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Fig 2

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ZDB-IMAGE-210817-11
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Figures for Bengani et al., 2021
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Fig 2 <italic toggle='yes'>TENM1</italic><sup>CRE</sup> alters enhancer function in the zebrafish brain by creating a repressive SIX3 binding site.

(A) A diagrammatic summary of the dual color fluorescence assay used in this study. The size of the human TENM1 element is provided in the left hand panel in base pairs (bp) (B) Human and mouse (TENM1CRE/Tenm1CRE) sequences are shown with the variant base marked in blue, resulting in gain of SIX3/SIX6 and HDX binding sites in TENM1CRE and Six6 and Hdx binding sites in Tenm1CRE. (C) mRNA in situ hybridization showing expression of tenm1 in midbrain, hindbrain and neural tube during embryonic development in wild-type zebrafish. (D-E) Dual color fluorescent transgenic assay in zebrafish with wild-type (Wt) and mutant TENM1CRE driving eGFP and mCherry expression respectively. Loss of enhancer activity is observed in midbrain and hindbrain with the mutant TENM1CRE allele. Further examples of embryos for different stable lines are shown in S34 Fig in S1 File. (F-E) six3 knockdown rescues the effect of the mutant variant on the activity of TENM1CRE. Control morpholino injected embryos show loss of reporter activity in midbrain and hindbrain by mutant allele, where the mutation creates a Six3 binding site (E). Knockdown of Six3 rescues the activity of mutant allele in the midbrain and hindbrain (F). MB: Midbrain; HB: Hindbrain; NT: Neural tube; hpf: Hours post fertilization.

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