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Fig. 4. PTK7 and LRP6 protein depletion gives identical phenotypes. (A) Embryos were injected at the one-cell stage with PTK7-MO (13 ng) and RNA encoding full-length tagged VSVG-LRP6 (1.5 ng) protein. ACs removed at the blastula stage were grown to gastrula st.10.5. Total protein was isolated from 18 ACs per group for western analysis. The VSVG antibody recognizes ectopically expressed LRP6 protein. (B) Embryos were injected at the one-cell stage with either the PTK7-MO (25 ng) or the LRP6-MO (25 ng). Embryos were grown to gastrula st.10.5. Total protein was isolated from ten embryos per group for western analysis. The Sp1490 antibody recognizes endogenous LRP6 protein. Whereas LRP6 protein levels were reduced, the PTK7-MO did not reduce lrp6 mRNA levels (not shown). (C) Embryos were injected at the one-cell stage with the PTK7-MO (lanes 4,8-10) and RNA encoding the Wnt3a (5 pg, lanes 5,7-10) and full-length VSVG-LRP6 (0.5 ng, lanes 6-10) proteins. These embryos were than co-injected with increasing doses of PTK7-encoding RNA (0.2 ng, 1 ng; lanes 9,10). ACs removed at the blastula stage were grown to gastrula st.10.5, when total RNA was isolated from six control (lane 2) and 18 ACs from each group (lanes 3-10). HoxD1 gene expression was measured by sqRT-PCR. His4 and PTK7 gene expression are controls for loading and injection, respectively. (D) Left panel: sqRT-PCR to pools of six gastrula (st.10.5) embryos (lane 2) injected at the one-cell stage with PTK7-MO (10 ng, lane 3) or LRP6-MO (20 ng, lane 4). Siamois, Xnr3 and HoxD1 expression was compared in the morphants. PTK7 mRNA levels are unaltered by protein knockdown. Center panel: sqRT-PCR to pools of six neurula (st.17) sibling embryos shown in the left panel. Nrp1 (pan-neural marker) and PTK7 mRNA levels are unaltered by protein knockdown. Right panel: Embryos were injected at the one-cell stage with PTK7-MO (10 ng) or LRP6-MO (20 ng). Four posterior neural markers were analyzed by in situ hybridization. Neurula-stage embryos (st.17), viewed dorsally, anterior is on top. Krox20 expression (a) is normal in 100% of the control embryos (n=10), perturbed (b) in 100% of the PTK7-morphant embryos (n=12) and (c) 100% of the LRP6 morphants (n=10). HoxB3 expression (d) is normal in all of the controls (n=10) and perturbed (e) in 100% of the PTK7 morphants (n=11) and (f) 100% of the LRP6 morphants (n=10). N-tub expression is normal (g) in 82% of the controls (n=11), and perturbed (h) in 100% of the PTK7 morphants (n=12) and (i) 100% of the LRP6 morphants (n=10). Snail2 expression is normal (j) in all controls (n=11) and perturbed (k) in 67% all of the PTK7 morphants (n=12) and (l) 100% of the LRP6 morphants (n=12). |
