specifically to ASP cytonemes for paracrine functions. Thus, GPI-anchored FGF organizes both source and recipient cells and self-regulates its cytoneme-mediated tissue-specific dispersion.

ARVCF catenin controls force production during vertebrate convergent extension

The design of an animal's body plan is encoded in the genome, and the execution of this program is a mechanical progression involving coordinated movement of proteins, cells, and whole tissues. Thus, a challenge to understanding morphogenesis is connecting events that occur across various length scales. Here, we describe how a poorly characterized adhesion effector, Arvcf catenin, controls Xenopus head-to-tail axis extension. We find that Arvcf is required for axis extension within the intact organism but not within isolated tissues. We show that the organism-scale phenotype results from a defect in tissue-scale force production. Finally, we determine that the force defect results from the dampening of the pulsatile recruitment of cell adhesion and cytoskeletal proteins to membranes. These results provide a comprehensive understanding of Arvcf function during axis extension and produce an insight into how a cellular-scale defect in adhesion results in an organism-scale failure of development.

Computational Systems Biology of Morphogenesis.

Extracting mechanistic knowledge from the spatial and temporal phenotypes of morphogenesis is a current challenge due to the complexity of biological regulation and their feedback loops. Furthermore, these regulatory interactions are also linked to the biophysical forces that shape a developing tissue, creating complex interactions responsible for emergent patterns and forms. Here we show how a computational systems biology approach can aid in the understanding of morphogenesis from a mechanistic perspective. This methodology integrates the modeling of tissues and whole-embryos with dynamical systems, the reverse engineering of parameters or even whole equations with machine learning, and the generation of precise computational predictions that can be tested at the bench. To implement and perform the computational steps in the methodology, we present user-friendly tools, computer code, and guidelines. The principles of this methodology are general and can be adapted to other model organisms to extract mechanistic knowledge of their morphogenesis.

STUDY ON THE IN VITRO MORPHOGENETIC RESPONSE FROM SHOOT AND CALLUS CULTURES OF Blepharis maderaspatensis (L.) Heyne ex. Roth

Blepharis maderaspatensis (L.) Heyne ex. Roth is a short-term plant which contains many important secondarycompounds with high medicinal value. Currently, most of the researches focus on chemical composition and pharmacological activity, but the source of raw materials is very limited. In this study, the first step is transferring the samples from nature into in vitro culture conditions to understand the effects of the factors related to shooting and callus morphogenesis was performed, the first node from shoots apical meristem was isolated and sterilized with 1.5% NaOCl for 20 minutes to achieve high efficiency with 86.11% sterile samples and 85.56% shoot growth rate after 2 weeks of culture on MS medium. The shoot generation from axillary shoots was continued to be investigated with the highest number of shoots formed on MS medium supplemented with BA (1 mg / l) showed 1.53 shoots/implant which the height and the number of leavesare 3.65cm and 6.67, respectively. Besides, the formation of callus from leaves of MS medium supplemented with 2.4 - D (0.25 mg / l) achieved the rate of 66.67% of cultured samples, forming good callus after 4 weeks of culture. The results of the study not only contribute importantly to understanding morphogenesis for micropropagation purposes but also serve as the scientific database for further studies at the cellular and molecular levels of this plant.

Cohort-based multiscale analysis of hemodynamic-driven growth and remodeling of the embryonic pharyngeal arch arteries.

Growth and remodeling of the primitive pharyngeal arch artery (PAA) network into the extracardiac great vessels is poorly understood but a major source of clinically serious malformations. Undisrupted blood flow is required for normal PAA development, yet specific relationships between hemodynamics and remodeling remain largely unknown. Meeting this challenge is hindered by the common reductionist analysis of morphology to single idealized models, where in fact structural morphology varies substantially. Quantitative technical tools that allow tracking of morphological and hemodynamic changes in a population-based setting are essential to advancing our understanding of morphogenesis. Here, we have developed a methodological pipeline from high-resolution nano-computed tomography imaging and live-imaging flow measurements to multiscale pulsatile computational models. We combine experimental-based computational models of multiple PAAs to quantify hemodynamic forces in the rapidly morphing Hamburger Hamilton (HH) stage HH18, HH24 and HH26 embryos. We identify local morphological variation along the PAAs and their association with specific hemodynamic changes. Population-level mechano-morphogenic variability analysis is a powerful strategy for identifying stage-specific regions of well and poorly tolerated morphological and/or hemodynamic variation that may protect or initiate cardiovascular malformations.

Inverse tissue mechanics of cell monolayer expansion.

Living tissues undergo deformation during morphogenesis. In this process, cells generate mechanical forces that drive the coordinated cell motion and shape changes. Recent advances in experimental and theoretical techniques have enabled in situ measurement of the mechanical forces, but the characterization of mechanical properties that determine how these forces quantitatively affect tissue deformation remains challenging, and this represents a major obstacle for the complete understanding of morphogenesis. Here, we proposed a non-invasive reverse-engineering approach for the estimation of the mechanical properties, by combining tissue mechanics modeling and statistical machine learning. Our strategy is to model the tissue as a continuum mechanical system and to use passive observations of spontaneous tissue deformation and force fields to statistically estimate the model parameters. This method was applied to the analysis of the collective migration of Madin-Darby canine kidney cells, and the tissue flow and force were simultaneously observed by the phase contrast imaging and traction force microscopy. We found that our monolayer elastic model, whose elastic moduli were reverse-engineered, enabled a long-term forecast of the traction force fields when given the tissue flow fields, indicating that the elasticity contributes to the evolution of the tissue stress. Furthermore, we investigated the tissues in which myosin was inhibited by blebbistatin treatment, and observed a several-fold reduction in the elastic moduli. The obtained results validate our framework, which paves the way to the estimation of mechanical properties of living tissues during morphogenesis.

Tension, contraction and tissue morphogenesis.

D'Arcy Thompson was a proponent of applying mathematical and physical principles to biological systems, an approach that is becoming increasingly common in developmental biology. Indeed, the recent integration of quantitative experimental data, force measurements and mathematical modeling has changed our understanding of morphogenesis - the shaping of an organism during development. Emerging evidence suggests that the subcellular organization of contractile cytoskeletal networks plays a key role in force generation, while on the tissue level the spatial organization of forces determines the morphogenetic output. Inspired by D'Arcy Thompson's On Growth and Form, we review our current understanding of how biological forms are created and maintained by the generation and organization of contractile forces at the cell and tissue levels. We focus on recent advances in our understanding of how cells actively sculpt tissues and how forces are involved in specific morphogenetic processes.

Viscoelastic Dissipation Stabilizes Cell Shape Changes during Tissue Morphogenesis

Tissue morphogenesis relies on the production ofactive cellular forces. Understanding how such forces are mechanically converted into cell shape changes is essential to our understanding of morphogenesis. Here, we use myosin II pulsatile activity during Drosophila embryogenesis to study how transient forces generate irreversible cell shape changes. Analyzing the dynamics of junction shortening and elongation resulting from myosin II pulses, we find that long pulses yield less reversible deformations, typically a signature of dissipative mechanics. This is consistent with a simple viscoelastic description, which we use to model individual shortening and elongation events. The model predicts that dissipation typically occurs on the minute timescale, a timescale commensurate with that of force generation by myosin II pulses. We test this estimate by applying time-controlled forces on junctions with optical tweezers. Finally, we show that actin turnover participates in dissipation, as reducing it pharmacologically increases the reversibility of contractile events. Our results argue that active junctional deformation is stabilized by actin-dependent dissipation. Hence, tissue morphogenesis requires coordination between force generation and dissipation.

From morphogen to morphogenesis and back.

A long-term aim of the life sciences is to understand how organismal shape is encoded by the genome. An important challenge is to identify mechanistic links between the genes that control cell-fate decisions and the cellular machines that generate shape, therefore closing the gap between genotype and phenotype. The logic and mechanisms that integrate these different levels of shape control are beginning to be described, and recently discovered mechanisms of cross-talk and feedback are beginning to explain the remarkable robustness of organ assembly. The 'full-circle' understanding of morphogenesis that is emerging, besides solving a key puzzle in biology, provides a mechanistic framework for future approaches to tissue engineering.

Quantitative methods to study epithelial morphogenesis and polarity.

Morphogenesis of an epithelial tissue emerges from the behavior of its constituent cells,including changes in shape, rearrangements, and divisions. In many instances the directionality of these cellular events is controlled by the polarized distribution of specific molecular components. In recent years, our understanding of morphogenesis and polarity highly benefited from advances in genetics, microscopy, and image analysis. They now make it possible to measure cellular dynamics and polarity with unprecedented precision for entire tissues throughout their development. Here we review recent approaches to visualize and measure cell polarity and tissue morphogenesis. The chapter is organized like an experiment. We first discuss the choice of cell and polarity reporters and describe the use of mosaics to reveal hidden cell polarities orlocal morphogenetic events. Then, we outline application-specific advantages and disadvantages of different microscopy techniques and image projection algorithms. Next, we present methods to extract cell outlinesto measure cell polarity and detect cellular events underlying morphogenesis. Finally, we bridge scales by presenting approaches to quantify the specific contribution of each cellular event to global tissue deformation. Taken together, we provide an in-depth description of available tools and theoretical concepts to quantitatively study cell polarity and tissue morphogenesis over multiple scales.

Towards an understanding of morphogenesis in metropolitan street-networks

We propose a GIS-based method to enable the understanding of how global street-network properties emerge from the temporal accumulation of individual street-network increments. The method entails the adoption of quantitative descriptions of individual street-patterns and of classification algorithms, in order to obtain numerically defined typomorphologies, which may then be statistically associated with the numerical outputs of street-network analysis. We apply the method to the case of Oporto Metropolitan Area, whose development we observed over 60 years. We isolate each increment of development entailing the creation of new streets (4208 objects), we quantify the morphology of their street-layouts, and we classify them into typomorphologies with clustering techniques. Through the investigation of the temporal and spatial frequencies of those typomorphologies, we assess their impacts on the street-networks of a set of selected civil-parishes of the metropolitan region, demonstrating that different typomorphological frequencies result in also different global street-network properties. We conclude by summarising the advantages of the method to generic urban morphological research and by suggesting that it may also contribute to inform bottom-up metropolitan spatial planning.

A practical guide to hydrogels for cell culture.

There is growing appreciation of the role that the extracellular environment plays in regulating cell behavior. Mechanical, structural, and compositional cues, either alone or in concert, can drastically alter cell function. Biomaterials, and particularly hydrogels, have been developed and implemented to present defined subsets of these cues for investigating countless cellular processes as a means of understanding morphogenesis, aging, and disease. Although most scientists concede that standard cell culture materials (tissue culture plastic and glass) do a poor job of recapitulating native cellular milieus, there is currently a knowledge barrier for many researchers in regard to the application of hydrogels for cell culture. Here, we introduce hydrogels to those who may be unfamiliar with procedures to culture and study cells with these systems, with a particular focus on commercially available hydrogels.

Self-shaping of oil droplets via the formation of intermediate rotator phases upon cooling.

Revealing the chemical and physical mechanisms underlying symmetry breaking and shape transformations is key to understanding morphogenesis. If we are to synthesize artificial structures with similar control and complexity to biological systems, we need energy- and material-efficient bottom-up processes to create building blocks of various shapes that can further assemble into hierarchical structures. Lithographic top-down processing allows a high level of structural control in microparticle production but at the expense of limited productivity. Conversely, bottom-up particle syntheses have higher material and energy efficiency, but are more limited in the shapes achievable. Linear hydrocarbons are known to pass through a series of metastable plastic rotator phases before freezing. Here we show that by using appropriate cooling protocols, we can harness these phase transitions to control the deformation of liquid hydrocarbon droplets and then freeze them into solid particles, permanently preserving their shape. Upon cooling, the droplets spontaneously break their shape symmetry several times, morphing through a series of complex regular shapes owing to the internal phase-transition processes. In this way we produce particles including micrometre-sized octahedra, various polygonal platelets, O-shapes, and fibres of submicrometre diameter, which can be selectively frozen into the corresponding solid particles. This mechanism offers insights into achieving complex morphogenesis from a system with a minimal number of molecular components.

Silica bioreplication preserves three-dimensional spheroid structures of human pluripotent stem cells and HepG2 cells.

Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus, they are increasingly employed as models for cancer and drug research, as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than those in 2D cultures, and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore, SBR preserves extracellular matrix-like materials and cellular proteins. These findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells, providing a pathway to enable our understanding of morphogenesis in 3D cultures.

Colloquium: Mechanical formalisms for tissue dynamics.

The understanding of morphogenesis in living organisms has been renewed by tremendous progress in experimental techniques that provide access to cell scale, quantitative information both on the shapes of cells within tissues and on the genes being expressed. This information suggests that our understanding of the respective contributions of gene expression and mechanics, and of their crucial entanglement, will soon leap forward. Biomechanics increasingly benefits from models, which assist the design and interpretation of experiments, point out the main ingredients and assumptions, and ultimately lead to predictions. The newly accessible local information thus calls for a reflection on how to select suitable classes of mechanical models. We review both mechanical ingredients suggested by the current knowledge of tissue behaviour, and modelling methods that can help generate a rheological diagram or a constitutive equation. We distinguish cell scale ("intra-cell") and tissue scale ("inter-cell") contributions. We recall the mathematical framework developed for continuum materials and explain how to transform a constitutive equation into a set of partial differential equations amenable to numerical resolution. We show that when plastic behaviour is relevant, the dissipation function formalism appears appropriate to generate constitutive equations; its variational nature facilitates numerical implementation, and we discuss adaptations needed in the case of large deformations. The present article gathers theoretical methods that can readily enhance the significance of the data to be extracted from recent or future high throughput biomechanical experiments.

Morphometrics, 3D Imaging, and Craniofacial Development.

Recent studies have shown how volumetric imaging and morphometrics can add significantly to our understanding of morphogenesis, the developmental basis for variation, and the etiology of structural birth defects. On the other hand, the complex questions and diverse imaging data in developmental biology present morphometrics with more complex challenges than applications in virtually any other field. Meeting these challenges is necessary in order to understand the mechanistic basis for variation in complex morphologies. This chapter reviews the methods and theory that enable the application of modern landmark-based morphometrics to developmental biology and craniofacial development, in particular. We discuss the theoretical foundations of morphometrics as applied to development and review the basic approaches to the quantification of morphology. Focusing on geometric morphometrics, we discuss the principal statistical methods for quantifying and comparing morphological variation and covariation structure within and among groups. Finally, we discuss the future directions for morphometrics in developmental biology that will be required for approaches that enable quantitative integration across the genotype-phenotype map.

Capturing structure and function in an embryonic heart with biophotonic tools.

Disturbed cardiac function at an early stage of development has been shown to correlate with cellular/molecular, structural as well as functional cardiac anomalies at later stages culminating in the congenital heart defects (CHDs) that present at birth. While our knowledge of cellular and molecular steps in cardiac development is growing rapidly, our understanding of the role of cardiovascular function in the embryo is still in an early phase. One reason for the scanty information in this area is that the tools to study early cardiac function are limited. Recently developed and adapted biophotonic tools may overcome some of the challenges of studying the tiny fragile beating heart. In this chapter, we describe and discuss our experience in developing and implementing biophotonic tools to study the role of function in heart development with emphasis on optical coherence tomography (OCT). OCT can be used for detailed structural and functional studies of the tubular and looping embryo heart under physiological conditions. The same heart can be rapidly and quantitatively phenotyped at early and again at later stages using OCT. When combined with other tools such as optical mapping (OM) and optical pacing (OP), OCT has the potential to reveal in spatial and temporal detail the biophysical changes that can impact mechanotransduction pathways. This information may provide better explanations for the etiology of the CHDs when interwoven with our understanding of morphogenesis and the molecular pathways that have been described to be involved. Future directions for advances in the creation and use of biophotonic tools are discussed.

Mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development.

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine(1). Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought(1). The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth(2). Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for <em>Ex Vivo</em> Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.