Neural Crest Precursors Research Articles

Neural crest precursors from the skin are the primary source of directly reprogrammed neurons

Direct reprogramming involves the conversion of differentiated cell types without returning to an earlier developmental state. Here, we explore how heterogeneity in developmental lineage and maturity of the starting cell population contributes to direct reprogramming using the conversion of murine fibroblasts into neurons. Our hypothesis is that a single lineage of cells contributes to most reprogramming and that a rare elite precursor with intrinsic bias is the source of reprogrammed neurons. We find that nearly all reprogrammed neurons are derived from the neural crest (NC) lineage. Moreover, when rare proliferating NC precursors are selectively ablated, there is a large reduction in the number of reprogrammed neurons. Previous interpretations of this paradigm are that it demonstrates a cell fate conversion across embryonic germ layers (mesoderm to ectoderm). Our interpretation is that this is actually directed differentiation of a neural lineage stem cell in the skin that has intrinsic bias to produce neuronal progeny.

SOX10 Loss Sensitizes Melanoma Cells to Cytokine-Mediated Inflammatory Cell Death.

These data support a role for SOX10 expression altering the response to T cell-mediated cell death and contribute to a broader understanding of the interaction between immune cells and melanoma cells.

Exposure to Tetrabutylammonium Bromide Impairs Cranial Neural Crest Specification, Neurogenic Program, and Brain Morphogenesis.

Tetrabutylammonium bromide (TBAB) is a widely used industrial reagent and is commonly found in our aquatic ecosystem as an industrial byproduct. In humans, the ingestion of TBAB causes severe neurological impairments and disorders such as vertigo, hallucinations, and delirium. Yet, the extent of environmental risk and TBAB toxicity to human health is poorly understood. In this study, we aim to determine the developmental toxicity of TBAB using zebrafish embryos as a model and provide novel insights into the mechanism of action of such chemicals on neurodevelopment and the overall embryonic program. Our results show that exposure to TBAB results in impaired development of the brain, inner ear, and pharyngeal skeletal elements in the zebrafish embryo. TBAB treatment resulted in aberrations in the specification of the neural crest precursors, hindbrain segmentation, and otic neurogenesis. TBAB treatment also induced a surge in apoptosis in the head, tail, and trunk regions of the developing embryo. Long-term TBAB exposure resulted in cardiac edema and craniofacial defects. Further, in silico molecular docking analysis indicated that TBAB binds to AMPA receptors and modulates neural developmental genes such as olfactomedin and acetylcholinesterase in the embryonic brain. To summarize, our study highlights the novel effects of TBAB on embryonic brain formation and segmentation, ear morphogenesis, and craniofacial skeletal development.

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.

Single-cell multiome sequencing clarifies enteric glial diversity and identifies an intraganglionic population poised for neurogenesis

The enteric nervous system (ENS) consists of glial cells (EGCs) and neurons derived from neural crest precursors. EGCs retain capacity for large-scale neurogenesis in culture, and invivo lineage tracing has identified neurons derived from glial cells in response to inflammation. We thus hypothesize that EGCs possess a chromatin structure poised for neurogenesis. We use single-cell multiome sequencing to simultaneously assess transcription and chromatin accessibility in EGCs undergoing spontaneous neurogenesis in culture, as well as small intestine myenteric plexus EGCs. Cultured EGCs maintain open chromatin at genomic loci accessible in neurons, and neurogenesis from EGCs involves dynamic chromatin rearrangements with a net decrease in accessible chromatin. A subset of invivo EGCs, highly enriched within the myenteric ganglia and that persist into adulthood, have a gene expression program and chromatin state consistent with neurogenic potential. These results clarify the mechanisms underlying EGC potential for neuronal fate transition.

Differentiation and on axon-guidance chip culture of human pluripotent stem cell-derived peripheral cholinergic neurons for airway neurobiology studies.

Airway cholinergic nerves play a key role in airway physiology and disease. In asthma and other diseases of the respiratory tract, airway cholinergic neurons undergo plasticity and contribute to airway hyperresponsiveness and mucus secretion. We currently lack human in vitro models for airway cholinergic neurons. Here, we aimed to develop a human in vitro model for peripheral cholinergic neurons using human pluripotent stem cell (hPSC) technology. hPSCs were differentiated towards vagal neural crest precursors and subsequently directed towards functional airway cholinergic neurons using the neurotrophin brain-derived neurotrophic factor (BDNF). Cholinergic neurons were characterized by ChAT and VAChT expression, and responded to chemical stimulation with changes in Ca2+ mobilization. To culture these cells, allowing axonal separation from the neuronal cell bodies, a two-compartment PDMS microfluidic chip was subsequently fabricated. The two compartments were connected via microchannels to enable axonal outgrowth. On-chip cell culture did not compromise phenotypical characteristics of the cells compared to standard culture plates. When the hPSC-derived peripheral cholinergic neurons were cultured in the chip, axonal outgrowth was visible, while the somal bodies of the neurons were confined to their compartment. Neurons formed contacts with airway smooth muscle cells cultured in the axonal compartment. The microfluidic chip developed in this study represents a human in vitro platform to model neuro-effector interactions in the airways that may be used for mechanistic studies into neuroplasticity in asthma and other lung diseases.

Epigenetic and pharmacological control of pigmentation via Bromodomain Protein 9 (BRD9).

Lineage-specific differentiation programs are activated by epigenetic changes in chromatin structure. Melanin-producing melanocytes maintain a gene expression program ensuring appropriate enzymatic conversion of metabolites into the pigment, melanin, and transfer to surrounding cells. During neuroectodermal development, SMARCA4 (BRG1), the catalytic subunit of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes, is essential for lineage specification. SMARCA4 is also required for development of multipotent neural crest precursors into melanoblasts, which differentiate into pigment-producing melanocytes. In addition to the catalytic domain, SMARCA4 and several SWI/SNF subunits contain bromodomains which are amenable to pharmacological inhibition. We investigated the effects of pharmacological inhibitors of SWI/SNF bromodomains on melanocyte differentiation. Strikingly, treatment of murine melanoblasts and human neonatal epidermal melanocytes with selected bromodomain inhibitors abrogated melanin synthesis and visible pigmentation. Using functional genomics, iBRD9, a small molecule selective for the bromodomain of BRD9 was found to repress pigmentation-specific gene expression. Depletion of BRD9 confirmed a requirement for expression of pigmentation genes in the differentiation program from melanoblasts into pigmented melanocytes and in melanoma cells. Chromatin immunoprecipitation assays showed that iBRD9 disrupts the occupancy of BRD9 and the catalytic subunit SMARCA4 at melanocyte-specific loci. These data indicate that BRD9 promotes melanocyte pigmentation whereas pharmacological inhibition of BRD9 is repressive.

The Polycomb group gene rnf2 is essential for central and enteric neural system development in zebrafish.

The development of central nervous system (CNS) and enteric nervous system (ENS) is under precise and strict control in vertebrates. Whether and how the Polycomb repressive complex 1 (PRC1) is involved in it remain unclear. To investigate the role of PRC1 in the nervous system development, using CRISPR/Cas9 technology, we have generated mutant zebrafish lines for the rnf2 gene which encodes Ring1b, the enzymatic component of the PRC1 complex. We show that rnf2 loss of function leads to abnormal migration and differentiation of neural crest and neural precursor cells. rnf2 mutant embryos exhibit aganglionosis, in which the hindgut is devoid of neurons. In particular, the formation of 5-HT serotonin neurons and myelinating glial cells is defective. Furthermore, ectopic expression of ENS marker genes is observed in forebrain of rnf2 mutant embryos. These findings suggest that the rnf2 gene plays an important role in the migration and differentiation of neural precursor cells, and its absence leads to abnormal development of ENS and CNS in zebrafish.

Neural Crest‐Specific Elp1 Deletion Disrupts Trigeminal Ganglion Development in a Model of Familial Dysautonomia

Neurons in the trigeminal ganglion relay sensory information from the face and oral cavity to the brain. The trigeminal ganglion is unique in its complexity, containing a mixed population of modality‐specific (i.e., touch, pain) neurons derived from both neural crest and placodal precursors. The identity of trigeminal ganglion neurons is associated with the expression of Trk neurotrophin receptors, which are required for long‐term neuronal function and survival. Within sensory ganglia, nociceptive neurons that detect pain or temperature generally express TrkA, while mechanoreceptors responding to touch express TrkB or TrkC. However, the processes that guide the emergence and maintenance of distinct neuronal subpopulations in the trigeminal ganglion are poorly understood. We recently uncovered a role for Elongator complex protein 1 (Elp1) in the maintenance of TrkA‐expressing trigeminal ganglion nociceptors. Mutation of ELP1 causes Familial Dysautonomia (FD), a fatal neuropathy characterized, in part, by trigeminal sensory deficits, including impaired facial nociception. In a mouse model of FD, where Elp1 is deleted from neural crest derivatives (Elp1 CKO), we found that Elp1 is required for proper trigeminal nerve morphology. Moreover, loss of Elp1 reduces TrkA levels and enhances death of TrkA neurons during embryogenesis, explaining the facial sensory impairments experienced by FD patients. Given the targeted effects on TrkA neurons in Elp1 CKO, we hypothesized that most TrkA neurons in the trigeminal ganglion arise from the neural crest, while TrkB and TrkC neurons originate from placode cells. To address this, we first examined whether individual Trk receptors are co‐expressed with the transcription factor Six1, which has previously been used to identify placodal neurons in the trigeminal ganglion. We discovered that Six1 is expressed in waves, marking newly differentiated trigeminal ganglion neurons regardless of presumptive placode or neural crest origin. Without bona fide immunohistochemical lineage markers, a Wnt1‐Cre reporter mouse was used to identify neural crest (Wnt1‐Cre recombined) versus placode (not recombined) derivatives. In support of our hypothesis, we observed a majority of TrkA neurons in the trigeminal ganglion had previously undergone Cre recombination, indicating trigeminal ganglion nociceptors are largely neural crest‐derived. In contrast, most TrkB and TrkC neurons were not targeted for recombination, confirming trigeminal ganglion mechanoreceptors generally arise from placodes. Lastly, the majority of Six1‐expressing cells were, indeed, Wnt1‐Cre recombined once neural crest neuronal differentiation commenced in the trigeminal ganglion, confirming that Six1 is not exclusively expressed by placodal neurons at later stages. Future studies will expand upon this work by crossing the Wnt1‐Cre reporter with Elp1 CKO to evaluate how Elp1 regulates the development and interactions of neural crest versus placodal derivatives within the trigeminal ganglion. Together, these findings provide novel insight into the origin and maintenance of trigeminal ganglion neurons and enhance our understanding of the pathogenesis of FD and other sensory nerve disorders.

Schwann Cells Are Required for Efficient Corneal Wound Healing

BACKGROUND: Corneal nerves play a crucial role in maintaining corneal health, which includes regulation of activity of limbal stem cell (LSC). Their loss leads to neurotrophic keratopathy (NK), with corneal ulceration, scarring, and ultimately, blindness.1 Having identified nerve-ensheathing Schwann cells (SC) in the corneal limbus, we hypothesize that SCs, via paracrine interaction with LSC, play a key role in corneal epithelial maintenance and healing. In this study we wanted to (1) Define the role of SCs in corneal healing; (2) Determine the paracrine interaction between the limbal SCs and LSC. METHODS: (1) Local corneal ablation of SCs was induced in a genetically modified mouse where the topical application of tamoxifen induced SCs apoptosis.2 The corneal epithelium was then removed with an Amoils brush under anesthesia and fluorescein was used to assess healing over 4 days. (2) We performed single-cell RNA expression analysis of 10,000 cells derived from dissociated rat limbus with droplet-based high throughput 10× Genomics to identify ~3000 genes.3 We used the data to predict possible ligand-receptor interactions between the limbal SCs and LSC. RESULTS: (1) Ablation of SCs impaired corneal wound healing in mouse cornea, suggesting the involvement of SC in innervation-dependent corneal epithelial recovery. (2) Genomic analysis suggested the presence of paracrine crosstalk between SCs and LSCs, and relevant downstream intracellular signaling events in LSCs. The latter included activation of Notch signaling and VEGF-mediated cell migration and inhibition of apoptosis.4 Further expression analysis comparing the limbal region of healthy and wounded corneas indicated significant changes in the expression of jag1, Pdgfa, Tgfb1, and Ptn genes by SCs. All of these genes could potentially play a role in corneal recovery. CONCLUSIONS: Our findings (i) describe the presence of a high volume of SCs at the limbus, located in close spatial vicinity to LSCs, (ii) demonstrate the importance of the limbal SCs for corneal wound healing, and (iii) suggest the presence of paracrine SC-LSC interaction that may be responsible for the limbal nerve-mediated activation of LSCs during homeostasis or the epithelial wound healing after injury. These findings suggest new therapeutic targets for treating NK. REFERENCES: 1. Dua HS, Said DG, Messmer EM, et al. Neurotrophic keratopathy. Prog Retin Eye Res. 2018 Sep;66:107-131. 2. Johnston APW, Naska S, Jones K, et al. Sox2-mediated regulation of adult neural crest precursors and skin repair. Stem Cell Rep. 2013;1(1):38–45. doi:10.1016/j.stemcr.2013.04.004 3. Yuzwa SA, Borrett MJ, Innes BT, et al. Developmental emergence of adult neural stem cells as revealed by single-cell transcriptional profiling. Cell Rep. 2017;21(13):3970–3986. doi:10.1016/j.celrep.2017.12.017 4. Lindsell CE, Shawber CJ, Boulter J, et al. Jagged: A mammalian ligand that activates notch1. Cell. 1995;80(6):909–917. doi:10.1016/0092-8674(95)90294-5

A comprehensive series of Irx cluster mutants reveals diverse roles in facial cartilage development.

Proper function of the vertebrate skeleton requires the development of distinct articulating embryonic cartilages. Irx transcription factors are arranged in co-regulated clusters that are expressed in the developing skeletons of the face and appendages. IrxB cluster genes are required for the separation of toes in mice and formation of the hyoid joint in zebrafish, yet whether Irx genes have broader roles in skeletal development remains unclear. Here, we perform a comprehensive loss-of-function analysis of all 11 Irx genes in zebrafish. We uncover conserved requirements for IrxB genes in formation of the fish and mouse scapula. In the face, we find a requirement for IrxAb genes and irx7 in formation of anterior neural crest precursors of the jaw, and for IrxBa genes in formation of endodermal pouches and gill cartilages. We also observe extensive joint loss and cartilage fusions in animals with combinatorial losses of Irx clusters, with in vivo imaging revealing that at least some of these fusions arise through inappropriate chondrogenesis. Our analysis reveals diverse roles for Irx genes in the formation and later segmentation of the facial skeleton.

Influence of Sox protein SUMOylation on neural development and regeneration.

SRY-related HMG-box (Sox) transcription factors are known to regulate central nervous system development and are involved in several neurological diseases. Post-translational modification of Sox proteins is known to alter their functions in the central nervous system. Among the different types of post-translational modification, small ubiquitin-like modifier (SUMO) modification of Sox proteins has been shown to modify their transcriptional activity. Here, we review the mechanisms of three Sox proteins in neuronal development and disease, along with their transcriptional changes under SUMOylation. Across three species, lysine is the conserved residue for SUMOylation. In Drosophila, SUMOylation of SoxN plays a repressive role in transcriptional activity, which impairs central nervous system development. However, deSUMOylation of SoxE and Sox11 plays neuroprotective roles, which promote neural crest precursor formation in Xenopus and retinal ganglion cell differentiation as well as axon regeneration in the rodent. We further discuss a potential translational therapy by SUMO site modification using AAV gene transduction and Clustered regularly interspaced short palindromic repeats-Cas9 technology. Understanding the underlying mechanisms of Sox SUMOylation, especially in the rodent system, may provide a therapeutic strategy to address issues associated with neuronal development and neurodegeneration.

Haploinsufficiency of SF3B2 causes craniofacial microsomia

Craniofacial microsomia (CFM) is the second most common congenital facial anomaly, yet its genetic etiology remains unknown. We perform whole-exome or genome sequencing of 146 kindreds with sporadic (n = 138) or familial (n = 8) CFM, identifying a highly significant burden of loss of function variants in SF3B2 (P = 3.8 × 10−10), a component of the U2 small nuclear ribonucleoprotein complex, in probands. We describe twenty individuals from seven kindreds harboring de novo or transmitted haploinsufficient variants in SF3B2. Probands display mandibular hypoplasia, microtia, facial and preauricular tags, epibulbar dermoids, lateral oral clefts in addition to skeletal and cardiac abnormalities. Targeted morpholino knockdown of SF3B2 in Xenopus results in disruption of cranial neural crest precursor formation and subsequent craniofacial cartilage defects, supporting a link between spliceosome mutations and impaired neural crest development in congenital craniofacial disease. The results establish haploinsufficient variants in SF3B2 as the most prevalent genetic cause of CFM, explaining ~3% of sporadic and ~25% of familial cases.

Clonal analysis and dynamic imaging identify multipotency of individual Gallus gallus caudal hindbrain neural crest cells toward cardiac and enteric fates

Neural crest stem cells arising from caudal hindbrain (often called cardiac and posterior vagal neural crest) migrate long distances to form cell types as diverse as heart muscle and enteric ganglia, abnormalities of which lead to common congenital birth defects. Here, we explore whether individual caudal hindbrain neural crest precursors are multipotent or predetermined toward these particular fates and destinations. To this end, we perform lineage tracing of chick neural crest cells at single-cell resolution using two complementary approaches: retrovirally mediated multiplex clonal analysis and single-cell photoconversion. Both methods show that the majority of these neural crest precursors are multipotent with many clones producing mesenchymal as well as neuronal derivatives. Time-lapse imaging demonstrates that sister cells can migrate in distinct directions, suggesting stochasticity in choice of migration path. Perturbation experiments further identify guidance cues acting on cells in the pharyngeal junction that can influence this choice; loss of CXCR4 signaling results in failure to migrate to the heart but no influence on migration toward the foregut, whereas loss of RET signaling does the opposite. Taken together, the results suggest that environmental influences rather than intrinsic information govern cell fate choice of multipotent caudal hindbrain neural crest cells.

Molecular neural crest cell markers enable discrimination of organophosphates in the murine cardiac embryonic stem cell test.

The cardiac embryonic stem cell test (ESTc) originally used the differentiation of beating cardiomyocytes for embryotoxicity screenings of compounds. However, the ESTc consists of a heterogeneous cell population, including neural crest (NC) cells, which are important contributors to heart development in vivo. Molecular markers for NC cells were investigated to explore if this approach improved discrimination between structurally related chemicals, using the three organophosphates (OP): chlorpyrifos (CPF), malathion (MLT), and triphenyl phosphate (TPP). To decrease the test duration and to improve the objective quantification of the assay read-out, gene transcript biomarkers were measured on study day 4 instead of the traditional cardiomyocyte beating assessment at day 10. Gene expression profiling and immunocytochemistry were performed using markers for pluripotency, proliferation and cardiomyocyte and NC differentiation. Cell proliferation was also assessed by measurements of embryoid body (EB) size and total protein quantification (day 7). Exposure to the OPs resulted in similar patterns of inhibition of beating cardiomyocyte differentiation and of myosin protein expression on day 10. However, these three chemically related compounds induced distinctive effects on NC cell differentiation, indicated by changes in expression levels of the NC precursor (Msx2), NC marker (Ap2α), and epithelial to mesenchymal transition (EMT; Snai2) gene transcripts. This study shows that investigating NC markers can provide added value for ESTc outcome profiling and may enhance the applicability of this assay for the screening of structurally related test chemicals.

Bone marrow-derived neural crest precursors improve nerve defect repair partially through secreted trophic factors

BackgroundEmerging evidence suggests that neural crest-derived cells (NCCs) present important functions in peripheral nerve regeneration to correct the insufficiency of autogenous Schwann cells. Postmigratory NCCs have been successfully isolated from adult rat bone marrow in our previous work. In this study, we aim to provide neural crest-derived Schwann cell precursors (SCPs) for repair of nerve defects in adult rats, and partially reveal the mechanisms involved in neuroregeneration of cell therapy.MethodsA clonal cell line of neural crest precursors of rat bone marrow origin (rBM-NCPs) with SCP identity was expanded in adherent monolayer culture to ensure the stable cell viability of NCPs and potentiate the repair of nerve defects after rBM-NCPs implantation based on tissue engineering nerve grafts (TENG). Here the behavioral, morphological, and electrophysiological detection was performed to evaluate the therapy efficacy. We further investigated the treatment with NCP-conditioned medium (NCP-CM) to sensory neurons after exposure to oxygen-glucose-deprivation (OGD) and partially compared the expression of trophic factor genes in rBM-NCPs with that in mesenchymal stem cells of bone marrow origin (rBM-MSCs).ResultsIt was showed that the constructed TENG with rBM-NCPs loaded into silk fibroin fiber scaffolds/chitosan conduits repaired 10-mm long sciatic nerve defects more efficiently than conduits alone. The axonal regrowth, remyelination promoted the reinnervation of the denervated hind limb muscle and skin and thereby alleviated muscle atrophy and facilitated the rehabilitation of motor and sensory function. Moreover, it was demonstrated that treatment with NCP-CM could restore the cultured primary sensory neurons after OGD through trophic factors including epidermal growth factor (EGF), platelet-derived growth factor alpha (PDGFα), ciliary neurotrophic factor (CNTF), and vascular endothelial growth factor alpha (VEGFα).ConclusionsIn summary, our findings indicated that monolayer-cultured rBM-NCPs cell-based therapy might effectively repair peripheral nerve defects partially through secreted trophic factors, which represented the secretome of rBM-NCPs differing from that of rBM-MSCs.

Paraganglioma vulvovaginal primario. Reporte de caso

El paraganglioma es una neoplasia neuroendocrina extraadrenal derivada de precursores de la cresta neural, que surgen en asociación con los ganglios autónomos y, en ocasiones, también son denominados feocromocitomas extraadrenales. Se definen por su sitio anatómico y si son o no hormonalmente funcionales. Los paragangliomas son neoplasias poco frecuentes del tracto genital femenino y pueden aparecer en ovarios, útero o cuello uterino. Los que aparecen en la región vulvovaginal son extremadamente raros. Las manifestaciones clínicas dependen de la secreción no regulada de catecolaminas y la localización. El diagnóstico se basa en hallazgos morfológicos e inmunohistoquímicos. La resección quirúrgica es un tratamiento primario para este tumor, ya que no responde bien a la quimio-radioterapia. Debido a su baja frecuencia y sintomatología inespecífica, puede mostrar dificultades para realizar el diagnóstico. Se presenta un caso de paraganglioma vulvovaginal primario.

A zebrafish FASD model for congenital heart defects

Mothers who drink during pregnancy expose their developing babies to ethanol, and excessive drinking often produces devastating birth defects collectively termed fetal alcohol spectrum disorder (FASD). These defects include craniofacial defects, cognitive impairment, sensorimotor disabilities and organ deformities, including various congenital heart defects (CHDs), particularly septal and conotruncal defects. Genesis mechanisms for FASD‐associated CHDs are not well understood. Using a zebrafish model, experiments tested whether alcohol interferes with different heart development events or the multiple cardiac precursors that may contribute to CHD genesis. These experiments showed that ethanol affects various cardiac regulatory networks in multiple steps of cardiogenesis (specification, myocardial migration, differentiation, looping, chamber morphogenesis, and endocardial cushion formation). Retinoic acid and folic acid co‐treatment with ethanol had differential rescue effects on FASD‐induced cardiac defects. Early (first heart field) and late‐added (second heart field and cardiac neural crest precursors) cardiac precursors were reduced by ethanol exposure. Experiments showed dysregulation of BMP, Notch and other signaling pathways during atrioventricular valve formation produced persistent valve defects. Together, these results show that embryonic ethanol exposure disrupts critical cardiac morphogenic events and reduce cardiac precursor contributions to the growing heart. Additional mechanistic studies are needed to identify key events that can be targeted therapeutically.Support or Funding InformationNIH/NIAAA AA0022396 and AA026711This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RNA-seq of Isolated Chromaffin Cells Highlights the Role of Sex-Linked and Imprinted Genes in Adrenal Medulla Development

Adrenal chromaffin cells and sympathetic neurons synthesize and release catecholamines, and both cell types are derived from neural crest precursors. However, they have different developmental histories, with sympathetic neurons derived directly from neural crest precursors while adrenal chromaffin cells arise from neural crest-derived cells that express Schwann cell markers. We have sought to identify the genes, including imprinted genes, which regulate the development of the two cell types in mice. We developed a method of separating the two cell types as early as E12.5, using differences in expression of enhanced yellow fluorescent protein driven from the tyrosine hydroxylase gene, and then used RNA sequencing to confirm the characteristic molecular signatures of the two cell types. We identified genes differentially expressed by adrenal chromaffin cells and sympathetic neurons. Deletion of a gene highly expressed by adrenal chromaffin cells, NIK-related kinase, a gene on the X-chromosome, results in reduced expression of adrenaline-synthesizing enzyme, phenyl-N-methyl transferase, by adrenal chromaffin cells and changes in cell cycle dynamics. Finally, many imprinted genes are up-regulated in chromaffin cells and may play key roles in their development.

The myelin protein PMP2 is regulated by SOX10 and drives melanoma cell invasion.

The transcription factor sex determining region Y-box 10 (SOX10) plays a key role in the development of melanocytes and glial cells from neural crest precursors. SOX10 is involved in melanoma initiation, proliferation, invasion, and survival. However, specific mediators which impart its oncogenic properties remain widely unknown. To identify target genes of SOX10, we performed RNA sequencing after ectopic expression of SOX10 in human melanoma cells. Among nine differentially regulated genes, peripheral myelin protein 2 (PMP2) was consistently upregulated in several cell lines. Direct regulation of PMP2 by SOX10 was shown by chromatin immunoprecipitation, electrophoretic mobility shift, and luciferase reporter assays. Moreover, a coregulation of PMP2 by SOX10 and early growth response 2 in melanoma cells was found. Phenotypical investigation demonstrated that PMP2 expression can increase melanoma cell invasion. As PMP2 protein was detected only in a subset of melanoma cell lines, it might contribute to melanoma heterogeneity.