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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.


Triple staining of a pupal wing tissue.The tissue was stained with Hoechst 33342 for nuclei, BODIPY FL Thapsigargin for ER, and MitoTracker Orange for mitochondria. White arrows indicate endosome-like or autophagosome-like unstained structures. (a) Low magnification view. (b) High magnification view. Original scanning images were acquired with 1.0 μm intervals, and those 23 images were stacked to produce the final image of (b) and (d). (c) Hoechst 33342 staining. A part of the image shown in (a) was enlarged and its contrast was enhanced. Right and left images are identical except that nuclei with possible circular arrangements are highlighted in white. (d) 3D image of an epithelial tissue.
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pone.0128332.g003: Triple staining of a pupal wing tissue.The tissue was stained with Hoechst 33342 for nuclei, BODIPY FL Thapsigargin for ER, and MitoTracker Orange for mitochondria. White arrows indicate endosome-like or autophagosome-like unstained structures. (a) Low magnification view. (b) High magnification view. Original scanning images were acquired with 1.0 μm intervals, and those 23 images were stacked to produce the final image of (b) and (d). (c) Hoechst 33342 staining. A part of the image shown in (a) was enlarged and its contrast was enhanced. Right and left images are identical except that nuclei with possible circular arrangements are highlighted in white. (d) 3D image of an epithelial tissue.

Mentions: We examined triple staining patterns of the larval wing imaginal disks (n = 4; n designates the number of individuals observed hereafter) (Fig 2) and the pupal wing tissues (n = 4) (Fig 3) using Hoechst 33342 for nuclei, BODIPY FL thapsigargin for ER, and MitoTracker Orange for mitochondria. Although staining patterns were somewhat uneven in the tissue, all three dyes stained the tissues well.


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

Ohno Y, Otaki JM - PLoS ONE (2015)

Triple staining of a pupal wing tissue.The tissue was stained with Hoechst 33342 for nuclei, BODIPY FL Thapsigargin for ER, and MitoTracker Orange for mitochondria. White arrows indicate endosome-like or autophagosome-like unstained structures. (a) Low magnification view. (b) High magnification view. Original scanning images were acquired with 1.0 μm intervals, and those 23 images were stacked to produce the final image of (b) and (d). (c) Hoechst 33342 staining. A part of the image shown in (a) was enlarged and its contrast was enhanced. Right and left images are identical except that nuclei with possible circular arrangements are highlighted in white. (d) 3D image of an epithelial tissue.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128332.g003: Triple staining of a pupal wing tissue.The tissue was stained with Hoechst 33342 for nuclei, BODIPY FL Thapsigargin for ER, and MitoTracker Orange for mitochondria. White arrows indicate endosome-like or autophagosome-like unstained structures. (a) Low magnification view. (b) High magnification view. Original scanning images were acquired with 1.0 μm intervals, and those 23 images were stacked to produce the final image of (b) and (d). (c) Hoechst 33342 staining. A part of the image shown in (a) was enlarged and its contrast was enhanced. Right and left images are identical except that nuclei with possible circular arrangements are highlighted in white. (d) 3D image of an epithelial tissue.
Mentions: We examined triple staining patterns of the larval wing imaginal disks (n = 4; n designates the number of individuals observed hereafter) (Fig 2) and the pupal wing tissues (n = 4) (Fig 3) using Hoechst 33342 for nuclei, BODIPY FL thapsigargin for ER, and MitoTracker Orange for mitochondria. Although staining patterns were somewhat uneven in the tissue, all three dyes stained the tissues well.

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.