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Fig. 1. PTK7 protein is required for posterior neural cell fate specification. (A) Left panel: sqRT-PCR to pools of ten early-neurula (st.12) embryos injected with 15 ng Meis3-MO at the one-cell stage. HoxD1 expression inhibition is as a positive control for Meis3-MO activity. His4 controls for RNA levels. A control PCR omitting reverse transcriptase (-RT) was performed on RNA isolated from control embryos in all experiments shown. Right panel: sqRT-PCR to pools of five neurula (st.19) embryos injected with 22 ng Meis3-MO at the one-cell stage. Krox20 expression inhibition is a positive control for Meis3-MO activity. EF1α controls for RNA levels. (B) sqRT-PCR to pools of six early-neurula (st.12.5) embryos injected with 10-20 ng PTK7-MO at the one-cell stage. (C) Left panel: embryos were injected into one blastomere at either the one- or two-cell stage with PTK7-MO (12 or 6 ng, respectively). Four posterior cell-type markers were analyzed by in situ hybridization. Neurula embryos (st.17), viewed dorsally, anterior is on top. Arrows and dashed line mark the dorsal midline; two-cell-stage embryos injected on right side. Krox20 expression (a) is normal in 100% of the control embryos (n=21) and perturbed (b,c) in 78% of the PTK7-morphant embryos (n=46). HoxB3 expression (d) is normal in all of the controls (n=23) and perturbed (e,f) in 98% of the PTK7 morphants (n=46). N-tub expression is normal (g) in all controls (n=23) and perturbed (h,i) in 95% of the morphants (n=44). Snail2 expression is normal (j) in all controls (n=20) and perturbed (k,l) in 87% of the morphants (n=46). Right panel: sqRT-PCR to pools of six neurula (st.19) embryos injected at the one-cell stage with PTK7-MO (10 or 20 ng). (D) Upper panel: sqRT-PCR to pools of eight neurula (st.17) embryos injected at the one-cell stage with PTK7-MO (12 ng) and RNA encoding the human PTK7 protein (100 pg). Lower panel: sqRT-PCR to pools of seven neurula (st.16) embryos injected at the one-cell stage with PTK7-MO (10 ng) and RNA encoding the Meis3 protein (1.1 ng). |
