Schwann cell precursors: a hub of neural crest development.

Schwann cell precursors (SCPs) are transient glial progenitors that are important for the formation of late neural crest derivatives, yet their heterogeneity and developmental potential remain incompletely understood. In this issue, Kastriti, Faure, von Ahsen et al (2022) use comprehensive single-cell RNA sequencing analyses to identify a transient "hub" state common to SCPs and neural crest cells (NCCs), revealing a striking similarity of SCPs to late migrating NCCs. These results raise important questions about the potential role of such a state in adult tissue regeneration and tumourigenesis.

Extracellular sodium regulates fibroblast growth factor 23 (FGF23) formation.

The bone-derived hormone fibroblast growth factor-23 (FGF23) has recently received much attention due to its association with chronic kidney disease and cardiovascular disease progression. Extracellular sodium concentration ([Na+]) plays a significant role in bone metabolism. Hyponatremia (lower serum [Na+]) has recently been shown to be independently associated with FGF23 levels in patients with chronic systolic heart failure. However, nothing is known about the direct impact of [Na+] on FGF23 production. Here, we show that an elevated [Na+] (+20 mM) suppressed FGF23 formation, whereas low [Na+] (-20 mM) increased FGF23 synthesis in the osteoblast-like cell lines UMR-106 and MC3T3-E1. Similar bidirectional changes in FGF23 abundance were observed when osmolality was altered by mannitol but not by urea, suggesting a role of tonicity in FGF23 formation. Moreover, these changes in FGF23 were inversely proportional to the expression of NFAT5 (nuclear factor of activated T cells-5), a transcription factor responsible for tonicity-mediated cellular adaptations. Furthermore, arginine vasopressin, which is often responsible for hyponatremia, did not affect FGF23 production. Next, we performed a comprehensive and unbiased RNA-seq analysis of UMR-106 cells exposed to low versus high [Na+], which revealed several novel genes involved in cellular adaptation to altered tonicity. Additional analysis of cells with Crisp-Cas9-mediated NFAT5 deletion indicated that NFAT5 controls numerous genes associated with FGF23 synthesis, thereby confirming its role in [Na+]-mediated FGF23 regulation. In line with these in vitro observations, we found that hyponatremia patients have higher FGF23 levels. Our results suggest that [Na+] is a critical regulator of FGF23 synthesis.

Injury and stress responses of adult neural crest-derived cells.

Multipotent neural crest cells can self-renew and give rise to a plethora of neural and non-neural cell types in the vertebrate embryo. Intriguingly, cells reminiscent of such neural crest stem cells (NCSCs) have also been isolated from various postnatal and adult neural crest (NC)-derived structures. However, it has been debated whether NCSC-like cells in the adult correspond to 'in vitro artefacts' emerging upon isolation or fulfil a physiological role in vivo. Here, we discuss recent findings indicating that in different adult NC derivatives, injury or stress responses induce a NCSC-like state, presumably by reprogramming differentiated cells such as Schwann cells. Thereby, injury or stress appear to endow NC-derived cells with the capacity to generate new cell types during the repair process; in addition, injury can activate a repair program in adult NC-derived cells, which promotes tissue repair or regeneration by paracrine signalling. Thus, there is increasing evidence that NCSC-like cells in NC derivatives represent an in vivo state implicated in distinct physiological functions in the adult organism.

Probing transcription-specific outputs of ß-catenin in vivo.

ß-Catenin, apart from playing a cell-adhesive role, is a key nuclear effector of Wnt signaling. Based on activity assays in Drosophila, we generated mouse strains where the endogenous ß-catenin protein is replaced by mutant forms, which retain the cell adhesion function but lack either or both of the N- and the C-terminal transcriptional outputs. The C-terminal activity is essential for mesoderm formation and proper gastrulation, whereas N-terminal outputs are required later during embryonic development. By combining the double-mutant ß-catenin with a conditional null allele and a Wnt1-Cre driver, we probed the role of Wnt/ß-catenin signaling in dorsal neural tube development. While loss of ß-catenin protein in the neural tube results in severe cell adhesion defects, the morphology of cells and tissues expressing the double-mutant form is normal. Surprisingly, Wnt/ß-catenin signaling activity only moderately regulates cell proliferation, but is crucial for maintaining neural progenitor identity and for neuronal differentiation in the dorsal spinal cord. Our model animals thus allow dissecting signaling and structural functions of ß-catenin in vivo and provide the first genetic tool to generate cells and tissues that entirely and exclusively lack canonical Wnt pathway activity.

Cre-driver lines used for genetic fate mapping of neural crest cells in the mouse: An overview.

The neural crest is one of the embryonic structures with the broadest developmental potential in vertebrates. Morphologically, neural crest cells emerge during neurulation in the dorsal folds of the neural tube before undergoing an epithelial-to-mesenchymal transition (EMT), delaminating from the neural tube, and migrating to multiple sites in the growing embryo. Neural crest cells generate cell types as diverse as peripheral neurons and glia, melanocytes, and so-called mesectodermal derivatives that include craniofacial bone and cartilage and smooth muscle cells in cardiovascular structures. In mice, the fate of neural crest cells has been determined mainly by means of transgenesis and genome editing technologies. The most frequently used method relies on the Cre-loxP system, in which expression of Cre-recombinase in neural crest cells or their derivatives genetically enables the expression of a Cre-reporter allele, thus permanently marking neural crest-derived cells. Here, we provide an overview of the Cre-driver lines used in the field and discuss to what extent these lines allow precise neural crest stage and lineage-specific fate mapping.

Antagonistic cross-regulation between Sox9 and Sox10 controls an anti-tumorigenic program in melanoma.

Melanoma is the most fatal skin cancer, but the etiology of this devastating disease is still poorly understood. Recently, the transcription factor Sox10 has been shown to promote both melanoma initiation and progression. Reducing SOX10 expression levels in human melanoma cells and in a genetic melanoma mouse model, efficiently abolishes tumorigenesis by inducing cell cycle exit and apoptosis. Here, we show that this anti-tumorigenic effect functionally involves SOX9, a factor related to SOX10 and upregulated in melanoma cells upon loss of SOX10. Unlike SOX10, SOX9 is not required for normal melanocyte stem cell function, the formation of hyperplastic lesions, and melanoma initiation. To the contrary, SOX9 overexpression results in cell cycle arrest, apoptosis, and a gene expression profile shared by melanoma cells with reduced SOX10 expression. Moreover, SOX9 binds to the SOX10 promoter and induces downregulation of SOX10 expression, revealing a feedback loop reinforcing the SOX10 low/SOX9 high ant,m/ii-tumorigenic program. Finally, SOX9 is required in vitro and in vivo for the anti-tumorigenic effect achieved by reducing SOX10 expression. Thus, SOX10 and SOX9 are functionally antagonistic regulators of melanoma development.

Ezh2 is required for neural crest-derived cartilage and bone formation.

The emergence of craniofacial skeletal elements, and of the jaw in particular, was a crucial step in the evolution of higher vertebrates. Most facial bones and cartilage are generated during embryonic development by cranial neural crest cells, while an osteochondrogenic fate is suppressed in more posterior neural crest cells. Key players in this process are Hox genes, which suppress osteochondrogenesis in posterior neural crest derivatives. How this specific pattern of osteochondrogenic competence is achieved remains to be elucidated. Here we demonstrate that Hox gene expression and osteochondrogenesis are controlled by epigenetic mechanisms. Ezh2, which is a component of polycomb repressive complex 2 (PRC2), catalyzes trimethylation of lysine 27 in histone 3 (H3K27me3), thereby functioning as transcriptional repressor of target genes. Conditional inactivation of Ezh2 does not interfere with localization of neural crest cells to their target structures, neural development, cell cycle progression or cell survival. However, loss of Ezh2 results in massive derepression of Hox genes in neural crest cells that are usually devoid of Hox gene expression. Accordingly, craniofacial bone and cartilage formation is fully prevented in Ezh2 conditional knockout mice. Our data indicate that craniofacial skeleton formation in higher vertebrates is crucially dependent on epigenetic regulation that keeps in check inhibitors of an osteochondrogenic differentiation program.

Wnt/ß-catenin signaling regulates sequential fate decisions of murine cortical precursor cells.

The fate of neural progenitor cells (NPCs) is determined by a complex interplay of intrinsic programs and extrinsic signals, very few of which are known. ß-Catenin transduces extracellular Wnt signals, but also maintains adherens junctions integrity. Here, we identify for the first time the contribution of ß-catenin transcriptional activity as opposed to its adhesion role in the development of the cerebral cortex by combining a novel ß-catenin mutant allele with conditional inactivation approaches. Wnt/ß-catenin signaling ablation leads to premature NPC differentiation, but, in addition, to a change in progenitor cell cycle kinetics and an increase in basally dividing progenitors. Interestingly, Wnt/ß-catenin signaling affects the sequential fate switch of progenitors, leading to a shortened neurogenic period with decreased number of both deep and upper-layer neurons and later, to precocious astrogenesis. Indeed, a genome-wide analysis highlighted the premature activation of a corticogenesis differentiation program in the Wnt/ß-catenin signaling-ablated cortex. Thus, ß-catenin signaling controls the expression of a set of genes that appear to act downstream of canonical Wnt signaling to regulate the stage-specific production of appropriate progenitor numbers, neuronal subpopulations, and astroglia in the forebrain.

A dual role for SOX10 in the maintenance of the postnatal melanocyte lineage and the differentiation of melanocyte stem cell progenitors.

During embryogenesis, the transcription factor, Sox10, drives the survival and differentiation of the melanocyte lineage. However, the role that Sox10 plays in postnatal melanocytes is not established. We show in vivo that melanocyte stem cells (McSCs) and more differentiated melanocytes express SOX10 but that McSCs remain undifferentiated. Sox10 knockout (Sox10(fl); Tg(Tyr::CreER)) results in loss of both McSCs and differentiated melanocytes, while overexpression of Sox10 (Tg(DctSox10)) causes premature differentiation and loss of McSCs, leading to hair graying. This suggests that levels of SOX10 are key to normal McSC function and Sox10 must be downregulated for McSC establishment and maintenance. We examined whether the mechanism of Tg(DctSox10) hair graying is through increased expression of Mitf, a target of SOX10, by asking if haploinsufficiency for Mitf (Mitf(vga9) ) can rescue hair graying in Tg(DctSox10) animals. Surprisingly, Mitf(vga9) does not mitigate but exacerbates Tg(DctSox10) hair graying suggesting that MITF participates in the negative regulation of Sox10 in McSCs. These observations demonstrate that while SOX10 is necessary to maintain the postnatal melanocyte lineage it is simultaneously prevented from driving differentiation in the McSCs. This data illustrates how tissue-specific stem cells can arise from lineage-specified precursors through the regulation of the very transcription factors important in defining that lineage.

Distinct adhesion-independent functions of ß-catenin control stage-specific sensory neurogenesis and proliferation.

BACKGROUND: ß-catenin plays a central role in multiple developmental processes. However, it has been difficult to study its pleiotropic effects, because of the dual capacity of ß-catenin to coordinate cadherin-dependent cell adhesion and to act as a component of Wnt signal transduction. To distinguish between the divergent functions of ß-catenin during peripheral nervous system development, we made use of a mutant allele of ß-catenin that can mediate adhesion but not Wnt-induced TCF transcriptional activation. This allele was combined with various conditional inactivation approaches. RESULTS: We show that of all peripheral nervous system structures, only sensory dorsal root ganglia require ß-catenin for proper formation and growth. Surprisingly, however, dorsal root ganglia development is independent of cadherin-mediated cell adhesion. Rather, both progenitor cell proliferation and fate specification are controlled by ß-catenin signaling. These can be divided into temporally sequential processes, each of which depends on a different function of ß-catenin. CONCLUSIONS: While early stage proliferation and specific Neurog2- and Krox20-dependent waves of neuronal subtype specification involve activation of TCF transcription, late stage progenitor proliferation and Neurog1-marked sensory neurogenesis are regulated by a function of ß-catenin independent of TCF activation and adhesion. Thus, switching modes of ß-catenin function are associated with consecutive cell fate specification and stage-specific progenitor proliferation.

Loss of Ezh2 promotes a midbrain-to-forebrain identity switch by direct gene derepression and Wnt-dependent regulation.

BACKGROUND: Precise spatiotemporal control of gene expression is essential for the establishment of correct cell numbers and identities during brain development. This process involves epigenetic control mechanisms, such as those mediated by the polycomb group protein Ezh2, which catalyzes trimethylation of histone H3K27 (H3K27me3) and thereby represses gene expression. RESULTS: Herein, we show that Ezh2 plays a crucial role in the development and maintenance of the midbrain. Conditional deletion of Ezh2 in the developing midbrain resulted in decreased neural progenitor proliferation, which is associated with derepression of cell cycle inhibitors and negative regulation of Wnt/ß-catenin signaling. Of note, Ezh2 ablation also promoted ectopic expression of a forebrain transcriptional program involving derepression of the forebrain determinants Foxg1 and Pax6. This was accompanied by reduced expression of midbrain markers, including Pax3 and Pax7, as a consequence of decreased Wnt/ß-catenin signaling. CONCLUSION: Ezh2 is required for appropriate brain growth and maintenance of regional identity by H3K27me3-mediated gene repression and control of canonical Wnt signaling.

HDAC1 and HDAC2 control the specification of neural crest cells into peripheral glia.

Schwann cells, the myelinating glia of the peripheral nervous system (PNS), originate from multipotent neural crest cells that also give rise to other cells, including neurons, melanocytes, chondrocytes, and smooth muscle cells. The transcription factor Sox10 is required for peripheral glia specification. However, all neural crest cells express Sox10 and the mechanisms directing neural crest cells into a specific lineage are poorly understood. We show here that histone deacetylases 1 and 2 (HDAC1/2) are essential for the specification of neural crest cells into Schwann cell precursors and satellite glia, which express the early determinants of their lineage myelin protein zero (P0) and/or fatty acid binding protein 7 (Fabp7). In neural crest cells, HDAC1/2 induced expression of the transcription factor Pax3 by binding and activating the Pax3 promoter. In turn, Pax3 was required to maintain high Sox10 levels and to trigger expression of Fabp7. In addition, HDAC1/2 were bound to the P0 promoter and activated P0 transcription. Consistently, in vivo genetic deletion of HDAC1/2 in mouse neural crest cells led to strongly decreased Sox10 expression, no detectable Pax3, virtually no satellite glia, and no Schwann cell precursors in dorsal root ganglia and peripheral nerves. Similarly, in vivo ablation of Pax3 in the mouse neural crest resulted in strongly reduced expression of Sox10 and Fabp7. Therefore, by controlling the expression of Pax3 and the concerted action of Pax3 and Sox10 on their target genes, HDAC1/2 direct the specification of neural crest cells into peripheral glia.

Temporal control of neural crest lineage generation by Wnt/ß-catenin signaling.

Wnt/ß-catenin signaling controls multiple steps of neural crest development, ranging from neural crest induction, lineage decisions, to differentiation. In mice, conditional ß-catenin inactivation in premigratory neural crest cells abolishes both sensory neuron and melanocyte formation. Intriguingly, the generation of melanocytes is also prevented by activation of ß-catenin in the premigratory neural crest, which promotes sensory neurogenesis at the expense of other neural crest derivatives. This raises the question of how Wnt/ß-catenin signaling regulates the formation of distinct lineages from the neural crest. Using various Cre lines to conditionally activate ß-catenin in neural crest cells at different developmental stages, we show that neural crest cell fate decisions in vivo are subject to temporal control by Wnt/ß-catenin. Unlike in premigratory neural crest, ß-catenin activation in migratory neural crest cells promotes the formation of ectopic melanoblasts, while the production of most other lineages is suppressed. Ectopic melanoblasts emerge at sites of neural crest target structures and in many tissues usually devoid of neural crest-derived cells. ß-catenin activation at later stages in glial progenitors or in melanoblasts does not lead to surplus melanoblasts, indicating a narrow time window of Wnt/ß-catenin responsiveness during neural crest cell migration. Thus, neural crest cells appear to be multipotent in vivo both before and after emigration from the neural tube but adapt their response to extracellular signals in a temporally controlled manner.

Persistent Wnt/ß-catenin signaling determines dorsalization of the postnatal subventricular zone and neural stem cell specification into oligodendrocytes and glutamatergic neurons.

In the postnatal and adult central nervous system (CNS), the subventricular zone (SVZ) of the forebrain is the main source of neural stem cells (NSCs) that generate olfactory neurons and oligodendrocytes (OLs), the myelinating cells of the CNS. Here, we provide evidence of a primary role for canonical Wnt/ß-catenin signaling in regulating NSC fate along neuronal and oligodendroglial lineages in the postnatal SVZ. Our findings demonstrate that glutamatergic neuronal precursors (NPs) and oligodendrocyte precursors (OPs) are derived strictly from the dorsal SVZ (dSVZ) microdomain under the control of Wnt/ß-catenin, whereas GABAergic NPs are derived mainly from the lateral SVZ (lSVZ) microdomain independent of Wnt/ß-catenin. Transcript analysis of microdissected SVZ microdomains revealed that canonical Wnt/ß-catenin signaling was more pronounced in the dSVZ microdomain. This was confirmed using the ß-catenin-activated Wnt-reporter mouse and by pharmacological stimulation of Wnt/ß-catenin by infusion of the specific glycogen synthase kinase 3ß inhibitor, AR-A014418, which profoundly increased the generation of cycling cells. In vivo genetic/pharmacological stimulation or inhibition of Wnt/ß-catenin, respectively, increased and decreased the differentiation of dSVZ-NSCs into glutamatergic NPs, and had a converse effect on GABAergic NPs. Activation of Wnt/ß-catenin dramatically stimulated the generation of OPs, but its inhibition had no effect, indicating other factors act in concert with Wnt/ß-catenin to fine tune oligodendrogliogenesis in the postnatal dSVZ. These results demonstrate a role for Wnt/ß-catenin signaling within the dorsal microdomain of the postnatal SVZ, in regulating the genesis of glutamatergic neurons and OLs.

Smad4 and Trim33/Tif1γ redundantly regulate neural stem cells in the developing cortex.

During central nervous system (CNS) development, proliferation and differentiation of neural stem cells (NSCs) have to be regulated in a spatio-temporal fashion. Here, we report different branches of the transforming growth factor ß (TGFß) signaling pathway to be required for the brain area-specific control of NSCs. In the midbrain, canonical TGFß signaling via Smad4 regulates the balance between proliferation and differentiation of NSCs. Accordingly, Smad4 deletion resulted in horizontal expansion of NSCs due to increased proliferation, decreased differentiation, and decreased cell cycle exit. In the developing cortex, however, ablation of Smad4 alone did not have any effect on proliferation and differentiation of NSCs. In contrast, concomitant mutation of both Smad4 and Trim33 led to an increase in proliferative cells in the ventricular zone due to decreased cell cycle exit, revealing a functional redundancy of Smad4 and Trim33. Furthermore, in Smad4-Trim33 double mutant embryos, cortical NSCs generated an excess of deep layer neurons concurrent with a delayed and reduced production of upper layer neurons and, in addition, failed to undergo the neurogenic to gliogenic switch at the right developmental stage. Thus, our data disclose that in different regions of the developing CNS different aspects of the TGFß signaling pathway are required to ensure proper development.

Monitoring melanoma patients on treatment reveals a distinct macrophage population driving targeted therapy resistance.

Resistance to targeted therapy remains a major clinical challenge in melanoma. To uncover resistance mechanisms, we perform single-cell RNA sequencing on fine-needle aspirates from resistant and responding tumors of patients undergoing BRAFi/MEKi treatment. Among the genes most prominently expressed in resistant tumors is POSTN, predicted to signal to a macrophage population associated with targeted therapy resistance (TTR). Accordingly, tumors from patients with fast disease progression after therapy exhibit high POSTN expression levels and high numbers of TTR macrophages. POSTN polarizes human macrophages toward a TTR phenotype and promotes resistance to targeted therapy in a melanoma mouse model, which is associated with a phenotype change in intratumoral macrophages. Finally, polarized TTR macrophages directly protect human melanoma cells from MEKi-induced killing via CD44 receptor expression on melanoma cells. Thus, interfering with the protective activity of TTR macrophages may offer a strategy to overcome resistance to targeted therapy in melanoma.

Neural crest progenitors and stem cells: from early development to adulthood.

In the vertebrate embryo, the neural crest forms transiently in the dorsal neural primordium to yield migratory cells that will invade nearly all tissues and later, will differentiate into bones and cartilages, neurons and glia, endocrine cells, vascular smooth muscle cells and melanocytes. Due to the amazingly diversified array of cell types it produces, the neural crest is an attractive model system in the stem cell field. We present here in vivo and in vitro studies of single cell fate, which led to the discovery and the characterization of stem cells in the neural crest of avian and mammalian embryos. Some of the key issues in neural crest cell diversification are discussed, such as the time of segregation of mesenchymal vs. neural/melanocytic lineages, and the origin and close relationships between the glial and melanocytic lineages. An overview is also provided of the diverse types of neural crest-like stem cells and progenitors, recently identified in a growing number of adult tissues in animals and humans. Current and future work, in which in vivo lineage studies and the use of injury models will complement the in vitro culture analysis, should help in unraveling the properties and function of neural crest-derived progenitors in development and disease.

Neuronal Nogo-A regulates neurite fasciculation, branching and extension in the developing nervous system.

Wiring of the nervous system is a multi-step process involving complex interactions of the growing fibre with its tissue environment and with neighbouring fibres. Nogo-A is a membrane protein enriched in the adult central nervous system (CNS) myelin, where it restricts the capacity of axons to grow and regenerate after injury. During development, Nogo-A is also expressed by neurons but its function in this cell type is poorly known. Here, we show that neutralization of neuronal Nogo-A or Nogo-A gene ablation (KO) leads to longer neurites, increased fasciculation, and decreased branching of cultured dorsal root ganglion neurons. The same effects are seen with antibodies against the Nogo receptor complex components NgR and Lingo1, or by blocking the downstream effector Rho kinase (ROCK). In the chicken embryo, in ovo injection of anti-Nogo-A antibodies leads to aberrant innervation of the hindlimb. Genetic ablation of Nogo-A causes increased fasciculation and reduced branching of peripheral nerves in Nogo-A KO mouse embryos. Thus, Nogo-A is a developmental neurite growth regulatory factor with a role as a negative regulator of axon-axon adhesion and growth, and as a facilitator of neurite branching.

Mesenchymal stem cells and neural crest stem cells from adult bone marrow: characterization of their surprising similarities and differences.

The generation of neuronal cells from stem cells obtained from adult bone marrow is of significant clinical interest in order to design new cell therapy protocols for several neurological disorders. The recent identification in adult bone marrow of stem cells derived from the neural crest stem cells (NCSC) might explain the neuronal phenotypic plasticity shown by bone marrow cells. However, little information is available about the nature of these cells compared to mesenchymal stem cells (MSC), including their similarities and differences. In this paper, using transcriptomic as well as proteomic technologies, we compared NCSC to MSC and stromal nestin-positive cells, all of them isolated from adult bone marrow. We demonstrated that the nestin-positive cell population, which was the first to be described as able to differentiate into functional neurons, was a mixed population of NCSC and MSC. More interestingly, we demonstrated that MSC shared with NCSC the same ability to truly differentiate into Tuj1-positive cells when co-cultivated with paraformaldehyde-fixed cerebellar granule neurons. Altogether, those results suggest that both NCSC and MSC can be considered as important tools for cellular therapies in order to replace neurons in various neurological diseases.