Limits...
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.

Show MeSH
Lifeact-mTurquoise2 localizes to the nuclear boundary and exhibits a ‘flash’ of Lifeact during nuclear disassembly. A) Time series of a living cell undergoing cell division. Just prior to nuclear disassembly, accumulation of Lifeact is apparent (A2.0-6.0) and during prometaphase disappears with a ‘flash’ (A6.5-7.5). See main text for a more detailed description of the full cleavage cycle. Scale bar 10 μm. B) When the cell rounds up during metaphase-anaphase Lifeact-mTurquoise2 fluorescence appears to increase at the cell boundary through time (different time points are indicated by colored lines). The average profiles perpendicular to the plasma membrane region going from the intracellular space (left) to the extracellular space (right) from the region depicted in A8.0 (yellow line) are normalized to the peak value of the profile at A8.0, show for this cell an increase of about 2.5 fold. C) Quantification of the Lifeact fluorescence at the nuclear boundary from 0.0-6.0 min. The profiles show the averaged normalized fluorescence perpendicular to the nuclear boundary going from nucleoplasm (left) to the cytoplasm (right).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Lifeact-mTurquoise2 localizes to the nuclear boundary and exhibits a ‘flash’ of Lifeact during nuclear disassembly. A) Time series of a living cell undergoing cell division. Just prior to nuclear disassembly, accumulation of Lifeact is apparent (A2.0-6.0) and during prometaphase disappears with a ‘flash’ (A6.5-7.5). See main text for a more detailed description of the full cleavage cycle. Scale bar 10 μm. B) When the cell rounds up during metaphase-anaphase Lifeact-mTurquoise2 fluorescence appears to increase at the cell boundary through time (different time points are indicated by colored lines). The average profiles perpendicular to the plasma membrane region going from the intracellular space (left) to the extracellular space (right) from the region depicted in A8.0 (yellow line) are normalized to the peak value of the profile at A8.0, show for this cell an increase of about 2.5 fold. C) Quantification of the Lifeact fluorescence at the nuclear boundary from 0.0-6.0 min. The profiles show the averaged normalized fluorescence perpendicular to the nuclear boundary going from nucleoplasm (left) to the cytoplasm (right).

Mentions: To better characterize cellular dynamics of Lifeact-mTurquoise2 during a cleavage event, we concentrated on visualizing individual cells with sufficient zoom and time resolution (Figure 3, Additional file 3: Video S3a, b). In this time series (images were captured approximately every 30–40 seconds) we were able to observe several distinct phases of the N. vectensis cell cycle. Figure 3A shows an image sequence of one typical cell prior to (Figure 3A, 0:00) and during division (Figure 3A, 20:13 arrows indicate axis of division). At 2:35, we first observe an accumulation of Lifeact at the nuclear boundary and at the same time, centrosomes become visible as regions of increased cytoplasmic Lifeact-mTurquoise2 which form adjacent to the nucleus (Figure 3A, 3:55 arrow). As we previously stated, the non-specific labeling of spindle fibers and centrosomes appearing in FP injections, can be used to identify these structures. The accumulation of Lifeact-mTurquoise2 in a ring around the nucleus continues over a time course of about 4 minutes. After this phase (reminiscent of prophase), we observe a distinct deformation of the circular structure around the nucleus (Figure 3A, 8:48 arrows), which starts at the position of the presumptive centrosomes and often results in a donut shaped nucleus. (also see Additional file 1: Video S1A, Additional file 3: Video S3a). During this phase, we observe that the centrosomes move further away from the nucleus and that in the time course of about two minutes the nucleus disassembles, during which the nucleus elongates and a fibrous network of Lifeact is visible (Figure 3A 13:22).Figure 3


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)

Lifeact-mTurquoise2 localizes to the nuclear boundary and exhibits a ‘flash’ of Lifeact during nuclear disassembly. A) Time series of a living cell undergoing cell division. Just prior to nuclear disassembly, accumulation of Lifeact is apparent (A2.0-6.0) and during prometaphase disappears with a ‘flash’ (A6.5-7.5). See main text for a more detailed description of the full cleavage cycle. Scale bar 10 μm. B) When the cell rounds up during metaphase-anaphase Lifeact-mTurquoise2 fluorescence appears to increase at the cell boundary through time (different time points are indicated by colored lines). The average profiles perpendicular to the plasma membrane region going from the intracellular space (left) to the extracellular space (right) from the region depicted in A8.0 (yellow line) are normalized to the peak value of the profile at A8.0, show for this cell an increase of about 2.5 fold. C) Quantification of the Lifeact fluorescence at the nuclear boundary from 0.0-6.0 min. The profiles show the averaged normalized fluorescence perpendicular to the nuclear boundary going from nucleoplasm (left) to the cytoplasm (right).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Lifeact-mTurquoise2 localizes to the nuclear boundary and exhibits a ‘flash’ of Lifeact during nuclear disassembly. A) Time series of a living cell undergoing cell division. Just prior to nuclear disassembly, accumulation of Lifeact is apparent (A2.0-6.0) and during prometaphase disappears with a ‘flash’ (A6.5-7.5). See main text for a more detailed description of the full cleavage cycle. Scale bar 10 μm. B) When the cell rounds up during metaphase-anaphase Lifeact-mTurquoise2 fluorescence appears to increase at the cell boundary through time (different time points are indicated by colored lines). The average profiles perpendicular to the plasma membrane region going from the intracellular space (left) to the extracellular space (right) from the region depicted in A8.0 (yellow line) are normalized to the peak value of the profile at A8.0, show for this cell an increase of about 2.5 fold. C) Quantification of the Lifeact fluorescence at the nuclear boundary from 0.0-6.0 min. The profiles show the averaged normalized fluorescence perpendicular to the nuclear boundary going from nucleoplasm (left) to the cytoplasm (right).
Mentions: To better characterize cellular dynamics of Lifeact-mTurquoise2 during a cleavage event, we concentrated on visualizing individual cells with sufficient zoom and time resolution (Figure 3, Additional file 3: Video S3a, b). In this time series (images were captured approximately every 30–40 seconds) we were able to observe several distinct phases of the N. vectensis cell cycle. Figure 3A shows an image sequence of one typical cell prior to (Figure 3A, 0:00) and during division (Figure 3A, 20:13 arrows indicate axis of division). At 2:35, we first observe an accumulation of Lifeact at the nuclear boundary and at the same time, centrosomes become visible as regions of increased cytoplasmic Lifeact-mTurquoise2 which form adjacent to the nucleus (Figure 3A, 3:55 arrow). As we previously stated, the non-specific labeling of spindle fibers and centrosomes appearing in FP injections, can be used to identify these structures. The accumulation of Lifeact-mTurquoise2 in a ring around the nucleus continues over a time course of about 4 minutes. After this phase (reminiscent of prophase), we observe a distinct deformation of the circular structure around the nucleus (Figure 3A, 8:48 arrows), which starts at the position of the presumptive centrosomes and often results in a donut shaped nucleus. (also see Additional file 1: Video S1A, Additional file 3: Video S3a). During this phase, we observe that the centrosomes move further away from the nucleus and that in the time course of about two minutes the nucleus disassembles, during which the nucleus elongates and a fibrous network of Lifeact is visible (Figure 3A 13:22).Figure 3

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