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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:EGFPreporter 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. jCombining 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 + 2.0drlenhancer 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