XB-IMG-176106
Xenbase Image ID: 176106
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Fig. 2. The cPTK7 622–1070 and cPTK 726–1070 proteins rescue NTDs and posterior neural cell fates in PTK7 knockdown/morphant embryos
A. Embryos were examined for morphological rescue at neurula (stage 17–18, top two rows of panels) and tailbud (stage 24–26, lower two panels). Embryos were injected with 12 ng PTK7-MO and rescued by co-injection of 100–400 pg of cPTK7 622–1070 protein encoding RNA. Four representative embryos are shown per injected group. The control embryos (CE) are the two panels of four on the left side (upper four: neurula stage and lower four: tailbud stage). The middle two panels of four are the PTK7-MO injected embryos. The two panels of four on the right are the PTK7-MO/cPTK7 622–1070 co-injected rescued embryos. At neurula stages, stronger neural folding is observed in the PTK7-MO/cPTK7 622–1070 co-injected embryos versus the PTK7-MO injected embryos (upper-right four panels, versus upper-center four panels). At tailbud stages the embryos are less squat and more elongated the in the PTK7-MO/cPTK7 622–1070 co-injected embryos versus the PTK7-MO injected embryos (lower right four panels, versus lower center four panels). Rescue experiment statistics and data bar graphs are shown in Fig. 2C.
B. Embryos were examined for morphological rescue at neurula (stage 17–18, top two rows of panels) and tailbud (stage 24–26, lower two panels). Embryos were injected with 12 ng PTK7-MO and rescued by co-injection of 100–400 pg of cPTK7 726–1070 protein encoding RNA. Four representative embryos are shown per injected group. The control embryos (CE) are the two panels of four on the left side (upper four: neurula stage and lower four: tailbud stage). The middle two panels of four are the PTK7-MO injected embryos. The two panels of four on the right are the PTK7-MO/cPTK7 726–1070 co-injected rescued embryos. At neurula stages, stronger neural folding is observed in the PTK7-MO/cPTK7 726–1070 co-injected embryos versus the PTK7-MO injected embryos (upper-right four panels, versus upper-center four panels). At tailbud stages the embryos are less squat and more elongated the in the PTK7-MO/cPTK7 726–1070 co-injected embryos versus the PTK7-MO injected embryos (lower right four panels, versus lower center four panels). Rescue experiment statistics and data bar graphs are shown in Fig. 2C.
C. Statistical analysis of embryos shown in Fig. 2A–B. Embryos were scored at tailbud stage for rescue as described in the text. Control embryos (CE) n = 434, 95% normal neural plate folding; PTK7-MO injected embryos (MO) n = 390, 6% normal neural plate folding; PTK7-MO/PTK7 full-length protein co-injected embryos (MO + PTK7) n = 418, 61% normal neural plate folding; PTK7-MO/cPTK7 622–1070 protein co-injected embryos (MO+622) n = 296, 50% normal neural plate folding; cPTK7 726–1070 protein co-injected embryos (MO+726) n = 250, 42% normal neural plate folding;
D. Control embryos (lane 2) were injected at the one-cell stage with PTK7-MO (12 ng, lane 3) or the PTK7-MO and RNA encoding cPTK7 622–1070 protein (400 pg, lane 4) Total RNA was isolated from pools of eight neurula (st.17) embryos from each group. Gene expression was analysed by Semi-quantitative (sq)RT-PCR analysis to: krox20, hoxb3 (hindbrain), n-tub (primary neuron), sox2 and ptk7 (panneural). –RT was performed (lane 1) to total RNA from the control uninjected embryos. EF1α is as positive control for RNA levels. Markers are rescued by the cPTK7 622–1070 protein (lanes 2–4)
E. Control embryos (lane 2) were injected at the one-cell stage with PTK7-MO (12 ng, lane 3) or the PTK7-MO and RNA encoding the full-length human PTK7 protein (50 pg, lane 4), which serves as a positive control for rescue (ref. 11). In parallel, the PTK7-MO was co-injected with increasing concentrations of RNA encoding the cPTK7 726–1070 protein (100 pg, 200 pg, 400 pg, lanes 5–7). Total RNA was isolated from pools of eight neurula (st.17) embryos from each injected group and sqRT-PCR was performed to analyse expression of the krox20, hoxb3 (hindbrain), n-tub (primary neuron), xanf1 (forebrain), sox2 and ptk7 (panneural) genes. –RT was performed (lane 1) to total RNA from the control uninjected embryos. ODC serves as positive control for RNA levels. Markers are rescued by either the full-length PTK7 or cPTK7 726–1070 proteins (compare lane 3–4 to 5–7).
Image published in: Lichtig H et al. (2019) Copyright © 2019. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |