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. 2019 Feb 12;10:708. doi: 10.1038/s41467-018-07953-w

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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. 3 — vars−/− larvae display severe developmental phenotype with early lethality. a Spatiotemporal expression patterns of vars by whole-mount RNA in situ hybridization at 18, 24, 36, and 72 hpf. BA Branchial Arches; H Hindbrain; HICM Hematopoietic Intermediate Cell Mass; Ints Intestine; Liv Liver; MBH Midbrain-Hindbrain boundary; M Midbrain; R Retina. Scale bars = 200 μm b RT-qPCR data demonstrating the expression of total vars in vars+/+, vars+/−, and vars−/− larvae at 3 and 5 dpf. Values are mean of three separate experiments performed in triplicate. c Kaplan–Meier survival curve of vars+/+ (n = 37), vars+/− (n = 82), and vars−/− (n = 34) larvae. d Graph illustrating changes in loss-of-posture and touch response of vars−/− larvae throughout the life span. Only the surviving larvae were included. e Representative lateral and dorsal bright-field images of 3 and 5 dpf vars+/+, vars+/−, and vars−/− larvae (scale bar, 500 μm). Pericardial edema, small eye, and periocular swelling around the eye were marked with arrows. f H&E histological staining of paraffin-embedded coronary sections from the forebrain of 1–5 dpf vars+/+, vars+/−, and vars−/− larvae (scale bar, 100 μm). Magnification of the disruption in the organization of the brain and the eye for 5 dpf vars+/+, vars+/−, and vars−/− was marked with black and green stripped line, respectively. Red arrows point out some structural abnormalities. D diencephalon; IPL inner plexiform layer; L lens; MC mandibular cartilage; ON optic nerve; OPL outer plexiform layer; R&C rods and cones; RGC retinal ganglion cell; T trabecula. g–i Comparison of the individual measurements for head size g, brain size h, and eye size i for vars+/+, vars+/−, and vars−/− at 3 dpf (n = 9, n = 20 and n = 11, respectively) and 5 dpf (n = 9, n = 14 and n = 24, respectively). In b and g–i one-way ANOVA with Tukey’s multiple comparisons test was used. Values are mean of three separate experiments. In c log-rank (Mantel-Cox) test was used. Error bars represent SD. Significant values are noted as ***p < 0.001 and ****p < 0.0001