ZFIN Image: Savage et al., 2019, Fig. 3

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Figure Caption

Fig. 3

Injection of sgRNAs targeting tmem33 into stable Tg(fli1a:dCas9;cryaa:CFP)^sh512 embryos recapitulates knockdown of tmem33. a dCas9 is expressed within the developing vasculature (red arrowhead) including SeAs (black arrowheads) in ~50% of progeny from a Tg(fli1a:dCas9;cryaa:Cerulean)^sh512/ + outcross at 26 hpf. At this stage the dominant cryaa:Ceruleanmarker is not expressed. b dCas9 is not expressed within the developing vasculature in CFP^-Tg(fli1a:dCas9;cryaa:Cerulean)^sh512 transgenic embryos at 50 hpf. Probe trapping in notochord is highlighted (blue arrowhead). c dCas9 is expressed within the dorsal aorta (red arrowhead) and SeAs (black arrowheads) in CFP⁺Tg(fli1a:dCas9;cryaa:Cerulean)^sh512 transgenic embryos at 50 hpf. Probe trapping in notochord is highlighted (blue arrowhead). d–g Colocalisation of dCas9 and GFP in 72 hpf fli1a:dCas9; fli1a:EGFP embryo indicates EC restricted expression of dCas9 (white arrowheads) Scale bars 20 μm. h, i Tg(fli1a:dCas9;cryaa:Cerulean) embryos injected with sgRNA targeting dll4 phenocopies ectopic vascular looping within the DLAV previously observed in dll4 morphants and mutants at 3 dpf (white arrowheads). j Tg(fli1a:dCas9;cryaa:Cerulean)-positive embryos injected with sgRNAs targeting dll4 display increased DLAV diameter (unpaired t-test ***p = < 0.001; t = 4.203 DF = 35; 2 repeats; n = 9 embryos per group). Scale bars 50 µm. k-mTg(fli1a:dCas9;cryaa:Cerulean) embryos injected with sgRNAs targeting tmem33 display reduced SeA length (yellow arrowheads highlight normal SeAs, white arrowheads highlight delayed SeAs) at 30 hpf (unpaired t-test ***p = < 0.0001; t = 6.716 DF = 62; 3 repeats; n = 9–12 embryos per group). Scale bars 50 µm. n–p Tg(fli1a:dCas9;cryaa:Cerulean) embryos injected with sgRNAs targeting tmem33 display absent parachordal lymphangioblasts (red arrowhead) and reduced DLAV continuity (yellow arrowheads) (unpaired t-test ***p = < 0.0001; t = 6.399 DF = 56; 3 repeats; n = 9–11 embryos per group). Scale bars 50 µm. q–s Tg(fli1a:dCas9;cryaa:Cerulean) embryos injected with sgRNAs targeting tmem33 display no significant difference in glomerular area, compared with Tg(fli1a:dCas9;cryaa:Cerulean)-negative siblings embryos (unpaired t-test, ns = not significant; t = 0.4048; DF = 74; 3 repeats; n = 12–13 embryos per group). Scale bars 200 µm. Source data are provided as a Source Data file

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Acknowledgments

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