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Fig. 3 – SHH, BMP4 and MRF4 are misexpressed in WSTF knockdown embryos. A: BMP4 expression in the neural plate analyzed by whole mount in situ hybridization at late neurulation (stage 17; dorsal view, anterior at top) in embryos injected with WSTF morpholino at the two-cell stage. BMP4 expression is expanded beyond its normal domain on the WSTF knockdown side (left) compared to the uninjected control side (right). B: Whole mount in situ hybridization displaying normal expression pattern of BMP4 compared to global WSTF knockdown embryos at late tailbud stage (stage 40; side view, anterior to left). One-cell stage embryos were injected with either WSTF MO (bottom) or WSTF-inverse MO (INV, top). Inverse MO globally injected embryos display normal BMP4 expression in the hindbrain, developing heart, and faintly in the otic vesicle. Global WSTF knockdowns display decreased BMP4 anterior expression as well as aberrant expression in dorsal tissues. C: Whole mount in situ hybridization displaying normal expression of pattern SHH compared to global WSTF knockdowns at late tailbud stage (stage 36–37; side view, anterior to left). One-cell stage embryos were injected with either WSTF MO (bottom) or WSTF-inverse MO (top). Globally injected inverse MO embryos display SHH expression in the head and notochord. Global WSTF knockdown embryos display decreased SHH expression throughout. D: Global WSTF knockdown embryo (right) displaying cyclopia/holoprosencephaly compared to globally injected inverse MO embryo (left) at stage 45 (dorsal views of heads, anterior at top). Arrows indicate location of eye(s). One-cell stage embryos were injected with either WSTF MO or WSTF-inverse MO. E: Real time RTPCR results for BMP4 and SHH graphed as a percentage of normal expression. BMP4 (stage 37) is significantly over-expressed and SHH expression (stage 15) is significantly reduced in WSTF knockdown embryos injected at the one-cell stage. Data are the average of 3–4 independent experiments and standard errors are shown. F: MRF4 expression in a stage 15 embryo (dorsal view, anterior at top) injected with WSTF MO at the two-cell stage. The injected side (left) displays a significant decrease in normal MRF4 expression in the developing somites. Scale bars: 0.5 mm (A and F) and 1 mm (B–D). |
