Fig. 3. Cell-mixing experiment. (A-C) After multiple intraperitoneal BrdU injections, almost all cells derived from En double mutants were positive for BrdU (B) when attached to a coverslip after 1 hour. (C) Example of double-labelled TH-positive neurons (arrow) (TH, green; BrdU, red). (D-F) In the mixture of En double mutant cells and control, no TH-positive, BrdU-labelled cells were present after 3 days. (G) Number of TH-positive cells after 24 hours and 72 hours in vitro: control (wild type), mixture of heterozygote (En1^+/–;En2^–/–) and homozygote (En2^–/–). In controls, the number of mDA neurons decreased only slightly between 24 hours and 72 hours. Regardless of culture conditions, the numbers of TH-positive cells derived from engrailed double mutants decreased almost to zero after 72 hours from a baseline of an average of 45 TH-positive mutant cells at 24 hours. n=12 for each bar (Student’s t-test, P<0.001). Error bars indicate s.d. Scale bars: 20 µm.