Trunk Neural Crest Research Articles

Comparison of human pluripotent stem cell differentiation protocols to generate neuroblastoma tumors

Neuroblastoma is the most common pediatric extracranial solid tumor and is derived from trunk neural crest cells (tNCC) and its progenitor sympathoadrenal (SA) cells. While human pluripotent stem cell (PSC) models of neuroblastoma have been described, the PSC were differentiated using protocols that made neural crest cells, but not specifically the trunk subtype. Here, we compared four recent protocols to differentiate pluripotent stem cells (PSC) toward SA cells and examined their efficiency at generating SA cells along with earlier cell states (neuromesodermal progenitors [NMP], tNCC), as well as generating MYCN-driven tumors. Interestingly, the protocols that created cells with the highest level of NMP markers did not produce cells with the highest tNCC or SA cell markers. We identified a protocol that consistently produced cells with the highest level of SA markers using two PSC lines of different genders. This protocol also generated tumors with the highest level of PHOX2B, a marker of neuroblastoma. Transcriptionally, however, each protocol generates tumors that resemble neuroblastoma. Two of the protocols repeatedly produced adrenergic neuroblastoma whereas the other two protocols were ambiguous. Thus, we identified a protocol that reliably generates adrenergic neuroblastoma.

Conditional heterozygous loss of kit receptor tyrosine kinase in neural crest cell lineage is associated with midline cleft lip and bifid nose deformity

ObjectivesThe receptor tyrosine kinase Kit is expressed in cells derived from the trunk neural crest (NC), such as melanocytes; however, its role in cranial NC cell development is not fully understood. MethodsWe investigated the effects of the heterozygous loss of Kit in NC cells during embryonic development by mating Kit2lox/+ mice with Wnt1-Cre mice to produce Wnt1-Cre; Kit2lox/+ embryos. In addition, Wnt1-Cre mice were mated with Rosa26R-yellow fluorescent protein (YFP) mice to visualize the tissue regions expressing Cre recombinase. Histological studies of the craniofacial regions of these mice were performed using samples from embryonic day (E) 12.5 and postnatal day (P) 1. Cellular apoptosis and proliferation were both analyzed through the immunostaining of tissue sections collected on E13.5 and E14.5 using anti-cleaved caspase 3 (CC3) to detect apoptosis and anti-Ki67 to detect proliferation. Cells from YFP-positive tissue regions of the facial areas of Wnt1-Cre; Kit+/+; Rosa26R–YFP embryos and Wnt1-Cre; Kit2lox/+; Rosa26R–YFP embryos collected on E12.5 and E15.5 were cultured and evaluated for cell proliferation. ResultsCompared with control littermates, Wnt1-Cre; Kit2lox/+ embryos exhibited midline cleft lip and bifid nose deformities. Substantial early (P1) postnatal lethality was observed in Wnt1-Cre; Kit2lox/+ mice, with none surviving to 3 weeks of age. YFP-positive cells from the maxillary regions of Wnt1-Cre; Kit2lox/+; Rosa26R–YFP embryos exhibited defective cell growth and self-renewal in vitro. ConclusionConditional heterozygous loss of Kit in Wnt1-Cre; Kit2lox/+ embryos is associated with craniofacial dysplasia and exhibit defective NC development in vitro and in vivo.

Genomic mosaicism reveals developmental organization of trunk neural crest-derived ganglia.

The neural crest generates numerous cell types, but conflicting results leave developmental origins unresolved. Here using somatic mosaic variants as cellular barcodes, we infer embryonic clonal dynamics of trunk neural crest, focusing on the sensory and sympathetic ganglia. From three independent adult neurotypical human donors, we identified 1,278 mosaic variants using deep whole-genome sequencing, then profiled allelic fractions in 187 anatomically dissected ganglia. We found a massive rostrocaudal spread of progenitor clones specific to sensory or sympathetic ganglia, which unlike in the brain, showed robust bilateral distributions. Computational modeling suggested neural crest progenitor fate specification preceded delamination from neural tube. Single-cell multiomic analysis suggested both neurons and glia contributed to the rostrocaudal clonal organization. CRISPR barcoding in mice and live imaging in quail embryos confirmed these clonal dynamics across multiple somite levels. Our findings reveal an evolutionarily conserved clonal spread of cells populating peripheral neural ganglia.

A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

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 β-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.

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 neurodevelopmentmodels 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.

Generation of Functional and Mature Sympathetic Neurons from Human Pluripotent Stem Cells via a Neuroepithelial Route.

The sympathetic nervous system (SNS) is a crucial branch of the autonomic nervous system (ANS) that is responsible for regulating visceral function and various physiological processes. Dysfunction of the SNS can lead to various diseases, such as hypertension and metabolic disorders. However, obtaining sympathetic neurons from human tissues for research is challenging. The current research aimed at recapitulating the process of human sympathetic neuron development and achieved the successful establishment of a stepwise, highly efficient in vitro differentiation protocol. This protocol facilitated the generation of functional and mature sympathetic neurons from human pluripotent stem cells (hPSCs) using a chemical-defined induction medium. Initially, each differentiation stage was refined to derive sympathoadrenal progenitors (SAPs) from hPSCs through neural epithelial cells (NECs) and trunk neural crest stem cells (NCSCs). hPSC-derived SAPs could be expanded in vitro for at least 12 passages while maintaining the expression of SAP-specific transcription factors and neuronal differentiation potency. SAPs readily generated functional sympathetic neurons (SymNs) when cultured in the neuronal maturation medium for 3-4 weeks. These SymNs expressed sympathetic markers, exhibited electrophysiological properties, and secreted sympathetic neurotransmitters. More importantly, we further demonstrated that hPSC-derived SymNs can efficiently regulate the adipogenesis of human adipose-derived stem cells (ADSCs) and lipid metabolism in vitro. In conclusion, our study provided a simple and robust protocol for generating functional sympathetic neurons from hPSCs, which may be an invaluable tool in unraveling the mechanisms of SNS-related diseases.

Autonomous and non-cell autonomous role of cilia in structural birth defects in mice.

Ciliopathies are associated with wide spectrum of structural birth defects (SBDs), indicating important roles for cilia in development. Here, we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140, an intraflagellar transport (IFT) protein regulating ciliogenesis. Ift140-deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula (TEF), randomized heart looping, congenital heart defects (CHDs), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAGGCre-ER deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD were not observed with 4 Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest-mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathies.

Revealing the developmental origin and lineage predilection of neural progenitors within human bone marrow via single-cell analysis: implications for regenerative medicine

BackgroundHuman bone marrow stromal cells (BMSCs) are an easily accessible and expandable progenitor population with the capacity to generate neural cell types in addition to mesoderm. Lineage tracing studies in transgenic animals have indicated Nestin + BMSCs to be descended from the truncal neural crest. Single-cell analysis provides a means to identify the developmental origin and identity of human BMSC-derived neural progenitors when lineage tracing remains infeasible. This is a prerequisite towards translational application.MethodsWe attained transcriptomic profiles of embryonic long bone, adult human bone marrow, cultured BMSCs and BMSC-derived neurospheres. Integrated scRNAseq analysis was supplemented by characterization of cells during culture expansion and following provision of growth factors and signalling agonists to bias lineage.ResultsReconstructed pseudotime upon the integrated dataset indicated distinct neural and osteogenic differentiation trajectories. The starting state towards the neural differentiation trajectory consisted of Nestin + /MKI67 + BMSCs, which could also be diverted towards the osteogenic trajectory via a branch point. Nestin + /PDGFRA + BMSCs responded to neurosphere culture conditions to generate a subpopulation of cells with a neuronal phenotype according to marker expression and gene ontogeny analysis that occupied the end state along the neural differentiation trajectory. Reconstructed pseudotime also revealed an upregulation of BMP4 expression during culture of BMSC-neurospheres. This provided the rationale for culture supplementation with the BMP signalling agonist SB4, which directed progenitors to upregulate Pax6 and downregulate Nestin.ConclusionsThis study suggested BMSCs originating from truncal neural crest to be the source of cells within long bone marrow possessing neural differentiation potential. Unravelling the transcriptomic dynamics of BMSC-derived neural progenitors promises to enhance differentiation efficiency and safety towards clinical application in cell therapy and disease modelling.

Glucose oxidation drives trunk neural crest cell development and fate.

Bioenergetic metabolism is a key regulator of cellular function and signaling, but how it can instruct the behavior of cells and their fate during embryonic development remains largely unknown. Here, we investigated the role of glucose metabolism in the development of avian trunk neural crest cells (NCCs), a migratory stem cell population of the vertebrate embryo. We uncovered that trunk NCCs display glucose oxidation as a prominent metabolic phenotype, in contrast to what is seen for cranial NCCs, which instead rely on aerobic glycolysis. In addition, only one pathway downstream of glucose uptake is not sufficient for trunk NCC development. Indeed, glycolysis, mitochondrial respiration and the pentose phosphate pathway are all mobilized and integrated for the coordinated execution of diverse cellular programs, epithelial-to-mesenchymal transition, adhesion, locomotion, proliferation and differentiation, through regulation of specific gene expression. In the absence of glucose, the OXPHOS pathway fueled by pyruvate failed to promote trunk NCC adaptation to environmental stiffness, stemness maintenance and fate-decision making. These findings highlight the need for trunk NCCs to make the most of the glucose pathway potential to meet the high metabolic demands appropriate for their development.

First person – Nioosha Nekooie Marnany

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Nioosha Nekooie Marnany is first author on ‘ Glucose oxidation drives trunk neural crest cell development and fate’, published in JCS. Nioosha conducted the research described in this article while a PhD student in Dr Sylvie Dufour and Dr Jean-Loup Duband's lab at Institut Mondor de Recherches Biomédicales, Université Paris-Est Créteil, France. Currently, she is a postdoc in the ‘Institut de Biologie, Ecole Normale Supérieure (IBENS)' in Paris, at Hervé Le Hire's lab where she is working on the relationship between the exon junction complex (EJC) and metabolism.

Ancient vertebrate dermal armor evolved from trunk neural crest

Bone is an evolutionary novelty of vertebrates, likely to have first emerged as part of ancestral dermal armor that consisted of osteogenic and odontogenic components. Whether these early vertebrate structures arose from mesoderm or neural crest cells has been a matter of considerable debate. To examine the developmental origin of the bony part of the dermal armor, we have performed in vivo lineage tracing in the sterlet sturgeon, a representative of nonteleost ray-finned fish that has retained an extensive postcranial dermal skeleton. The results definitively show that sterlet trunk neural crest cells give rise to osteoblasts of the scutes. Transcriptional profiling further reveals neural crest gene signature in sterlet scutes as well as bichir scales. Finally, histological and microCT analyses of ray-finned fish dermal armor show that their scales and scutes are formed by bone, dentin, and hypermineralized covering tissues, in various combinations, that resemble those of the first armored vertebrates. Taken together, our results support a primitive skeletogenic role for the neural crest along the entire body axis, that was later progressively restricted to the cranial region during vertebrate evolution. Thus, the neural crest was a crucial evolutionary innovation driving the origin and diversification of dermal armor along the entire body axis.

Mutations in cdon and boc affect trunk neural crest cell migration and slow-twitch muscle development in zebrafish.

The transmembrane proteins cdon and boc are implicated in regulating hedgehog signaling during vertebrate development. Recent work showing roles for these genes in axon guidance and neural crest cell migration suggest that cdon and boc may play additional functions in regulating directed cell movements. We use newly generated and existing mutants to investigate a role for cdon and boc in zebrafish neural crest cell migration. We find that single mutant embryos exhibit normal neural crest phenotypes, but that neural crest migration is strikingly disrupted in double cdon;boc mutant embryos. We further show that this migration phenotype is associated with defects in the differentiation of slow-twitch muscle cells, and the loss of a Col1a1a-containing extracellular matrix, suggesting that neural crest defects may be a secondary consequence to defects in mesoderm development. Combined, our data add to a growing literature showing that cdon and boc act synergistically to promote hedgehog signaling during vertebrate development, and suggest that the zebrafish can be used to study the function of hedgehog receptor paralogs.

Description of trunk neural crest migration and peripheral nervous system formation in the Egyptian cobra Naja haje haje

The neural crest is a stem cell population that forms in the neurectoderm of all vertebrates and gives rise to a diverse set of cells such as sensory neurons, Schwann cells and melanocytes. Neural crest development in snakes is still poorly understood. From the point of view of evolutionary and comparative anatomy is an interesting topic given the unique anatomy of snakes. The aim of the study was to characterize how trunk neural crest cells (TNCC) migrate in the developing elapid snake Naja haje haje and consequently, look at the beginnings of development of neural crest derived sensory ganglia (DRG) and spinal nerves. We found that trunk neural crest and DRG development in Naja haje haje is like what has been described in other vertebrates and the colubrid snake strengthening our knowledge on the conserved mechanisms of neural crest development across species. Here we use the marker HNK1 to follow the migratory behavior of TNCC in the elapid snake Naja haje haje through stages 1–6 (1–9 days postoviposition). We observed that the TNCC of both snake species migrate through the rostral portion of the somite, a pattern also conserved in birds and mammals. The development of cobra peripheral nervous system, using neuronal and glial markers, showed the presence of spectrin in Schwann cell precursors and of axonal plexus along the length of the cobra embryos. In conclusion, cobra embryos show strong conserved patterns in TNCC and PNS development among vertebrates.

Reprogramming of trunk neural crest to a cranial crest-like identity alters their transcriptome and developmental potential

Neural crest cells along the body axis of avian embryos differ in their developmental potential, such that the cranial neural crest forms cartilage and bone whereas the trunk neural crest is unable to do so. Previous studies have identified a cranial crest-specific subcircuit that can imbue the trunk neural crest with the ability to form cartilage after grafting to the head. Here, we examine transcriptional and cell fate changes that accompany this reprogramming. First, we examined whether reprogrammed trunk neural crest maintain the ability to form cartilage in their endogenous environment in the absence of cues from the head. The results show that some reprogrammed cells contribute to normal trunk neural crest derivatives, whereas others migrate ectopically to the forming vertebrae and express cartilage markers, thus mimicking heterotypically transplanted cranial crest cells. We find that reprogrammed trunk neural crest upregulated more than 3000 genes in common with cranial neural crest, including numerous transcriptional regulators. In contrast, many trunk neural crest genes are downregulated. Together, our findings show that reprogramming trunk neural crest with cranial crest subcircuit genes alters their gene regulatory program and developmental potential to be more cranial crest-like.

HPSC-derived sacral neural crest enables rescue in a severe model of Hirschsprung's disease.

The enteric nervous system (ENS) is derived from both the vagal and sacral component of the neural crest (NC). Here, we present the derivation of sacral ENS precursors from human PSCs via timed exposure to FGF, WNT, and GDF11, which enables posterior patterning and transition from posterior trunk to sacral NC identity, respectively. Using a SOX2::H2B-tdTomato/T::H2B-GFP dual reporter hPSC line, we demonstrate that both trunk and sacral NC emerge from a double-positive neuro-mesodermal progenitor (NMP). Vagal and sacral NC precursors yield distinct neuronal subtypes and migratory behaviors invitro and invivo. Remarkably, xenografting of both vagal and sacral NC lineages is required to rescue a mouse model of total aganglionosis, suggesting opportunities in the treatment of severe forms of Hirschsprung's disease.

Characteristic tetraspanin expression patterns mark various tissues during early Xenopus development.

The tetraspanins (Tspans) constitute a family of cell surface proteins with four transmembrane domains. Tspans have been found on the plasma membrane and on exosomes of various organelles. Reports on the function of Tspans during the early development of Xenopus have mainly focused on the expression of uroplakins in gametes. Although the roles of extracellular vesicles (EVs) including exosomes have been actively analyzed in cancer research, the contribution of EVs to early development is not well understood. This is because the diffusivity of EVs is not compatible with a very strict developmental process. In this study, we analyzed members of the Tspan family in early development of Xenopus. Expression was prominent in specific organs such as the notochord, eye, cranial neural crest cells (CNCs), trunk neural crest cells, placodes, and somites. We overexpressed several combinations of Tspans in CNCs in vitro and in vivo. Changing the partner changed the distribution of fluorescent-labeled Tspans. Therefore, it is suggested that expression of multiple Tspans in a particular tissue might produce heterogeneity of intercellular communication, which has not yet been recognized.

Analysis of Integrin-Dependent Melanoblast Migration During Development.

The neural crest is a transient embryonic structure that gives rise to a number of important cell types and tissues, including most of the peripheral and enteric nervous systems, pigment-producing skin cells known as melanocytes, and many craniofacial structures. Melanoblasts, the precursors of melanocytes, are derived from the so-called trunk neural crest cells. These cells delaminate and migrate along a dorsolateral pathway to colonize their final destination in the skin, and consequently, defects in melanoblast migration result in pigmentation defects. Studying melanocyte migration is a topic of great interest due to the involvement of melanocytes in highly metastatic skin cancer. A role for integrin-mediated adhesion is well established in neural crest migration, and our recent work has provided direct evidence for a key role for integrin-based adhesion in melanocyte migration. Imaging of melanoblast migration in the context of intact skin has proven to be a particularly powerful tool to study integrin-based adhesion during melanoblast migration. Here, we describe the use of skin explants combined with genetically encoded markers for melanocytes and high-resolution live imaging as a powerful and informative approach to analyze melanoblast migration in an ex vivo context.

The cardiac neural crest gene MafB ectopically directs CXCR4 expression in the trunk neural crest

The cardiac neural crest is a subpopulation of cells arising from the caudal hindbrain. The delaminated cardiac neural crest cells migrate to the heart using the CXCR/SDF1 chemokine signaling system. These cells contribute to the formation of the cardiovascular system, including the septation of the outflow tract, which is unique to these cells. Here, we investigated the effect of ectopic expression of the cardiac neural crest gene MafB on trunk neural crest cells. First, we found that MafB has the potential to activate its own cis-regulatory element in enteric and trunk neural crest cells but not in cranial neural crest cells. Forced expression of two cardiac neural crest genes, Ets1 and Sox8, together with or without MafB, induced ectopic Sox10E2 enhancer activity in the trunk region. Finally, we uncovered that the expression of MafB, Ets1 and Sox8 can induce ectopic CXCR4 expression in the trunk neural crest cells, resulting in acquisition of responsiveness to the SDF1 signal. These results demonstrate that MafB, Ets1 and Sox8 are critical components for generation of the identity of the cardiac neural crest, especially the cell migration property.

Early anteroposterior regionalisation of human neural crest is shaped by a pro-mesodermal factor.

The neural crest (NC) is an important multipotent embryonic cell population and its impaired specification leads to various developmental defects, often in an anteroposterior (A-P) axial level-specific manner. The mechanisms underlying the correct A-P regionalisation of human NC cells remain elusive. Recent studies have indicated that trunk NC cells, the presumed precursors of childhood tumour neuroblastoma, are derived from neuromesodermal-potent progenitors of the postcranial body. Here we employ human embryonic stem cell differentiation to define how neuromesodermal progenitor (NMP)-derived NC cells acquire a posterior axial identity. We show that TBXT, a pro-mesodermal transcription factor, mediates early posterior NC/spinal cord regionalisation together with WNT signalling effectors. This occurs by TBXT-driven chromatin remodelling via its binding in key enhancers within HOX gene clusters and other posterior regulator-associated loci. This initial posteriorisation event is succeeded by a second phase of trunk HOX gene control that marks the differentiation of NMPs toward their TBXT-negative NC/spinal cord derivatives and relies predominantly on FGF signalling. Our work reveals a previously unknown role of TBXT in influencing posterior NC fate and points to the existence of temporally discrete, cell type-dependent modes of posterior axial identity control.