Figure 4

Early dynamics of NB loss on Smed-H2B(RNAi) animals. (a-t) WMISH of Smedwi1 (a,e,i,m,q), Smed-pcna (b,f,j,n,r), Smedtud-1 (c,g,k,o,s) and Smedwi-2 (d,h,l,p,t) in control(RNAi) (a-d) and Smed-H2B(RNAi) animals 1 (e-h), 3 (i-l) and 5 (m-p) days after RNAi, and irradiated animals (q-t). Only one control(RNAi) time point (20 days) is shown since no differences were detected among them or non-irradiated controls. Most signals located in NBs disappeared progressively for all markers (e-p). Almost no signals were detected 5 days after RNAi for the NB-specific markers Smedwi-1 and Smed-pcna (m,n), similar to irradiated animals (q,r). The expression in the CNS of Smedtud-1 (o) and Smedwi-2 (p) was not eliminated by Smed-H2B RNAi, similar to irradiated animals (s,t). (u) Quantification of the level of expression by quantitative RT-PCR of Smedwi-1, Smed-pcna, Smedtud-1 and Smedwi-2 in Smed-H2B(RNAi) animals 1, 3 and 5 days after RNAi (left), normalized expression and relative to control(RNAi) samples and in irradiated animals (right), normalized expression and relative to non-irradiated animals. Error bars represent standard deviation. The expression level of all markers is downregulated after NB ablation by Smed-H2B RNAi or irradiation, but a considerable portion of the expression of Smedtud-1 and Smedwi-2 is still detected after NB ablation. (v) Quantification of mitosis by counting of phospho-histone-3 (H3P)-positive cells in WMIHC on control(RNAi) and Smed-H2B(RNAi) animals 1, 3 and 5 days after RNAi (N = 5 per time point). Smed-H2B(RNAi) animals have significantly reduced numbers of mitotic cells 5 days after RNAi. (w-z) WMIHC with an anti-H3P counterstained with nuclear staining (nuclei/anti-H3P) in control(RNAi) (w) and Smed-H2B(RNAi) animals 1 (x), 3 (y) and 5 (z) days after RNAi. The mitosis of Smed-H2B(RNAi) animals tended to disappear progressively (z). (a-t,w-z) Anterior is to the left. Scale bars: 500 μm.