Fig. 3. Barhl2 induces apoptosis in Xenopus neuroectoderm. (A) Stage 15 embryos. (a) Embryo injected with Xbarhl2AS and immunostained using anti-phospho-histone H3 antibody. (b,c) In situ hybridization using Xsox3 as a probe on embryos injected with: (b) Xbarhl2AS and treated with HU; (c) human BCL2 (500 pg). (B) TUNEL staining of stage 15 embryos representative of those injected with mouse Barhl2 (20 pg) (a), Xbarhl2 (20 pg) (b) and mouse Barhl2 (10 pg) (c). (a,b) Anterior views, dorsal upwards; (c) dorsal view, anterior upwards. (C) Number of TUNEL⁺ cells were counted on the injected side and compared to the number on the control side at stage 15. The results were assessed by Student's t-test. Results are shown as mean±s.e.m. Injection of mouse Barhl2 increased apoptosis 2.6 times (10 pg to 25 pg) (n=70, P=3.10^–8), as does Xbarhl2 (20 pg, n=20, P=9.10^–5). Injection of human BCL2 (500 pg) reduced apoptosis (n=35, P=0.002), and the effect of Barhl2 (10 pg) was reversed by co-injection of human BCL2 (500 pg) (n=40, P>0.01). Injection of mouse Barhl2FIL had no effect (n=28, P>0.01). (D) In situ hybridization using Xsox3 as a probe of stage 14 embryos injected with: (a) mouse Barhl2 (10 pg), 82%; (b) human BCL2 (500 pg) 89%; and (c) mouse Barhl2 (10 pg) and human BCL2 (500 pg), 60%.