Figure 2

RNAi against par-1 in B. malayi early embryos. Stained embryos from untreated (A, C) or par-1 hsiRNA-treated (B, D) worms are shown. In embryos from untreated control worms (A, C) divisions at the 2-cell stage are asynchronous and the anterior blastomere divides first in a transverse orientation giving rise to anterior blastomere a (ABa; tapered arrowhead) and anterior blastomere b (ABb; compact arrowhead). The spindle (star-like structure) in the posterior blastomere (P1; asterisk) rotates to align along the anterior-posterior axis. In contrast, in embryos from par-1 hsiRNA-treated worms (B, D) divisions at the 2-cell stage become synchronous as evidenced by the star-like spindles, and the posterior spindle in P1 fails to rotate, remaining transverse as in the anterior blastomere. Immunostaining with α-tubulin antibody (A, B). Merge of α-tubulin (red) with actin (green) as revealed by phalloidin staining (C, D). Anterior to the left. A ~2 kb fragment of the B. malayi par-1 locus (corresponding to ~500 bp cDNA) was amplified with primers: par-1F 5'-TAATACGACTCACTATAG G GGAGAGGAATCTTGCCAACGG-3' and par-1R 5'-TAATACGACTCACTATAG G GAACTGCTTGTGCAGATGCGC-3' where the T7 promoter sequence is underlined and followed by a GG nucleotide (see Methods). An annealing temperature of 59°C was used in the PCR. The PCR fragment amplified from gDNA was transcribed and RNA diced to hsiRNA as described. Adult females were soaked for 2 days in 1 μM par-1 hsiRNA and tissues fixed on the third day. Control females were kept in the same conditions and included worms cultured without hsiRNA or with a comparable concentration of Lit28i polylinker ShortCut siRNA mix.