Fig. 5. Absence of caspase activation during ENS degeneration induced by inactivation of GFR1. (A) Timecourse analysis of ENS degeneration after administration of 4-OHT (at 0 h). The gut samples collected at different time points were doubly stained by GFP immunohistochemistry and TUNEL. Note the progressive loss of GFP-positive cells in cKO colon 24-36 hours after administration of 4-OHT. TUNEL staining was observed in the colon 24-27 hours after 4-OHT treatment. GFP staining persisted in control colon (bottom). (B) Immunostaining of GFP (green) and Phox2b (red). Nearly complete overlap between GFP and Phox2b signals indicates that GFP predominantly labels enteric neurons and glia, but not smooth muscle cells, in the E16 distal colon. (C) Histochemical visualization of activated caspase-3-(green) and TUNEL-positive (red) cells. In embryonic DRGs, activated-caspase-3-positive cells outnumbered TUNEL-positive cells, and there was a significant overlap between those two cell populations. Caspase activity was not detected in any of the TUNEL-positive cells in cKO colon at any point during the timecourse of loss of GFP⁺ cells. Scale bars: 20 µm.