Fig. 4. Depletion of At-sog by dsRNA injection results in unseparated limb buds. (A-C) Flat preparations of DNA-stained stage 9 embryos derived from females injected with At-sog dsRNA (A,B) or gfp dsRNA (C). The limb bud defects were classified as severe (A) and mild (B) (see Materials and methods). (D,E) Comparison of At-sog expression by whole-mount in situ hybridization in stage 9 embryos derived from females injected with At-sog (D) or gfp (E) dsRNA. (F-P) Time course of phenotype expression in limb buds following dsRNA injection. Each graph refers to one female injected with At-sog (F-M) or gfp (N-P) dsRNA. Each vertical bar indicates the relative numbers of embryos exhibiting severe (red), mild (yellow), and normal (blue) phenotype in each egg sac. Unfertilized eggs or embryos exhibiting non-specific defects before germ band formation were not considered. The day when the first dsRNA injection was performed was defined as day 0. The numbers in parentheses indicate how many times dsRNA injection was performed. Asterisks indicate unhealthy egg sacs (see Materials and methods). Eggs from the egg sacs labeled D,E,Q in graphs F and N were used for the experiments shown in D,E,Q, respectively. (Q) RT-PCR comparison of the levels of At-sog, At-sim, ef1 and histone H3 transcripts in At-sog RNAi (sog Ri) and untreated (u.t.) embryos at stage 9. `+' and `-' indicate reactions with and without reverse transcriptase, respectively. Note that faint bands of At-sog and At-sim transcripts are visible in the `+' lanes of the At-sog RNAi sample. Ch, chelicerae; Pp, pedipalps; L1-L4, first to fourth walking legs; sog Ri, At-sog RNAi, gfp Ri, gfp RNAi. Scale bars: 100 µm.