<i>krox20</i> expression and enhancer dynamics.

<p>(A) Analysis of <i>krox20</i> expression by in situ hybridization at the indicated somite stages (s) in wild type (<i>krox20</i>^<i>+/+</i>) or <i>krox20</i> null (<i>krox20</i>^{<i>fh227/fh227</i>}) backgrounds. (B) Analysis of <i>GFP</i> expression by in situ hybridization at the indicated stages in 6 transgenic lines carrying GFP reporter constructs in which the different putative <i>krox20</i> enhancers have been inserted. Positions of r3, r4 and r5 are shown. Neural crest cells migrating from r5/r6 are indicated by arrowheads.</p

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

Physical interactions within the <i>Krox20</i> locus.

<p><b>(A)</b> Alignment of data in the <i>Krox20</i> and adjacent loci from Hi-C in ES cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006903#pgen.1006903.ref011" target="_blank">11</a>], 4C-seq in E9.5 whole mouse embryos, using the <i>Krox20</i> and <i>Nrbf2</i> promoters as viewpoints (this work, 2 biological replicates) and CTCF ChIP-seq in E14.5 mouse brain (ENCODE, [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006903#pgen.1006903.ref058" target="_blank">58</a>]). <b>(B)</b> Zoom in on the <i>Krox20</i> locus, showing 4C-seq data from the <i>Krox20</i> promoter, element A, element B and element C as viewpoints. CTCF ChIP-seq data in E14.5 mouse brain (ENCODE) are indicated below. Signals from simultaneously processed E9.5 whole embryo (dark blue) and E8.5 embryo head (light blue) samples are shown. On the right, normalized distributions of the 4C-seq signals in different genomic regions are indicated. TADs as defined in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006903#pgen.1006903.ref007" target="_blank">7</a>] or by our additional analysis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006903#pgen.1006903.s002" target="_blank">S2 Fig</a>) are indicated above, with dashed lines in the graphs demarcating TAD boundaries. Genes (black/red), <i>cis</i>-regulatory elements (orange) and genomic coordinates are indicated below each set of data. Arrowheads above each 4C track pinpoint viewpoints.</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

A model for <i>Krox20</i> regulation and the dual function of element C.

<p>(<b>A</b>) Schematic representation of the regulation of <i>Krox20</i> in r3. Three situations are envisaged in wild type embryos. Left: silent locus. If both element C and the new enhancer (NE) are inactive, no expression occurs. Middle: early expression phase. At this stage, elements C and NE have been bound by their respective transcription factors and have initiated the expression of <i>Krox20</i> via their classical enhancer functions. Nevertheless, element C has not yet been unlocked (decompacted) element A and/or the concentration of the KROX20 protein has not reached high enough levels to allow the establishment of a stable feedback loop with a significant probability. Right: late expression phase. Via its potentiator function, element C has unlocked element A, which can bind the KROX20 protein, which has now accumulated at a high enough concentration. Activation of enhancer A establishes the autoregulatory loop. <b>(B)</b> Three mutations that disrupt the positive feedback loop are presented at late expression phase. Left: mutation of the KROX20 protein preventing binding to element A. Middle: mutation of element A, preventing the binding of the KROX20 protein. Right: mutation of element C, preventing unlocking of element A.</p

Phenotypic traits of mutants at birth: impaired oro-buccal behavior and increased tidal volume

<p><b>Copyright information:</b></p><p>Taken from "Distinct roles of and in the development of rhythmic neural networks controlling inspiratory depth, respiratory frequency, and jaw opening"</p><p>http://www.neuraldevelopment.com/content/2/1/19</p><p>Neural Development 2007;2():19-19.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2098766.</p><p></p> Plethsymographic recordings of wild-type (top), and heterozygous (middle) and homozygous (bottom) mutant mice at P0. Inspiration is upward. Note that in mice, there is a two-fold increase in tidal volume compared with and wild-type littermates, whereas the frequency is the same (about 110 breaths/minute). Individual data relating tidal volume (V, abscissa) and number (nb) of jaw openings (ordinates) at P0.1. Each symbol corresponds to one animal. Black triangles are for mutants (b, c), open circles represent mutants (c) and open squares correspond to wild-type animals (b). Note that mutants can be separated from other genotypes at P0.1, due to their two-fold increased tidal volume and their reduced number of jaw openings. Broken lines indicate the values used to calculate penetrance of the phenotype (V, all data inferior to mean – 1 standard deviation; jaw openings, all data superior to mean + 1 standard deviation)