Neural Crest-derived Lineages Research Articles

The transcription factor Sox10 is an essential determinant of branching morphogenesis and involution in the mouse mammary gland

The high mobility group-domain containing transcription factor Sox10 is an essential regulator of developmental processes and homeostasis in the neural crest, several neural crest-derived lineages and myelinating glia. Recent studies have also implicated Sox10 as an important factor in mammary stem and precursor cells. Here we employ a series of mouse mutants with constitutive and conditional Sox10 deficiencies to show that Sox10 has multiple functions in the developing mammary gland. While there is no indication for a requirement of Sox10 in the specification of the mammary placode or descending mammary bud, it is essential for both the prenatal hormone-independent as well as the pubertal hormone-dependent branching of the mammary epithelium and for proper alveologenesis during pregnancy. It furthermore acts in a dosage-dependent manner. Sox10 also plays a role during the involution process at the end of the lactation period. Whereas its effect on epithelial branching and alveologenesis are likely causally related to its function in mammary stem and precursor cells, this is not the case for its function during involution where Sox10 seems to work at least in part through regulation of the miR-424(322)/503 cluster.

From neural crest cells to melanocytes: cellular plasticity during development and beyond.

Here, we review melanocyte development and how the embryonic melanoblast, although specified to become a melanocyte, is prone to cellular plasticity and is not fully committed to the melanocyte lineage. Even fully differentiated and pigment-producing melanocytes do not always have a stable phenotype. The gradual lineage restriction of neural crest cells toward the melanocyte lineage is determined by both cell-intrinsic and extracellular signals in which differentiation and pathfinding ability reciprocally influence each other. These signals are leveraged by subtle differences in timing and axial positioning. The most extensively studied migration route is the dorsolateral path between the dermomyotome and the prospective epidermis, restricted to melanoblasts. In addition, the embryonic origin of the skin dermis through which neural crest derivatives migrate may also affect the segregation between melanogenic and neurogenic cells in embryos. It is widely accepted that, irrespective of the model organism studied, the immediate precursor of both melanoblast and neurogenic populations is a glial-melanogenic bipotent progenitor. Upon exposure to different conditions, melanoblasts may differentiate into other neural crest-derived lineages such as neuronal cells and vice versa. Key factors that regulate melanoblast migration and patterning will regulate melanocyte homeostasis during different stages of hair cycling in postnatal hair follicles.

Top-Down Inhibition of BMP Signaling Enables Robust Induction of hPSCs Into Neural Crest in Fully Defined, Xeno-free Conditions.

SummaryDefects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. However, the protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have now found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can also affect the outcome of differentiation protocols.

The MITF, p.E318K Variant, as a Risk Factor for Pheochromocytoma and Paraganglioma.

The microphthalmia-associated transcription factor (MITF) regulates the survival, proliferation, and differentiation of neural crest-derived lineages. Recent studies reported an increased risk of melanoma in individuals carrying the rare variant MITF, p.E318K (rs149617956). Whether this variant plays a role in other neural crest-derived tumors is unknown. In the present study, we aimed at determining the prevalence of the MITF, p.E318K variant, in a well-characterized French cohort of pheochromocytomas/paragangliomas (PCC/PGL). Genomic DNA from 555 unrelated patients with PCC/PGL was genotyped for the p.E318K variant in MITF using Sanger sequencing. The prevalence of the mutation in the PCC/PGL cohort was compared with a population-based sample of 2348 ethnically matched controls. We identified seven carriers (five patients with sporadic PCCs, two with PGLs). The prevalence of the MITF, p.E318K variant, was higher in the PCC/PGL cohort than in controls, and appears to be a significant risk factor (odds ratio, 3.19; 95% confidence interval, 1.34-7.59; P = .005). Noteworthy, two patients were homozygous for the p.E318K risk allele, a patient with metastatic PCC and an SDHB-mutated patient with PGL. Our results indicate that the germline variant MITF, p.E318K is associated with an increased risk of other neural crest-derived tumors such as PCC/PGL.

Su2033 Deficits in Neural Crest-Derived Lineages and GI Motility in the Ganglionated Regions of Murine Hirschsprung Enteric Nervous System

Su2033 Deficits in Neural Crest-Derived Lineages and GI Motility in the Ganglionated Regions of Murine Hirschsprung Enteric Nervous System

Anatomic Modifications in the Enteric Nervous System of JF1 Mice with the Classic Piebald Mutation

The Japanese Fancy Mouse 1 (JF1) has a characteristic coat color similar to a very old mutant, piebald. The mutation in JF1 and the classic piebald was previously thought to be the same recessive allele in the endothelin B receptor gene (Ednrb) according to the haplotype pattern, which is insufficient for this conclusion. In this study, we identified the same insertion of a retroposon-like element in intron 1 of the Ednrb gene in JF1 as in the classic piebald mutation by PCR. Further, we investigated whether the intestine shows neuronal intestinal malformations such as hypoganglionosis and immaturity of ganglion cells by histochemical staining. Though it has been assumed that the defect of neural crest-derived lineages is restricted to melanocytes in JF1, we found that the enteric innervation and neuronal density were impaired throughout the whole colon in JF1 mice.

Plasticité phénotypique des mélanocytes et des cellules de Schwann

Melanocytes, the pigmented cells of the skin, and the glial Schwann cells lining peripheral nerves are developmentally derived from an early and transient ectodermal structure of the vertebrate embryo, the neural crest, which is also at the origin of multiple neural and non-neural cell types. Besides melanocytes and neural cells of the peripheral nervous system, the neural crest cells give rise to mesenchymal cell types in the head, which form most of the craniofacial skeleton, dermis, fat tissue and vascular musculo-connective components. How such a wide diversity of differentiation fates is established during embryogenesis and is later maintained in adult tissues are among key questions in developmental and stem cell biology. The analysis of the developmental potentials of single neural crest cells cultured in vitro led to characterizing multipotent stem/progenitor cells as well as more restricted precursors in the early neural crest of avian and mammalian embryos. Data support a hierarchical model of the diversification of neural crest lineages through progressive restrictions of multipotent stem cell potentials driven by local environmental factors. In particular, melanocytes and glial Schwann cells were shown to arise from a common bipotent progenitor, which depends upon the peptide endothelin-3 for proliferation and self-renewal ability. In vivo, signaling by endothelin-3 and its receptor is also required for the early development of melanocytes and proper pigmentation of the vertebrate body. It is generally assumed that, after lineage specification and terminal differentiation, specialized cell types, like the melanocytes and Schwann cells, do not change their identity. However, this classic notion that somatic cell differentiation is a stable and irreversible process has been challenged by emerging evidence that dedifferentiation can occur in different biological systems through nuclear transfer, cell fusion, epigenetic modifications and ectopic gene expression. This review considers the issue of whether neural crest-derived lineages are endowed with some phenotypic plasticity. Emphasis is put on the ability of pigment cells and Schwann cells to dedifferentiate and reprogram their fate in vitro. To address this question, we have studied the clonal progeny of differentiated Schwann cells and melanocytes after their isolation from the sciatic nerve and the back skin of quail embryos, respectively. When stimulated to proliferate in vitro in the presence of endothelin-3, both cell types were able to dedifferentiate and produce alternative neural crest-derived cell lineages. Individual Schwann cells isolated by FACS, using a glial-specific surface marker, gave rise in culture to pigment cells and myofibroblasts/smooth muscle cells. Treatment of the cultures with endothelin-3 was required for Schwann cell conversion into melanocytes, which involved acquisition of multipotency. Moreover, Schwann cell plasticity could also be induced in vivo: following transplantation into the branchial arch of a young chick host embryo, dedifferentiating Schwann cells were able to integrate the forming head structures of the host and, specifically, to contribute smooth muscle cells to the wall of cranial blood vessels. We also analyzed the in vitro behavior of individual pigment cells obtained by microdissection and enzymatic treatment of quail epidermis at embryonic and hatching stages. In single cell cultures treated with endothelin-3, pigment cells strongly proliferated while rapidly dedifferentiating into unpigmented cells, leading to the formation of large colonies that comprised glial cells and myofibroblasts in addition to melanocytes. By serially subcloning these primary colonies, we could efficiently propagate a bipotent glial-melanocytic precursor that is generated in the progeny of the melanocytic founder. These data therefore suggest that pigment cells have the ability to revert back to the state of self-renewing neural crest-like progenitors. Altogether, these studies have shown that Schwann cells and pigment cells display an unstable status of differentiation, which can be disclosed if these differentiated cells are displaced out of their native tissue. When challenged with new environmental conditions in vitro, differentiated Schwann cells and pigment cells can reacquire stem cell properties of their neural crest ancestors. Notably, such reprogramming was achieved through the effect of a single exogenous factor and without the need of any induced genetic modification. Deciphering the cellular and molecular mechanisms that regulate the plasticity and maintenance of neural crest-derived differentiated cells is likely to be an important step towards the understanding of the neurocristopathies and cancers that target neural crest derivatives in humans.

Mice with DNA repair gene Ercc1 deficiency in a neural crest lineage are a model for late-onset Hirschsprung disease

The Ercc1 gene is essential for nucleotide excision repair and is also important in recombination repair and the repair of interstrand crosslinks. We have previously used a floxed Ercc1 allele with a keratinocyte-specific Cre recombinase transgene to inactivate Ercc1 in the epidermal layer of the skin and so generate a mouse model for UV-induced non-melanoma skin cancer. Now, in an attempt to generate a model for UV-induced melanoma, we have used the floxed Ercc1 allele in combination with a Cre transgene under the control of the tyrosinase gene promoter to produce mice with Ercc1-deficient melanocytes that are hypersensitive to UV irradiation. These animals developed normally, but died when 4–6 months old with severe colonic obstruction. Melanocytes are derived from the neural crest and the tyrosinase promoter is also expressed in additional neural crest-derived lineages, including the progenitors of the parasympathetic nervous system that innervates the gastrointestinal tract and controls gut peristalsis. A functional enteric nervous system developed in floxed Ercc1 mice with the tyrosinase Cre transgene, but was found to have degenerated in the colons of affected mice. We suggest that accumulating unrepaired endogenous DNA damage in the Ercc1-deficient colonic parasympathetic ganglia leads to the degeneration of this network and results in a colonic obstructive disorder that resembles late-onset Hirschsprung disease in man.

Glutamate‐dependent transcriptional control in Bergmann glia: Sox10 as a repressor

Glutamate (Glu) is the major excitatory transmitter in the vertebrate brain. Ligand-gated and G protein-coupled Glu receptors present in glial cells are presumably involved in neuronal function. Activation of Bergmann glial Glu receptors triggers a membrane to nuclei signaling cascade that regulates gene expression at the transcriptional and translational levels. Sry-related high-mobility group box (Sox10), a member of the conserved high-mobility group box transcription factor family is expressed in neural crest stem cells and in a subset of neural crest-derived lineages that include glial, but not neuronal cells. To gain insight into the role of Sox10 in Bergmann glial cells, we explored its expression and regulation. We demonstrate herein that Sox10 is expressed in Bergmann glial cells and that its DNA binding activity, mRNA, and protein levels as well as its transcriptional behavior augments upon the activation of metabotropic Glu receptors. Increase in Sox10-DNA complexes and Sox10 mRNA and protein levels were found upon exposure to Glu. Over-expression of Sox10 leads to transcriptional repression in reporter gene assays and in one of its target genes: the chick kainate binding protein gene. These findings add a new perspective into glial glutamatergic signaling and suggest the participation of Sox10 in cerebellar glutamatergic transactions. Bergmann glial cells, glutamate, metabotropic glutamate receptors, signaling, Sox10, transcriptional control.

Lineage-specific responses to reduced embryonic Pax3 expression levels

Pax3 is an essential paired- and homeodomain-containing transcription factor that is necessary for closure of the neural tube, and morphogenesis of the migratory neural crest and myoblast lineages. Homozygous loss-of-function mutation results in mid-gestational lethality with defects in myogenesis, neural tube closure and neural crest-derived lineages including melanocytes, Schwann cells and insufficient mesenchymal cells to septate the cardiac outflow tract. To address the function of Pax3 in later fetal stages and in specific adult tissues, we generated a floxed Pax3 allele (Pax3flox). An intermediate allele (Pax3neo) was produced via creation of the floxed allele, in which the TK-neo(R) cassette is present between exons 5 and 6. It was deduced to be a hypomorph, as Pax3 protein expression is reduced by 80% and homozygote hypomorphs die postnatally. To assess the consequences of reduced Pax3 levels on the various Pax3-expressing lineages and to determine the underlying cause of lethality, we examined Pax3 spatiotemporal expression and the resultant defects. Defective limb and tongue musculature were observed and lethality was due to an inability to suckle. However, the heart, diaphragm, trunk musculature, as well as the various neural crest-derived lineages and neural tube were all unaffected by reduced Pax3 levels. Significantly, elevated levels of the related Pax7 protein were present in unaffected neural tube and epaxial somatic component. The limb and tongue myogenic defects were found to be due to a significant increase in apoptosis within the somites that leads to a paucity of migratory hypaxial myoblasts. These effects were attributed to the hypomorphic effect of the Pax3neo allele, as removal of the TK-neo(R) cassette completely relieves the hypomorphic effect, as 100% of the Pax3flox/flox mice were normal. These data demonstrate a lineage-specific response to ∼80% loss of Pax3 protein expression, with myogenesis of limb and tongue being most sensitive to reduced Pax3 levels. Thus, we have established that there are different minimum threshold requirements for Pax3 within different Pax3-expressing lineages.

Hypomorphic Sox10 alleles reveal novel protein functions and unravel developmental differences in glial lineages

The transcription factor Sox10 regulates early neural crest development, specification of neural crest-derived lineages and terminal differentiation of oligodendrocytes in the central nervous system. Here, we generated two novel hypomorphic Sox10 alleles in the mouse. Mutant mice either expressed a Sox10 protein with a triple alanine substitution in the dimerization domain, or a Sox10 protein with a deletion in the central portion that we define as a cell-specific transactivation domain. Phenotypic analysis revealed important roles for a functional dimerization domain and the newly defined novel transactivation domain in melanocyte and enteric nervous system development, whereas early neural crest development and oligodendrocyte differentiation were surprisingly little disturbed in both mutants. Unique requirements were additionally detected for the novel transactivation domain in satellite glia differentiation and during Schwann cell myelination, whereas DNA-dependent dimerization was needed for immature Schwann cells to enter the promyelinating stage. These two hypomorphic alleles thus uncover novel functions of Sox10 in satellite glia and Schwann cells during late developmental stages and reveal important developmental differences between these two types of peripheral glia and oligodendrocytes regarding their reliance on Sox10.

EDNRB/EDN3 and Hirschsprung disease type II.

The study of vertebrate pigmentary anomalies has greatly improved our understanding of melanocyte biology. One such disorder, Waardenburg syndrome (WS), is a mendelian trait characterized by hypopigmentation and sensorineural deafness. It is commonly subdivided into four types (WS1-4), defined by the presence or absence of additional symptoms. WS type 4 (WS4), or Shah-Waardenburg syndrome, is also known as Hirschsprung disease Type II (HSCR II) and is characterized by an absence of epidermal melanocytes and enteric ganglia. Mutations in the genes encoding the endothelin type-B receptor (EDNRB) and its physiological ligand endothelin 3 (EDN3) are now known to account for the majority of HSCR II patients. Null mutations in the mouse genes Ednrb and Edn3 have identified a key role for this pathway in the normal development of melanocytes and other neural crest-derived lineages. The pleiotropic effects of genes in this pathway, on melanocyte and enteric neuron development, have been clarified by the embryologic identification of their common neural crest (NC) ancestry. EDNRB and EDN3 are transiently expressed in crest-derived melanoblast and neuroblast precursors, and in the surrounding mesenchymal cells, respectively. The influence of EDNRB-mediated signaling on the emigration, migration, proliferation, and differentiation of melanocyte and enteric neuron precursors, in vivo and in vitro has recently been the subject of great scrutiny. A major emergent theme is that EDN3-induced signaling prevents the premature differentiation of melanocyte and enteric nervous system precursors and is essential between 10 and 12.5 days post-coitum. We review the present understanding of pigment cell development in the context of EDNRB/EDN3--a receptor-mediated pathway with pleiotropic effects.

Parental origin-specific developmental defects in mice with uniparental disomy for chromosome 12.

Genetic analysis has shown that the distal portion of mouse chromosome 12 is imprinted; however, the developmental roles of imprinted genes in this region are not known. We have therefore generated conceptuses with uniparental disomy for chromosome 12, in which both copies of chromosome 12 are either paternally or maternally derived (pUPD12 and mUPD12, respectively). Both types of UPD12 result in embryos that are non-viable and that exhibit distinct developmental abnormalities. Embryos with pUPD12 die late in gestation, whereas embryos with mUPD12 can survive to term but die perinatally. The mUPD12 conceptuses are invariably growth-retarded while pUPD12 conceptuses exhibit placentomegaly. Skeletal muscle maturation defects are evident in both types of UPD12. In addition, embryos with paternal UPD12 have costal cartilage defects and hypo-ossification of mesoderm-derived bones. In embryos with mUPD12, the development of the neural crest-derived middle ear ossicles is defective. Some of these anomalies are consistent with those seen with uniparental disomies of the orthologous chromosome 14 region in humans. Thus, imprinted genes on chromosome 12 are essential for viability, the regulation of prenatal growth, and the development of mesodermal and neural crest-derived lineages.

Environmental signals and neural crest cells

Cell lineage analysis in both the central and peripheral nervous system of vertebrates has revealed that many neural progenitor cells are multipotent. These observations have raised the general issue of when and how such multipotent progenitors generate their various differentiated progeny. The environment of these progenitors controls the cell lineage decisions in the neural crest. This review considers the roles of the environmental signals in the context of the development of several different neural crest-derived lineages.

Plasticity of neuroblastoma tumor cells to differentiate along a fetal adrenal ganglionic lineage predicts for improved patient survival.

We have recently presented a model of human adrenal medullary histogenesis that incorporates all neural crest-derived lineages (chromaffin, sustentacular, and ganglionic) known to compose this tissue. To determine if neuroblastomas correspond to the arrested maturation of embryonal adrenal medullary cells, we evaluated the expression of adrenal medullary developmental markers in 81 neuroblastoma tumors. We found that patterns of chromaffin-related gene expression in these tumors correlated exactly with the patterns observed during maturation of adrenal medullary cells (P2 < 10(-5). In a multivariate Cox proportional hazards analysis of developmental marker expression and other well-recognized prognostic variables, evidence of maturation along a fetal ganglionic lineage, as monitored by HNK-1 immunoreactivity (relative risk of 6.42, P2 = 0.0001), and age at diagnosis (relative risk of 5.05, P2 = 0.0042) were independent and significant prognostic indicators of patient survival. These studies demonstrate that neuroblastomas correspond to embryonal adrenal medullary cells arrested at recognizable stages during development, and that evidence of maturation along a fetal ganglionic lineage appears to have major importance in predicting patient survival.

Mouse mutations and human disorders are paired

Pax3 is an essential paired- and homeodomain-containing transcription factor that is necessary for closure of the neural tube, and morphogenesis of the migratory neural crest and myoblast lineages. Homozygous loss-of-function mutation results in mid-gestational lethality with defects in myogenesis, neural tube closure and neural crest-derived lineages including melanocytes, Schwann cells and insufficient mesenchymal cells to septate the cardiac outflow tract. To address the function of Pax3 in later fetal stages and in specific adult tissues, we generated a floxed Pax3 allele (Pax3flox). An intermediate allele (Pax3neo) was produced via creation of the floxed allele, in which the TK-neo(R) cassette is present between exons 5 and 6. It was deduced to be a hypomorph, as Pax3 protein expression is reduced by 80% and homozygote hypomorphs die postnatally. To assess the consequences of reduced Pax3 levels on the various Pax3-expressing lineages and to determine the underlying cause of lethality, we examined Pax3 spatiotemporal expression and the resultant defects. Defective limb and tongue musculature were observed and lethality was due to an inability to suckle. However, the heart, diaphragm, trunk musculature, as well as the various neural crest-derived lineages and neural tube were all unaffected by reduced Pax3 levels. Significantly, elevated levels of the related Pax7 protein were present in unaffected neural tube and epaxial somatic component. The limb and tongue myogenic defects were found to be due to a significant increase in apoptosis within the somites that leads to a paucity of migratory hypaxial myoblasts. These effects were attributed to the hypomorphic effect of the Pax3neo allele, as removal of the TK-neo(R) cassette completely relieves the hypomorphic effect, as 100% of the Pax3flox/flox mice were normal. These data demonstrate a lineage-specific response to ∼ 80% loss of Pax3 protein expression, with myogenesis of limb and tongue being most sensitive to reduced Pax3 levels. Thus, we have established that there are different minimum threshold requirements for Pax3 within different Pax3-expressing lineages.