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. 2015 Jun 15;10(6):e0129242. doi: 10.1371/journal.pone.0129242

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© 2015 Yuri et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Fig 2 — (A) Aggregates were reconstituted from embryonic kidneys at different developmental stages, starting from E11.5 up to E15.5, and immuno-stained for Six2 (green) and DBA (red) after 7 days in culture. Aggregates from E11.5 (a,f,k) and 12.5 (b,g,l), showed similar characteristics in Six2⁺-NPC maintenance and UB branching morphogenesis. In contrast, aggregates from later developmental stages, i.e., at E13.5 (c,h,m), 14.5 (d,i,n) and 15.5 (e,j,o), showed less well maintenance of Six2⁺-NPC and failure of UB cells to reorganize into orderly branching structures. (Scale bar = 500 μm). (B) (a) A representative E12.5 aggregate after 7 days in culture was immuno-stained for NPC marker, Six2 (green), UB marker, DBA (red) and DNA (blue). Abundant Six2⁺ NPC were seen surrounding UB structures, with Six2^-/DBA^- cells in adjacent areas. (b) EdU (5-ethynyl-2’-deoxyuridine) staining of a representative E12.5 aggregate showed Six2⁺/EdU⁺ cells in MM areas that were both close to and distant from the UB structure (white arrow). In contrast, DBA⁺/EdU⁺ cells were detected mostly in UB tips surrounded by Six2⁺-NPC cells (white arrowhead). (c-e) A representative aggregate was immuno-stained for: (c) renal vesicle marker, Lef1 (green)/UB marker, DBA (red); (d) proximal tubule marker, LTL (red)/epithelial marker, pancytokeratin (blue)/NPC marker, Six2 (green); (e) podocyte marker, podocalyxin (green)/UB marker, DBA (red), and showed the development of Lef⁺-renal vesicle like structures (c), LTL⁺- (d) and podocalyxin⁺-(e) epithelial structures. (Scale bar = 200 μm)