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Fig. 2. 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
-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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