Neural crest patterning: autoregulatory and crest-specific elements co-operate for Krox20 transcriptional control

International audienceNeural crest patterning constitutes an important element in the control of the morphogenesis of craniofacial structures. Krox20, a transcription factor gene that plays a critical role in the development of the segmented hindbrain, is expressed in rhombomeres (r) 3 and 5 and in a stream of neural crest cells migrating from r5 toward the third branchial arch. We have investigated the basis of the specific neural crest expression of Krox20 and identified a cis-acting enhancer element (NCE) located 26 kb upstream of the gene that is conserved between mouse, man and chick and can recapitulate the Krox20 neural crest pattern in transgenic mice. Functional dissection of the enhancer revealed the presence of two conserved Krox20 binding sites mediating direct Krox20 autoregulation in the neural crest. In addition, the enhancer included another essential element containing conserved binding sites for high mobility group (HMG) box proteins and which responded to factors expressed throughout the neural crest. Consistent with this the NCE was strongly activated in vitro by Sox10, a crest-specific HMG box protein, in synergism with Krox20, and the inactivation of Sox10 prevented the maintenance of Krox20 expression in the migrating neural crest. These results suggest that the dependency of the enhancer on both crest- (Sox10) and r5- (Krox20) specific factors limits its activity to the r5-derived neural crest. This organisation also suggests a mechanism for the transfer and maintenance of rhombomere-specific gene expression from the hindbrain neuroepithelium to the emerging neural crest and may be of more general significance for neural crest patterning

Symmetry breaking in the embryonic skin triggers directional and sequential plumage patterning

International audienceThe development of an organism involves the formation of patterns from initially homogeneous surfaces in a reproducible manner. Simulations of various theoretical models recapitulate final states of natural patterns, yet drawing testable hypotheses from those often remains difficult. Consequently, little is known about pattern-forming events. Here, we surveyed plumage patterns and their emergence in Galliformes, ratites, passerines, and penguins, together representing the three major taxa of the avian phylogeny, and built a unified model that not only reproduces final patterns but also intrinsically generates shared and varying directionality, sequence, and duration of patterning. We used in vivo and ex vivo experiments to test its parameter-based predictions. We showed that directional and sequential pattern progression depends on a species-specific prepattern: an initial break in surface symmetry launches a travelling front of sharply defined, oriented domains with self-organising capacity. This front propagates through the timely transfer of increased cell density mediated by cell proliferation, which controls overall patterning duration. These results show that universal mechanisms combining prepatterning and self-organisation govern the timely emergence of the plumage pattern in birds

Cooperation, cis-interactions, versatility and evolutionary plasticity of multiple cis-acting elements underlie krox20 hindbrain regulation.

Cis-regulation plays an essential role in the control of gene expression, and is particularly complex and poorly understood for developmental genes, which are subject to multiple levels of modulation. In this study, we performed a global analysis of the cis-acting elements involved in the control of the zebrafish developmental gene krox20. krox20 encodes a transcription factor required for hindbrain segmentation and patterning, a morphogenetic process highly conserved during vertebrate evolution. Chromatin accessibility analysis reveals a cis-regulatory landscape that includes 6 elements participating in the control of initiation and autoregulatory aspects of krox20 hindbrain expression. Combining transgenic reporter analyses and CRISPR/Cas9-mediated mutagenesis, we assign precise functions to each of these 6 elements and provide a comprehensive view of krox20 cis-regulation. Three important features emerged. First, cooperation between multiple cis-elements plays a major role in the regulation. Cooperation can surprisingly combine synergy and redundancy, and is not restricted to transcriptional enhancer activity (for example, 4 distinct elements cooperate through different modes to maintain autoregulation). Second, several elements are unexpectedly versatile, which allows them to be involved in different aspects of control of gene expression. Third, comparative analysis of the elements and their activities in several vertebrate species reveals that this versatility is underlain by major plasticity across evolution, despite the high conservation of the gene expression pattern. These characteristics are likely to be of broad significance for developmental genes

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

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> 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

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

<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

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