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First published online 17 November 2004
doi: 10.1242/dev.01534

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In vivo evidence for short- and long-range cell communication in cranial neural crest cells

Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA

View larger version (125K): [in a new window]   Fig. 1. Typical confocal sections through a chick embryo show the dense streams of cranial neural crest cells and various features of cell shapes and extensions within the migratory streams. (A) This typical embryo was co-injected with Gap43-tagged EGFP (green) and MRFP (red) to label the cell plasma membranes and nuclei, respectively, and re-incubated for 15 hours. Neural crest cells are seen along the migratory routes in dense streams emanating from r4 and r6, and individual cells leaving from r7. Many of the cells have nearly reached the destination branchial arches (ba2-ba4). The individual boxes highlight regions that are magnified in the remainder of the figure. (B) Cells within the r4 stream are relatively densely packed. Some of the individual Gap43-labeled cells reveal filopodial extensions, which overlap with neighboring MRFP-labeled cells. The Gap43-labeled cells also show the difference in potential extent to which an individual cell has contacts with other neural crest cells in the stream. (C) Interestingly, some neural crest cells from r5 travel laterally to the edge of r5 and then move in the anteroposterior direction toward a neighboring migratory stream along the edge of the otic vesicle. (D) Neural crest cells that have nearly reached the branchial arch destinations are very closely packed within the migratory stream. The protruding extensions of the cells appear to stretch out from the cell body such that an individual cell may contact a cell that is not within its local neighborhood. (E) A closer look at individual cells within the stream reveals that an individual cell may extend many thin filopodial protrusions. Scale bars: 50 µm in D; 10 µm in E. ov, otic vesicle.

View larger version (66K): [in a new window]   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.

View larger version (37K): [in a new window]   Fig. 3. Neural crest cells display a wide variety of cell shape and number and length of filopodial and lamellipodial extensions. Static confocal images of typical neural crest cells within migratory streams show different features, including numerous long and short filopodia extending from all areas around the neural crest cell and shorter lamellipodial protrusions of various shapes extending from the cell body. (A) A typical hairy cell displays many long and short filopodia. The extensions course off in many different directions and cover a wide region in the environment near the cell body. The length of a filopodium may extend up to 100 µm. The embryo was injected with Gap43-EGFP, electroporated and re-incubated for 12 hours. (B) A typical bipolar cell within a neural crest cell stream (r4 stream). The cell displays a long, forward extending filopodium that intertwines around local and non-local neighboring neural crest cells. The trailing filopodium may extend as long as the forward protrusion. The embryo was co-injected with Gap43-EGFP (green) and H2B-MRFP (red) and electroporated to label the cell plasma membranes and nuclei, respectively, and re-incubated for 12 hours. (C) The neural crest cell displays an outstretched filopodium in the direction of the destination branchial arch along the flow of the migratory stream (r6 stream). The filopodium on this cell has many substructures along its length that resemble spines. These shorter protrusions are directed perpendicularly from the main filopodial branch. The embryo was injected with Gap43-EGFP, electroporated and re-incubated for 12 hours. Scale bars: 10 µm.

View larger version (29K): [in a new window]   Fig. 4. Quantitative analysis. (A) Quantitative analysis of typical neural crest migratory streams (n=8) shows that different cell shapes (bipolar versus hairy) are distributed in different positions along the stream. The graph plots the percent of hairy versus bipolar cells at the front (distal), middle and back (proximal) of typical neural crest cell streams as a percentage of the total number of cells in the stream. The majority of bipolar cells are located in the middle of the migratory stream, while there are almost three times as many hairy cells versus bipolar cells at the front of the stream. At the back portion of a stream, the percentage of hairy and bipolar cells are nearly equal. (B) Further quantitative analysis of the directional distribution and length of the filopodia on hairy versus bipolar cells is shown in the second set of graphs. A sample of hairy and bipolar cells (n=16) within typical migratory streams were selected and the filopodia lengths and spatial distributions were measured. Bipolar cells have the feature of having few long filopodia that typically extend into the first two quadrants, forward (toward the branchial arch) and trailing (toward the neural tube). In comparison, hairy cells have a large number of filopodia extending from nearly all aspects of the cell's circumference, seen in thick lines in all four quadrants. The filopodia are slightly longer in the directions toward the branchial arches and the neural tube. (C) A typical migrating neural crest cell was analyzed for the dynamic cell shape changes. The quantitative analysis of the area of a trailing cell, plotted as a function of time, reveals that the area of the cell does not increase monotonically in the direction of motion, but oscillates slightly.

View larger version (86K): [in a new window]   Fig. 5. Neural crest cell-cell contact via filopodia may alter the trajectory of a trailing cell. Selected images from a typical time-lapse imaging session show neural crest cells within the r6 stream. The direction of migration is to the right. Three individual neural crest cells are highlighted (green, red and blue), with the blue cell and the green cell positioned downstream (distal) to the red cell. First, the trailing cell (red) senses the local environment with short filopodial extensions (t=0). The downstream cell ahead in the r6 stream (blue) is migrating toward the branchial arch. The red cell extends a filopodium in the direction of the blue cell (t=1 hour). The blue-colored cell moves slightly backward. Then, the filopodium from the red cell makes contact with the blue cell, and elicits a response from the blue cell in the form of a short filopodial extension in the reverse direction of travel (t=1 hour 5 minutes). Both the red and blue cells retract their filopodia (t=1 hour 30 minutes). The red cell then begins to migrate in the direction of the point of contact with the blue cell (t=3 hours) and migrates further downstream to catch up with the blue cell. The images in the left column have been embossed from the raw data in Adobe Photoshop 7.0 to bring out the edges of the cells. The images in the right-hand column are tracings over the cells from the raw data. The + sign (t=3 hours) marks the original location of the red cell before contacting and migrating toward the blue cell. Scale bar: 30 µm.

View larger version (142K): [in a new window]   Fig. 6. Neural crest cells within a stream may be connected by long, thin cellular processes. In a typical cell migration stream, many neural crest cells appear to be connected by long, thin cellular processes. (A) Two neural crest cells appear to be linked by a long, thin cellular process (arrow) that stretches between two cells and intertwines around a cell. A fragmented cellular process lies adjacent to one of the cells (arrowhead). This image represents the projection of 30 confocal slices at 1 µm intervals. (B) A 3D rendering of the confocal z-stack reveals the long, thin cellular link (arrow) and short-range contacts. The fragments of a broken cellular process are also shown (arrowhead). (C) A close-up of the 3D rendering of the z-stack and rotation reveals the actual physical connection of the cellular process between the two cells (arrow). The z-stack has been turned by approximately 30 degrees around the y-axis. The embryo was injected with Gap43-EGFP (green) and H2B-MRFP (red) into the neural tube, electroporated and re-incubated for 12 hours. Scale bar: 30 µm.

View larger version (144K): [in a new window]   Fig. 7. The neural crest cell-cell long-range contact develops as two neighboring cells move apart. Selected images taken from a typical time-lapse confocal imaging session showing the interaction between two neighboring neural crest cells that move apart and continue to maintain contact. Initially (t=0), the red cell is migrating within the stream, moving laterally from r6 (not shown, but to the left in the figure). The red cell undergoes cell division to produce a progeny (blue cell) (t=15 minutes). As the blue cell moves away (t=15 minutes), a thin cellular process is maintained (arrow). As the blue cell continues to move laterally (t=30 minutes), the length of the cellular process increases (arrow). Also, the green neighboring cell continues to migrate in the lateral direction. The contact between the red and blue cells lengthens until it breaks at an arbitrary point (t=60 minutes), leaving remnants (arrow). The red cell begins to move toward the blue cell. The blue cell continues to move in the lateral direction and is nearly out of the field of view (t=90 minutes). The green cell migrates near to the location of the previous position of the blue cell and (t=120 minutes) the red cell makes contact with the green cell. The diameter of the red cell at (t=0) is about 10 µm.

View larger version (36K): [in a new window]   Fig. 8. Schematic of the neural crest cell streams and different forms of contact between two neural crest cells, which lead to directional movement. (A) Tracking a lead cell. Two neural crest cells (1 and 2) within a migratory stream may have short filopodia around the circumference of the cell. The contact is initiated when one of the cells (1) extends a filopodium in the direction of the other cell (2) and makes contact. The filopodium from the trailing cell then tracks the position (back end) of the downstream cell as the rest of the trailing cell body follows. (B) Contact, retraction, forward movement. Two migrating neural crest cells within a stream may come into contact with each other when a filopodium of the trailing cell (1) extends and contacts a downstream cell (2). The filopodium retracts and then the trailing cell moves forward to a new position (1') near the location of the contact as the downstream cell (2) moves away. (C) Tethered contact. Two neighboring cells (1 and 2) may begin to move apart from each other. As the cells move apart, or, as shown here, one of the cells (2) moves away, the cells maintain a filopodial connection. As the length of the filopodium grows, it breaks at an arbitrary point, leaving fragments in the extracellular matrix. The trailing cell (1) may stay in its location or move in the direction of the former neighboring cell (2). (D) Our previous view of neural crest cells within migratory streams (for example, the stream forming adjacent to r4) based on DiI cell labeling revealing round neural crest cells with short filopodia. (E) Our working model of neural crest cells within migratory streams based on imaging of cells with fusion protein expressing constructs targeted to the plasma membrane and cytoskeletal elements to reveal multiple cell-cell connections with short and long filopodia.