Figure 4.
Knock-down of RPTPs by in ovo RNAi in spinal cord affects the growth of a dorsal nerve in the limb. Spinal cords were electroporated with dsRNA targeting ChAT (n = 16), Axonin-1 (Ax-1; n = 16), PTP-σ (n = 30), PTP-δ (n = 27), or PTPRO (n = 37) and allowed to develop for 2 d before fixation and fluorescent staining with anti-neurofilament. Control embryos (n = 25) were only windowed, and PBS embryos (n = 19) were electroporated with PBS. A, Representative photomicrographs showing dorsal views of whole-mount embryos treated with PBS (left 2 panels) or PTPRO dsRNA (right 2 panels). The left (treated) dorsal nerve was visually compared with the right (untreated) dorsal nerve (arrowheads). Embryos were categorized as unaffected (u) or affected (a); affected embryos had much smaller or missing nerves (top), or showed major changes in fasciculation and branching (bottom). Scale bar, 0.5 mm. B, Quantification of dorsal nerve effect. The percentage of affected embryos is shown for each treatment group. Eighty to 100% of control embryos [control (Con), PBS, ChAT, Ax-1] fall into the unaffected category. However, the majority of embryos treated with dsRNA targeting the RPTPs were affected. A two-sided Fisher's exact test was used to compare treatment groups with ChAT. *Significantly different, p < 0.01. ***Significantly different, p < 0.001. The most severely affected phenotypes (missing or greatly truncated nerves) were never seen with untouched, PBS, ChAT, or axonin-1 embryos; percentages of severely affected embryos were as follows: PTP-σ, 23%; PTP-δ, 26%; PTPRO, 49% (data not shown).