On the evolutionary origins and regionalization of the neural crest.

The neural crest is a vertebrate-specific embryonic stem cell population that gives rise to a vast array of cell types throughout the animal body plan. These cells are first born at the edges of the central nervous system, from which they migrate extensively and differentiate into multiple cellular derivatives. Given the unique set of structures these cells comprise, the origin of the neural crest is thought to have important implications for the evolution and diversification of the vertebrate clade. In jawed vertebrates, neural crest cells exist as distinct subpopulations along the anterior-posterior axis. These subpopulations differ in terms of their respective differentiation potential and cellular derivatives. Thus, the modern neural crest is characterized as multipotent, migratory, and regionally segregated throughout the embryo. Here, we retrace the evolutionary origins of the neural crest, from the appearance of conserved regulatory circuitry in basal chordates to the emergence of neural crest subpopulations in higher vertebrates. Finally, we discuss a stepwise trajectory by which these cells may have arisen and diversified throughout vertebrate evolution.

Evolution of the new head by gradual acquisition of neural crest regulatory circuits.

The neural crest, an embryonic stem-cell population, is a vertebrate innovation that has been proposed to be a key component of the 'new head', which imbued vertebrates with predatory behaviour1,2. Here, to investigate how the evolution of neural crest cells affected the vertebrate body plan, we examined the molecular circuits that control neural crest development along the anteroposterior axis of a jawless vertebrate, the sea lamprey. Gene expression analysis showed that the cranial subpopulation of the neural crest of the lamprey lacks most components of a transcriptional circuit that is specific to the cranial neural crest in amniotes and confers the ability to form craniofacial cartilage onto non-cranial neural crest subpopulations3. Consistent with this, hierarchical clustering analysis revealed that the transcriptional profile of the lamprey cranial neural crest is more similar to the trunk neural crest of amniotes. Notably, analysis of the cranial neural crest in little skate and zebrafish embryos demonstrated that the transcriptional circuit that is specific to the cranial neural crest emerged via the gradual addition of network components to the neural crest of gnathostomes, which subsequently became restricted to the cephalic region. Our results indicate that the ancestral neural crest at the base of the vertebrate lineage possessed a trunk-like identity. We propose that the emergence of the cranial neural crest, by progressive assembly of an axial-specific regulatory circuit, allowed the elaboration of the new head during vertebrate evolution.

A regulatory sub-circuit downstream of Wnt signaling controls developmental transitions in neural crest formation.

The process of cell fate commitment involves sequential changes in the gene expression profiles of embryonic progenitors. This is exemplified in the development of the neural crest, a migratory stem cell population derived from the ectoderm of vertebrate embryos. During neural crest formation, cells transition through distinct transcriptional states in a stepwise manner. The mechanisms underpinning these shifts in cell identity are still poorly understood. Here we employ enhancer analysis to identify a genetic sub-circuit that controls developmental transitions in the nascent neural crest. This sub-circuit links Wnt target genes in an incoherent feedforward loop that controls the sequential activation of genes in the neural crest lineage. By examining the cis-regulatory apparatus of Wnt effector gene AXUD1, we found that multipotency factor SP5 directly promotes neural plate border identity, while inhibiting premature expression of specification genes. Our results highlight the importance of repressive interactions in the neural crest gene regulatory network and illustrate how genes activated by the same upstream signal become temporally segregated during progressive fate restriction.

Heterodimerization of TFAP2 pioneer factors drives epigenomic remodeling during neural crest specification.

Cell fate commitment involves the progressive restriction of developmental potential. Recent studies have shown that this process requires not only shifts in gene expression but also an extensive remodeling of the epigenomic landscape. To examine how chromatin states are reorganized during cellular specification in an in vivo system, we examined the function of pioneer factor TFAP2A at discrete stages of neural crest development. Our results show that TFAP2A activates distinct sets of genomic regions during induction of the neural plate border and specification of neural crest cells. Genomic occupancy analysis revealed that the repertoire of TFAP2A targets depends upon its dimerization with paralogous proteins TFAP2C and TFAP2B. During gastrula stages, TFAP2A/C heterodimers activate components of the neural plate border induction program. As neurulation begins, TFAP2A trades partners, and TFAP2A/B heterodimers reorganize the epigenomic landscape of progenitor cells to promote neural crest specification. We propose that this molecular switch acts to drive progressive cell commitment, remodeling the epigenomic landscape to define the presumptive neural crest. Our findings show how pioneer factors regulate distinct genomic targets in a stage-specific manner and highlight how paralogy can serve as an evolutionary strategy to diversify the function of the regulators that control embryonic development.

Post-transcriptional tuning of FGF signaling mediates neural crest induction.

Ectodermal patterning is required for the establishment of multiple components of the vertebrate body plan. Previous studies have demonstrated that precise combinations of extracellular signals induce distinct ectodermal cell populations, such as the neural crest and the neural plate. Yet, we still lack understanding of how the response to inductive signals is modulated to generate the proper transcriptional output in target cells. Here we show that posttranscriptional attenuation of fibroblast growth factor (FGF) signaling is essential for the establishment of the neural crest territory. We found that neural crest progenitors display elevated expression of DICER, which promotes enhanced maturation of a set of cell-type-specific miRNAs. These miRNAs collectively target components of the FGF signaling pathway, a central player in the process of neural induction in amniotes. Inactivation of this posttranscriptional circuit results in a fate switch, in which neural crest cells are converted into progenitors of the central nervous system. Thus, the posttranscriptional attenuation of signaling systems is a prerequisite for proper segregation of ectodermal cell types. These findings demonstrate how posttranscriptional repression may alter the activity of signaling systems to generate distinct spatial domains of progenitor cells.

Neural crest metabolism: At the crossroads of development and disease.

The neural crest is a migratory stem cell population that contributes to various tissues and organs during vertebrate embryonic development. These cells possess remarkable developmental plasticity and give rise to many different cell types, including chondrocytes, osteocytes, peripheral neurons, glia, melanocytes, and smooth muscle cells. Although the genetic mechanisms underlying neural crest development have been extensively studied, many facets of this process remain unexplored. One key aspect of cellular physiology that has gained prominence in the context of embryonic development is metabolic regulation. Recent discoveries in neural crest biology suggest that metabolic regulation may play a central role in the formation, migration, and differentiation of these cells. This possibility is further supported by clinical studies that have demonstrated a high prevalence of neural crest anomalies in babies with congenital metabolic disorders. Here, we examine why neural crest development is prone to metabolic disruption and discuss how carbon metabolism regulates developmental processes like epithelial-to-mesenchymal transition (EMT) and cell migration. Finally, we explore how understanding neural crest metabolism may inform upon the etiology of several congenital birth defects.

Evolution of vertebrates as viewed from the crest.

The origin of vertebrates was accompanied by the advent of a novel cell type: the neural crest. Emerging from the central nervous system, these cells migrate to diverse locations and differentiate into numerous derivatives. By coupling morphological and gene regulatory information from vertebrates and other chordates, we describe how addition of the neural-crest-specification program may have enabled cells at the neural plate border to acquire multipotency and migratory ability. Analysis of the topology of the neural crest gene regulatory network can serve as a useful template for understanding vertebrate evolution, including elaboration of neural crest derivatives.

A systems-level approach reveals new gene regulatory modules in the developing ear.

The inner ear is a complex vertebrate sense organ, yet it arises from a simple epithelium, the otic placode. Specification towards otic fate requires diverse signals and transcriptional inputs that act sequentially and/or in parallel. Using the chick embryo, we uncover novel genes in the gene regulatory network underlying otic commitment and reveal dynamic changes in gene expression. Functional analysis of selected transcription factors reveals the genetic hierarchy underlying the transition from progenitor to committed precursor, integrating known and novel molecular players. Our results not only characterize the otic transcriptome in unprecedented detail, but also identify new gene interactions responsible for inner ear development and for the segregation of the otic lineage from epibranchial progenitors. By recapitulating the embryonic programme, the genes and genetic sub-circuits discovered here might be useful for reprogramming naïve cells towards otic identity to restore hearing loss.

The molecular basis of neural crest axial identity.

The neural crest is a migratory cell population that contributes to multiple tissues and organs during vertebrate embryonic development. It is remarkable in its ability to differentiate into an array of different cell types, including melanocytes, cartilage, bone, smooth muscle, and peripheral nerves. Although neural crest cells are formed along the entire anterior-posterior axis of the developing embryo, they can be divided into distinct subpopulations based on their axial level of origin. These groups of cells, which include the cranial, vagal, trunk, and sacral neural crest, display varied migratory patterns and contribute to multiple derivatives. While these subpopulations have been shown to be mostly plastic and to differentiate according to environmental cues, differences in their intrinsic potentials have also been identified. For instance, the cranial neural crest is unique in its ability to give rise to cartilage and bone. Here, we examine the molecular features that underlie such developmental restrictions and discuss the hypothesis that distinct gene regulatory networks operate in these subpopulations. We also consider how reconstructing the phylogeny of the trunk and cranial neural crest cells impacts our understanding of vertebrate evolution.

Establishing neural crest identity: a gene regulatory recipe.

The neural crest is a stem/progenitor cell population that contributes to a wide variety of derivatives, including sensory and autonomic ganglia, cartilage and bone of the face and pigment cells of the skin. Unique to vertebrate embryos, it has served as an excellent model system for the study of cell behavior and identity owing to its multipotency, motility and ability to form a broad array of cell types. Neural crest development is thought to be controlled by a suite of transcriptional and epigenetic inputs arranged hierarchically in a gene regulatory network. Here, we examine neural crest development from a gene regulatory perspective and discuss how the underlying genetic circuitry results in the features that define this unique cell population.

Transcriptome analysis reveals novel players in the cranial neural crest gene regulatory network.

The neural crest is an embryonic stem cell population that gives rise to a multitude of derivatives. In particular, the cranial neural crest (CNC) is unique in its ability to contribute to both facial skeleton and peripheral ganglia. To gain further insight into the molecular underpinnings that distinguish the CNC from other embryonic tissues, we have utilized a CNC-specific enhancer as a tool to isolate a pure, region-specific NC subpopulation for transcriptional profiling. The resulting data set reveals previously unknown transcription factors and signaling pathways that may influence the CNC's ability to migrate and/or differentiate into unique derivatives. To elaborate on the CNC gene regulatory network, we evaluated the effects of knocking down known neural plate border genes and early neural crest specifier genes on selected neural crest-enriched transcripts. The results suggest that ETS1 and SOX9 may act as pan-neural crest regulators of the migratory CNC. Taken together, our analysis provides unprecedented characterization of the migratory CNC transcriptome and identifies new links in the gene regulatory network responsible for development of this critical cell population.

DNA methyltransferase 3B regulates duration of neural crest production via repression of Sox10.

Neural crest stem cells arise within the central nervous system but then undergo an epithelial-to-mesenchymal transition to migrate away and contribute to the peripheral nervous system and craniofacial skeleton. Here we show that DNA methyltransferase 3B (DNMT3B) is responsible for the loss of competence of dorsal neural tube cells to generate emigrating neural crest cells. DNMT3B knockdown results in up-regulation of neural crest markers, prolonged neural crest emigration, and subsequent precocious neuronal differentiation of the trigeminal ganglion. We find that DNMT3B binds to the promoter of Sox10, known to be important for neural crest emigration and lineage acquisition. Bisulfite sequencing further reveals methylation of the Sox10 promoter region upon cessation of emigration in normal embryos, whereas this mark is reduced after DNMT3B loss. Taken together, these results reveal the importance of DNA methylation in regulating the ability of neural tube cells to produce neural crest cells and the timing of peripheral neuron differentiation.

Evolutionarily conserved role for SoxC genes in neural crest specification and neuronal differentiation.

Members of the Sox family of transcription factors play a variety of critical developmental roles in both vertebrates and invertebrates. Whereas SoxBs and SoxEs are involved in neural and neural crest development, respectively, far less is known about members of the SoxC subfamily. To address this from an evolutionary perspective, we compare expression and function of SoxC genes in neural crest cells and their derivatives in lamprey (Petromyzon marinus), a basal vertebrate, to frog (Xenopus laevis). Analysis of transcript distribution reveals conservation of lamprey and X. laevis SoxC expression in premigratory neural crest, branchial arches, and cranial ganglia. Moreover, morpholino-mediated loss-of-function of selected SoxC family members demonstrates essential roles in aspects of neural crest development in both organisms. The results suggest important and conserved functions of SoxC genes during vertebrate evolution and a particularly critical, previously unrecognized role in early neural crest specification.

Insights into neural crest development and evolution from genomic analysis.

The neural crest is an excellent model system for the study of cell type diversification during embryonic development due to its multipotency, motility, and ability to form a broad array of derivatives ranging from neurons and glia, to cartilage, bone, and melanocytes. As a uniquely vertebrate cell population, it also offers important clues regarding vertebrate origins. In the past 30 yr, introduction of recombinant DNA technology has facilitated the dissection of the genetic program controlling neural crest development and has provided important insights into gene regulatory mechanisms underlying cell migration and differentiation. More recently, new genomic approaches have provided a platform and tools that are changing the depth and breadth of our understanding of neural crest development at a "systems" level. Such advances provide an insightful view of the regulatory landscape of neural crest cells and offer a new perspective on developmental as well as stem cell and cancer biology.

Dynamic and differential regulation of stem cell factor FoxD3 in the neural crest is Encrypted in the genome.

The critical stem cell transcription factor FoxD3 is expressed by the premigratory and migrating neural crest, an embryonic stem cell population that forms diverse derivatives. Despite its important role in development and stem cell biology, little is known about what mediates FoxD3 activity in these cells. We have uncovered two FoxD3 enhancers, NC1 and NC2, that drive reporter expression in spatially and temporally distinct manners. Whereas NC1 activity recapitulates initial FoxD3 expression in the cranial neural crest, NC2 activity recapitulates initial FoxD3 expression at vagal/trunk levels while appearing only later in migrating cranial crest. Detailed mutational analysis, in vivo chromatin immunoprecipitation, and morpholino knock-downs reveal that transcription factors Pax7 and Msx1/2 cooperate with the neural crest specifier gene, Ets1, to bind to the cranial NC1 regulatory element. However, at vagal/trunk levels, they function together with the neural plate border gene, Zic1, which directly binds to the NC2 enhancer. These results reveal dynamic and differential regulation of FoxD3 in distinct neural crest subpopulations, suggesting that heterogeneity is encrypted at the regulatory level. Isolation of neural crest enhancers not only allows establishment of direct regulatory connections underlying neural crest formation, but also provides valuable tools for tissue specific manipulation and investigation of neural crest cell identity in amniotes.

Structural shifts of aldehyde dehydrogenase enzymes were instrumental for the early evolution of retinoid-dependent axial patterning in metazoans.

Aldehyde dehydrogenases (ALDHs) catabolize toxic aldehydes and process the vitamin A-derived retinaldehyde into retinoic acid (RA), a small diffusible molecule and a pivotal chordate morphogen. In this study, we combine phylogenetic, structural, genomic, and developmental gene expression analyses to examine the evolutionary origins of ALDH substrate preference. Structural modeling reveals that processing of small aldehydes, such as acetaldehyde, by ALDH2, versus large aldehydes, including retinaldehyde, by ALDH1A is associated with small versus large substrate entry channels (SECs), respectively. Moreover, we show that metazoan ALDH1s and ALDH2s are members of a single ALDH1/2 clade and that during evolution, eukaryote ALDH1/2s often switched between large and small SECs after gene duplication, transforming constricted channels into wide opened ones and vice versa. Ancestral sequence reconstructions suggest that during the evolutionary emergence of RA signaling, the ancestral, narrow-channeled metazoan ALDH1/2 gave rise to large ALDH1 channels capable of accommodating bulky aldehydes, such as retinaldehyde, supporting the view that retinoid-dependent signaling arose from ancestral cellular detoxification mechanisms. Our analyses also indicate that, on a more restricted evolutionary scale, ALDH1 duplicates from invertebrate chordates (amphioxus and ascidian tunicates) underwent switches to smaller and narrower SECs. When combined with alterations in gene expression, these switches led to neofunctionalization from ALDH1-like roles in embryonic patterning to systemic, ALDH2-like roles, suggesting functional shifts from signaling to detoxification.

Histone lactylation couples cellular metabolism with developmental gene regulatory networks.

Embryonic cells exhibit diverse metabolic states. Recent studies have demonstrated that metabolic reprogramming drives changes in cell identity by affecting gene expression. However, the connection between cellular metabolism and gene expression remains poorly understood. Here we report that glycolysis-regulated histone lactylation couples the metabolic state of embryonic cells with chromatin organization and gene regulatory network (GRN) activation. We found that lactylation marks genomic regions of glycolytic embryonic tissues, like the neural crest (NC) and pre-somitic mesoderm. Histone lactylation occurs in the loci of NC genes as these cells upregulate glycolysis. This process promotes the accessibility of active enhancers and the deployment of the NC GRN. Reducing the deposition of the mark by targeting LDHA/B leads to the downregulation of NC genes and the impairment of cell migration. The deposition of lactyl-CoA on histones at NC enhancers is supported by a mechanism that involves transcription factors SOX9 and YAP/TEAD. These findings define an epigenetic mechanism that integrates cellular metabolism with the GRNs that orchestrate embryonic development.

ASCL1 promotes Scrt2 expression in the neural tube.

ASCL1 is a transcription factor that directs neural progenitors towards lineage differentiation. Although many of the molecular mechanisms underlying its action have been described, several of its targets remain unidentified. We identified in the chick genome a putative enhancer (cE1) upstream of the transcription factor Scratch2 (Scrt2) locus with a predicted heterodimerization motif for ASCL1 and POU3F2. In this study, we investigated the role of ASCL1 and this enhancer in regulating the expression of the Scrt2 in the embryonic spinal cord. We confirmed that cE1 region interacted with the Scrt2 promoter. cE1 was sufficient to mediate ASCL1-driven expression in the neural tube through the heterodimerization sites. Moreover, Scrt2 expression was inhibited when we removed cE1 from the genome. These findings strongly indicate that ASCL1 regulates Scrt2 transcription in the neural tube through cE1.

Gag proteins encoded by endogenous retroviruses are required for zebrafish development.

Transposable elements (TEs) make up the bulk of eukaryotic genomes and examples abound of TE-derived sequences repurposed for organismal function. The process by which TEs become coopted remains obscure because most cases involve ancient, transpositionally inactive elements. Reports of active TEs serving beneficial functions are scarce and often contentious due to difficulties in manipulating repetitive sequences. Here we show that recently active TEs in zebrafish encode products critical for embryonic development. Knockdown and rescue experiments demonstrate that the endogenous retrovirus family BHIKHARI-1 (Bik-1) encodes a Gag protein essential for mesoderm development. Mechanistically, Bik-1 Gag associates with the cell membrane and its ectopic expression in chicken embryos alters cell migration. Similarly, depletion of BHIKHARI-2 Gag, a relative of Bik-1, causes defects in neural crest development in zebrafish. We propose an "addiction" model to explain how active TEs can be integrated into conserved developmental processes.

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.