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Live Cell Imaging of Butterfly Pupal and Larval Wings In Vivo.

Ohno Y, Otaki JM - PLoS ONE (2015)

Bottom Line: Previously, we successfully recorded real-time in vivo images of developing butterfly wings over time at the tissue level.Furthermore, larval cells were flat, whereas pupal cells were vertically elongated as deep as 130 μm.From 60 μm to 80 μm in depth, horizontal connections between these clusters were observed.

View Article: PubMed Central - PubMed

Affiliation: The BCPH Unit of Molecular Physiology, Department of Chemistry, Biology and Marine Science, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa, 903-0213, Japan.

ABSTRACT
Butterfly wing color patterns are determined during the late larval and early pupal stages. Characterization of wing epithelial cells at these stages is thus critical to understand how wing structures, including color patterns, are determined. Previously, we successfully recorded real-time in vivo images of developing butterfly wings over time at the tissue level. In this study, we employed similar in vivo fluorescent imaging techniques to visualize developing wing epithelial cells in the late larval and early pupal stages 1 hour post-pupation. Both larval and pupal epithelial cells were rich in mitochondria and intracellular networks of endoplasmic reticulum, suggesting high metabolic activities, likely in preparation for cellular division, polyploidization, and differentiation. Larval epithelial cells in the wing imaginal disk were relatively large horizontally and tightly packed, whereas pupal epithelial cells were smaller and relatively loosely packed. Furthermore, larval cells were flat, whereas pupal cells were vertically elongated as deep as 130 μm. In pupal cells, many endosome-like or autophagosome-like structures were present in the cellular periphery down to approximately 10 μm in depth, and extensive epidermal feet or filopodia-like processes were observed a few micrometers deep from the cellular surface. Cells were clustered or bundled from approximately 50 μm in depth to deeper levels. From 60 μm to 80 μm in depth, horizontal connections between these clusters were observed. The prospective eyespot and marginal focus areas were resistant to fluorescent dyes, likely because of their non-flat cone-like structures with a relatively thick cuticle. These in vivo images provide important information with which to understand processes of epithelial cell differentiation and color pattern determination in butterfly wings.

No MeSH data available.


Deep serial cross-sections of pupal epithelial cells triple-stained with Hoechst 33342, BODIPY FL Thapsigargin, and MitoTracker Orange.Numbers at the right upper corner indicate the depth from the apical cellular surface. White arrows indicate a large cell, likely a prospective scale cell. Asterisks indicate a large endosome-like or autophagosome-like unstained structure (see Fig 6). Red dots within a nucleus are not explainable. Also see S1 Movie.
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pone.0128332.g005: Deep serial cross-sections of pupal epithelial cells triple-stained with Hoechst 33342, BODIPY FL Thapsigargin, and MitoTracker Orange.Numbers at the right upper corner indicate the depth from the apical cellular surface. White arrows indicate a large cell, likely a prospective scale cell. Asterisks indicate a large endosome-like or autophagosome-like unstained structure (see Fig 6). Red dots within a nucleus are not explainable. Also see S1 Movie.

Mentions: To further characterize the pupal cells, we made detailed optical sections, down to approximately 30 μm from the surface of the tissue using the same set of fluorescent indicators as in Figs 2 and 3 (n = 3) (Fig 5; S1 Movie). Epidermal feet were rich down to 5 μm, below which cellular diameters gradually became smaller and the number of epidermal feet connections decreased. The number of endosome-like or autophagosome-like bodies also decreased, but some of them became larger in deep levels at least down to 12 μm. Nuclei were also elongated down to approximately 20 μm in many cells. Indeed, at deep levels, the intracellular space was occupied mostly by a nucleus and endosome-like or autophagosome-like bodies.


Live Cell Imaging of Butterfly Pupal and Larval Wings In Vivo.

Ohno Y, Otaki JM - PLoS ONE (2015)

Deep serial cross-sections of pupal epithelial cells triple-stained with Hoechst 33342, BODIPY FL Thapsigargin, and MitoTracker Orange.Numbers at the right upper corner indicate the depth from the apical cellular surface. White arrows indicate a large cell, likely a prospective scale cell. Asterisks indicate a large endosome-like or autophagosome-like unstained structure (see Fig 6). Red dots within a nucleus are not explainable. Also see S1 Movie.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481267&req=5

pone.0128332.g005: Deep serial cross-sections of pupal epithelial cells triple-stained with Hoechst 33342, BODIPY FL Thapsigargin, and MitoTracker Orange.Numbers at the right upper corner indicate the depth from the apical cellular surface. White arrows indicate a large cell, likely a prospective scale cell. Asterisks indicate a large endosome-like or autophagosome-like unstained structure (see Fig 6). Red dots within a nucleus are not explainable. Also see S1 Movie.
Mentions: To further characterize the pupal cells, we made detailed optical sections, down to approximately 30 μm from the surface of the tissue using the same set of fluorescent indicators as in Figs 2 and 3 (n = 3) (Fig 5; S1 Movie). Epidermal feet were rich down to 5 μm, below which cellular diameters gradually became smaller and the number of epidermal feet connections decreased. The number of endosome-like or autophagosome-like bodies also decreased, but some of them became larger in deep levels at least down to 12 μm. Nuclei were also elongated down to approximately 20 μm in many cells. Indeed, at deep levels, the intracellular space was occupied mostly by a nucleus and endosome-like or autophagosome-like bodies.

Bottom Line: Previously, we successfully recorded real-time in vivo images of developing butterfly wings over time at the tissue level.Furthermore, larval cells were flat, whereas pupal cells were vertically elongated as deep as 130 μm.From 60 μm to 80 μm in depth, horizontal connections between these clusters were observed.

View Article: PubMed Central - PubMed

Affiliation: The BCPH Unit of Molecular Physiology, Department of Chemistry, Biology and Marine Science, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa, 903-0213, Japan.

ABSTRACT
Butterfly wing color patterns are determined during the late larval and early pupal stages. Characterization of wing epithelial cells at these stages is thus critical to understand how wing structures, including color patterns, are determined. Previously, we successfully recorded real-time in vivo images of developing butterfly wings over time at the tissue level. In this study, we employed similar in vivo fluorescent imaging techniques to visualize developing wing epithelial cells in the late larval and early pupal stages 1 hour post-pupation. Both larval and pupal epithelial cells were rich in mitochondria and intracellular networks of endoplasmic reticulum, suggesting high metabolic activities, likely in preparation for cellular division, polyploidization, and differentiation. Larval epithelial cells in the wing imaginal disk were relatively large horizontally and tightly packed, whereas pupal epithelial cells were smaller and relatively loosely packed. Furthermore, larval cells were flat, whereas pupal cells were vertically elongated as deep as 130 μm. In pupal cells, many endosome-like or autophagosome-like structures were present in the cellular periphery down to approximately 10 μm in depth, and extensive epidermal feet or filopodia-like processes were observed a few micrometers deep from the cellular surface. Cells were clustered or bundled from approximately 50 μm in depth to deeper levels. From 60 μm to 80 μm in depth, horizontal connections between these clusters were observed. The prospective eyespot and marginal focus areas were resistant to fluorescent dyes, likely because of their non-flat cone-like structures with a relatively thick cuticle. These in vivo images provide important information with which to understand processes of epithelial cell differentiation and color pattern determination in butterfly wings.

No MeSH data available.