A Sox10 enhancer element common to the otic placode and neural crest is activated by tissue-specific paralogs

The otic placode, a specialized region of ectoderm, gives rise to components of the inner ear and shares many characteristics with the neural crest, including expression of the key transcription factor Sox10. Here, we show that in avian embryos, a highly conserved cranial neural crest enhancer, Sox10E2, also controls the onset of Sox10 expression in the otic placode. Interestingly, we show that different combinations of paralogous transcription factors (Sox8, Pea3 and cMyb versus Sox9, Ets1 and cMyb) are required to mediate Sox10E2 activity in the ear and neural crest, respectively. Mutating their binding motifs within Sox10E2 greatly reduces enhancer activity in the ear. Moreover, simultaneous knockdown of Sox8, Pea3 and cMyb eliminates not only the enhancer-driven reporter expression, but also the onset of endogenous Sox10 expression in the ear. Rescue experiments confirm that the specific combination of Myb together with Sox8 and Pea3 is responsible for the onset of Sox10 expression in the otic placode, as opposed to Myb plus Sox9 and Ets1 for neural crest Sox10 expression. Whereas SUMOylation of Sox8 is not required for the initial onset of Sox10 expression, it is necessary for later otic vesicle formation. This new role of Sox8, Pea3 and cMyb in controlling Sox10 expression via a common otic/neural crest enhancer suggests an evolutionarily conserved function for the combination of paralogous transcription factors in these tissues of distinct embryological origin

Dissecting early regulatory relationships in the lamprey neural crest gene network

The neural crest, a multipotent embryonic cell type, originates at the border between neural and nonneural ectoderm. After neural tube closure, these cells undergo an epithelial–mesenchymal transition, migrate to precise, often distant locations, and differentiate into diverse derivatives. Analyses of expression and function of signaling and transcription factors in higher vertebrates has led to the proposal that a neural crest gene regulatory network (NC-GRN) orchestrates neural crest formation. Here, we interrogate the NC-GRN in the lamprey, taking advantage of its slow development and basal phylogenetic position to resolve early inductive events, 1 regulatory step at the time. To establish regulatory relationships at the neural plate border, we assess relative expression of 6 neural crest network genes and effects of individually perturbing each on the remaining 5. The results refine an upstream portion of the NC-GRN and reveal unexpected order and linkages therein; e.g., lamprey AP-2 appears to function early as a neural plate border rather than a neural crest specifier and in a pathway linked to MsxA but independent of ZicA. These findings provide an ancestral framework for performing comparative tests in higher vertebrates in which network linkages may be more difficult to resolve because of their rapid development

Ancient Pbx-Hox signatures define hundreds of vertebrate developmental enhancers

Background: Gene regulation through cis-regulatory elements plays a crucial role in development and disease. A major aim of the post-genomic era is to be able to read the function of cis-regulatory elements through scrutiny of their DNA sequence. Whilst comparative genomics approaches have identified thousands of putative regulatory elements, our knowledge of their mechanism of action is poor and very little progress has been made in systematically de-coding them. Results: Here, we identify ancient functional signatures within vertebrate conserved non-coding elements (CNEs) through a combination of phylogenetic footprinting and functional assay, using genomic sequence from the sea lamprey as a reference. We uncover a striking enrichment within vertebrate CNEs for conserved binding-site motifs of the Pbx-Hox hetero-dimer. We further show that these predict reporter gene expression in a segment specific manner in the hindbrain and pharyngeal arches during zebrafish development. Conclusions: These findings evoke an evolutionary scenario in which many CNEs evolved early in the vertebrate lineage to co-ordinate Hox-dependent gene-regulatory interactions that pattern the vertebrate head. In a broader context, our evolutionary analyses reveal that CNEs are composed of tightly linked transcription-factor binding-sites (TFBSs), which can be systematically identified through phylogenetic footprinting approaches. By placing a large number of ancient vertebrate CNEs into a developmental context, our findings promise to have a significant impact on efforts toward de-coding gene-regulatory elements that underlie vertebrate development, and will facilitate building general models of regulatory element evolution

Assembling Neural Crest Regulatory Circuits into a Gene Regulatory Network

The neural crest is a multipotent stem cell–like population that gives rise to a wide range of derivatives in the vertebrate embryo including elements of the craniofacial skeleton and peripheral nervous system as well as melanocytes. The neural crest forms in a series of regulatory steps that include induction and specification of the prospective neural crest territory–neural plate border, specification of bona fide neural crest progenitors, and differentiation into diverse derivatives. These individual processes during neural crest ontogeny are controlled by regulatory circuits that can be assembled into a hierarchical gene regulatory network (GRN). Here we present an overview of the GRN that orchestrates the formation of cranial neural crest cells. Formulation of this network relies on information largely inferred from gene perturbation studies performed in several vertebrate model organisms. Our representation of the cranial neural crest GRN also includes information about direct regulatory interactions obtained from the cis-regulatory analyses performed to date, which increases the resolution of the architectural circuitry within the network

Genomic code for Sox10 activation reveals a key regulatory enhancer for cranial neural crest

The neural crest is a multipotent, stem cell-like population that migrates extensively in the embryo and forms a wide array of derivatives, ranging from neurons to melanocytes and cartilage. Analyses of the gene regulatory network driving neural crest development revealed Sox10 as one of the earliest neural crest-specifying genes, cell-autonomously driving delamination and directly regulating numerous downstream effectors and differentiation gene batteries. In search of direct inputs to the neural crest specifier module, we dissected the chick Sox10 genomic region and isolated two downstream regulatory regions with distinct spatiotemporal activity. A unique element, Sox10E2 represents the earliest-acting neural crest cis-regulatory element, critical for initiating Sox10 expression in newly formed cranial, but not vagal and trunk neural crest. A second element, Sox10E1, acts in later migrating vagal and trunk crest cells. Deep characterization of Sox10E2 reveals Sox9, Ets1, and cMyb as direct inputs mediating enhancer activity. ChIP, DNA-pull down, and gel-shift assays demonstrate their direct binding to the Sox10E2 enhancer in vivo, whereas mutation of their corresponding binding sites, or inactivation of the three upstream regulators, abolishes both reporter and endogenous Sox10 expression. Using cis-regulatory analysis as a tool, our study makes critical connections within the neural crest gene regulatory network, thus being unique in establishing a direct link of upstream effectors to a key neural crest specifier

A Reporter Assay in Lamprey Embryos Reveals Both Functional Conservation and Elaboration of Vertebrate Enhancers

The sea lamprey is an important model organism for investigating the evolutionary origins of vertebrates. As more vertebrate genome sequences are obtained, evolutionary developmental biologists are becoming increasingly able to identify putative gene regulatory elements across the breadth of the vertebrate taxa. The identification of these regions makes it possible to address how changes at the genomic level have led to changes in developmental gene regulatory networks and ultimately to the evolution of morphological diversity. Comparative genomics approaches using sea lamprey have already predicted a number of such regulatory elements in the lamprey genome. Functional characterisation of these sequences and other similar elements requires efficient reporter assays in lamprey. In this report, we describe the development of a transient transgenesis method for lamprey embryos. Focusing on conserved non-coding elements (CNEs), we use this method to investigate their functional conservation across the vertebrate subphylum. We find instances of both functional conservation and lineage-specific functional evolution of CNEs across vertebrates, emphasising the utility of functionally testing homologous CNEs in their host species

Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution

Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ~500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms

DNA methyltransferase3A as a molecular switch mediating the neural tube-to-neural crest fate transition

Here, we explore whether silencing via promoter DNA methylation plays a role in neural versus neural crest cell lineage decisions. We show that DNA methyltransferase3A (DNMT3A) promotes neural crest specification by directly mediating repression of neural genes like Sox2 and Sox3. DNMT3A is expressed in the neural plate border, and its knockdown causes ectopic Sox2 and Sox3 expression at the expense of neural crest markers. In vivo chromatin immunoprecipitation of neural folds demonstrates that DNMT3A specifically associates with CpG islands in the Sox2 and Sox3 promoter regions, resulting in their repression by methylation. Thus, DNMT3A functions as a molecular switch, repressing neural to favor neural crest cell fate

Expression of Sympathetic Nervous System Genes in Lamprey Suggests Their Recruitment for Specification of a New Vertebrate Feature

The sea lamprey is a basal, jawless vertebrate that possesses many neural crest derivatives, but lacks jaws and sympathetic ganglia. This raises the possibility that the factors involved in sympathetic neuron differentiation were either a gnathostome innovation or already present in lamprey, but serving different purposes. To distinguish between these possibilities, we isolated lamprey homologues of transcription factors associated with peripheral ganglion formation and examined their deployment in lamprey embryos. We further performed DiI labeling of the neural tube combined with neuronal markers to test if neural crest-derived cells migrate to and differentiate in sites colonized by sympathetic ganglia in jawed vertebrates. Consistent with previous anatomical data in adults, our results in lamprey embryos reveal that neural crest cells fail to migrate ventrally to form sympathetic ganglia, though they do form dorsal root ganglia adjacent to the neural tube. Interestingly, however, paralogs of the battery of transcription factors that mediate sympathetic neuron differentiation (dHand, Ascl1 and Phox2b) are present in the lamprey genome and expressed in various sites in the embryo, but fail to overlap in any ganglionic structures. This raises the intriguing possibility that they may have been recruited during gnathostome evolution to a new function in a neural crest derivative

Expression and function of transcription factor cMyb during cranial neural crest development

The transcription factor cMyb has well known functions in vertebrate hematopoiesis, but little was known about its distribution or function at early developmental stages. Here, we show that cMyb transcripts are present at the neural plate during gastrulation in chick embryos. cMyb expression then resolves to the cranial neural folds and is maintained in early migrating cranial neural crest cells during and after neurulation. Morpholino-mediated knock-down of cMyb reduces expression of Pax7 and Twist at the neural plate border, as well as reducing expression of neural crest specifier genes Snail2 and Sox10 and completely eliminating expression of Ets1. On the other hand, its loss results in abnormal maintenance of Zic1, but little or no effect on other neural crest specifier genes like FoxD3 or Sox9. These results place cMyb in a critical hierarchical position within the cranial neural crest cell gene regulatory network, likely directly inhibiting Zic1 and upstream of Ets1 and some, but not all, neural crest specifier genes