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Megason, Sean

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Megason

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Megason, Sean
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    A bioelectrical phase transition patterns the first vertebrate heartbeats
    (Springer Science and Business Media LLC, 2023-09-27) Jia, Bill; Qi, Yitong; Wong-Campos, J. David; Megason, Sean; Cohen, Adam E.
    A regular heartbeat is essential to vertebrate life. In the mature heart this function is driven by an anatomically localized pacemaker. In contrast, pacemaking capability is broadly distributed in the early embryonic heart(1–3), raising the question of how tissue-scale organization of activity is first established and then maintained during embryonic development. The initial transition of the heart from silent to beating has never been characterized at the timescale of individual electrical events, and the structure in space and time of the early heartbeats remains poorly understood. Using all-optical electrophysiology, we captured the very first zebrafish heartbeat and analyzed the development of cardiac excitability and conduction around this singular event. The first beats appeared suddenly, had irregular inter-beat intervals, propagated coherently across the primordial heart, and emanated from loci that varied between animals and over time. The bioelectrical dynamics were well described by a noisy saddle-node on invariant circle (SNIC) bifurcation with action potential upstroke driven by CaV1.2. Our work shows how gradual and largely asynchronous development of single-cell bioelectrical properties produces a stereotyped and robust tissue-scale transition from quiescence to coordinated beating.
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    Lamellar projections in the endolymphatic sac act as a relief valve to regulate inner ear pressure
    (eLife Sciences Publications, Ltd, 2018) Swinburne, Ian; Mosaliganti, Kishore R; Upadhyayula, Srigokul; Liu, Tsung-Li; Hildebrand, David; Tsai, Tony Y -C; Chen, Anzhi; Al-Obeidi, Ebaa; Fass, Anna K; Malhotra, Samir; Engert, Florian; Lichtman, Jeff; Kirchausen, Tomas; Betzig, Eric; Megason, Sean
    The inner ear is a fluid-filled closed-epithelial structure whose function requires maintenance of an internal hydrostatic pressure and fluid composition. The endolymphatic sac (ES) is a dead-end epithelial tube connected to the inner ear whose function is unclear. ES defects can cause distended ear tissue, a pathology often seen in hearing and balance disorders. Using live imaging of zebrafish larvae, we reveal that the ES undergoes cycles of slow pressure-driven inflation followed by rapid deflation. Absence of these cycles in lmx1bb mutants leads to distended ear tissue. Using serial-section electron microscopy and adaptive optics lattice light-sheet microscopy, we find a pressure relief valve in the ES comprised of partially separated apical junctions and dynamic overlapping basal lamellae that separate under pressure to release fluid. We propose that this lmx1-dependent pressure relief valve is required to maintain fluid homeostasis in the inner ear and other fluid-filled cavities.
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    Nanog-like Regulates Endoderm Formation through the Mxtx2-Nodal Pathway
    (Elsevier, 2012) Xu, Cong; Fan, Zi Peng; Müller, Patrick; Fogley, Rachel; DiBiase, Anthony; Trompouki, Eirini; Unternaehrer, Juli; Xiong, Fengzhu; Torregroza, Ingrid; Evans, Todd; Megason, Sean; Daley, George; Schier, Alexander; Young, Richard A.; Zon, Leonard
    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 identi- fied 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 extraembry- onic yolk syncytial layer (YSL), which produces Nodal, required for endoderm induction. We exam- ined 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.
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    An efficient, scalable, and adaptable framework for solving generic systems of level-set PDEs
    (Frontiers Media S.A., 2013) Mosaliganti, Kishore R.; Gelas, Arnaud; Megason, Sean
    In the last decade, level-set methods have been actively developed for applications in image registration, segmentation, tracking, and reconstruction. However, the development of a wide variety of level-set PDEs and their numerical discretization schemes, coupled with hybrid combinations of PDE terms, stopping criteria, and reinitialization strategies, has created a software logistics problem. In the absence of an integrative design, current toolkits support only specific types of level-set implementations which restrict future algorithm development since extensions require significant code duplication and effort. In the new NIH/NLM Insight Toolkit (ITK) v4 architecture, we implemented a level-set software design that is flexible to different numerical (continuous, discrete, and sparse) and grid representations (point, mesh, and image-based). Given that a generic PDE is a summation of different terms, we used a set of linked containers to which level-set terms can be added or deleted at any point in the evolution process. This container-based approach allows the user to explore and customize terms in the level-set equation at compile-time in a flexible manner. The framework is optimized so that repeated computations of common intensity functions (e.g., gradient and Hessians) across multiple terms is eliminated. The framework further enables the evolution of multiple level-sets for multi-object segmentation and processing of large datasets. For doing so, we restrict level-set domains to subsets of the image domain and use multithreading strategies to process groups of subdomains or level-set functions. Users can also select from a variety of reinitialization policies and stopping criteria. Finally, we developed a visualization framework that shows the evolution of a level-set in real-time to help guide algorithm development and parameter optimization. We demonstrate the power of our new framework using confocal microscopy images of cells in a developing zebrafish embryo.
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    Improved Long-Term Imaging of Embryos with Genetically Encoded α-Bungarotoxin
    (Public Library of Science, 2015) Swinburne, Ian; Mosaliganti, Kishore R.; Green, Amelia A.; Megason, Sean
    Rapid advances in microscopy and genetic labeling strategies have created new opportunities for time-lapse imaging of embryonic development. However, methods for immobilizing embryos for long periods while maintaining normal development have changed little. In zebrafish, current immobilization techniques rely on the anesthetic tricaine. Unfortunately, prolonged tricaine treatment at concentrations high enough to immobilize the embryo produces undesirable side effects on development. We evaluate three alternative immobilization strategies: combinatorial soaking in tricaine and isoeugenol, injection of α-bungarotoxin protein, and injection of α-bungarotoxin mRNA. We find evidence for co-operation between tricaine and isoeugenol to give immobility with improved health. However, even in combination these anesthetics negatively affect long-term development. α-bungarotoxin is a small protein from snake venom that irreversibly binds and inactivates acetylcholine receptors. We find that α-bungarotoxin either as purified protein from snakes or endogenously expressed in zebrafish from a codon-optimized synthetic gene can immobilize embryos for extended periods of time with few health effects or developmental delays. Using α-bungarotoxin mRNA injection we obtain complete movies of zebrafish embryogenesis from the 1-cell stage to 3 days post fertilization, with normal health and no twitching. These results demonstrate that endogenously expressed α-bungarotoxin provides unprecedented immobility and health for time-lapse microscopy.
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    Myc and Fgf Are Required for Zebrafish Neuromast Hair Cell Regeneration
    (Public Library of Science, 2016) Lee, Sang; Huang, Mingqian; Obholzer, Nikolaus D.; Sun, Shan; Li, Wenyan; Petrillo, Marco; Dai, Pu; Zhou, Yi; Cotanche, Douglas A.; Megason, Sean; Li, Huawei; Chen, Zheng-Yi
    Unlike mammals, the non-mammalian vertebrate inner ear can regenerate the sensory cells, hair cells, either spontaneously or through induction after hair cell loss, leading to hearing recovery. The mechanisms underlying the regeneration are poorly understood. By microarray analysis on a chick model, we show that chick hair cell regeneration involves the activation of proliferation genes and downregulation of differentiation genes. Both MYC and FGF are activated in chick hair cell regeneration. Using a zebrafish lateral line neuromast hair cell regeneration model, we show that the specific inhibition of Myc or Fgf suppresses hair cell regeneration, demonstrating that both pathways are essential to the process. Rapid upregulation of Myc and delayed Fgf activation during regeneration suggest a role of Myc in proliferation and Fgf in differentiation. The dorsal-ventral pattern of fgfr1a in the neuromasts overlaps with the distribution of hair cell precursors. By laser ablation, we show that the fgfr1a-positive supporting cells are likely the hair cell precursors that directly give rise to new hair cells; whereas the anterior-posterior fgfr1a-negative supporting cells have heightened proliferation capacity, likely to serve as more primitive progenitor cells to replenish lost precursors after hair cell loss. Thus fgfr1a is likely to mark compartmentalized supporting cell subtypes with different capacities in renewal proliferation and hair cell regeneration. Manipulation of c-MYC and FGF pathways could be explored for mammalian hair cell regeneration.
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    Multibow: Digital Spectral Barcodes for Cell Tracing
    (Public Library of Science, 2015) Xiong, Fengzhu; Obholzer, Nikolaus D.; Noche, Ramil R.; Megason, Sean
    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.
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    Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy
    (The American Society for Cell Biology, 2016) Aguet, François; Upadhyayula, Srigokul; Gaudin, Raphaël; Chou, Yi-ying; Cocucci, Emanuele; He, Kangmin; Chen, Bi-Chang; Mosaliganti, Kishore; Pasham, Mithun; Skillern, Wesley; Legant, Wesley R.; Liu, Tsung-Li; Findlay, Greg; Marino, Eric; Danuser, Gaudenz; Megason, Sean; Betzig, Eric; Kirchhausen, Tom
    Membrane remodeling is an essential part of transferring components to and from the cell surface and membrane-bound organelles and for changes in cell shape, which are particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging and mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and noninvasive illumination of the newly developed lattice light-sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and found that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and was relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light-sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and multicellular assemblies.
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    ACME: Automated Cell Morphology Extractor for Comprehensive Reconstruction of Cell Membranes
    (Public Library of Science, 2012) Mosaliganti, Kishore R.; Noche, Ramil; Xiong, Fengzhu; Swinburne, Ian; Megason, Sean
    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).
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    Attenuation of Notch and Hedgehog Signaling Is Required for Fate Specification in the Spinal Cord
    (Public Library of Science, 2012) Huang, Peng; Xiong, Fengzhu; Megason, Sean; Schier, Alexander
    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-Agduhr″ (KA″) interneurons from lateral floor plate (LFP) progenitors. In vivo cell tracking demonstrates that KA″ 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 KA″ cells lose Hh response upon differentiation. Hh signaling is required in LFP progenitors for KA″ fate specification, but prolonged Hh signaling interferes with KA″ 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 KA″ 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.