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. 2017 Aug 7;7:7442. doi: 10.1038/s41598-017-06637-7

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© The Author(s) 2017

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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Knockdown of CD146 disrupts lymphangiogenesis in zebrafish. (a,b) The effects of cd146 or vegfc knockdown on PL formation. Tg (fli1a: EGFP) embryos, injected with control, cd146 or vegfc specific MO, were harvested at 60 hpf. Confocal images showed normal PL (red arrows) and impaired PL (red stars) in (a). Scale bars: 50 μm. The percentages of embryos with normal PL cells (normal) and impaired PL string formation (impaired PLs) were shown in (b). (c,d) The effects of cd146 or vegfc knockdown on TD formation. Tg (fli1a: EGFP) embryos, injected with control, cd146 or vegfc specific MO, were harvested at 5 dpf. Confocal images showed normal TD (white arrows) and abnormal TD (white stars) in (c). Scale bars: 30 μm. The percentages of embryos with different phenotypes of TD formation were shown in (d). Relative to the normal embryos, the TD length decreased to 30–90% were classified as weak, and those decreased to 10–30% were classified as severe. (e,f) The effects of cd146 knockdown on TD function. Tg (flk1: mcherry) zebrafish embryos injected with control or cd146 specific MO and FITC-dextran was injected subcutaneously in the tail of those fishes. The embryos were harvested at 5 dpf. Confocal images showed functional TD (yellow arrows) and dysfunctional TD (yellow stars) in (e). Scale bars: 50 μm. The percentages of embryos with different phenotypes of TD formation were shown in (f). The criterion used to classify the TD formation was the same as that used in (d). In all panels, the head of the embryo faces left and dorsal is up.