Interaction among cpn1, VEGF/Notch, and BMP signals. In the trunk region, the expression level of cpn1 was decreased in SU5416- (B,F) and DAPT (C,H)-treated embryos. In the tail region, the ectopic expression of cpn1 was observed in SU5416- (B,G) or DAPT-treated (C,I) embryos compared with that in DMSO-control embryos (A,D,E). (D–I) Transverse sections of the embryo trunk and tail region from A, B, and C. (J–R) Using DM and DMH1 to specify inactive BMP signals. In the trunk region, the expression level of cpn1 remained unchanged after DM (K,O) or DMH1 (L,R) treatment compared with that in DMSO control (J,M). In the tail region, the expression of cpn1 exhibited no differences in either DM (K,P) or DMH1 (L,R) treatment compared with that in DMSO controls (J,N). (M–R) Transverse sections of the embryo trunk and tail regions from J-L. Values on the top right indicate the number of embryos exhibiting phenotype per total number of embryos analyzed from 2 indepenedent experiments. Scale bars in A-C, J-L are 200 µm. (S) Relative expression of Notch downstream target gridlock and BMP regulated targets id1, eve1, gata2 and msx1b. The expression of id1 is downregulated while the BMP signal is blocked by DM treatment compared to DMSO as a positive control. Data are represented as means ± S.D. ***Refers to p < 0.001, **refers to p < 0.01 and *refers to p < 0.05 by an unpaired Student’s t-test. (T) Western blot analysis from two independent experiments showed reduced phosphorylation of Smad1/5/8 and ERK1/2 in cpn1 morphants compared to control, and β-actin serves as a loading control.
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