Fig. 5

The impact of zTM-b knockdown in heart and blood cell formation. Wild-type and Tg ( mpx:eGFP) embryos were injected with TM-b MO with/without co-injecting plasmids encoding human TM and examined for the development of heart and blood cells.  a- d The cardiac ejection fraction and cardiac output of larvae at 3 dpf were calculated from the change in the dimension of cardiac chamber (the circled area in ( a)) in the serial video still images (lateral view with anterior to the right) as described in Materials and Methods.  e,  f Embryos at 1 dpf were subjected to WISH for the distribution of  cmlc2transcripts, a cardiac primordium marker. Embryos displaying signal with decreased intensity or spatially aberrant distribution (heart tube with mid-line positioning or right-jogging) were categorized as abnormal and quantified.  g,  h The development of embryonic right-left patterning was evaluated at 12 hpf and categorized into normal, small, absent and multiple based on the number and morphology of Kupffer’s vesicle (arrows).  i,  j Embryos at 3 dpf were stained with o’-dianisidine for hemoglobin distribution. Larvae displaying weaker, dispersed or ectopic signals in common cardinal veins (arrowheads in left panel) and/or absence of posterior blood island (arrowheads in the right panel) were categorized as abnormal and quantified.  k,  l Tg ( mpx:eGFP) embryos with/without TM-b knockdown were imaged at 2 dpf and scored from 1 to 5 points based on the intensity of green fluorescence, which represented the quantity of  mpx-positive leukocytes (mainly neutrophils). Data were collected from at least 3 independent experiments with the total embryo numbers of 11–84 for each group. WT, un-injected wild-type embryos; MO, embryos injected with TM-b MO; hTM, human thrombomodulin; CTL, Tg ( mpx:eGFP) embryos without MO injection; A, atrium; V, ventricle; EDA, end-diastolic area; ESA, end-systolic area