What is bad in cancer is good in the embryo: importance of EMT in neural crest development.

Although the epithelial to mesenchymal transition (EMT) is famous for its role in cancer metastasis, it also is a normal developmental event in which epithelial cells are converted into migratory mesenchymal cells. A prime example of EMT during development occurs when neural crest (NC) cells emigrate from the neural tube thus providing an excellent model to study the principles of EMT in a nonmalignant environment. NC cells start life as neuroepithelial cells intermixed with precursors of the central nervous system. After EMT, they delaminate and begin migrating, often to distant sites in the embryo. While proliferating and maintaining multipotency and cell survival the transitioning neural crest cells lose apicobasal polarity and the basement membrane is broken down. This review discusses how these events are coordinated and regulated, by series of events involving signaling factors, gene regulatory interactions, as well as epigenetic and post-transcriptional modifications. Even though the series of events involved in NC EMT are well known, the sequence in which these steps take place remains a subject of debate, raising the intriguing possibility that, rather than being a single event, neural crest EMT may involve multiple parallel mechanisms.

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.

A versatile gene trap to visualize and interrogate the function of the vertebrate proteome.

We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins generated by the integration of an internal citrine exon. Cre-mediated conditional mutagenesis is enabled by heterotypic lox sites that delete Citrine and "flip" in its place mCherry with a polyadenylation signal, resulting in a truncated fusion protein. Inducing recombination with Cerulean-Cre results in fusion proteins that often mislocalize, exhibit mutant phenotypes, and dramatically knock down wild-type transcript levels. FRT sites in the vector enable targeted genetic manipulation of the trapped loci in the presence of Flp recombinase. Thus, the FlipTrap captures the functional proteome, enabling the visualization of full-length fluorescent fusion proteins and interrogation of function by conditional mutagenesis and targeted genetic manipulation.

Caldesmon regulates actin dynamics to influence cranial neural crest migration in Xenopus.

Caldesmon (CaD) is an important actin modulator that associates with actin filaments to regulate cell morphology and motility. Although extensively studied in cultured cells, there is little functional information regarding the role of CaD in migrating cells in vivo. Here we show that nonmuscle CaD is highly expressed in both premigratory and migrating cranial neural crest cells of Xenopus embryos. Depletion of CaD with antisense morpholino oligonucleotides causes cranial neural crest cells to migrate a significantly shorter distance, prevents their segregation into distinct migratory streams, and later results in severe defects in cartilage formation. Demonstrating specificity, these effects are rescued by adding back exogenous CaD. Interestingly, CaD proteins with mutations in the Ca(2+)-calmodulin-binding sites or ErK/Cdk1 phosphorylation sites fail to rescue the knockdown phenotypes, whereas mutation of the PAK phosphorylation site is able to rescue them. Analysis of neural crest explants reveals that CaD is required for the dynamic arrangements of actin and, thus, for cell shape changes and process formation. Taken together, these results suggest that the actin-modulating activity of CaD may underlie its critical function and is regulated by distinct signaling pathways during normal neural crest migration.

Human ESC-derived neural crest model reveals a key role for SOX2 in sensory neurogenesis.

The transcription factor SOX2 is widely known to play a critical role in the central nervous system; however, its role in peripheral neurogenesis remains poorly understood. We recently developed an hESC-based model in which migratory cells undergo epithelial to mesenchymal transition (EMT) to acquire properties of neural crest (NC) cells. In this model, we found that migratory NC progenitors downregulate SOX2, but then start re-expressing SOX2 as they differentiate to form neurogenic dorsal root ganglion (DRG)-like clusters. SOX2 downregulation was sufficient to induce EMT and resulted in massive apoptosis when neuronal differentiation was induced. In vivo, downregulation of SOX2 in chick and mouse NC cells significantly reduced the numbers of neurons within DRG. We found that SOX2 binds directly to NGN1 and MASH1 promoters and is required for their expression. Our data suggest that SOX2 plays a key role for NGN1-dependent acquisition of neuronal fates in sensory ganglia.

Early acquisition of neural crest competence during hESCs neuralization.

BACKGROUND: Neural crest stem cells (NCSCs) are a transient multipotent embryonic cell population that represents a defining characteristic of vertebrates. The neural crest (NC) gives rise to many derivatives including the neurons and glia of the sensory and autonomic ganglia of the peripheral nervous system, enteric neurons and glia, melanocytes, and the cartilaginous, bony and connective tissue of the craniofacial skeleton, cephalic neuroendocrine organs, and some heart vessels. METHODOLOGY/PRINCIPAL FINDINGS: We present evidence that neural crest (NC) competence can be acquired very early when human embryonic stem cells (hESCs) are selectively neuralized towards dorsal neuroepithelium in the absence of feeder cells in fully defined conditions. When hESC-derived neurospheres are plated on fibronectin, some cells emigrate onto the substrate. These early migratory Neural Crest Stem Cells (emNCSCs) uniformly upregulate Sox10 and vimentin, downregulate N-cadherin, and remodel F-actin, consistent with a transition from neuroepithelium to a mesenchymal NC cell. Over 13% of emNCSCs upregulate CD73, a marker of mesenchymal lineage characteristic of cephalic NC and connexin 43, found on early migratory NC cells. We demonstrated that emNCSCs give rise in vitro to all NC lineages, are multipotent on clonal level, and appropriately respond to developmental factors. We suggest that human emNCSC resemble cephalic NC described in model organisms. Ex vivo emNCSCs can differentiate into neurons in Ret.k(-) mouse embryonic gut tissue cultures and transplanted emNCSCs incorporate into NC-derived structures but not CNS tissues in chick embryos. CONCLUSIONS/SIGNIFICANCE: These findings will provide a framework for further studying early human NC development including the epithelial to mesenchymal transition during NC delamination.

Altering Glypican-1 levels modulates canonical Wnt signaling during trigeminal placode development.

Glypicans are conserved cell surface heparan sulfate proteoglycans expressed in a spatiotemporally regulated manner in many developing tissues including the nervous system. Here, we show that Glypican-1 (GPC1) is expressed by trigeminal placode cells as they ingress and contribute to trigeminal sensory neurons in the chick embryo. Either expression of full-length or truncated GPC1 in vivo causes defects in trigeminal gangliogenesis in a manner that requires heparan sulfate side chains. This leads to either abnormal placodal differentiation or organization, respectively, with near complete loss of the ophthalmic (OpV) trigeminal ganglion in the most severe cases after overexpression of full-length GPC1. Interestingly, modulating GPC1 alters levels of endogenous Wnt signaling activity in the forming trigeminal ganglion, as indicated by Wnt reporter expression. Accordingly, GPC1 overexpression phenocopies Wnt inhibition in causing loss of OpV placodal neurons. Furthermore, increased Wnt activity rescues the effects of GPC1 overexpression. Taken together, these results suggest that appropriate levels of GPC1 are essential for proper regulation of canonical Wnt signaling during differentiation and organization of trigeminal placodal cells into ganglia.

Histone demethylase JmjD2A regulates neural crest specification.

The neural crest is a multipotent stem cell-like population that is induced during gastrulation, but only acquires its characteristic morphology, migratory ability, and gene expression profile after neurulation. This raises the intriguing possibility that precursors are actively maintained by epigenetic influences in a stem cell-like state. Accordingly, we report that dynamic histone modifications are critical for proper temporal control of neural crest gene expression in vivo. The histone demethylase, JumonjiD2A (JmjD2A/KDM4A), is expressed in the forming neural folds. Loss of JmjD2A function causes dramatic downregulation of neural crest specifier genes analyzed by multiplex NanoString and in situ hybridization. Importantly, in vivo chromatin immunoprecipitation reveals direct stage-specific interactions of JmjD2A with regulatory regions of neural crest genes, and associated temporal modifications in methylation states of lysine residues directly affected by JmjD2A activity. Our findings show that chromatin modifications directly control neural crest genes in vertebrate embryos via modulating histone methylation.

Evidence for the prepattern/cooption model of vertebrate jaw evolution.

The appearance of jaws was a turning point in vertebrate evolution because it allowed primitive vertebrates to capture and process large, motile prey. The vertebrate jaw consists of separate dorsal and ventral skeletal elements connected by a joint. How this structure evolved from the unjointed gill bar of a jawless ancestor is an unresolved question in vertebrate evolution. To understand the developmental bases of this evolutionary transition, we examined the expression of 12 genes involved in vertebrate pharyngeal patterning in the modern jawless fish lamprey. We find nested expression of Dlx genes, as well as combinatorial expression of Msx, Hand and Gsc genes along the dorso-ventral (DV) axis of the lamprey pharynx, indicating gnathostome-type pharyngeal patterning evolved before the appearance of the jaw. In addition, we find that Bapx and Gdf5/6/7, key regulators of joint formation in gnathostomes, are not expressed in the lamprey first arch, whereas Barx, which is absent from the intermediate first arch in gnathostomes, marks this domain in lamprey. Taken together, these data support a new scenario for jaw evolution in which incorporation of Bapx and Gdf5/6/7 into a preexisting DV patterning program drove the evolution of the jaw by altering the identity of intermediate first-arch chondrocytes. We present this "Pre-pattern/Cooption" model as an alternative to current models linking the evolution of the jaw to the de novo appearance of sophisticated pharyngeal DV patterning.

The odontode explosion: the origin of tooth-like structures in vertebrates.

Essentially we show recent data to shed new light on the thorny controversy of how teeth arose in evolution. Essentially we show (a) how teeth can form equally from any epithelium, be it endoderm, ectoderm or a combination of the two and (b) that the gene expression programs of oral versus pharyngeal teeth are remarkably similar. Classic theories suggest that (i) skin denticles evolved first and odontode-inductive surface ectoderm merged inside the oral cavity to form teeth (the 'outside-in' hypothesis) or that (ii) patterned odontodes evolved first from endoderm deep inside the pharyngeal cavity (the 'inside-out' hypothesis). We propose a new perspective that views odontodes as structures sharing a deep molecular homology, united by sets of co-expressed genes defining a competent thickened epithelium and a collaborative neural crest-derived ectomesenchyme. Simply put, odontodes develop 'inside and out', wherever and whenever these co-expressed gene sets signal to one another. Our perspective complements the classic theories and highlights an agenda for specific experimental manipulations in model and non-model organisms.

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.

Insights into the organization of dorsal spinal cord pathways from an evolutionarily conserved raldh2 intronic enhancer.

Comparative studies of the tetrapod raldh2 (aldh1a2) gene, which encodes a retinoic acid (RA) synthesis enzyme, have led to the identification of a dorsal spinal cord enhancer. Enhancer activity is directed dorsally to the roof plate and dorsal-most (dI1) interneurons through predicted Tcf- and Cdx-homeodomain binding sites and is repressed ventrally via predicted Tgif homeobox and ventral Lim-homeodomain binding sites. Raldh2 and Math1/Cath1 expression in mouse and chicken highlights a novel, transient, endogenous Raldh2 expression domain in dI1 interneurons, which give rise to ascending circuits and intraspinal commissural interneurons, suggesting roles for RA in the ontogeny of spinocerebellar and intraspinal proprioceptive circuits. Consistent with expression of raldh2 in the dorsal interneurons of tetrapods, we also found that raldh2 is expressed in dorsal interneurons throughout the agnathan spinal cord, suggesting ancestral roles for RA signaling in the ontogenesis of intraspinal proprioception.

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.

Pax2 and Pea3 synergize to activate a novel regulatory enhancer for spalt4 in the developing ear.

The transcription factor spalt4 is a key early-response gene in otic placode induction. Here, we characterize the cis-regulatory regions of spalt4 responsible for activation of its expression in the developing otic placode and report the isolation of a novel core enhancer. Identification and mutational analysis of putative transcription factor binding sites reveal that Pea3, a downstream effector of FGF signaling, and Pax2 directly activate spalt4 during ear development. Morpholino-mediated knock-down of each factor reduces or eliminates reporter expression. In contrast, combined over-expression of Pea3 and Pax2 drives ectopic reporter expression, suggesting that they function synergistically. These studies expand the gene regulatory network underlying early otic development by identifying direct inputs that mediate spalt4 expression.

N-cadherin acts in concert with Slit1-Robo2 signaling in regulating aggregation of placode-derived cranial sensory neurons.

Vertebrate cranial sensory ganglia have a dual origin from the neural crest and ectodermal placodes. In the largest of these, the trigeminal ganglion, Slit1-Robo2 signaling is essential for proper ganglion assembly. Here, we demonstrate a crucial role for the cell adhesion molecule N-cadherin and its interaction with Slit1-Robo2 during gangliogenesis in vivo. A common feature of chick trigeminal and epibranchial ganglia is the expression of N-cadherin and Robo2 on placodal neurons and Slit1 on neural crest cells. Interestingly, N-cadherin localizes to intercellular adherens junctions between placodal neurons during ganglion assembly. Depletion of N-cadherin causes loss of proper ganglion coalescence, similar to that observed after loss of Robo2, suggesting that the two pathways might intersect. Consistent with this possibility, blocking or augmenting Slit-Robo signaling modulates N-cadherin protein expression on the placodal cell surface concomitant with alteration in placodal adhesion. Lack of an apparent change in total N-cadherin mRNA or protein levels suggests post-translational regulation. Co-expression of N-cadherin with dominant-negative Robo abrogates the Robo2 loss-of-function phenotype of dispersed ganglia, whereas loss of N-cadherin reverses the aberrant aggregation induced by increased Slit-Robo expression. Our study suggests a novel mechanism whereby N-cadherin acts in concert with Slit-Robo signaling in mediating the placodal cell adhesion required for proper gangliogenesis.

Semaphorin3A/neuropilin-1 signaling acts as a molecular switch regulating neural crest migration during cornea development.

Cranial neural crest cells migrate into the periocular region and later contribute to various ocular tissues including the cornea, ciliary body and iris. After reaching the eye, they initially pause before migrating over the lens to form the cornea. Interestingly, removal of the lens leads to premature invasion and abnormal differentiation of the cornea. In exploring the molecular mechanisms underlying this effect, we find that semaphorin3A (Sema3A) is expressed in the lens placode and epithelium continuously throughout eye development. Interestingly, neuropilin-1 (Npn-1) is expressed by periocular neural crest but down-regulated, in a manner independent of the lens, by the subpopulation that migrates into the eye and gives rise to the cornea endothelium and stroma. In contrast, Npn-1 expressing neural crest cells remain in the periocular region and contribute to the anterior uvea and ocular blood vessels. Introduction of a peptide that inhibits Sema3A/Npn-1 signaling results in premature entry of neural crest cells over the lens that phenocopies lens ablation. Furthermore, Sema3A inhibits periocular neural crest migration in vitro. Taken together, our data reveal a novel and essential role of Sema3A/Npn-1 signaling in coordinating periocular neural crest migration that is vital for proper ocular development.

Myosin-X is critical for migratory ability of Xenopus cranial neural crest cells.

The neural crest is a highly migratory cell population, unique to vertebrates, that forms much of the craniofacial skeleton and peripheral nervous system. In exploring the cell biological basis underlying this behavior, we have identified an unconventional myosin, myosin-X (Myo10) that is required for neural crest migration. Myo10 is highly expressed in both premigratory and migrating cranial neural crest (CNC) cells in Xenopus embryos. Disrupting Myo10 expression using antisense morpholino oligonucleotides leads to impaired neural crest migration and subsequent cartilage formation, but only a slight delay in induction. In vivo grafting experiments reveal that Myo10-depleted CNC cells migrate a shorter distance and fail to segregate into distinct migratory streams. Finally, in vitro cultures and cell dissociation-reaggregation assays suggest that Myo10 may be critical for cell protrusion and cell-cell adhesion. These results demonstrate an essential role for Myo10 in normal cranial neural crest migration and suggest a link to cell-cell interactions and formation of processes.

Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease.

The events that convert adherent epithelial cells into individual migratory cells that can invade the extracellular matrix are known collectively as epithelial-mesenchymal transition (EMT). Throughout evolution, the capacity of cells to switch between these two cellular states has been fundamental in the generation of complex body patterns. Here, we review the EMT events that build the embryo and further discuss two prototypical processes governed by EMT in amniotes: gastrulation and neural crest formation. Cells undergo EMT to migrate and colonize distant territories. Not surprisingly, this is also the mechanism used by cancer cells to disperse throughout the body.

Molecular and tissue interactions governing induction of cranial ectodermal placodes.

Whereas neural crest cells are the source of the peripheral nervous system in the trunk of vertebrates, the "ectodermal placodes," together with neural crest, form the peripheral nervous system of the head. Cranial ectodermal placodes are thickenings in the ectoderm that subsequently ingress or invaginate to make important contributions to cranial ganglia, including epibranchial and trigeminal ganglia, and sensory structures, the ear, nose, lens, and adenohypophysis. Recent studies have uncovered a number of molecular signals mediating induction and differentiation of placodal cells. Here, we described recent advances in understanding the tissue interactions and signals underlying induction and neurogenesis of placodes, with emphasis on the trigeminal and epibranchial. Important roles of Fibroblast Growth Factors, Platelet Derived Growth Factors, Sonic Hedgehog, TGFbeta superfamily members, and Wnts are discussed.

Chapter 1. Gene regulatory networks in neural crest development and evolution.

The neural crest is a multipotent migratory embryonic cell population that is present in all vertebrates, but missing from basal chordates. In this chapter, we discuss recent work in amphioxus, ascidians, lamprey, and gnathostomes that reflects the current state of knowledge of the evolutionary origin of this fascinating cell population. We summarize recent evidence for the ongoing diversification of the neural crest in several vertebrate species, with particular reference to studies in nontraditional vertebrate model organisms.