Primary transcript staining of rho and epsilon genes.

<p>A) Schematic diagram of chicken beta globin locus. B,C) rho (B) and epsilon (C) cytoplasmic probes stain in all blood cells at stage HH10. D,E) Intron probe against rho (D) or epsilon (E) reveals three types of blood cell staining: negative, single positive and double positive. Left: Sections of HH10 embryo; right: Sections of HH13 embryo.</p

Schematic diagram of 10 possible combinations for rho and epsilon expression and their relative percentages.

<p>rho(−/−)epsilon(−/−) is not counted. rho(+/−)epsilon(+/−) data includes both scenarios. The statistics on ++/−− and +−/−+ in the text includes data from this and additional analysis focusing only on rho(+/−)epsilon(+/−) nuclei.</p

Percentages of nuclei with −/−, +/− or +/+ staining for <i>rho</i> and <i>epsilon</i> single intron staining at HH10.

<p>Percentages of nuclei with −/−, +/− or +/+ staining for <i>rho</i> and <i>epsilon</i> single intron staining at HH10.</p

Double staining with rho (green) and epsilon (red) probes reveal significant colocalization (merge). A: ++/++; B: ++/+−; C: ++/−+; D: ++/−−.

<p>Double staining with rho (green) and epsilon (red) probes reveal significant colocalization (merge). A: ++/++; B: ++/+−; C: ++/−+; D: ++/−−.</p

Tracking of labeled cells.

<p>Labeled cells (dividing and post-division) were tracked throughout the time-lapse imaging process and embryos were processed for anti-GFP staining immediately after the last frame. Successfully tracked and matched daughter pairs are shown in this example (whole-mount, anti-GFP stained embryo). The area shown is located in the right-lateral and posterior region of an HH10 embryo. Each daughter pair is also marked with the time of observed mitosis (e.g., d161 represents the division observed at the 161th minute of filming). Three green highlighted stripes (i, ii and iii) indicate regions of sections shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001228#pone-0001228-g006" target="_blank">Fig. 6A and B</a>.</p

Most divisions have daughter cells of the same fate.

<p>A) Section view of three regions indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001228#pone-0001228-g005" target="_blank">Figure 5</a> (i, ii and iii). Three divisions (d149, d161 and d328) give rise to 2 BC pairs (d149 shown in Fig. 6Ai; d328 shown in Fig. 6Aiii) and one EC pair (d161 shown in Fig. 6Ai and 6Aii). B) Magnified view of blue highlighted region in A. Arrowheads: ECs; Arrows: BCs. C) Occasional hemangioblast-type divisions are seen, represented by d266 (with d266a becoming EC and d266b becoming BC). D) Section view of highlighted regions in C (i and ii). Black arrows: ECs; Red arrows: BCs.</p

Mitotic profile from stage HH4 to HH10 by phospho-S10-H3 staining.

<p>A) HH4; B) HH5; C) HH6. Arrows indicate mitotic cells in forming blood islands; D) HH7; E) HH8; F) HH9; G) HH10. Arrows in D–G indicate dividing blood island cells.</p

Percentage of mitotic cells in ventral mesoderm population.

<p>A) Numbers of mitotic cells scored in 19 embryos from HH4-10. For HH4-5 embryos, cells in the lateral half of the mesoderm population from the posterior half the embryo are scored. For HH6-10 embryos, mesoderm cells in blood-island forming lateral region are scored. B) Statistical representation of mitotic index in ventral mesoderm population from HH4-10.</p

Summary of mitoses observed during time-lapse imaging.

<p>A) Three examples of scored mitoses throughout 11-hour live imaging. Images were captured with the frequency of one frame per minute. Multiple dots in a given frame represent multiple mitoses. B) Division rate (as a percentage of total labeled cells) in any given 100 frames (100 minutes). C) Summary.</p

Time-lapse imaging of cell divisions in the ventral mesoderm population.

<p>A) Imaging set-up. B) Overview of a field of ventral mesoderm cells with a few dozen labeled cells. C) Two cells marked in B (red and white arrowheads) undergo division. Actual film was taken with one minute intervals.</p