A mobile insulator system to detect and disrupt cis-regulatory landscapes in vertebrates.

In multicellular organisms, cis-regulation controls gene expression in space and time. Despite the essential implication of cis-regulation in the development and evolution of organisms and in human diseases, our knowledge about regulatory sequences largely derives from analyzing their activity individually and outside their genomic context. Indeed, the contribution of these sequences to the expression of their target genes in their genomic context is still largely unknown. Here we present a novel genetic screen designed to visualize and interrupt gene regulatory landscapes in vertebrates. In this screen, based on the random insertion of an engineered Tol2 transposon carrying a strong insulator separating two fluorescent reporter genes, we isolated hundreds of zebrafish lines containing insertions that disrupt the cis-regulation of tissue-specific expressed genes. We therefore provide a new easy-to-handle tool that will help to disrupt and chart the regulatory activity spread through the vast noncoding regions of the vertebrate genome.

Early evolution of the T-box transcription factor family.

Developmental transcription factors are key players in animal multicellularity, being members of the T-box family that are among the most important. Until recently, T-box transcription factors were thought to be exclusively present in metazoans. Here, we report the presence of T-box genes in several nonmetazoan lineages, including ichthyosporeans, filastereans, and fungi. Our data confirm that Brachyury is the most ancient member of the T-box family and establish that the T-box family diversified at the onset of Metazoa. Moreover, we demonstrate functional conservation of a homolog of Brachyury of the protist Capsaspora owczarzaki in Xenopus laevis. By comparing the molecular phenotype of C. owczarzaki Brachyury with that of homologs of early branching metazoans, we define a clear difference between unicellular holozoan and metazoan Brachyury homologs, suggesting that the specificity of Brachyury emerged at the origin of Metazoa. Experimental determination of the binding preferences of the C. owczarzaki Brachyury results in a similar motif to that of metazoan Brachyury and other T-box classes. This finding suggests that functional specificity between different T-box classes is likely achieved by interaction with alternative cofactors, as opposed to differences in binding specificity.

Differences in enhancer activity in mouse and zebrafish reporter assays are often associated with changes in gene expression.

BACKGROUND: Phenotypic evolution in animals is thought to be driven in large part by differences in gene expression patterns, which can result from sequence changes in cis-regulatory elements (cis-changes) or from changes in the expression pattern or function of transcription factors (trans-changes). While isolated examples of trans-changes have been identified, the scale of their overall contribution to regulatory and phenotypic evolution remains unclear. RESULTS: Here, we attempt to examine the prevalence of trans-effects and their potential impact on gene expression patterns in vertebrate evolution by comparing the function of identical human tissue-specific enhancer sequences in two highly divergent vertebrate model systems, mouse and zebrafish. Among 47 human conserved non-coding elements (CNEs) tested in transgenic mouse embryos and in stable zebrafish lines, at least one species-specific expression domain was observed in the majority (83%) of cases, and 36% presented dramatically different expression patterns between the two species. Although some of these discrepancies may be due to the use of different transgenesis systems in mouse and zebrafish, in some instances we found an association between differences in enhancer activity and changes in the endogenous gene expression patterns between mouse and zebrafish, suggesting a potential role for trans-changes in the evolution of gene expression. CONCLUSIONS: In total, our results: (i) serve as a cautionary tale for studies investigating the role of human enhancers in different model organisms, and (ii) suggest that changes in the trans environment may play a significant role in the evolution of gene expression in vertebrates.

sox21a directs lateral line patterning by modulating FGF signaling.

The development of organs composed by repeated functional units is, in many cases, accomplished by the transition of cells from a progenitor to a differentiation domain, triggering a reiterated developmental program. Yet, how these discrete fields are formed during development is still a largely unresolved question. The posterior lateral line (pLL), a sensory organ present in fish and amphibians, develops from a primordium that migrates along the flanks of the animal periodically depositing neuromasts, the pLL functional units. In zebrafish (Danio rerio), the developmental program of the pLL is triggered by the transit of progenitor cells from a Wnt to a Fgf signaling domain. It has been proposed that these two fields are defined by the antagonistic activity of these two signaling pathways, but how they are formed and maintained is still an open question in the development of the pLL. In this work, we show that sox21a, an HMG -box transcription factor, is expressed within the Fgf domain. We demonstrate that, while the Fgf signaling pathway do not control sox21a, knockdown of sox21a causes impairment of Fgf signaling, expansion of the Wnt signaling domain and disruption of neuromast development. These results suggest that sox21a is a key player in the pLL primordium patterning, fine-tuning the border of the Fgf and Wnt signaling domains.

Heartbeat-driven pericardiac fluid forces contribute to epicardium morphogenesis.

BACKGROUND: Hydrodynamic forces play a central role in organ morphogenesis. The role of blood flow in shaping the developing heart is well established, but the role of fluid forces generated in the pericardial cavity surrounding the heart is unknown. Mesothelial cells lining the pericardium generate the proepicardium (PE), the precursor cell population of the epicardium, the outer layer covering the myocardium, which is essential for its maturation and the formation of the heart valves and coronary vasculature. However, there is no evidence from in vivo studies showing how epicardial precursor cells reach and attach to the heart. RESULTS: Using optical tools for real-time analysis in the zebrafish, including high-speed imaging and optical tweezing, we show that the heartbeat generates pericardiac fluid advections that drive epicardium formation. These flow forces trigger PE formation and epicardial progenitor cell release and motion. The pericardial flow also influences the site of PE cell adhesion to the myocardium. We additionally identify novel mesothelial sources of epicardial precursors and show that precursor release and adhesion occur both through pericardiac fluid advections and through direct contact with the myocardium. CONCLUSIONS: Two hydrodynamic forces couple cardiac development and function: first, blood flow inside the heart, and second, the pericardial fluid advections outside the heart identified here. This pericardiac fluid flow is essential for epicardium formation and represents the first example of hydrodynamic flow forces controlling organogenesis through an action on mesothelial cells.