Crlr acts upstream of vegf in modulating arterial differentiation in zebrafish. Std-MO–injected embryos (A,C,E) and crlr-MO1–injected embryos (B,D,F) were analyzed at 15-ss for shh, vegf, and kdra expression by ISH (panels A-F; embryos are anterior to the left and lateral to the top). No changes in shh notochord expression (panel B; n = 15) and in endothelial progenitor kdra expression (panel F; n = 13) were observed in crlr morphants when compared with controls (A,E). Note the proper expression and localization of the kdra staining in crlr morphant embryos (black arrowhead in panel F) when compared with control embryos (black arrowhead in panel E). In contrast, somitic vegf expression was reduced in 20 of 32 morphants examined (panel D; black arrowhead). No changes in gene expression were instead observed in control std-MO–injected embryos when compared with uninjected embryos (n = 25, data not shown). (G) Total RNA was extracted at 15-ss from 40 embryos per group, and shh, vegf, and kdra expression were evaluated by real-time RT-PCR. Data in triplicate were normalized for zebrafish β-actin expression and represent the percentage change in morphant embryos relative to controls. Similar results were obtained in 4 independent experiments (*P < .05 vs controls; Student t test). (H) ephrin-B2a expression was assessed at 26 hpf in the DA of the trunk of GFP mRNA–injected control embryos (n = 30), crlr morphants (n = 25), vegf₁₂₁ mRNA–injected crlr morphants (n = 25), and GS402-treated crlr morphants (n = 25) and data were expressed as percentage of positive embryos. The results are the mean plus or minus SD of 3 independent experiments (*P < .05 vs crlr morphants, Student t test). (I,J) representative images of the trunk of crlr morphant (I) and vegf₁₂₁ mRNA-injected crlr morphant (J) embryos showing the ability of vegf overexpression to rescue ephrin-B2a expression in the DA of crlr morphants (black arrowhead).