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First published online 8 December 2004
doi: 10.1242/dev.01553

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Imaging neural crest cell dynamics during formation of dorsal root ganglia and sympathetic ganglia

¹ Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT 59717, USA
² Stowers Institute for Medical Research, Kansas City, MO 64110, USA

View larger version (49K): [in a new window]   Fig. 1. Imaging perspectives in sagittal explants. (A) Schematic showing the migratory routes of neural crest cells from the dorsal neural tube to the DRG (pink) and SG (blue). (B,C) Typical views of a sagittal explant containing GFP-labeled neural crest cells. (B) Medial view; (C) lateral view. Scale bar: 25 µm. D, dorsal; da, dorsal aorta; DRG, dorsal root ganglia; nc, notochord; nt, neural tube; SG, sympathetic ganglia; so, somite; V, ventral.

View larger version (147K): [in a new window]   Fig. 2. Aggregating neural crest cells as they form the DRG anlagen. Embryos were injected at HH stage 10 with GFP coding vector pMES and sagittal explant mounted at embryonic day 3.5. (A) Neural crest cells migrating from three axial levels through the rostral somite, with arrowhead indicating cells between two DRG anlagen. Double-ended arrow indicates dorsal (D) and ventral (V) directions. (B) High magnification of area indicated by arrowhead in (A). Cells from DRG1 and DRG2 are in contact with each other. (C-D) Cell tracking of a single neural crest cell that initially resided in one DRG and then migrated to the DRG immediately posterior. Scale bars: 20 µm in A; 40 µm in C-E.

View larger version (54K): [in a new window]   Fig. 3. Migrating neural crest cells forming chains through the rostral sclerotome, extending from the DRG to the dorsal aorta. (A) Lateral view of sagittal explant of pMES fluorescently labeled neural crest cells overlaid on brightfield image. (B) Fluorescence of labeled neural crest cells. (C-F) Single chain extending from neural tube to dorsal aorta. (C) GFP-labeled neural crest cells. (D) Embossed image of chain. (E) Fluorescence and embossed overlay. (F) Individual cells within chain colored separately to define cell boundaries. (G-J) Lateral view of sagittal explant of eGFP fluorescently labeled neural crest cells. (G) Fluorescence of single chain. (H) Embossed image of chain. (I) Individual cells colored separately and overlaid on embossed image. (J) Depth coding of entire chain. Arrow indicates dorsal (D) and ventral (V) directions. Scale bars: 50 µm in A,B; 20 µm in C-F; 20 µm in G-J. nt, neural tube; sg, sympathetic ganglia.

View larger version (57K): [in a new window]   Fig. 4. Filopodial contact is important for neural crest migration through the rostral somite. (A-D) A cell positioned midway in a chain extends filopodia into the caudal somite and touches a cell in a chain in a different axial level. The cell maintains filopodial contact with its chain neighbors and returns to its original chain. (E-H) A cell attempts to extend a filopodium into the caudal somite but all connections are lost with its neighbors in the chain. This cell roams in a disoriented, undirected manner through the rostral and caudal somite. Scale bars: 30 µm in A-D; 30 µm in E-H. cs, caudal somite; D, dorsal; DRG, dorsal root ganglia; SG, sympathetic ganglia; V, ventral.

View larger version (98K): [in a new window]   Fig. 5. Early migrating neural crest cells, but not later migrating cells, can reorient and reverse their initial direction of movement. (A-C) Single neural crest cells tracked in teal and red, were able to reverse their original direction through the rostral sclerotome and populate a different structure than that they were originally headed toward. (D) Summary of their pathways are shown using vector diagrams. (E-H) Cells tracked in yellow and pink try to reverse their direction but are forced back in their original direction. (F) Vector diagrams show seven individually tracked cells that were unable to reverse their migration direction. Scale bar: 30 µm. D, dorsal; drg, dorsal root ganglia; sg, sympathetic ganglia; V, ventral.

View larger version (105K): [in a new window]   Fig. 6. Distribution of neural crest cells as they arrive in the vicinity of the dorsal aorta in a medial explant. (A) Cells initially distributed adjacent to the dorsal aorta. Double-ended arrow indicates dorsal (D) and ventral (V) directions. Dotted circles indicate where SG anlagen will form. (B) High magnification view of cells between developing SG at a later time point in this explant. Dashed line indicates somite boundary. Scale bars: 25 µm. C, caudal somite; R, rostral somite.

View larger version (74K): [in a new window]   Fig. 7. Time-lapse analysis of SG formation in chick trunk sagittal explant imaged medially. Fluorescently labeled neural crest cells in a sagittal explant mounted at embryonic day 3.5. (A) Medial image of neural crest cells dispersed along dorsal aorta forming the SG anlagen. Unbroken lines indicate somite borders; double-ended arrow indicates dorsal (D) and ventral (V) directions. The behavior of cells in the interganglionic space (arrow) between two adjacent SG is illustrated in B-G and B'-G'. (B-G) Segregation of neural crest cells dispersed adjacent to the dorsal aorta into discrete SG anlagen. B'-G' are embossed images of B-G. Broken lines indicate somite borders. All time is in hours. Scale bar: 50 µm.

View larger version (28K): [in a new window]   Fig. 8. High-resolution analysis of single cell dynamics during SG segregation. (A) Static outline of cells as they segregate into separate SG. Red and blue outlines represent two cells imaged at high resolution situated in the interganglionic space during SG segregation. The red and blue cells separate and join two separate SG. Inset shows orientation of cells between SG1 and SG2. Green highlighted region corresponds to the area in which the green bar measurements were taken for B. (B) Quantification of the extent of cell-cell contact during the segregation process. Blue bars represent the amount of contact (length of cell membrane in µm) the blue cell has with SG#1; green bars represent the amount of contact between the red and blue cell in the interganglionic space; red bars represent the amount of contact the red cell has with SG#2.

View larger version (56K): [in a new window]   Fig. 9. Neural crest cells migrate through the sclerotome and can change axial levels once at the dorsal aorta. (A) Medial view of migrating neural crest cells shown at higher resolution in B-G. (B-C) Single cell, tracked through time, that migrates through the somite at one axial level. (D-E) This cell establishes filopodial contact with a cell in the SG anlagen immediately caudal to its axial level. (F) Cell moves caudally in the direction of the established contact. (G) This cell ends up populating the SG anlagen caudal to its original axial level of migration. Scale bar: 20 µm. D, dorsal; DRG, dorsal root ganglia; SG, sympathetic ganglia; so, somite; V, ventral