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Open Access Methodology article

An effective assay for high cellular resolution time-lapse imaging of sensory placode formation and morphogenesis

Celia E Shiau12*, Raman M Das1 and Kate G Storey1*

Author Affiliations

1 Neural Development Group, Division of Cell & Developmental Biology, College of Life Science, University of Dundee, Dundee DD1 5EH, Scotland, UK

2 Current Address: Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA

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BMC Neuroscience 2011, 12:37  doi:10.1186/1471-2202-12-37

Published: 9 May 2011

Additional files

Additional file 1:

Time-lapse movie of a representative cranial slice in bright-field. The image sequence is taken from the middle slice of a z-stack over the duration of the time-lapse imaging (8.4 hours). For each z-stack, a bright-field image was taken at the middle slice as control to monitor integrity of tissue in parallel with the fluorescence imaging. Images were taken at 3-minute intervals at 20×, and shown at 6.5 fps. ecto, ectoderm; mes, mesenchyme; nt, neural tube.

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Additional file 2:

Time-lapse movie of a dividing cell labeled by H2B-RFP undergoing a parallel cleavage in the basal side of the placodal ectoderm. Metaphase plate rotates prior to division (arrow). 72 minutes in real time taken at 3-minute intervals; 24 um z-stack; 20×; 6.5 fps.

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Additional file 3:

Time-lapse movie of a dividing cell labeled by H2B-RFP undergoing a perpendicular cleavage in the apical side of the placodal ectoderm. 60 minutes in real time taken at 3-minute intervals; 24 um z-stack; 20×; 6.5 fps.

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Additional file 4:

Time-lapse imaging of cell divisions (dotted lines around cells) in the trigeminal placodal ectoderm as labeled by nuclear H2B-RFP and membrane GFP. Dotted white line demarcate basal edge of ectoderm. Yellow arrow points to a placodal cell that divides prior to ingression and a different placodal cell (blue arrow) undergoes division after it is already in the mesenchyme. Video covers 336 minutes of development in real time captured at 3-minute intervals; 45 um z-stack; 20×; 7.5 fps.

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Additional file 5:

Same as Movie 4 but showing only the H2B-RFP channel for clearer visualization of only nuclei, 6.5 fps.

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Additional file 6:

Time-lapse imaging of the cranial slice in Figure 3C showing only nuclear H2B-RFP labeled ectoderm and ectoderm-derived cells to follow movements of placodal cell bodies within the ectoderm and during delamination. Two examples of placodal cells that move from within the ectoderm are each marked by a different colored dot (red and green) starting at a time point when the cell can initially be tracked. Tracking of the green cell begins after it has already begun migration. Some cells move in different directions before appearing to follow a path to the site of exit on the basal side of the epithelium (see red cell). 210 minutes in real time taken at 3-minute intervals; 45 um z-stack; 20×; 10 fps.

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Additional file 7:

Time-lapse imaging of actively ingressing cells labeled by membrane GFP and nuclear H2B-RFP from the placodal ectoderm. Movie shows highly interacting placodal cells as they stream out of a discrete exit point on the basal side of the epithelium, forming contacts among themselves and with already ingressed ganglionic placodal neurons. Imaging shows 336 minutes of development captured at 3-minute intervals; 45 um z-stack; 20×; 6.5 fps.

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Additional file 8:

Same as Movie 6 but showing only the membrane GFP channel for clearer visualization of the cell morphology.

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Additional file 9:

Time-lapse imaging of placodal neurons labeled by membrane GFP and nuclear H2B-RFP in the ganglionic anlage show intimate contacts by placodal processes. Movie shows highly dynamic interactions at sites of axon-axon contacts which eventually make a connection at time of growth cone collapse. 336 minutes in real time taken at 3-minute intervals; 45 um z-stack; 20×; 6.5 fps.

Format: AVI Size: 3.9MB Download file

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