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Fig. 2. Depth coding and 3D reconstruction of neighboring neural crest cells
reveals contacts between neighboring cells. (A-C) Depth coding (in
z-height) reconstruction of a confocal z-stack through
neighboring neural crest cells in a live fluorescently-labeled (Gap43-EGFP and
H2B MRFP) chick embryo emphasizes the filopodial connections between cells in
a migrating r6 stream near the third branchial arch (ba3). The
z-stack represents the neighboring cells shown in
Fig. 1E. (A) A migrating cell
(asterisk) has connections with two different migrating cells (arrow and
arrowhead). The depth code shows the depth to the filopodia and the different
parts of the cell, and z-height differences between each filopodium.
The connections are very different; one cell is making a double connection
with two filopodia (arrow; top left corner) while the other cell is stretching
out a thicker filopodium, which is making a side connection with a neighboring
cell (arrowhead; bottom right corner). (B) A magnified view reveals that the
contacts are in the same z-plane (green-to-green and light
blue-to-green) between the two cells. (C) A magnified view of the side
connection (arrowhead) between the two neighboring cells shows that the two
cells may not be in contact with one another, but that there may be as much as
14 µm in z-height difference. The color code ranges from 0 (blue)
to 14 µm (red). (D-F) 3D rendering of the same confocal z-stack.
(D) The main cell (asterisk) has multiple filopodial connections between two
different cells while migrating. The first connection (arrow) shows two
outstretched filopodia contacting a neighboring neural crest cell. The thick
connection (arrowhead) shows a possible connection between the neighboring
cells toward the bottom right corner. (E) A magnified view reveals the local
contacts (arrow). (F) Surprisingly, the 3D reconstruction and rotation of the
data stack shows there is a separate branch of the extending filopodium that
is definitely in contact with the neighboring cell (arrowhead). This contact
was not visible in either the flat projection or the depth coding of the
z-stack. Scale bars: 10 µm.
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