Dataset Information

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

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 ?m. In pupal cells, many endosome-like or autophagosome-like structures were present in the cellular periphery down to approximately 10 ?m 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 ?m in depth to deeper levels. From 60 ?m to 80 ?m 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.

SUBMITTER: Ohno Y

PROVIDER: S-EPMC4481267 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Publications

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

Ohno Yoshikazu Y Otaki Joji M JM

PloS one 20150624 6

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 l ...[more]

PMID: 26107809

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Project description:Butterfly wings are covered with regularly arranged single-colored scales that are formed at the pupal stage. Understanding pupal wing development is therefore crucial to understand wing color pattern formation. Here, we successfully employed real-time in vivo imaging techniques to observe pupal hindwing development over time in the blue pansy butterfly, Junonia orithya. A transparent sheet of epithelial cells that were not yet regularly arranged was observed immediately after pupation. Bright-field imaging and autofluorescent imaging revealed free-moving hemocytes and tracheal branches of a crinoid-like structure underneath the epithelium. The wing tissue gradually became gray-white, epithelial cells were arranged regularly, and hemocytes disappeared, except in the bordering lacuna, after which scales grew. The dynamics of the epithelial cells and scale growth were also confirmed by fluorescent imaging. Fluorescent in vivo staining further revealed that these cells harbored many mitochondria at the surface of the epithelium. Organizing centers for the border symmetry system were apparent immediately after pupation, exhibiting a relatively dark optical character following treatment with fluorescent dyes, as well as in autofluorescent images. The wing tissue exhibited slow and low-frequency contraction pulses with a cycle of approximately 10 to 20 minutes, mainly occurring at 2 to 3 days postpupation. The pulses gradually became slower and weaker and eventually stopped. The wing tissue area became larger after contraction, which also coincided with an increase in the autofluorescence intensity that might have been caused by scale growth. Examination of the pattern of color development revealed that the black pigment was first deposited in patches in the central areas of an eyespot black ring and a parafocal element. These results of live in vivo imaging that covered wide wing area for a long time can serve as a foundation for studying the cellular dynamics of living wing tissues in butterflies.

Project description:Hox genes regionalize the animal body axis by modifying complex morphogenetic and differentiation processes during development. The transformation of wings into halteres by the Hox gene Ultrabithorax (Ubx) in Drosophila melanogaster presents an excellent model system to study the transcriptional networks that control such complex developmental programmes. We have employed an inducible misexpression system to switch on Ubx in the wing epithelium at successive larval, prepupal and pupal stages, and have used microarray expression profiling to identify the primary transcriptional responses to Ubx. We find that Ubx regulates hundreds of downstream genes, mostly in a subtle manner. These targets are largely distinct at the different stages of appendage development and diversification. We have generated an experimental fly line combining the nabGal4NP3537-driver, a tub-GAL80ts transgene, and a UAS-UbxIa transgene (the control line was carrying a UAS-eGFP transgene instead). Our core microarray analysis has involved comparison of the transcriptional profile of experimental wings carrying the UAS-UbxIa transgene with that of control wings carrying the UAS-eGFP transgene. Pairwise comparisons have been carried out at three successive developmental stages, in particular at (i) the third instar larval wandering stage, about 4hrs before puparium formation at 29˚C, (ii) the prepupal stage, 6hrs after puparium formation (APF) at 29˚C, and (iii) the early pupal stage, 16hrs APF at 29˚C. Moreover, pairwise comparisons have been carried out with samples developed exclusively at 19˚C (UbxIa or eGFP expression OFF), as well as with samples collected at 16hrs after the temperature shift from 19 to 29˚C (UbxIa or eGFP expression ON). This has allowed us to distinguish the Ubx-dependent effects from the intrinsic expression differences between the fly lines used, and from the temperature-induced responses. We have carried out 4 biological replicates for each condition making a total of 48 hybridizations to Affymetrix Drosophila Genome 2.0 arrays.

Dataset Information

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

Publications

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

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