Biomedical subjects

Brenda J Rongish

Publications and source records attributed to Brenda J Rongish.

10 recordsLinked to original sources

PubMed2006

Mesodermal cell displacements during avian gastrulation are due to both individual cell-autonomous and convective tissue movements.

Gastrulation is a fundamental process in early development that results in the formation of three primary germ layers. During avian gastrulation, presumptive mesodermal cells in the dorsal epiblast ingress through a furrow called the primitive streak (PS), and subsequently move away from the PS and form adult tissues. The biophysical mechanisms driving mesodermal cell movements during gastrulation in amniotes, notably warm-blooded embryos, are not understood. Until now, a major challenge has been distinguishing local individual cell-autonomous (active) displacements from convective displacements caused by large-scale (bulk) morphogenetic tissue movements. To address this problem, we used multiscale, time-lapse microscopy and a particle image velocimetry method for computing tissue displacement fields. Immunolabeled fibronectin was used as an in situ marker for quantifying tissue displacements. By imaging fluorescently labeled mesodermal cells and surrounding extracellular matrix simultaneously, we were able to separate directly the active and passive components of cell displacement during gastrulation. Our results reveal the following: (i) Convective tissue motion contributes significantly to total cell displacement and must be subtracted to measure true cell-autonomous displacement; (ii) Cell-autonomous displacement decreases gradually after regression from the PS; and (iii) There is an increasing cranial-to-caudal (head-to-tail) cell-autonomous motility gradient, with caudal cells actively moving away from the PS faster than cranial cells. These studies show that, in some regions of the embryo, total mesodermal cell displacements are mostly due to convective tissue movements; thus, the data have profound implications for understanding cell guidance mechanisms and tissue morphogenesis in warm-blooded embryos.

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PubMed2006

New insights into extracellular matrix assembly and reorganization from dynamic imaging of extracellular matrix proteins in living osteoblasts.

The extracellular matrix (ECM) has been traditionally viewed as a static scaffold that supports cells and tissues. However, recent dynamic imaging studies suggest that ECM components are highly elastic and undergo continual movement and deformation. Latent transforming growth factor beta (TGFbeta) binding protein-1 (LTBP1) is an ECM glycoprotein that binds latent TGFbeta and regulates its availability and activity. LTBP1 initially co-distributes with fibronectin in the extracellular matrix of osteoblasts, and depends on fibronectin for its assembly. To gain further insights into the mechanisms of assembly of LTBP1 and its spatial and temporal interactions with fibronectin, we have performed dual fluorescence time-lapse imaging of these two proteins in living osteoblasts using fluorescent probes. Time-lapse movies showed surprisingly large fibril displacements associated with cellular movement as well as occasional breaking of LTBP1 or fibronectin-containing fibrils. Individual fibrils stretched to as much as 3.5 times or contracted to as much as one fourth of their original length. Motile cells appeared to actively mediate extracellular matrix assembly by adding 'globules' or 'packets' of matrix material onto existing fibrils. They also actively reorganized the extracellular matrix by shunting matrix material from one location to another and exchanging fibrillar material between fibrils. This cell-mediated matrix reorganization was primarily associated with the assembly and remodeling of the initial (early) matrix, whereas mature, established ECM was more stable. Displacement vector mapping showed that different matrix fibrillar networks within the same cultures can show different dynamic motion in response to cell movement and showed that the motion of fibrils was correlated with cell motion. These data suggest novel cell-mediated mechanisms for assembly and reorganization of the extracellular matrix and highlight a role for cell motility in the assembly process.

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PubMed2006

Electroporation and EGFP labeling of gastrulating quail embryos.

Labeling embryonic cells to trace their motion is a classical experimental approach with a host of techniques being used to mark live cells and tissues. Genetically engineered fluorescent protein vectors (DNA plasmids) are a recent technology well suited to time-resolved studies of cellular motion in live embryos. DNA plasmids encoding fluorescent proteins can be introduced into cells using several methods, including electroporation, a technique used widely for analysis of tissue culture and embryonic cells. Here we describe a technique designed to introduce DNA plasmids into early gastrulation stage quail embryos, ex ovo. The method is effective, and with practice enables an investigator to direct the vectors to relatively confined regions of gastrulating embryos. The required electroporation chamber can be fabricated from common laboratory materials. We anticipate that using this method of labeling cells in a warm-blooded embryo, during gastrulation, will be a fruitful means of studying subsequent embryogenesis.

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PubMed2006

Elastic fiber formation: a dynamic view of extracellular matrix assembly using timer reporters.

To study the dynamics of elastic fiber assembly, mammalian cells were transfected with a cDNA construct encoding bovine tropoelastin in frame with the Timer reporter. Timer is a derivative of the DsRed fluorescent protein that changes from green to red over time and, hence, can be used to distinguish new from old elastin. Using dynamic imaging microscopy, we found that the first step in elastic fiber formation is the appearance of small cell surface-associated elastin globules that increased in size with time (microassembly). The elastin globules are eventually transferred to pre-existing elastic fibers in the extracellular matrix where they coalesce into larger structures (macroassembly). Mechanical forces associated with cell movement help shape the forming, extracellular elastic fiber network. Time-lapse imaging combined with the use of Timer constructs provides unique tools for studying the temporal and spatial aspects of extracellular matrix formation by live cells.

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PubMed2006

Elastic fiber macro-assembly is a hierarchical, cell motion-mediated process.

Elastic fibers are responsible for the extensibility and resilience of many vertebrate tissues, and improperly assembled elastic fibers are implicated in a number of human diseases. It was recently demonstrated that in vitro, cells first secrete tropoelastin into a punctate pattern of globules. To study the dynamics of macroassembly, that is, the assembly of the secreted tropoelastin globules into elastic fibers, we utilized long-term time-lapse immunofluorescence imaging and a tropoelastin p Timer fusion protein, which shifts its fluorescence spectrum over time. Pulse-chase immunolabeling of the fibroblast-like RFL-6 cells demonstrates that tropoelastin globules aggregate in a hierarchical manner, creating progressively larger fibrillar structures. By analyzing the correlation between cell and extracellular matrix movements, we show that both the aggregation process and shaping the aggregates into fibrillar form is coupled to cell motion. We also show that the motion of non-adjacent cells becomes more coordinated as the physical size of elastin-containing aggregates increases. Our data imply that the formation of elastic fibers involves the concerted action and motility of multiple cells.

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PubMed2006

Extracellular matrix macroassembly dynamics in early vertebrate embryos.

This chapter focuses on the in vivo macroassembly dynamics of fibronectin and fibrillin-2--two prominent extracellular matrix (ECM) components, present in vertebrate embryos at the earliest stages of development. The ECM is an inherently dynamic structure with a well-defined position fate: ECM filaments are not only anchored to and move with established tissue boundaries, but are repositioned prior to the formation of new anatomical features. We distinguish two ECM filament relocation processes-each operating on different length scales. First, ECM filaments are moved by large-scale tissue motion, which rearranges major organ primordia within the embryo. The second type of motion, on the scale of the individual ECM filaments, is driven by local motility and protrusive activity of nearby cells. The motion decomposition is made practically possible by recent advances in microscopy and high-resolution particle image velocimetry algorithms. We demonstrate that both kinds of motion contribute substantially to the establishment of normal ECM structure, and both must be taken into account when attempting to understand ECM macroassembly during embryonic morphogenesis. The tissue-scale motion changes the local amount (density) and the tissue-level structure (e.g., orientation) of ECM fibers. Local reorganization includes filament assembly and the segregation of ECM into specific patterns. Local reorganization takes place most actively at Hensen's node and around the primitive streak. These regions are also sites of active cell migration, where fibrillin-2 and fibronectin are often colocalized in ECM globules, and new fibrillin-2 foci are deposited. During filament assembly, the globular patches of ECM are joined into larger linear structures in a hierarchical process: increasingly larger structures are created by the aggregation of smaller units. A future understanding of ECM assembly thus requires the study of the complex interactions between biochemical assembly steps, local cell action, and tissue motion.

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PubMed2005

A digital image-based method for computational tissue fate mapping during early avian morphogenesis.

The early stages of vertebrate development, encompassing gastrulation, segmentation, and caudal axis formation, presumably involve large (finite) morphogenetic deformations; however, there are few quantitative biomechanical data available for describing such large-scale or tissue-level deformations in the embryo. In this study, we present a new method for automated computational "tissue fate mapping," by combining a recently developed high-resolution time-lapse digital microscopy system for whole-avian embryo imaging with particle image velocimetry (PIV), a well-established digital image correlation technique for measuring continuum deformations. Tissue fate mapping, as opposed to classical cell fate mapping or other cell tracking methods, is used to track the spatiotemporal trajectories of arbitrary (virtual) tissue material points in various layers of the embryo, which can then be used to calculate finite morphogenetic deformation or strain maps. To illustrate the method, we present representative tissue fate and strain mapping data for normal early-stage quail embryos. These data demonstrate, to our knowledge, for the first time, large tissue-level deformations that are shared between different germ layers in the embryo, suggesting a more global morphogenetic patterning mechanism than had been previously appreciated.

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PubMed2004

Extracellular matrix dynamics during vertebrate axis formation.

The first evidence for the dynamics of in vivo extracellular matrix (ECM) pattern formation during embryogenesis is presented below. Fibrillin 2 filaments were tracked for 12 h throughout the avian intraembryonic mesoderm using automated light microscopy and algorithms of our design. The data show that these ECM filaments have a reproducible morphogenic destiny that is characterized by directed transport. Fibrillin 2 particles initially deposited in the segmental plate mesoderm are translocated along an unexpected trajectory where they eventually polymerize into an intricate scaffold of cables parallel to the anterior-posterior axis. The cables coalesce near the midline before the appearance of the next-formed somite. Moreover, the ECM filaments define global tissue movements with high precision because the filaments act as passive motion tracers. Quantification of individual and collective filament "behaviors" establish fate maps, trajectories, and velocities. These data reveal a caudally propagating traveling wave pattern in the morphogenetic movements of early axis formation. We conjecture that within vertebrate embryos, long-range mechanical tension fields are coupled to both large-scale patterning and local organization of the ECM. Thus, physical forces or stress fields are essential requirements for executing an emergent developmental pattern-in this case, paraxial fibrillin cable assembly.

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PubMed2004

Dynamic imaging of cell, extracellular matrix, and tissue movements during avian vertebral axis patterning.

Vertebrate axis patterning depends on cell and extracellular matrix (ECM) repositioning and proper cell-ECM interactions. However, there are few in vivo data addressing how large-scale tissue deformations are coordinated with the motion of local cell ensembles or the displacement of ECM constituents. Combining the methods of dynamic imaging and experimental biology allows both cell and ECM fate-mapping to be correlated with ongoing tissue deformations. These fate-mapping studies suggest that the axial ECM components "move" both as a composite meshwork and as autonomous particles, depending on the length scale being examined. Cells are also part of this composite, and subject to passive displacements resulting from tissue deformations. However, in contrast to the ECM, cells are self-propelled. The net result of cell and ECM displacements, along with proper ECM-cell adhesion, is the assembly of new tissue architecture. Data herein show that disruption of normal cell-ECM interactions during axis formation results in developmental abnormalities and a disorganization of the ECM. Our goal in characterizing the global displacement patterns of axial cells and ECM is to provide critical information regarding existing strain fields in the segmental plate and paraxial mesoderm. Deducing the mechanical influences on cell behavior is critical, if we are to understand vertebral axis patterning. Supplementary material for this article is available online at http://www.mrw.interscience.wiley.com/suppmat/1542-975X/suppmat/72/v72.266.html.

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PubMed2003

Culturing of avian embryos for time-lapse imaging.

Monitoring morphogenetic processes, at high resolution over time, has been a long-standing goal of many developmental cell biologists. It is critical to image cells in their natural environment whenever possible; however, imaging many warm-blooded vertebrates, especially mammals, is problematic. At early stages of development, birds are ideal for imaging, since the avian body plan is very similar to that of mammals. We have devised a culturing technique that allows for the acquisition of high-resolution differential interference contrast and epifluorescence images of developing avian embryos in a 4-D (3-D + time) system. The resulting information, from intact embryos, is derived from an area encompassing several millimeters, at micrometer resolution for up to 30 h.

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