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Supplemental Figure 4. D. rerio and X. laevis clp24 sequences and blood vascular effects in clp24 morpholino treated X. laevis embryos. A) D. rerio clp24 mRNA sequence. Black, bold text indicates a morpholino (MO) targeted against the 5untranslated region (clp24-UTR), blue underlined text indicates a MO (clp24-ATG)targeted against the translational start site (bold underlined) of clp24 mRNA. X. laevis partial mRNA sequence. Underlined is a MO targeted against the 5untranslated region, and in bold a MO targeted against the translational start site (bold underlined). B) Results from the experiments using the D. rerio clp24-ATG MO confirm the results obtained using the clp24-UTR MO, presented in Fig. 2. Toxic off-target effects of the second Clp24 MO assumably related to induction of a p53-dependent cell death pathway were attenuated by co-injection with a p53 morpholino as described (Robu et al. 2007). C-F) Control (C, D) and clp24 morpholino oligonucleotides (40 ng) (E, F) were injected into X. laevis embryos and the embryos were analyzed at st 33/34 by in situ hybridization with a msr probe staining the vasculature. At stage 32 msr was expressed in the heart and developing primary blood vessels. The clp24 morphants show less staining in the dorsal aorta (DA) and in the posterior cardinal vein (PCV), and have less intersomitic vessel (ISV) sprouts as compared to the control (3.67 0.39 (n=21) vs 4.1 0.33 (n=20), respectively), although this decrease did not reach statistical significance. |