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Junb knockdown in zebrafish causes failure in PL formation. (a) Expression analysis of cDNA isolated from EGFP + FAC-sorted cells from 24 hpf Tg(fli1:EGFP)y1 embryos. Junba and junbb are expressed in ECs at levels similar to the EC marker kdrl. The strong enrichment of kdrl in the GFP + and of the neuronal marker znp-1 in the GFP- fraction, respectively, proves a proper FAC-sorting. Error bars indicate Mean +/- SD (b) Wild type zebrafish embryos co-injected with 100 pg of a linearized junba:EGFP or junbb:EGFP reporter plasmid and 2 ng of the respective experimental or standard morpholino (std-MO) as indicated were assayed for reporter EGFP expression at 24 hpf. Embryos injected with either junb:EGFP-reporter construct and std-MO display strong EGFP expression, while either junba or junbb morpholino completely blocked the EGFP signal, respectively. Panel displays representative embryos from each injection group. (c) Left panel, bright field images of representative embryos at 72 hpf injected with the indicated morpholino: std-MO (4 ng, n = 104), junba-MO1 (2 ng, n = 115), junbb-MO1 (2 ng, n = 92), junba-MO1 + junbb-MO1 (2 ng + 2 ng, n = 108). Middle panel, confocal images of trunk vasculature in Tg(fli1:EGFP)y1 embryonic region marked by rectangle on the left. Right panel, enlarged view of trunk region marked by the yellow rectangle in the middle panel. DA, dorsal aorta; PCV, posterior cardinal vein; ISVs, intersegmental vessels; PL is marked with yellow arrows or if absent with asterisks. Panels are lateral view; dorsal is up, anterior to the left. Scale bars represent 500 µm (black) and 50 µm (white). (d,e) Percentage of PL defects in embryos shown in (c) at 48 and 72 hpf and for a non-overlapping second set of junb-specific translation-blocking MOs (MO-2). std-MO (n = 58), junba-MO2 + junbb-MO2 (n = 76). Sum of 16 hemisegments per embryo was categorized into groups with no, partial or complete absence of PL. ***P < 0.001, Mann-Whitney U-test. (e) Quantification of arterial and venous ISVs (aISV, vISV) given as % aISV and vISV in Tg(fli1:EGFP)y1 zebrafish embryos injected with indicated morpholinos at 72 hpf. Mean ± SD, n = 50-60 embryo/group. ***P < 0.001, Student’s t-test.
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