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. 2019 Aug 26;10:3857. doi: 10.1038/s41467-019-11561-7

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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. 5 — EomesA, FoxH1, and MixL1 together can induce the +2.0drl enhancer. a ChIP-seq tracks for EomesA. FoxH1, MixL1, and Smad2 in the drl locus. See text for details. Bottom depicts the +2.0drl intronic enhancer and the smaller, minimally specific region +2.4drl after removal of repeat sequences. b–e Constitutively-active VP16-EomesA boosts +2.0drl:EGFP reporter expression in its native territory. Compared to injection controls (b, c), microinjection of VP16-eomesA mRNA in +2.0drl:EGFP reporter transgenics enhances and prolongs EGFP expression in the native reporter expression domain (d, e). f–k Gastrulation-stage (shield to 75% epiboly) zebrafish embryos, lateral view left and animal view right, probed with EGFP ISH to detect expression of +2.0drl:EGFP. Compared to controls (n = 18/18) (f), embryos injected with mRNA encoding full-length eomesA (n = 16/26), foxh1 (n = 18/31), or mixl1 (n = 13/27) show enhanced +2.0drl:EGFP reporter activity (g–i), as indicated by arrowheads. The asterisk in g points out ectopic expression in the enveloping layer cells. j Combining mRNAs encoding full-length eomesA (e), foxh1 (f), and mixl1 (m) (e/f/m) triggers ectopic reporter expression also in dorsal blastomeres (n = 13/15, compared to native reporter expression pattern in f), an activity that also remained in embryos devoid of endoderm after sox32 morpholino injection (n = 7/17) (k). l–p Mixl1 acts on the +2.0drl enhancer as analyzed in Cas9 RNP-mediated crispants. l Schematic representation of the mixl1 and mezzo loci, with the individual sgRNAs for mutagenesis annotated (cc for CRISPR cutting, followed by sgRNA index). m–p mRNA in situ hybridization of EGFP expression in +2.0drl:EGFP embryos as crispant control (n = 30/30) (m), injected with Cas9 RNPs of (n) mixl1 ccB (n = 6/6), (o) mezzo ccA (n = 11/11), and (p) mixl1 ccB together with mezzo ccA sgRNA (n = 21/27); the resulting mosaic mixl1 ccB crispants show diminished +2.0drl reporter expression at late gastrulation, as pointed out by arrowheads. Lateral and animal views as indicated. Scale bar in (b) 500 μm, (c) 250 μm