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Tissue samples for biochemical analyses were snap frozen and
To determine what parts and which stages of the intermediate mesodermal (IM) endothelin-2 give rise to the UB cells, cell-lineage tracing experiments were performed with the fluorescent dye DiI. Noting that previous study [20], had shown that the IM cells adjacent to the most caudal somite at the 10-somite stage included the WD progenitors, the IM cells were labeled under various spatial and temporal conditions. When the IM cells adjacent to the caudal border of somitogenesis between the 6 and 12 somite stages (30–40 h after incubation) were labeled (Fig. 1A), the DiI-labeled cells migrated caudally and were spread through the entire part of the elongating WD at 24 h after labeling (E2.5) (Fig. 1B). The cells were specifically integrated into all WD epithelia (Fig. 1C). At 48 h (E3.5), the DiI-labeled WD expanded dorsally at its caudal terminal region that becomes a UB, indicated by expression of chicken Pax2 ( Fig. 1D–F). These results demonstrated that the UB progenitors exist in the IM cells of the neck-trunk boundary region adjacent to the caudal border of somitogenesis between the 6 and 12 somite stages and also showed that not only the WD cells but also the UB cells were exclusively derived from these IM cells. They are referred to as the chicken WD/UB progenitors.





 
 
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