Schematic of the <i>cis</i>-regulation of <i>krox20</i> expression in r3 and r5, illustrating differences between zebrafish and mouse.

<p><i>Cis</i>-acting elements are indicated by light blue boxes along the locus, with their position with respect to the site of transcription initiation underneath. The different types of activities of the elements are represented by arrows originating from the element: enhancer activities involved in the initiation of <i>krox20</i> expression are indicated by green arrows pointing toward the promoter, enhancer activities corresponding to direct autoregulation are indicated by blue arrows pointing back to the element and the potentiator activity of element C is represented by red arrows pointing toward element A. Question marks indicate that the activity is suspected, but not confirmed.</p

DNA accessibility and candidate enhancer sequences within and around the zebrafish <i>krox20</i> locus.

<p>UCSC genome browser view of the <i>krox20</i> locus showing gene positions (purple), repetitive sequences (black) and the sequences selected for enhancer activity tests (light blue), including those that showed activity (named A to F). Below are ATAC-seq data from experiments performed at the indicated stages, either on whole embryos (95% epiboly) or dissected hindbrain or posterior regions of the embryos (5s and 15s), as shown on the schematics on the right side. The seven mostly significant peaks located in non-coding sequences are highlighted in yellow. Underneath is a Vista browser view of sequence conservation between zebrafish and mouse (black) over the region.</p

Evolution of enhancer A activity in vertebrates.

<p>The orthologues of element A from 6 vertebrate species, zebrafish (zA), koi carp (kA), spotted gar (sA), <i>Xenopus laevis</i> (xA), chicken (cA) and mouse (mA) were transferred into a GFP reporter construct and the corresponding plasmids were used to generate zebrafish transgenic lines, as indicated. <i>GFP</i> expression was analysed by in situ hybridization at 8s in embryos from each line, either in wild type (WT) or <i>krox20</i> null (<i>krox20*</i>) backgrounds, the latter being obtained by injection of Cas9 protein together with guide RNAs targeting the coding sequence of Krox20’s zinc fingers. Positions of r3 and r5 are shown. A phylogenetic tree with the indication of the node time distances from the present in millions of years (MYA) is shown underneath.</p

Collaboration in <i>cis</i> between elements A and C for the control of autoregulation.

<p>Embryos carrying combinations of homozygous deletions of elements A (∆A), C (C*), D (D*), E (E*) and of heterozygous deletions of elements A (∆A/+) or C (∆C/+) were analysed for <i>krox20</i> expression by in situ hybridization at the indicated stages. The genotype (∆A/+ +/∆C) corresponds to heterozygous deletions of A and C affecting different chromosomes. Somatic deletions are indicated by the * symbol and positions of r3 and r5 are shown. Neural crest cells migrating from r5/r6 are indicated by an arrowhead.</p

Three enhancer elements cooperate for <i>krox20</i> positive autoregulation.

<p>(A) Analysis of the dependence on Krox20 of the enhancer elements affecting late <i>krox20</i> expression. Four transgenes consisting of GFP reporter constructs, in which the indicated <i>krox20</i> enhancers were inserted, were transferred into wild type (<i>krox20</i>^<i>+/+</i>) and <i>krox20</i> null (<i>krox20</i>^{<i>fh227/fh227</i>}) backgrounds and embryos were analysed for <i>GFP</i> expression by in situ hybridization in at the 12s stage. Positions of r3, r4 and r5 are shown. (B) Embryos carrying combinations of deletions affecting both alleles of elements A, D and/or E, as indicated, were analysed for <i>krox20</i> expression by in situ hybridization at the indicated stages. Somatic deletions are indicated by the * symbol and positions of r3 and r5 are shown. Neural crest cells migrating from r5/r6 are indicated by an arrowhead.</p

<i>krox20</i> r5 expression involves cooperation between three enhancer elements.

<p>Embryos carrying combinations of deletions affecting both alleles of elements B, A and/or C, as indicated, were analysed for <i>krox20</i> expression by in situ hybridization at the indicated stages. Somatic deletions are indicated by the * symbol and positions of r3 and r5 are shown.</p