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. 2018 Sep 17;8:13906. doi: 10.1038/s41598-018-32125-7

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© The Author(s) 2018

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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Cardiomyocyte-specific but not endothelial-specific inhibition of sox9b function results in hypoplastic atrioventricular cushions. Control larvae (a,c,d,d’; Tg(myl7:Gal4VP16;kdrl:GFP)) and larvae with cardiomyocyte-specific loss of sox9b function (b,d,e,e’; Tg(myl7:Gal4VP16;kdrl:GFP;UAS:dnsox9b-2A-tRFP)) were fixed at 80 hpf and processed for fluorescent immunohistochemistry using antibodies against activated leukocyte cell adhesion molecule (Alcam; purple). (a,b) Larvae were mounted ventrally in low melting point agarose and imaged at 40x magnification with a confocal microscope. (c,d) Boxed areas in a & b showing endocardial expressing Alcam, a marker of differentiated endocardial cushion cells. (a,c) AV cushions form normally in control larvae, as indicated by a coalescence of Alcam + endocardial cells at the junction between the atrium and ventricle (boxed area in a). (b,d) Larvae with in cardiomyocyte-specific inhibition of sox9b had hypoplastic cushions with Alcam + endocardial cells. White arrow indicates endocardium pushing through a hole in myocardium. Ventricle (V) and atrium (At) are abbreviated as indicated. Images are representative phenotypes, n = 5 per group. (e-f’) Control larvae (Tg(fli1a:Gal4ff;UAS:Kaede)) and larvae with endothelial-specific loss of sox9b function Tg(fli1a:Gal4ff;UAS:dnsox9b-2A-tRFP; UAS:Kaede)) were fixed at 120 hpf, processed for fluorescent immunohistochemistry using antibodies against Alcam, and scored for the presence of endothelial cushion. Endothelial cushions clearly formed in both control larvae (e) and larvae with endothelial-specific loss of sox9b function (f). Images are representative phenotypes, n = 8 per group.