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In vivo imaging of Nematostella vectensis embryogenesis and late development using fluorescent probes.

DuBuc TQ, Dattoli AA, Babonis LS, Salinas-Saavedra M, Röttinger E, Martindale MQ, Postma M - BMC Cell Biol. (2014)

Bottom Line: Utilizing fluorescent probes in vivo helped to identify a concentrated 'flash' of Lifeact-mTurquoise2 around the nucleus, immediately prior to cytokinesis in developing embryos.Moreover, Lifeact-mTurquoise2 expression in adult animals allowed the identification of various cell types as well as cellular boundaries.Finally, we present a clear methodology for the visualization of minute temporal events during cnidarian development.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Cnidarians are the closest living relatives to bilaterians and have been instrumental to studying the evolution of bilaterian properties. The cnidarian model, Nematostella vectensis, is a unique system in which embryology and regeneration are both studied, making it an ideal candidate to develop in vivo imaging techniques. Live imaging is the most direct way for quantitative and qualitative assessment of biological phenomena. Actin and tubulin are cytoskeletal proteins universally important for regulating many embryological processes but so far studies in Nematostella primarily focused on the localization of these proteins in fixed embryos.

Results: We used fluorescent probes expressed in vivo to investigate the dynamics of Nematostella development. Lifeact-mTurquoise2, a fluorescent cyan F-actin probe, can be visualized within microvilli along the cellular surface throughout embryonic development and is stable for two months after injection. Co-expression of Lifeact-mTurquoise2 with End-Binding protein1 (EB1) fused to mVenus or tdTomato-NLS allows for the visualization of cell-cycle properties in real time. Utilizing fluorescent probes in vivo helped to identify a concentrated 'flash' of Lifeact-mTurquoise2 around the nucleus, immediately prior to cytokinesis in developing embryos. Moreover, Lifeact-mTurquoise2 expression in adult animals allowed the identification of various cell types as well as cellular boundaries.

Conclusion: The methods developed in this manuscript provide an alternative protocol to investigate Nematostella development through in vivo cellular analysis. This study is the first to utilize the highly photo-stable florescent protein mTurquoise2 as a marker for live imaging. Finally, we present a clear methodology for the visualization of minute temporal events during cnidarian development.

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Localization of Lifeact-mTurquoise2 in microvilli and cell boundaries. A-B) Preserved embryos at early cleavage stages visualized using scanning electron microscopy, show microvilli on the cell surface. C) Similar stage preserved embryos stained using phallacidin to highlight filamentous-actin containing microvilli at the cells surface. D-E) During gastrulation, phallacidin can be used to visualize cellular boundaries along the outer surface of the gastrula. E) Zoomed in region of the ectoderm showing cellular boundaries. F-I) Cleavage stage embryos injected with the mRNA of Lifeact-mTurquoise2. F-G) Embryo approximately at the 32-cell stage, exhibits an increase in surface area contact between neighboring after cell cleavage. H-I) Embryo approximately at the 64-cell stage exhibits an increase in surface area contact between neighboring cells after cell cleavage. J-L) Gastrula stage embryos labeled through injection of Lifeact-mTurquoise2 RNA. J) Early gastrula where the endodermal plate is clearly visible by Lifeact-mTurquoise2 protein (red asterisk). K) Late gastrula where the endodermal plate has invaginated inward out of view (red asterisk – site of gastrulation). L) Magnification of cell boundaries clearly labeled with Lifeact-mTurquoise2 from late gastrulation stage embryo shown in K. M) Time series of an early embryo with a loose aggregate of cells with a flattened configuration that develops into a compact ball-shaped embryo, thereby increasing cell-cell contact (0.0-12.0 min, Additional file 7: Video S6).
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Fig5: Localization of Lifeact-mTurquoise2 in microvilli and cell boundaries. A-B) Preserved embryos at early cleavage stages visualized using scanning electron microscopy, show microvilli on the cell surface. C) Similar stage preserved embryos stained using phallacidin to highlight filamentous-actin containing microvilli at the cells surface. D-E) During gastrulation, phallacidin can be used to visualize cellular boundaries along the outer surface of the gastrula. E) Zoomed in region of the ectoderm showing cellular boundaries. F-I) Cleavage stage embryos injected with the mRNA of Lifeact-mTurquoise2. F-G) Embryo approximately at the 32-cell stage, exhibits an increase in surface area contact between neighboring after cell cleavage. H-I) Embryo approximately at the 64-cell stage exhibits an increase in surface area contact between neighboring cells after cell cleavage. J-L) Gastrula stage embryos labeled through injection of Lifeact-mTurquoise2 RNA. J) Early gastrula where the endodermal plate is clearly visible by Lifeact-mTurquoise2 protein (red asterisk). K) Late gastrula where the endodermal plate has invaginated inward out of view (red asterisk – site of gastrulation). L) Magnification of cell boundaries clearly labeled with Lifeact-mTurquoise2 from late gastrulation stage embryo shown in K. M) Time series of an early embryo with a loose aggregate of cells with a flattened configuration that develops into a compact ball-shaped embryo, thereby increasing cell-cell contact (0.0-12.0 min, Additional file 7: Video S6).

Mentions: F-actin can be detected in microvilli, protrusions of the cellular membrane [33,34]. In N. vectensis, these structures can be visualized by scanning electron microscopy during early embryonic development (Figure 5A,B). A similar structure is visible by confocal microscopy of early embryos labeled with the F-actin binding molecule, phallacidin (Figure 5C). Phallacidin labeled embryos during gastrulation highlight the separation of ectodermal and endodermal tissue (Figure 5D) and distinguishes cell boundaries along the ectodermal surface (Figure 5E). During early embryogenesis, Lifeact-mTurquoise2 localizes to microvilli along the cell surface, and highlights cellular boundaries providing a three dimensional shape when confocal images are stacked from images taken along the z-axis (Figure 5F). During early development Lifeact-mTurquoise2 can be used to visualize microvillar actin bundles along the cellular surface, which exhibit erratic swirling behavior along the cellular boundary (see Additional file 6: Video S5). Immediately prior to cleavage events, we found that embryos become more spherical in shape up to the point of cleavage at which the cells lose their round shape and appear to increase adjacent cell-cell contact (Figure 5F vs. G,H vs. I). Embryos in Figure 5F-G or H- I were taken from a time series of cleavage events, from very early development (approximately 32–64 cell stage). Embryos in F and H were taken after embryos that had recently divided, while G and I are what the embryo looks like before undergoing division. Generally, prior to division, the embryo as a whole exhibits a more compact or round shape with increased surface contact between neighboring cells than earlier stages with roughly equal numbers of cells (F and H, respectively). Although cell counts were not performed, during this phase of development, it has been reported that cell division occurs in a highly synchronous fashion [35].Figure 5


In vivo imaging of Nematostella vectensis embryogenesis and late development using fluorescent probes.

DuBuc TQ, Dattoli AA, Babonis LS, Salinas-Saavedra M, Röttinger E, Martindale MQ, Postma M - BMC Cell Biol. (2014)

Localization of Lifeact-mTurquoise2 in microvilli and cell boundaries. A-B) Preserved embryos at early cleavage stages visualized using scanning electron microscopy, show microvilli on the cell surface. C) Similar stage preserved embryos stained using phallacidin to highlight filamentous-actin containing microvilli at the cells surface. D-E) During gastrulation, phallacidin can be used to visualize cellular boundaries along the outer surface of the gastrula. E) Zoomed in region of the ectoderm showing cellular boundaries. F-I) Cleavage stage embryos injected with the mRNA of Lifeact-mTurquoise2. F-G) Embryo approximately at the 32-cell stage, exhibits an increase in surface area contact between neighboring after cell cleavage. H-I) Embryo approximately at the 64-cell stage exhibits an increase in surface area contact between neighboring cells after cell cleavage. J-L) Gastrula stage embryos labeled through injection of Lifeact-mTurquoise2 RNA. J) Early gastrula where the endodermal plate is clearly visible by Lifeact-mTurquoise2 protein (red asterisk). K) Late gastrula where the endodermal plate has invaginated inward out of view (red asterisk – site of gastrulation). L) Magnification of cell boundaries clearly labeled with Lifeact-mTurquoise2 from late gastrulation stage embryo shown in K. M) Time series of an early embryo with a loose aggregate of cells with a flattened configuration that develops into a compact ball-shaped embryo, thereby increasing cell-cell contact (0.0-12.0 min, Additional file 7: Video S6).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4264334&req=5

Fig5: Localization of Lifeact-mTurquoise2 in microvilli and cell boundaries. A-B) Preserved embryos at early cleavage stages visualized using scanning electron microscopy, show microvilli on the cell surface. C) Similar stage preserved embryos stained using phallacidin to highlight filamentous-actin containing microvilli at the cells surface. D-E) During gastrulation, phallacidin can be used to visualize cellular boundaries along the outer surface of the gastrula. E) Zoomed in region of the ectoderm showing cellular boundaries. F-I) Cleavage stage embryos injected with the mRNA of Lifeact-mTurquoise2. F-G) Embryo approximately at the 32-cell stage, exhibits an increase in surface area contact between neighboring after cell cleavage. H-I) Embryo approximately at the 64-cell stage exhibits an increase in surface area contact between neighboring cells after cell cleavage. J-L) Gastrula stage embryos labeled through injection of Lifeact-mTurquoise2 RNA. J) Early gastrula where the endodermal plate is clearly visible by Lifeact-mTurquoise2 protein (red asterisk). K) Late gastrula where the endodermal plate has invaginated inward out of view (red asterisk – site of gastrulation). L) Magnification of cell boundaries clearly labeled with Lifeact-mTurquoise2 from late gastrulation stage embryo shown in K. M) Time series of an early embryo with a loose aggregate of cells with a flattened configuration that develops into a compact ball-shaped embryo, thereby increasing cell-cell contact (0.0-12.0 min, Additional file 7: Video S6).
Mentions: F-actin can be detected in microvilli, protrusions of the cellular membrane [33,34]. In N. vectensis, these structures can be visualized by scanning electron microscopy during early embryonic development (Figure 5A,B). A similar structure is visible by confocal microscopy of early embryos labeled with the F-actin binding molecule, phallacidin (Figure 5C). Phallacidin labeled embryos during gastrulation highlight the separation of ectodermal and endodermal tissue (Figure 5D) and distinguishes cell boundaries along the ectodermal surface (Figure 5E). During early embryogenesis, Lifeact-mTurquoise2 localizes to microvilli along the cell surface, and highlights cellular boundaries providing a three dimensional shape when confocal images are stacked from images taken along the z-axis (Figure 5F). During early development Lifeact-mTurquoise2 can be used to visualize microvillar actin bundles along the cellular surface, which exhibit erratic swirling behavior along the cellular boundary (see Additional file 6: Video S5). Immediately prior to cleavage events, we found that embryos become more spherical in shape up to the point of cleavage at which the cells lose their round shape and appear to increase adjacent cell-cell contact (Figure 5F vs. G,H vs. I). Embryos in Figure 5F-G or H- I were taken from a time series of cleavage events, from very early development (approximately 32–64 cell stage). Embryos in F and H were taken after embryos that had recently divided, while G and I are what the embryo looks like before undergoing division. Generally, prior to division, the embryo as a whole exhibits a more compact or round shape with increased surface contact between neighboring cells than earlier stages with roughly equal numbers of cells (F and H, respectively). Although cell counts were not performed, during this phase of development, it has been reported that cell division occurs in a highly synchronous fashion [35].Figure 5

Bottom Line: Utilizing fluorescent probes in vivo helped to identify a concentrated 'flash' of Lifeact-mTurquoise2 around the nucleus, immediately prior to cytokinesis in developing embryos.Moreover, Lifeact-mTurquoise2 expression in adult animals allowed the identification of various cell types as well as cellular boundaries.Finally, we present a clear methodology for the visualization of minute temporal events during cnidarian development.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Cnidarians are the closest living relatives to bilaterians and have been instrumental to studying the evolution of bilaterian properties. The cnidarian model, Nematostella vectensis, is a unique system in which embryology and regeneration are both studied, making it an ideal candidate to develop in vivo imaging techniques. Live imaging is the most direct way for quantitative and qualitative assessment of biological phenomena. Actin and tubulin are cytoskeletal proteins universally important for regulating many embryological processes but so far studies in Nematostella primarily focused on the localization of these proteins in fixed embryos.

Results: We used fluorescent probes expressed in vivo to investigate the dynamics of Nematostella development. Lifeact-mTurquoise2, a fluorescent cyan F-actin probe, can be visualized within microvilli along the cellular surface throughout embryonic development and is stable for two months after injection. Co-expression of Lifeact-mTurquoise2 with End-Binding protein1 (EB1) fused to mVenus or tdTomato-NLS allows for the visualization of cell-cycle properties in real time. Utilizing fluorescent probes in vivo helped to identify a concentrated 'flash' of Lifeact-mTurquoise2 around the nucleus, immediately prior to cytokinesis in developing embryos. Moreover, Lifeact-mTurquoise2 expression in adult animals allowed the identification of various cell types as well as cellular boundaries.

Conclusion: The methods developed in this manuscript provide an alternative protocol to investigate Nematostella development through in vivo cellular analysis. This study is the first to utilize the highly photo-stable florescent protein mTurquoise2 as a marker for live imaging. Finally, we present a clear methodology for the visualization of minute temporal events during cnidarian development.

Show MeSH
Related in: MedlinePlus