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Fig. 6. Biochemical characterization of Cvl2 proteins. (A) SDS-PAGE
gel showing purified Cvl2-N (lanes 2, 5), Cvl2-CM (lanes 3, 6) and Cvl2-WT
(lanes 4, 7) proteins expressed in SF9 cells under reducing (lanes 2-4) and
non-reducing (lanes 5-7) conditions; bands in lane 4 correspond to the
uncleaved protein (NC), and the N- and C-terminal cleavage products.
(B) Biacore sensograms showing the binding of 50 (a), 100 (b), 300 (c)
nM Cvl2-N to immobilized Bmp2. (C) Sensograms showing binding of 8 (a),
25 (b) and 75 (c) nM Chordin to immobilized Bmp2. (D) Binding of
Chordin to Cvl2-N saturated immobilized Bmp2. At time zero, perfusion with 500
nM Cvl2-N was initiated. The saturation binding of Cvl2-N was set as zero.
After 120 seconds, perfusion was continued with 500 nM Cvl2-N plus Chordin. In
different cycles, 25 (b) and 75 (d) nM Chordin were applied. In between,
sensograms were recorded in the absence of Chordin (a,c). Perfusion with
buffer started at 240 seconds. The subtle shift upon perfusion with highly
concentrated Chordin (b,d) might result from a slow, equilibrium-driven
replacement of Cvl2-N by Chordin. (E-H) Western blots with anti-Myc
antibody. (E) Differential distribution of Cvl2 proteins in HEK 293 cell
cultures. Cells were transfected with empty vector (lanes 1,2), or plasmids
encoding Cvl2-WT (lanes 3-5) or Cvl2-N (lanes 6,7). Cleaved/associated Cvl2-WT
is characterized by a high molecular weight band in the absence of ME (lane
3), and a low molecular weight band in the presence of ME (lane 4). It is
found only in the supernatant, whereas uncleaved Cvl2-wt (high molecular
weight band in presence of ME) is found only in the ECM fraction (lanes 4,5).
Cvl2-N is present only in the supernatant (lanes 6,7). (F) Elution profiles of
Cvl2 proteins from heparin-coated sepharose beads. Cvl2-CM,
Cvl2-CM 393-396 and Cvl2-N display decreasing affinity to heparin. (G)
Removal of a putative heparin binding site (amino acids 393-396) from Cvl2-CM
leads to accumulation of the protein in the supernatant of HEK 293 cell
cultures. (H) In injected zebrafish embryos, Cvl2-CM and Cvl2-CM393-396
are present in similar amounts, although the dorsalizing effect of
Cvl2-CM393-396 is much weaker (compare with
Table 1).
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-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.
