MO-induced depletion of alk3a/b results in loss of endogenous ephb4a and disrupted circulation with no clear axial vessels. a-c. Whole mount in situ hybridization for dll4 and ephb4a in zebrafish after morpholino (MO)-mediated depletion of alk3a/b at 28hpf (a) and 36hpf (b). Graph (c) depicts observed expression of dll4 (blue) and ephb4a (green) in all embryos (at 28hpf, dll4 WT n=41 MO n=43, ephb4a WT n=32 MO=29; at 36 hpf, dll4 WT n=47 MO n=45, ephb4a WT n=46 MO=46), high levels of expression are represented by solid colour, weak levels by pattern and absent expression by solid white. Zebrafish embryos shown are representative, values on the bottom right indicate number of embryos with the displayed phenotype per total number of embryos analysed. d. Combined alk3a/alk3b knockdown resulted in the loss of a detectable axial vein at 48hpf as seen using the pan-vascular tg(kdlr:HRAS-mCherry) transgenic fish line. e. Graph depicts observed status of heartbeat and circulation loop in control (n=90) and alk3a/b MO injected (n=106) fish. Normal heartbeat is represented by solid red, no heartbeat by pattern red, normal circulation is represented by solid pink, the presence of a short loop shown by pattern and no circulation by solid white. Zebrafish embryos shown are representative, red bracket=dorsal aorta, white bracket=posterior cardinal and ventral vein, grey bracket=absent vein.
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