On Hemangioblasts in Chicken

Hemangioblasts are bi-potential precursors for blood and endothelial cells (BCs and ECs). Existence of the hemangioblast in vivo by its strict definition, i.e. a clonal precursor giving rise to these two cell types after division, is still debated. Using a combination of mitotic figure analysis, cell labeling and long-term cell tracing, we show that, in chicken, cell division does not play a major role during the entire ventral mesoderm differentiation process after gastrulation. One eighth of cells do undergo at least one round of division, but mainly give rise to daughter cells contributing to the same lineage. Approximately 7% of the dividing cells that contribute to either the BC or EC lineage meet the criteria of true hemangioblasts, with one daughter cell becoming a BC and the other an EC. Our data suggest that hemangioblast-type generation of BC/EC occurs, but is not used as a major mechanism during early chicken development. It remains unclear, however, whether hemangioblast-like progenitor cells play a more prominent role in later development

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

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

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

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

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

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