(a) Chick retinal cells after transfection with scrambled siRNA, stained with anti-PDI antibody (red) and the nuclear marker DAPI (blue). Scale bar 10 μM. (b) Stained as in a, after PDI knockdown using FITC-siRNA (green). Scale bar 10 μM. (c, d) P-half-sclerotome cells showing PDI expression (red) after transfection with scrambled FITC-siRNA (green) (c), and loss of PDI expression after knockdown with FITC-siRNA (green; d). Scale bar 10 μM. (e) Somite strip after injection of control scrambled siRNA, showing normal PNA staining in 5 P-half-sclerotomes along the A-P axis. P-half-sclerotome, P; Dermomyotome, DM; neural tube, NT. Scale bar 50 μM. (f, g) Somite strips stained for PNA after siRNA PDI knockdown. f, loss of PNA staining in three consecutive P-half-somites (left) compared with two normally stained P-half-somites (right); P, P-half-sclerotome; g, loss of PNA staining in two segments with residual spots of FITC-siRNA expression (arrows). Scale bars 50 μM. (h) Left, sagittal section of a stage 22 siRNA-transfected embryo, hybridized with a Tbx18 probe; regional expression in A-half-somites is unaltered. Scale bar 100 μM. Right, stage 22 PDI-siRNA transfected whole-mounted embryo, hybridized with an Uncx4.1 probe, showing diminished expression in 3 P-half-sclerotomes (arrows). Scale bar 100 μM. (i) Rescue of siRNA-induced ventral root/motor axon phenotype by co-injection in ovo of siRNA with PDI-expressing plasmid (1 μg or 2 μg as indicated). For each histogram bar, 10 consecutive somites in the injected region of each embryo were assessed (observer-blind to treatment condition) for the presence or absence of motor axons projecting abnormally into P-half-somite, using anti-TUJ1-HRP-stained whole-mounted embryos. (j) Sclerotome cells stained with HRP-labelled anti-PDI antibody in a whole-mounted embryo that received co-injected siRNA and plasmid (1 μg). Scale bar 5 μM. (k) Sclerotome cells stained with fluorescein-conjugated anti-FLAG-M1 antibody in a whole-mounted embryo that received co-injected siRNA and plasmid (1 μg). Scale bar 5 μM. l, PACMA 31 (200μM administered by direct injection to somites on one side in ovo) caused a significant increase in aberrant motor/ventral root axon sprouting compared with PACMA 56 injection. (m) PACMA 31 (200μM by direct injection in ovo) resulted in dorsal 'bridges' of sensory axons (arrows) interconnecting adjacent axon bundles, contrasting with the normal dorsal/sensory axon segmentation on the non-injected side (NT, neural tube; P, P-half-sclerotome). Scale bar 100 μM. (n) PACMA 31 (200μM by direct injection in ovo) showing the incidence of dorsal bridges of sensory axons in PACMA 31-injected embryos compared with their absence in PACMA 56-injected embryos (P56). (o) Assessment of ventral root A-P width after PACMA injection (200 μM) into somites in ovo. Sections of stage-21 TUJ1-stained embryos were blind-coded and assessed by fluorescence microscopy. Images were taken with QCapture Pro 6.0 and analyzed with ImageJ, measuring the A-P width of the ventral root at the most proximal position where constituent motor axons align in parallel (schematic inset upper right, above dashed line); n = 12 embryos (PACMA31), n = 9 embryos (PACMA56), n = 10 embryos (untreated).