|
Fig. 1 VE-cadherin morpholinos downregulate VE-cadherin expression in a dosage dependent manner.
(A) Schematic representation of zebrafish VE-cadherin/cdh5 gene with exons represented as black boxes. The positions of the morpholinos employed in this study are indicated with red arrows, and the primers used for the analysis of splice variants as black arrows (VE5F, e1F2 and VE3R). (B–C) Expression of VE-cadherin was analyzed by RT-PCR using two sets of primers. (B) Upon injection of high doses (16 ng) of VE-cadherin morpholinos, aberrant splicing of VE-cadherin was observed for both MO2 and MO3 when using primer pair VE5F-VE3R. Primer pair e1F2-VE3R allowed the detection of endogenous wild type VE-cadherin transcript in controls (Ctrl) and proved the absence of this transcript in MO2 injected embryos (left panel in B). This primer pair confirmed the altered splicing for MO3 injected embryos (right panel in B). Actin was used as an internal control. (C) Injection of low doses of morpholino (0.8 ng) caused partial knockdown of VE-cadherin. These embryos expressed both wild type VE-cadherin and aberrant spliced transcripts. (D–E) Histograms show the quantification of the effect of VE-cadherin morpholinos at 54 hpf. Both MO2 (D) and MO3 (E) cause increased severity of the phenotype increasing the doses injected. With low doses (0.8 ng) most of the embryos present circulation and hemorrhages (mild phenotype). Conversely, injection of high doses of either one morpholino (16 ng) cause a highly penetrant phenotype, with complete absence of blood circulation (severe phenotype). Intermediate amounts of morpholino lead to both mild and severe phenotype in different percentages. MO2 presented higher penetrance than MO3.