cvl2 is required for ventral specification and Bmp signaling. (A) Western blot; translation of myc-tagged cvl2 mRNA is suppressed by cvl2 ATG-MO. (B) Western blots, revealing strongly reduced pSmad1/5 levels in embryos of 80% epiboly stage after injection of cvl2 MO1 (upper panel) or co-injection of cvl2 MO1+2 (lower panel). (C,D) Whole-mount immunostaining, lateral view at 80% epiboly; cvl2 MO1 leads to a reduction of ventral α-pSmad1/5 immunoreactivity. (E,F) p-Smad1/5 intensity along dorsoventral axis of cvl2 morphant (gray in F) and control sibling (black in F) at 80% epiboly stage. Signal intensities were measured from vegetal views, along a yellow line indicated in E for one of the investigated controls (n=7). (G-X) Whole-mount in situ hybridization with probes indicated in lower left corners. (G-N) Eighty percent epiboly stage. (G,H,K-N) Lateral views; (I,J) vegetal views, dorsal rightwards. Ratios of affected embryos were: eve1, 33/39; cvl2, 66/71; gata2, 31/32; otx2, 45/53. (J) Only the ventral expression of cvl2 is reduced in the morphant, while expression in the prechordal plate is normal. (O,P) Ten-somite stage, view of tailbud, anterior leftwards; expression domain of the blood precursor marker gata1 is reduced in cvl2 morphant (29/37 embryos). (Q-X) Five-somite stage, anterior leftwards; the krox20 expression domains are ventrally expanded in cvl2 morphant (R, 53/68 embryos), but rescued to near wild-type condition in embryo co-injected with cvl2 MO and Drosophila cvl2 mRNA (T, 46/47 embryos). (U-X) Low doses of cvl2 MO1 enhance the mild dorsalization of tolloid/minifin mutants (40/43 in cross of two mfn homozygotes, 22/97 in cross of two mfn heterozygotes), while having no or a much weaker effect in wild-type embryos injected in parallel (0/33). Control embryos are injected with a mismatch cvl2 morpholino. Abbreviations: Coom, Coomassie Blue staining of gel loaded with same amounts of protein; PonRed, Ponceau Red staining of blots after immunoreaction.
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