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. 2019 Feb 13;10:732. doi: 10.1038/s41467-019-08590-7

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — tmem33 knockdown inhibits angiogenesis and localises to the ER in ECs. a–d tmem33 is expressed ubiquitously during segmentation, but displays enrichment in the pronephros (black arrowheads) and somite boundaries, which is more pronounced from 24 hpf. Pronephric expression is evident in 26 hpf transverse sections (black arrows). e–g Tmem33-EGFP protein localises to the nuclear envelope (blue arrowheads) and ER (white arrowheads) within the caudal artery in fli1a:DsRedEx2 embryos (Scale bars 1 µm). h–k tmem33 morphants injected with 0.4 ng morpholinos display delayed migration of Tg(fli1a:egfp) positive SeAs, which stall at the horizontal myoseptum (j, k, white arrowheads), compared with control Tg(fli1a:egfp) positive morphants (h, i), which begin to anastomose by 30 hpf (yellow arrowheads) (scale bars 50 µm). l–o By 48 hpf, Tg(fli1a:EGFP;−0.8flt1:RFP) tmem33 morphant SeAs complete dorsal migration, but display incomplete DLAV formation (n, o, yellow arrowheads) and lack lymphatic vasculature (red arrowheads). At 48 hpf Tg(fli1a:EGFP; −0.8flt1:RFP) control morphants display secondary angiogenesis (l, m, yellow arrowheads) and parachordal lymphangioblasts (red arrowhead) (scale bars 50 µm). p tmem33 morphants injected with 0.4 ng morpholinos display reduced SeA length at 30 hpf (t-test ****p = < 0.0001; t = 4.075; DF = 24. n = 3 repeats, 10 embryos per group). q tmem33 morphants injected with 0.4 ng morpholinos display incomplete formation of DLAV (t-test ****p = < 0.0001; t = 5.618; DF = 28. n = 3 repeats, 9 or 10 embryos per group). r, s Thoracic duct formation is impaired in tmem33 morphants injected with 0.4 ng morpholinos (white asterisks), compared with control morphants (white arrowheads) (scale bars 50 µm). DA, dorsal aorta; PCV, posterior cardinal vein. Source data are provided as a Source Data file