Neural crest cell division orientation and time and distance to first division. (A) Examples of typical dividing NC cells with an angle of division either parallel or perpendicular to the direction of migration. (B) NC cell dual color labeling (Gap43-GFP, green; H2B-mcherry, red) provided scoring of cell division orientation, with respect to the direction of migration. Graphs showing the percentage of parallel or perpendicular orientation angles in dividing lead and trailing NC cells. (C) Measurements of the time (top, n=36 divisions) and distance (bottom, n=35 divisions) to first NC cell division. Scale bars: 10 μm.

BrdU labeling reveals proliferating NC cells along the migratory pathway. (A-C) A typical transverse section through the hindbrain rhombomere 4 (r4) level of an E3.5 chick embryo during NC cell migration showing the dorsal region of the tissue section on one side of the embryo. (A,A′) BrdU-positive cells (red), (B,B′) GFP-labeled NC cells (green) and (C,C′) colocalized BrdU-positive NC cells (overlay) are shown. (A′-C′) Magnified views of a small subset of cells from A-C showing an example of a BrdU-positive NC cell (red and green signal) and BrdU-negative NC cell (green with no red signal). (C′) Line intensity profile through two neighboring cells in C over 0-21 μm (measured left to right) showing the bi-modal peaks of GFP (indicating both cells are GFP-positive NC cells) and single peak RFP (indicating only one of the cells has a red fluorescence signal and is BrdU positive). (D) The average percentage of BrdU-/GFP-positive NC cells measured along the migratory pathway (0-1000 μm) and binned into 100 μm intervals (squares; n=5 embryos, 2204 total cells). The black line represents the moving average of two neighboring measurements (interval of 400 μm) of BrdU-/GFP-positive NC cells (squares). The red line and shaded area represent the average percentage of BrdU-positive cells (±s.e.m.) in the trailing subpopulation (0-700 μm, 20.6±3%). The blue line and shaded area represent the average % BrdU-positive cells (±s.e.m.) in the lead population (700-1000 μm, 32.4±3%). Scale bars: 50 μm in A; 10 μm in A′.

Photoconversion cell labeling and tissue ablation show differences in NC cell proliferative activity depending on NC stream position, migratory phase and stream density. (A-F) Schematic and example images showing photoconversion of mKikGR-labeled NC cells within a typical cranial NC cell stream. (B,E) Small numbers of NC cells were photoconverted (dashed box) at +12 hours and +24 hours after labeling premigratory NC cells with mKikGR. (C,F) Embryos were re-incubated for an additional +12 hours and the total number of photoconverted cells and progeny were measured. (G) Graph showing the number of cells derived from photoconverted migrating NC cells and doubling times at two distinct time points and from either lead or trailing subpopulations of the NC migratory stream. DT, doubling time. (H) Photoconversion of single lead NC cells (at +12/+24 hours after mKikGR-labeling or premigratory cells) and number of cell progeny derived from single photoconverted NC cells (measured at +12 hours). (I) Schematic of tissue ablation of later emerging premigratory NC cells at the mid-hindbrain level and percentage of dividing NC cells per hour measured from in vivo confocal time-lapse imaging data (n=7 time-lapse imaging sessions). The embryos are the same in B,C and E,F. Scale bars: are 50 μm in B,C,E,F.

FACS and Ki-67 analysis reveal distinct cell cycle profiles of lead and trailing neural crest cells. (A,D) Schematic of the lead and trailing subregions of a typical cranial NC cell migratory stream selected for FACS and Ki-67 analysis. (B,C,E,F) Distribution of cells in G1 and G2/M phases of the cell cycle; cells in S phase are located between the two peaks and are represented by the hatched curve, as determined using Modfit software. (G,H) The cell cycle phase distribution of NC cells from both the lead and trailing subpopulations of a typical cranial NC cell migratory stream (n=8 at each time point). (I-K) The percentage of Ki-67 negative cells in the lead and trailing NC cell subpopulations (n=4 at 16 hours and n=3 at 32 hours), showing a significantly higher percentage of Ki67^- cells in the trailing population (P=0.041).