The loss of Fer kinase function resulted in intersegmental vessel (ISV) formation defects and loss of blood flow in zebrafish embryos. fer-MO1 injected embryos failed to properly organize the dorsal aorta (DA) and cardinal vein (CV) regions (A,B), and had fewer ISV structures form (white arrowheads), when compared to the control MO injected embryos (E,F). Additionally, at similar time points in the absence of Fer kinase, hematopoietic precursor cells formed in the region of the DA but failed to circulate (C,D) (control MO—(G,H)). Vasculature was visualized using fli1::nEGFP and hematopoietic cells were visualized using gata1::dsRed. In (C,D,G,H), punctate dots indicate stationary cells, while continuous lines indicate moving cells. Time points are at 30 somites (~28 hpf) for (A,C,E,G) and 48 hpf for (B,D,F,H). (Note: some cells in appear overexposed due to the abundance of overlapping cells in one region.) The quantitative PCR showed that the expression of hematopoietic precursor genes (scl, gata1, notch1a, notch1b, notch3, c-myb) were increased in the absence of Fer kinase activity (runx1 is decreased early but recovers by 30 somites), while genes expressed in the vascular endothelium remained generally unaffected (fli1, kdrl/flk1). The cell–cell adhesion complex component, cdh5 (VE-cadherin) expression was unaffected in the absence of Fer kinase (I). (black bars indicate Fer MO at 8 somites, gray bars indicate Fer MO at 28 somites, * indicate not done). The dotted line denotes the threshold for up-regulated expression, when normalized to the wildtype expression levels, which was set at a value of 1 on the y-axis.
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