scholarly journals Double-layered two-directional somatopleural cell migration during chicken body wall development revealed with local fluorescent tissue labeling

2021 ◽  
Author(s):  
Nobuyuki Sakamoto ◽  
Hirohiko Aoyama ◽  
Koji Ikegami
Keyword(s):  
Body Wall ◽  
Fluorescent Protein ◽  
Gene Transfection ◽  
Three Dimensional ◽  
Migration Behavior ◽  
Lateral Migration ◽  
Lateral Plate ◽  
Dimensional Distribution ◽  
Or Gene ◽  
Green Fluorescent

The ventral body wall is derived from the somatic layer of the lateral plate mesoderm, somatopleure, and somite. The primary ventral body wall is formed as a result of the lateral growth of the somatopleure. The secondary body wall is generated through the migration of somitic cells into the somatopleure. While it is reported that the cervical somatopleural cells migrate caudally to the thoracic region during body wall development, the migration of the thoracic somatopleural cells has not been elucidated. To investigate the migration behavior of the somatopleural cells in the thorax during chicken ventral body wall development, we labeled the thoracic somatopleural cells of one somite wide by DiI labeling or gene transfection of enhanced green fluorescent protein and observed the three-dimensional distribution of the labeled cells with the tissue-clearing technique FRUIT. Our labeling experiments revealed the rostral migration of the somatopleural cells into a deep part of the thoracic body wall in embryonic day 6.5 chickens. For embryonic day 8.5 chickens, these deep migrating somatopleural cells were found around the sternal ribs. Thus, we identified the double-layered two-directional migrating pathways of the somatopleural cells: the rostral migration of the deep somatopleural cells and the lateral migration of the superficial somatopleural cells. Our findings imply that the rostral migration of deep somatopleural cells and the lateral migration of superficial ones might be associated with the developing sternal ribs and the innervation of the thoracic cutaneous nerves, respectively.

scholarly journals Intercellular Calcium Waves in HeLa Cells Expressing GFP-labeled Connexin 43, 32, or 26

2000 ◽  
Vol 11 (5) ◽  
pp. 1815-1827 ◽  
Author(s):  
Koen Paemeleire ◽  
Patricia E. M. Martin ◽  
Sharon L. Coleman ◽  
Kevin E. Fogarty ◽  
Walter A. Carrington ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Hela Cells ◽  
Connexin 43 ◽  
High Speed ◽  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Dimensional Distribution ◽  
Green Fluorescent ◽  
Intracellular Pathway

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca2+ waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca2+]i associated with Ca2+waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca2+-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca2+ waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca2+]i changes were characterized by initiating Ca2+ puffs associated with the perinuclear ER. By contrast, in Cx–GFP-transfected cells and in the presence of apyrase, [Ca2+]i changes were propagated without initiating perinuclear Ca2+ puffs and were communicated between cells at the sites of the Cx–GFP gap junctions. The efficiency of Cx expression determined the extent of Ca2+ wave propagation. These results demonstrate that intercellular Ca2+ waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.

scholarly journals Microvascular Sprouting, Extension, and Creation of New Capillary Connections with Adaptation of the Neighboring Astrocytes in Adult Mouse Cortex under Chronic Hypoxia

2013 ◽  
Vol 34 (2) ◽  
pp. 325-331 ◽  
Author(s):  
Kazuto Masamoto ◽  
Hiroyuki Takuwa ◽  
Chie Seki ◽  
Junko Taniguchi ◽  
Yoshiaki Itoh ◽  
...  
Keyword(s):  
Adult Mouse ◽  
Fluorescent Protein ◽  
Vascular Endothelial Cells ◽  
Three Dimensional ◽  
Vascular Endothelial ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Mouse Cortex ◽  
Hypoxia Adaptation ◽  
Green Fluorescent

The present study aimed to determine the spatiotemporal dynamics of microvascular and astrocytic adaptation during hypoxia-induced cerebral angiogenesis. Adult C57BL/6J and Tie2-green fluorescent protein (GFP) mice with vascular endothelial cells expressing GFP were exposed to normobaric hypoxia for 3 weeks, whereas the three-dimensional microvessels and astrocytes were imaged repeatedly using two-photon microscopy. After 7 to14 days of hypoxia, a vessel sprout appeared from the capillaries with a bump-like head shape (mean diameter 14  μm), and stagnant blood cells were seen inside the sprout. However, no detectable changes in the astrocyte morphology were observed for this early phase of the hypoxia adaptation. More than 50% of the sprouts emerged from capillaries 60  μm away from the center penetrating arteries, which indicates that the capillary distant from the penetrating arteries is a favored site for sprouting. After 14 to 21 days of hypoxia, the sprouting vessels created a new connection with an existing capillary. In this phase, the shape of the new vessel and its blood flow were normalized, and the outside of the vessels were wrapped with numerous processes from the neighboring astrocytes. The findings indicate that hypoxia-induced cerebral angiogenesis provokes the adaptation of neighboring astrocytes, which may stabilize the blood–brain barrier in immature vessels.

Developmental derivation of vocal fold mucosa from human induced pluripotent stem cells

2019 ◽  
Author(s):  
Vlasta Lungova ◽  
Susan Thibeault
Keyword(s):  
Epithelial Cells ◽  
Pluripotent Stem Cells ◽  
Vocal Fold ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Stratified Squamous Epithelium ◽  
Three Dimensional Models ◽  
Induced Pluripotent ◽  
Green Fluorescent

Abstract Development of treatments for vocal dysphonia has been inhibited by lack of human vocal fold (VF) mucosa models because of difficulty in procuring VF epithelial cells, epithelial cells’ limited proliferative capacity and absence of cell lines. We report development of engineered VF mucosae from hiPSC, transfected via TALEN constructs for green fluorescent protein, that mimic development of VF epithelial cells in utero. Modulation of FGF signaling achieves stratified squamous epithelium from definitive and anterior foregut derived cultures. Robust culturing of these cells on collagen-fibroblast constructs produces three-dimensional models comparable to in vivo VF mucosa.

scholarly journals Phosphatidylinositol-4,5-Bisphosphate-Rich Plasma Membrane Patches Organize Active Zones of Endocytosis and Ruffling in Cultured Adipocytes

2004 ◽  
Vol 24 (20) ◽  
pp. 9102-9123 ◽  
Author(s):  
Shaohui Huang ◽  
Larry Lifshitz ◽  
Varsha Patki-Kamath ◽  
Richard Tuft ◽  
Kevin Fogarty ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Large Scale ◽  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Dimensional Space ◽  
Three Dimensional ◽  
Actin Dynamics ◽  
Ph Domain ◽  
Membrane Ruffling ◽  
Green Fluorescent

ABSTRACT A major regulator of endocytosis and cortical F-actin is thought to be phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] present in plasma membranes. Here we report that in 3T3-L1 adipocytes, clathrin-coated membrane retrieval and dense concentrations of polymerized actin occur in restricted zones of high endocytic activity. Ultrafast-acquisition and superresolution deconvolution microscopy of cultured adipocytes expressing an enhanced green fluorescent protein- or enhanced cyan fluorescent protein (ECFP)-tagged phospholipase Cδ1 (PLCδ1) pleckstrin homology (PH) domain reveals that these zones spatially coincide with large-scale PtdIns(4,5)P2-rich plasma membrane patches (PRMPs). PRMPs exhibit lateral dimensions exceeding several micrometers, are relatively stationary, and display extensive local membrane folding that concentrates PtdIns(4,5)P2 in three-dimensional space. In addition, a higher concentration of PtdIns(4,5)P2 in the membranes of PRMPs than in other regions of the plasma membrane can be detected by quantitative fluorescence microscopy. Vesicular structures containing both clathrin heavy chains and PtdIns(4,5)P2 are revealed immediately beneath PRMPs, as is dense F actin. Blockade of PtdIns(4,5)P2 function in PRMPs by high expression of the ECFP-tagged PLCδ1 PH domain inhibits transferrin endocytosis and reduces the abundance of cortical F-actin. Membrane ruffles induced by the expression of unconventional myosin 1c were also found to localize at PRMPs. These results are consistent with the hypothesis that PRMPs organize active PtdIns(4,5)P2 signaling zones in the adipocyte plasma membrane that in turn control regulators of endocytosis, actin dynamics, and membrane ruffling.

scholarly journals A New Model for Nuclear Envelope Breakdown

2001 ◽  
Vol 12 (2) ◽  
pp. 503-510 ◽  
Author(s):  
Mark Terasaki ◽  
Paul Campagnola ◽  
Melissa M. Rolls ◽  
Pascal A. Stein ◽  
Jan Ellenberg ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Nuclear Pore ◽  
Permeability Barrier ◽  
Second Phase ◽  
New Model ◽  
Nuclear Envelope Breakdown ◽  
Two Phases ◽  
Green Fluorescent

Nuclear envelope breakdown was investigated during meiotic maturation of starfish oocytes. Fluorescent 70-kDa dextran entry, as monitored by confocal microscopy, consists of two phases, a slow uniform increase and then a massive wave. From quantitative analysis of the first phase of dextran entry, and from imaging of green fluorescent protein chimeras, we conclude that nuclear pore disassembly begins several minutes before nuclear envelope breakdown. The best fit for the second phase of entry is with a spreading disruption of the membrane permeability barrier determined by three-dimensional computer simulations of diffusion. We propose a new model for the mechanism of nuclear envelope breakdown in which disassembly of the nuclear pores leads to a fenestration of the nuclear envelope double membrane.

scholarly journals Revealing a new fluorescence peak of the enhanced green fluorescent protein using three-dimensional fluorescence spectroscopy

RSC Advances ◽  
2019 ◽  
Vol 9 (40) ◽  
pp. 22853-22858 ◽  
Author(s):  
Nathalia Vieira dos Santos ◽  
Carolina Falaschi Saponi ◽  
Tamar Louise Greaves ◽  
Jorge Fernando Brandão Pereira
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Spectroscopy ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Green Fluorescent ◽  
Fluorescence Peak

Three-dimensional fluorescence spectroscopy as a powerful tool to identify a new fluorescence peak of Enhanced Green Fluorescent Protein (EGFP).

scholarly journals Human induced pluripotent stem cell-derived vocal fold mucosa mimics development and responses to smoke exposure

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Vlasta Lungova ◽  
Xia Chen ◽  
Ziyue Wang ◽  
Christina Kendziorski ◽  
Susan L. Thibeault
Keyword(s):  
Epithelial Cells ◽  
Vocal Fold ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Induced Pluripotent Stem Cell ◽  
Mucosal Inflammation ◽  
Three Dimensional Models ◽  
Green Fluorescent

Abstract Development of treatments for vocal dysphonia has been inhibited by lack of human vocal fold (VF) mucosa models because of difficulty in procuring VF epithelial cells, epithelial cells’ limited proliferative capacity and absence of cell lines. Here we report development of engineered VF mucosae from hiPSC, transfected via TALEN constructs for green fluorescent protein, that mimic development of VF epithelial cells in utero. Modulation of FGF signaling achieves stratified squamous epithelium from definitive and anterior foregut derived cultures. Robust culturing of these cells on collagen-fibroblast constructs produces three-dimensional models comparable to in vivo VF mucosa. Furthermore, we demonstrate mucosal inflammation upon exposure of these constructs to 5% cigarette smoke extract. Upregulation of pro-inflammatory genes in epithelium and fibroblasts leads to aberrant VF mucosa remodeling. Collectively, our results demonstrate that hiPSC-derived VF mucosa is a versatile tool for future investigation of genetic and molecular mechanisms underlying epithelium-fibroblasts interactions in health and disease.

Cell-sorting in aggregates of Dictyostelium discoideum

1999 ◽  
Vol 112 (22) ◽  
pp. 3923-3929 ◽  
Author(s):  
A. Nicol ◽  
W. Rappel ◽  
H. Levine ◽  
W.F. Loomis
Keyword(s):  
Cell Sorting ◽  
Fluorescent Protein ◽  
Cell Mass ◽  
Three Dimensional ◽  
Cell Movement ◽  
Time Lapse ◽  
Cell Types ◽  
Cell Type ◽  
Prespore Cell ◽  
Green Fluorescent

When Dictyostelium cells are induced to develop between a coverslip and a layer of agarose, they aggregate normally into groups containing up to a thousand cells but are then constrained to form disks only a few cells thick that appear to be equivalent to the three-dimensional mounds formed on top of agarose. Such vertically restricted aggregates frequently develop into elongated motile structures, the flattened equivalent of three-dimensional slugs. The advantage of using this system is that the restricted z-dimension enables direct microscopic visualization of most of the cells in the developing structure. We have used time lapse digital fluorescence microscopy of Dictyostelium strains expressing green fluorescent protein (GFP) under the control of either prestalk or prespore specific promoters to follow cell sorting in these flattened mounds. We find that prestalk and prespore cells expressing GFP arise randomly in early aggregates and then rotate rapidly around the disk mixed with the other cell type. After a few hours, the cell types sort out by a process which involves striking changes in relative cell movement. Once sorted, the cell types move independently of each other showing very little heterotypic adhesion. When a group of prestalk cells reaches the edge of the disk, it moves out and is followed by the prespore cell mass. We suggest that sorting may result from cell type specific changes in adhesion and the consequent disruption of movement in the files of cells that are held together by end-to-end adhesion.

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