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Intercellular bridges in vertebrate gastrulation.

Caneparo L, Pantazis P, Dempsey W, Fraser SE - PLoS ONE (2011)

Bottom Line: The developing zebrafish embryo has been the subject of many studies of regional patterning, stereotypical cell movements and changes in cell shape.To better study the morphological features of cells during gastrulation, we generated mosaic embryos expressing membrane attached Dendra2 to highlight cellular boundaries.These findings reveal a surprising feature of the cellular landscape in zebrafish embryos and open new possibilities for cell-cell communication during gastrulation, with implications for modeling, cellular mechanics, and morphogenetic signaling.

View Article: PubMed Central - PubMed

Affiliation: Beckman Institute and Division of Biology, California Institute of Technology, Pasadena, California, United States of America. caneparo@caltech.edu

ABSTRACT
The developing zebrafish embryo has been the subject of many studies of regional patterning, stereotypical cell movements and changes in cell shape. To better study the morphological features of cells during gastrulation, we generated mosaic embryos expressing membrane attached Dendra2 to highlight cellular boundaries. We find that intercellular bridges join a significant fraction of epiblast cells in the zebrafish embryo, reaching several cell diameters in length and spanning across different regions of the developing embryos. These intercellular bridges are distinct from the cellular protrusions previously reported as extending from hypoblast cells (1-2 cellular diameters in length) or epiblast cells (which were shorter). Most of the intercellular bridges were formed at pre-gastrula stages by the daughters of a dividing cell maintaining a membrane tether as they move apart after mitosis. These intercellular bridges persist during gastrulation and can mediate the transfer of proteins between distant cells. These findings reveal a surprising feature of the cellular landscape in zebrafish embryos and open new possibilities for cell-cell communication during gastrulation, with implications for modeling, cellular mechanics, and morphogenetic signaling.

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Membrane Dynamics of Intercellular Bridges.(A-A”’) Time-lapse rendering of photoconverted mDendra2 diffusing along the intercellular bridges (see also: Movie S4 and S6). (B-C’) Membrane transfer of photoconverted protein in embryos labeled with mDendra2. We photoconverted mDendra2 with a 405 nm light [7], which shifts the emission and excitation spectra. (B, B’) Non-converted mDendra2 positive cells are in green (B), and photoconverted cells are in red, with H2B-mCherry highlighting the nuclei (B’). The light square in (B) shows the region of photoconversion. (C-C’) The same cells are connected via an intercellular bridge. The pool of photoconverted mDendra2 reaches the cell at the opposite end of the intercellular bridge, indicated by the white arrowhead and white dotted outline. (D) Quantification of the dynamics — length as function of time — of a pool of photoconverted mDendra2 along the intercellular bridge (time in minute and length in µm). Scattered red and blue dots represent the raw data for mDendra2 and CD8-Dendra2, respectively. The curve fitting was performed using CurveFittingTool in MATLAB, and each curve has an R2 value of at least 0.9. Scale bar (A-C’): 10 µm.
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pone-0020230-g003: Membrane Dynamics of Intercellular Bridges.(A-A”’) Time-lapse rendering of photoconverted mDendra2 diffusing along the intercellular bridges (see also: Movie S4 and S6). (B-C’) Membrane transfer of photoconverted protein in embryos labeled with mDendra2. We photoconverted mDendra2 with a 405 nm light [7], which shifts the emission and excitation spectra. (B, B’) Non-converted mDendra2 positive cells are in green (B), and photoconverted cells are in red, with H2B-mCherry highlighting the nuclei (B’). The light square in (B) shows the region of photoconversion. (C-C’) The same cells are connected via an intercellular bridge. The pool of photoconverted mDendra2 reaches the cell at the opposite end of the intercellular bridge, indicated by the white arrowhead and white dotted outline. (D) Quantification of the dynamics — length as function of time — of a pool of photoconverted mDendra2 along the intercellular bridge (time in minute and length in µm). Scattered red and blue dots represent the raw data for mDendra2 and CD8-Dendra2, respectively. The curve fitting was performed using CurveFittingTool in MATLAB, and each curve has an R2 value of at least 0.9. Scale bar (A-C’): 10 µm.

Mentions: To verify that the intercellular bridges are continuous between pairs of cells we photoconverted mDendra2 in one cell body and followed the motions of the photoconverted pool towards the other cell (Figure 3 and Movie S6). In all cases, the mDendra2 moved along the length of the intercellular bridge, eventually reaching the plasma membrane of the sister cell (Figure 3B-C'). The photoconversion of mDendra permitted us to measure the time required for a tagged protein to move along the length of the intercellular bridge. The movement seems faster than expected by diffusion alone, reaching distances of 100 µm in about 30 minutes (3.4 µm/min,). This fast rate is not particular to mDendra2; Dendra2 anchored via CD8 (CD8-Dendra2) [9], [10] was found to move at rate of 3.3 µm/min. The movement of the membrane-linked Dendra2 probes seems linear with respect to time, suggesting that it is due to some active process rather than by diffusion alone (Figure 3D). This rate of transfer along the intercellular bridges is sufficiently fast to permit a role in cell-cell communication during gastrulation. In cultured mammalian cells, tunneling nanotubes have been shown to be capable of organelle transfer [18], retroviral infection [19] and immune surface receptor transfer [20]. The motion of membrane-linked Dendra2 along the intercellular bridges seen here offers up the intriguing possibility that the intercellular bridges perform similar biological functions in zebrafish embryogenesis.


Intercellular bridges in vertebrate gastrulation.

Caneparo L, Pantazis P, Dempsey W, Fraser SE - PLoS ONE (2011)

Membrane Dynamics of Intercellular Bridges.(A-A”’) Time-lapse rendering of photoconverted mDendra2 diffusing along the intercellular bridges (see also: Movie S4 and S6). (B-C’) Membrane transfer of photoconverted protein in embryos labeled with mDendra2. We photoconverted mDendra2 with a 405 nm light [7], which shifts the emission and excitation spectra. (B, B’) Non-converted mDendra2 positive cells are in green (B), and photoconverted cells are in red, with H2B-mCherry highlighting the nuclei (B’). The light square in (B) shows the region of photoconversion. (C-C’) The same cells are connected via an intercellular bridge. The pool of photoconverted mDendra2 reaches the cell at the opposite end of the intercellular bridge, indicated by the white arrowhead and white dotted outline. (D) Quantification of the dynamics — length as function of time — of a pool of photoconverted mDendra2 along the intercellular bridge (time in minute and length in µm). Scattered red and blue dots represent the raw data for mDendra2 and CD8-Dendra2, respectively. The curve fitting was performed using CurveFittingTool in MATLAB, and each curve has an R2 value of at least 0.9. Scale bar (A-C’): 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102083&req=5

pone-0020230-g003: Membrane Dynamics of Intercellular Bridges.(A-A”’) Time-lapse rendering of photoconverted mDendra2 diffusing along the intercellular bridges (see also: Movie S4 and S6). (B-C’) Membrane transfer of photoconverted protein in embryos labeled with mDendra2. We photoconverted mDendra2 with a 405 nm light [7], which shifts the emission and excitation spectra. (B, B’) Non-converted mDendra2 positive cells are in green (B), and photoconverted cells are in red, with H2B-mCherry highlighting the nuclei (B’). The light square in (B) shows the region of photoconversion. (C-C’) The same cells are connected via an intercellular bridge. The pool of photoconverted mDendra2 reaches the cell at the opposite end of the intercellular bridge, indicated by the white arrowhead and white dotted outline. (D) Quantification of the dynamics — length as function of time — of a pool of photoconverted mDendra2 along the intercellular bridge (time in minute and length in µm). Scattered red and blue dots represent the raw data for mDendra2 and CD8-Dendra2, respectively. The curve fitting was performed using CurveFittingTool in MATLAB, and each curve has an R2 value of at least 0.9. Scale bar (A-C’): 10 µm.
Mentions: To verify that the intercellular bridges are continuous between pairs of cells we photoconverted mDendra2 in one cell body and followed the motions of the photoconverted pool towards the other cell (Figure 3 and Movie S6). In all cases, the mDendra2 moved along the length of the intercellular bridge, eventually reaching the plasma membrane of the sister cell (Figure 3B-C'). The photoconversion of mDendra permitted us to measure the time required for a tagged protein to move along the length of the intercellular bridge. The movement seems faster than expected by diffusion alone, reaching distances of 100 µm in about 30 minutes (3.4 µm/min,). This fast rate is not particular to mDendra2; Dendra2 anchored via CD8 (CD8-Dendra2) [9], [10] was found to move at rate of 3.3 µm/min. The movement of the membrane-linked Dendra2 probes seems linear with respect to time, suggesting that it is due to some active process rather than by diffusion alone (Figure 3D). This rate of transfer along the intercellular bridges is sufficiently fast to permit a role in cell-cell communication during gastrulation. In cultured mammalian cells, tunneling nanotubes have been shown to be capable of organelle transfer [18], retroviral infection [19] and immune surface receptor transfer [20]. The motion of membrane-linked Dendra2 along the intercellular bridges seen here offers up the intriguing possibility that the intercellular bridges perform similar biological functions in zebrafish embryogenesis.

Bottom Line: The developing zebrafish embryo has been the subject of many studies of regional patterning, stereotypical cell movements and changes in cell shape.To better study the morphological features of cells during gastrulation, we generated mosaic embryos expressing membrane attached Dendra2 to highlight cellular boundaries.These findings reveal a surprising feature of the cellular landscape in zebrafish embryos and open new possibilities for cell-cell communication during gastrulation, with implications for modeling, cellular mechanics, and morphogenetic signaling.

View Article: PubMed Central - PubMed

Affiliation: Beckman Institute and Division of Biology, California Institute of Technology, Pasadena, California, United States of America. caneparo@caltech.edu

ABSTRACT
The developing zebrafish embryo has been the subject of many studies of regional patterning, stereotypical cell movements and changes in cell shape. To better study the morphological features of cells during gastrulation, we generated mosaic embryos expressing membrane attached Dendra2 to highlight cellular boundaries. We find that intercellular bridges join a significant fraction of epiblast cells in the zebrafish embryo, reaching several cell diameters in length and spanning across different regions of the developing embryos. These intercellular bridges are distinct from the cellular protrusions previously reported as extending from hypoblast cells (1-2 cellular diameters in length) or epiblast cells (which were shorter). Most of the intercellular bridges were formed at pre-gastrula stages by the daughters of a dividing cell maintaining a membrane tether as they move apart after mitosis. These intercellular bridges persist during gastrulation and can mediate the transfer of proteins between distant cells. These findings reveal a surprising feature of the cellular landscape in zebrafish embryos and open new possibilities for cell-cell communication during gastrulation, with implications for modeling, cellular mechanics, and morphogenetic signaling.

Show MeSH
Related in: MedlinePlus