A patterned human neural tube model using microfluidic gradients.

The human nervous system is a highly complex but organized organ. The foundation of its complexity and organization is laid down during regional patterning of the neural tube, the embryonic precursor to the human nervous system. Historically, studies of neural tube patterning have relied on animal models to uncover underlying principles. Recently, models of neurodevelopment based on human pluripotent stem cells, including neural organoids1-5 and bioengineered neural tube development models6-10, have emerged. However, such models fail to recapitulate neural patterning along both rostral-caudal and dorsal-ventral axes in a three-dimensional tubular geometry, a hallmark of neural tube development. Here we report a human pluripotent stem cell-based, microfluidic neural tube-like structure, the development of which recapitulates several crucial aspects of neural patterning in brain and spinal cord regions and along rostral-caudal and dorsal-ventral axes. This structure was utilized for studying neuronal lineage development, which revealed pre-patterning of axial identities of neural crest progenitors and functional roles of neuromesodermal progenitors and the caudal gene CDX2 in spinal cord and trunk neural crest development. We further developed dorsal-ventral patterned microfluidic forebrain-like structures with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic development of the human forebrain pallium and subpallium, respectively. Together, these microfluidics-based neurodevelopment models provide three-dimensional lumenal tissue architectures with in vivo-like spatiotemporal cell differentiation and organization, which will facilitate the study of human neurodevelopment and disease.

Neural crest origin of sympathetic neurons at the dawn of vertebrates.

The neural crest is an embryonic stem cell population unique to vertebrates1 whose expansion and diversification are thought to have promoted vertebrate evolution by enabling emergence of new cell types and structures such as jaws and peripheral ganglia2. Although jawless vertebrates have sensory ganglia, convention has it that trunk sympathetic chain ganglia arose only in jawed vertebrates3-8. Here, by contrast, we report the presence of trunk sympathetic neurons in the sea lamprey, Petromyzon marinus, an extant jawless vertebrate. These neurons arise from sympathoblasts near the dorsal aorta that undergo noradrenergic specification through a transcriptional program homologous to that described in gnathostomes. Lamprey sympathoblasts populate the extracardiac space and extend along the length of the trunk in bilateral streams, expressing the catecholamine biosynthetic pathway enzymes tyrosine hydroxylase and dopamine ß-hydroxylase. CM-DiI lineage tracing analysis further confirmed that these cells derive from the trunk neural crest. RNA sequencing of isolated ammocoete trunk sympathoblasts revealed gene profiles characteristic of sympathetic neuron function. Our findings challenge the prevailing dogma that posits that sympathetic ganglia are a gnathostome innovation, instead suggesting that a late-developing rudimentary sympathetic nervous system may have been characteristic of the earliest vertebrates.

Cranial suture lineage and contributions to repair of the mouse skull.

The cranial sutures are proposed to be a stem cell niche, harbouring skeletal stem cells that are directly involved in development, homeostasis and healing. Like the craniofacial bones, the sutures are formed from both mesoderm and neural crest. During cranial bone repair, neural crest cells have been proposed to be key players; however, neural crest contributions to adult sutures are not well defined, and the relative importance of suture proximity is unclear. Here, we use genetic approaches to re-examine the neural crest-mesoderm boundaries in the adult mouse skull. These are combined with calvarial wounding experiments suggesting that suture proximity improves the efficiency of cranial repair. Furthermore, we demonstrate that Gli1+ and Axin2+ skeletal stem cells are present in all calvarial sutures examined. We propose that the position of the defect determines the availability of neural crest-derived progenitors, which appear to be a key element in the repair of calvarial defects.

The people behind the papers - Paul Huber and Carole LaBonne.

The bromodomain and extra-terminal (BET) family of proteins reads epigenetic histone acetylation marks on the genome and regulates the transcriptional machinery. In their study, Carole LaBonne and colleagues reveal the role of BET protein activity in the maintenance of pluripotency and establishment of the neural crest in Xenopus laevis. To know more about their work, we spoke to the first author Paul Huber and the corresponding author Carole LaBonne, Developmental and Stem Cell Biologist at Northwestern University.

BET activity plays an essential role in control of stem cell attributes in Xenopus.

Neural crest cells are a stem cell population unique to vertebrate embryos that retains broad multi-germ layer developmental potential through neurulation. Much remains to be learned about the genetic and epigenetic mechanisms that control the potency of neural crest cells. Here, we examine the role that epigenetic readers of the BET (bromodomain and extra terminal) family play in controlling the potential of pluripotent blastula and neural crest cells. We find that inhibiting BET activity leads to loss of pluripotency at blastula stages and a loss of neural crest at neurula stages. We compare the effects of HDAC (an eraser of acetylation marks) and BET (a reader of acetylation) inhibition and find that they lead to similar cellular outcomes through distinct effects on the transcriptome. Interestingly, loss of BET activity in cells undergoing lineage restriction is coupled to increased expression of genes linked to pluripotency and prolongs the competence of initially pluripotent cells to transit to a neural progenitor state. Together these findings advance our understanding of the epigenetic control of pluripotency and the formation of the vertebrate neural crest.

SoxB1 transcription factors are essential for initiating and maintaining neural plate border gene expression.

SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis, we set out to determine whether SoxB1 transcription factors have a regulatory function in NPB formation. Here, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq, we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as potential Sox3 partners in regulating the formation of the NPB and show that their combined activity is needed for normal NPB gene expression. Together, these data identify a role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.

Zebrafish raptor mutation inhibits the activity of mTORC1, inducing craniofacial defects due to autophagy-induced neural crest cell death.

The mechanistic target of rapamycin (mTOR) coordinates metabolism and cell growth with environmental inputs. mTOR forms two functional complexes: mTORC1 and mTORC2. Proper development requires both complexes but mTORC1 has unique roles in numerous cellular processes, including cell growth, survival and autophagy. Here, we investigate the function of mTORC1 in craniofacial development. We created a zebrafish raptor mutant via CRISPR/Cas9, to specifically disrupt mTORC1. The entire craniofacial skeleton and eyes were reduced in size in mutants; however, overall body length and developmental timing were not affected. The craniofacial phenotype associates with decreased chondrocyte size and increased neural crest cell death. We found that autophagy is elevated in raptor mutants. Chemical inhibition of autophagy reduced cell death and improved craniofacial phenotypes in raptor mutants. Genetic inhibition of autophagy, via mutation of the autophagy gene atg7, improved facial phenotypes in atg7;raptor double mutants, relative to raptor single mutants. We conclude that finely regulated levels of autophagy, via mTORC1, are crucial for craniofacial development.

Piezo1 and Piezo2 collectively regulate jawbone development.

Piezo1 and Piezo2 are recently reported mechanosensory ion channels that transduce mechanical stimuli from the environment into intracellular biochemical signals in various tissues and organ systems. Here, we show that Piezo1 and Piezo2 display a robust expression during jawbone development. Deletion of Piezo1 in neural crest cells causes jawbone malformations in a small but significant number of mice. We further demonstrate that disruption of Piezo1 and Piezo2 in neural crest cells causes more striking defects in jawbone development than any single knockout, suggesting essential but partially redundant roles of Piezo1 and Piezo2. In addition, we observe defects in other neural crest derivatives such as malformation of the vascular smooth muscle in double knockout mice. Moreover, TUNEL examinations reveal excessive cell death in osteogenic cells of the maxillary and mandibular arches of the double knockout mice, suggesting that Piezo1 and Piezo2 together regulate cell survival during jawbone development. We further demonstrate that Yoda1, a Piezo1 agonist, promotes mineralization in the mandibular arches. Altogether, these data firmly establish that Piezo channels play important roles in regulating jawbone formation and maintenance.

A pre-vertebrate endodermal origin of calcitonin-producing neuroendocrine cells.

Vertebrate calcitonin-producing cells (C-cells) are neuroendocrine cells that secrete the small peptide hormone calcitonin in response to elevated blood calcium levels. Whereas mouse C-cells reside within the thyroid gland and derive from pharyngeal endoderm, avian C-cells are located within ultimobranchial glands and have been reported to derive from the neural crest. We use a comparative cell lineage tracing approach in a range of vertebrate model systems to resolve the ancestral embryonic origin of vertebrate C-cells. We find, contrary to previous studies, that chick C-cells derive from pharyngeal endoderm, with neural crest-derived cells instead contributing to connective tissue intimately associated with C-cells in the ultimobranchial gland. This endodermal origin of C-cells is conserved in a ray-finned bony fish (zebrafish) and a cartilaginous fish (the little skate, Leucoraja erinacea). Furthermore, we discover putative C-cell homologs within the endodermally-derived pharyngeal epithelium of the ascidian Ciona intestinalis and the amphioxus Branchiostoma lanceolatum, two invertebrate chordates that lack neural crest cells. Our findings point to a conserved endodermal origin of C-cells across vertebrates and to a pre-vertebrate origin of this cell type along the chordate stem.

Melanocyte lineage dynamics in development, growth and disease.

Melanocytes evolved to produce the melanin that gives colour to our hair, eyes and skin. The melanocyte lineage also gives rise to melanoma, the most lethal form of skin cancer. The melanocyte lineage differentiates from neural crest cells during development, and most melanocytes reside in the skin and hair, where they are replenished by melanocyte stem cells. Because the molecular mechanisms necessary for melanocyte specification, migration, proliferation and differentiation are co-opted during melanoma initiation and progression, studying melanocyte development is directly relevant to human disease. Here, through the lens of advances in cellular omic and genomic technologies, we review the latest findings in melanocyte development and differentiation, and how these developmental pathways become dysregulated in disease.

miR-203 secreted in extracellular vesicles mediates the communication between neural crest and placode cells required for trigeminal ganglia formation.

While interactions between neural crest and placode cells are critical for the proper formation of the trigeminal ganglion, the mechanisms underlying this process remain largely uncharacterized. Here, by using chick embryos, we show that the microRNA (miR)-203, whose epigenetic repression is required for neural crest migration, is reactivated in coalescing and condensing trigeminal ganglion cells. Overexpression of miR-203 induces ectopic coalescence of neural crest cells and increases ganglion size. By employing cell-specific electroporations for either miR-203 sponging or genomic editing using CRISPR/Cas9, we elucidated that neural crest cells serve as the source, while placode cells serve as the site of action for miR-203 in trigeminal ganglion condensation. Demonstrating intercellular communication, overexpression of miR-203 in the neural crest in vitro or in vivo represses an miR-responsive sensor in placode cells. Moreover, neural crest-secreted extracellular vesicles (EVs), visualized using pHluorin-CD63 vector, become incorporated into the cytoplasm of placode cells. Finally, RT-PCR analysis shows that small EVs isolated from condensing trigeminal ganglia are selectively loaded with miR-203. Together, our findings reveal a critical role in vivo for neural crest-placode communication mediated by sEVs and their selective microRNA cargo for proper trigeminal ganglion formation.

The development of brain pericytes requires expression of the transcription factor nkx3.1 in intermediate precursors.

Brain pericytes are one of the critical cell types that regulate endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. The genetic pathways guiding undifferentiated cells into mature pericytes are not well understood. We show here that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express the transcription factor nkx3.1 develop into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1-, foxf2a-, and cxcl12b-expressing pericyte precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. Cxcl12b-Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. Through genetic experiments, we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.

Disruption of DNA methylation-mediated cranial neural crest proliferation and differentiation causes orofacial clefts in mice.

Orofacial clefts of the lip and palate are widely recognized to result from complex gene-environment interactions, but inadequate understanding of environmental risk factors has stymied development of prevention strategies. We interrogated the role of DNA methylation, an environmentally malleable epigenetic mechanism, in orofacial development. Expression of the key DNA methyltransferase enzyme DNMT1 was detected throughout palate morphogenesis in the epithelium and underlying cranial neural crest cell (cNCC) mesenchyme, a highly proliferative multipotent stem cell population that forms orofacial connective tissue. Genetic and pharmacologic manipulations of DNMT activity were then applied to define the tissue- and timing-dependent requirement of DNA methylation in orofacial development. cNCC-specific Dnmt1 inactivation targeting initial palate outgrowth resulted in OFCs, while later targeting during palatal shelf elevation and elongation did not. Conditional Dnmt1 deletion reduced cNCC proliferation and subsequent differentiation trajectory, resulting in attenuated outgrowth of the palatal shelves and altered development of cNCC-derived skeletal elements. Finally, we found that the cellular mechanisms of cleft pathogenesis observed in vivo can be recapitulated by pharmacologically reducing DNA methylation in multipotent cNCCs cultured in vitro. These findings demonstrate that DNA methylation is a crucial epigenetic regulator of cNCC biology, define a critical period of development in which its disruption directly causes OFCs, and provide opportunities to identify environmental influences that contribute to OFC risk.

A time-resolved single-cell roadmap of the logic driving anterior neural crest diversification from neural border to migration stages.

Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of early molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification toward definitive neural crest. Here, we combine ultradense single-cell transcriptomes with machine-learning and large-scale transcriptomic and epigenomic experimental validation of selected trajectories, to provide the general principles and highlight specific features of the GRN underlying neural crest fate diversification from induction to early migration stages using Xenopus frog embryos as a model. During gastrulation, a transient neural border zone state precedes the choice between neural crest and placodes which includes multiple converging gene programs. During neurulation, transcription factor connectome, and bifurcation analyses demonstrate the early emergence of neural crest fates at the neural plate stage, alongside an unbiased multipotent-like lineage persisting until epithelial-mesenchymal transition stage. We also decipher circuits driving cranial and vagal neural crest formation and provide a broadly applicable high-throughput validation strategy for investigating single-cell transcriptomes in vertebrate GRNs in development, evolution, and disease.

Molecular Characterization of Nodose Ganglia Development Reveals a Novel Population of Phox2b+ Glial Progenitors in Mice.

The vagal ganglia, comprised of the superior (jugular) and inferior (nodose) ganglia of the vagus nerve, receive somatosensory information from the head and neck or viscerosensory information from the inner organs, respectively. Developmentally, the cranial neural crest gives rise to all vagal glial cells and to neurons of the jugular ganglia, while the epibranchial placode gives rise to neurons of the nodose ganglia. Crest-derived nodose glial progenitors can additionally generate autonomic neurons in the peripheral nervous system, but how these progenitors generate neurons is unknown. Here, we found that some Sox10+ neural crest-derived cells in, and surrounding, the nodose ganglion transiently expressed Phox2b, a master regulator of autonomic nervous system development, during early embryonic life. Our genetic lineage-tracing analysis in mice of either sex revealed that despite their common developmental origin and extreme spatial proximity, a substantial proportion of glial cells in the nodose, but not in the neighboring jugular ganglia, have a history of Phox2b expression. We used single-cell RNA-sequencing to demonstrate that these progenitors give rise to all major glial subtypes in the nodose ganglia, including Schwann cells, satellite glia, and glial precursors, and mapped their spatial distribution by in situ hybridization. Lastly, integration analysis revealed transcriptomic similarities between nodose and dorsal root ganglia glial subtypes and revealed immature nodose glial subtypes. Our work demonstrates that these crest-derived nodose glial progenitors transiently express Phox2b, give rise to the entire complement of nodose glial cells, and display a transcriptional program that may underlie their bipotent nature.

Small molecule-mediated reprogramming of Xenopus blastula stem cells to a neural crest state.

Neural crest cells are a stem cell population unique to vertebrates that give rise to a diverse array of derivatives, including much of the peripheral nervous system, pigment cells, cartilage, mesenchyme, and bone. Acquisition of these cells drove the evolution of vertebrates and defects in their development underlies a broad set of neurocristopathies. Moreover, studies of neural crest can inform differentiation protocols for pluripotent stem cells and regenerative medicine applications. Xenopus embryos are an important system for studies of the neural crest and have provided numerous insights into the signals and transcription factors that control the formation and later lineage diversification of these stem cells. Pluripotent animal pole explants are a particularly powerful tool in this system as they can be cultured in simple salt solution and instructed to give rise to any cell type including the neural crest. Here we report a protocol for small molecule-mediated induction of the neural crest state from blastula stem cells and validate it using transcriptome analysis and grafting experiments. This is an powerful new tool for generating this important cell type that will facilitate future studies of neural crest development and mutations and variants linked to neurocristopathies.

Complete persistence of the primary somatosensory system in zebrafish.

The somatosensory system detects peripheral stimuli that are translated into behaviors necessary for survival. Fishes and amphibians possess two somatosensory systems in the trunk: the primary somatosensory system, formed by the Rohon-Beard neurons, and the secondary somatosensory system, formed by the neural crest cell-derived neurons of the Dorsal Root Ganglia. Rohon-Beard neurons have been characterized as a transient population that mostly disappears during the first days of life and is functionally replaced by the Dorsal Root Ganglia. Here, I follow Rohon-Beard neurons in vivo and show that the entire repertoire remains present in zebrafish from 1-day post-fertilization until the juvenile stage, 15-days post-fertilization. These data indicate that zebrafish retain two complete somatosensory systems until at least a developmental stage when the animals display complex behavioral repertoires.

Claudin-3 in the non-neural ectoderm is essential for neural fold fusion in chicken embryos.

The neural tube, the embryonic precursor to the brain and spinal cord, begins as a flat sheet of epithelial cells, divided into non-neural and neural ectoderm. Proper neural tube closure requires that the edges of the neural ectoderm, the neural folds, to elevate upwards and fuse along the dorsal midline of the embryo. We have previously shown that members of the claudin protein family are required for the early phases of chick neural tube closure. Claudins are transmembrane proteins, localized in apical tight junctions within epithelial cells where they are essential for regulation of paracellular permeability, strongly involved in apical-basal polarity, cell-cell adhesion, and bridging the tight junction to cytoplasmic proteins. Here we explored the role of Claudin-3 (Cldn3), which is specifically expressed in the non-neural ectoderm. We discovered that depletion of Cldn3 causes folic acid-insensitive primarily spinal neural tube defects due to a failure in neural fold fusion. Apical cell surface morphology of Cldn3-depleted non-neural ectodermal cells exhibited increased membrane blebbing and smaller apical surfaces. Although apical-basal polarity was retained, we observed altered Par3 and Pals1 protein localization patterns within the apical domain of the non-neural ectodermal cells in Cldn3-depleted embryos. Furthermore, F-actin signal was reduced at apical junctions. Our data presents a model of spina bifida, and the role that Cldn3 is playing in regulating essential apical cell processes in the non-neural ectoderm required for neural fold fusion.

Tlx3 mediates neuronal differentiation and proper condensation of the developing trigeminal ganglion.

The trigeminal ganglion, the largest of the vertebrate cranial ganglia, is comprised of sensory neurons that relay sensations of pain, touch, and temperature to the brain. These neurons are derived from two embryonic cell types, the neural crest and ectodermal placodes, whose interactions are critical for proper ganglion formation. While the T-cell leukemia homeobox 3 (Tlx3) gene is known to be expressed in placodally-derived sensory neurons and necessary for their differentiation, little was known about Tlx3 expression and/or function in the neural crest-derived component of the developing trigeminal ganglion. By combining lineage labeling with in situ hybridization in the chick embryo, we show that neural crest-derived cells that contribute to the cranial trigeminal ganglion express Tlx3 at a time point that coincides with the onset of ganglion condensation. Importantly, loss of Tlx3 function in vivo diminishes the overall size and abundance of neurons within the trigeminal ganglion. Conversely, ectopic expression of Tlx3 in migrating cranial neural crest results in their premature neuronal differentiation. Taken together, our results demonstrate a critical role for Tlx3 in neural crest-derived cells during chick trigeminal gangliogenesis.

From neural tube to spinal cord: The dynamic journey of the dorsal neuroepithelium.

In a developing embryo, formation of tissues and organs is remarkably precise in both time and space. Through cell-cell interactions, neighboring progenitors coordinate their activities, sequentially generating distinct types of cells. At present, we only have limited knowledge, rather than a systematic understanding, of the underlying logic and mechanisms responsible for cell fate transitions. The formation of the dorsal aspect of the spinal cord is an outstanding model to tackle these dynamics, as it first generates the peripheral nervous system and is later responsible for transmitting sensory information from the periphery to the brain and for coordinating local reflexes. This is reflected first by the ontogeny of neural crest cells, progenitors of the peripheral nervous system, followed by formation of the definitive roof plate of the central nervous system and specification of adjacent interneurons, then a transformation of roof plate into dorsal radial glia and ependyma lining the forming central canal. How do these peripheral and central neural branches segregate from common progenitors? How are dorsal radial glia established concomitant with transformation of the neural tube lumen into a central canal? How do the dorsal radial glia influence neighboring cells? This is only a partial list of questions whose clarification requires the implementation of experimental paradigms in which precise control of timing is crucial. Here, we outline some available answers and still open issues, while highlighting the contributions of avian models and their potential to address mechanisms of neural patterning and function.