Figure 3.

Novel interactive process of spatiotemporally restricted focal apoptosis is required to form the primary WD-cloaca contact before UB budding. (A) Images show time course analysis of WD-cloaca junction in control embryos using whole-mount immunofluorescence three-dimensional confocal microscopy with E-cad (red, cloaca and WD) and Pax2 (green, WD and MetM) antibodies combined with TUNEL staining (white). Cyan dashed circles represent the putative WD-cloaca fusion site; there is no apoptosis at this site in cloaca before its contact with WD (left). Note persistent focal apoptosis in cloaca at its site of contact with WD beginning at embryonic day 10.5 and until they fuse (middle and right). At embryonic day 11.5 onwards, CND begins to show further increase in apoptosis as previously reported to pave the way for later ureter insertion. Bottom image illustrates views from different angle depicting WD-cloaca contact and apoptosis. (B) The schematic illustrates the wave of interactive apoptosis between WD and cloaca during embryonic day 9.5 to 11.0 from the results shown in (A). (C) Inhibiting apoptosis prevents WD-cloacal fusion in whole urinary tract cultures. Whole-mount immunofluorescence analysis with E-cad (magenta, WD and cloacal epithelia) and Pax2 (green, WD) antibodies using three-dimensional reconstructed confocal imaging of whole urinary tract culture (starting at embryonic day 10.0) with pan-caspase inhibitor z-vad-fmk was performed. Vehicle treatment (0.5% DMSO) shows WD fusion with cloacal epithelia after 24 hours of culture (top, three-dimensional rendering and slice views, yellow arrowhead shows contact point). On the other hand, 100 μM z-vad-fmk treatment shows WD laying over cloacal epithelia but no clear fusion (bottom, yellow arrowheads point to no clear fusion between the WD tip and cloaca).