Cranial Neural Crest-derived Cells Research Articles

Cell-cell interaction determines cell fate of mesoderm-derived cell in tongue development through Hh signaling.

Dysfunction of primary cilia leads to genetic disorder, ciliopathies, which shows various malformations in many vital organs such as brain. Multiple tongue deformities including cleft, hamartoma, and ankyloglossia are also seen in ciliopathies, which yield difficulties in fundamental functions such as mastication and vocalization. Here, we found these tongue anomalies in mice with mutation of ciliary protein. Abnormal cranial neural crest-derived cells (CNCC) failed to evoke Hh signal for differentiation of mesoderm-derived cells into myoblasts, which resulted in abnormal differentiation of mesoderm-derived cells into adipocytes. The ectopic adipose subsequently arrested tongue swelling formation. Ankyloglossia was caused by aberrant cell migration due to lack of non-canonical Wnt signaling. In addition to ciliopathies, these tongue anomalies are often observed as non-familial condition in human. We found that these tongue deformities could be reproduced in wild-type mice by simple mechanical manipulations to disturb cellular processes which were disrupted in mutant mice. Our results provide hints for possible future treatment in ciliopathies.

Dental pulp stem cells as a therapy for congenital entero-neuropathy

Hirschsprung’s disease is a congenital entero-neuropathy that causes chronic constipation and intestinal obstruction. New treatments for entero-neuropathy are needed because current surgical strategies have limitations5. Entero-neuropathy results from enteric nervous system dysfunction due to incomplete colonization of the distal intestine by neural crest-derived cells. Impaired cooperation between the enteric nervous system and intestinal pacemaker cells may also contribute to entero-neuropathy. Stem cell therapy to repair these multiple defects represents a novel treatment approach. Dental pulp stem cells derived from deciduous teeth (dDPSCs) are multipotent cranial neural crest-derived cells, but it remains unknown whether dDPSCs have potential as a new therapy for entero-neuropathy. Here we show that intravenous transplantation of dDPSCs into the Japanese Fancy-1 mouse, an established model of hypoganglionosis and entero-neuropathy, improves large intestinal structure and function and prolongs survival. Intravenously injected dDPSCs migrate to affected regions of the intestine through interactions between stromal cell-derived factor-1α and C-X-C chemokine receptor type-4. Transplanted dDPSCs differentiate into both pacemaker cells and enteric neurons in the proximal colon to improve electrical and peristaltic activity, in addition to their paracrine effects. Our findings indicate that transplanted dDPSCs can differentiate into different cell types to correct entero-neuropathy-associated defects.

Lifelong single-cell profiling of cranial neural crest diversification in zebrafish

The cranial neural crest generates a huge diversity of derivatives, including the bulk of connective and skeletal tissues of the vertebrate head. How neural crest cells acquire such extraordinary lineage potential remains unresolved. By integrating single-cell transcriptome and chromatin accessibility profiles of cranial neural crest-derived cells across the zebrafish lifetime, we observe progressive and region-specific establishment of enhancer accessibility for distinct fates. Neural crest-derived cells rapidly diversify into specialized progenitors, including multipotent skeletal progenitors, stromal cells with a regenerative signature, fibroblasts with a unique metabolic signature linked to skeletal integrity, and gill-specific progenitors generating cell types for respiration. By retrogradely mapping the emergence of lineage-specific chromatin accessibility, we identify a wealth of candidate lineage-priming factors, including a Gata3 regulatory circuit for respiratory cell fates. Rather than multilineage potential being established during cranial neural crest specification, our findings support progressive and region-specific chromatin remodeling underlying acquisition of diverse potential.

Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques

Choroidal melanocytes (HCMs) are melanin-producing cells in the vascular uvea of the human eye (iris, ciliary body and choroid). These cranial neural crest-derived cells migrate to populate a mesodermal microenvironment, and display cellular functions and extracellular interactions that are biologically distinct to skin melanocytes. HCMs (and melanins) are important in normal human eye physiology with roles including photoprotection, regulation of oxidative damage and immune responses. To extend knowledge of cytoplasmic melanins and melanosomes in label-free HCMs, a non-invasive ‘fit-free’ approach, combining 2-photon excitation fluorescence lifetimes and emission spectral imaging with phasor plot segmentation was applied. Intracellular melanin-mapped FLIM phasors showed a linear distribution indicating that HCM melanins are a ratio of two fluorophores, eumelanin and pheomelanin. A quantitative histogram of HCM melanins was generated by identifying the image pixel fraction contributed by phasor clusters mapped to varying eumelanin/pheomelanin ratio. Eumelanin-enriched dark HCM regions mapped to phasors with shorter lifetimes and longer spectral emission (580–625 nm) and pheomelanin-enriched lighter pigmented HCM regions mapped to phasors with longer lifetimes and shorter spectral emission (550–585 nm). Overall, we demonstrated that these methods can identify and quantitatively profile the heterogeneous eumelanins/pheomelanins within in situ HCMs, and visualize melanosome spatial distributions, not previously reported for these cells.

Head to Knee: Cranial Neural Crest-Derived Cells as Promising Candidates for Human Cartilage Repair.

A large array of therapeutic procedures is available to treat cartilage disorders caused by trauma or inflammatory disease. Most are invasive and may result in treatment failure or development of osteoarthritis due to extensive cartilage damage from repeated surgery. Despite encouraging results of early cell therapy trials that used chondrocytes collected during arthroscopic surgery, these approaches have serious disadvantages, including morbidity associated with cell harvesting and low predictive clinical outcomes. To overcome these limitations, adult stem cells derived from bone marrow and subsequently from other tissues are now considered as preferred sources of cells for cartilage regeneration. Moreover, with new evidence showing that the choice of cell source is one of the most important factors for successful cell therapy, there is growing interest in neural crest-derived cells in both the research and clinical communities. Neural crest-derived cells such as nasal chondrocytes and oral stem cells that exhibit chondrocyte-like properties seem particularly promising in cartilage repair. Here, we review the types of cells currently available for cartilage cell therapy, including articular chondrocytes and various mesenchymal stem cells, and then highlight recent developments in the use of neural crest-derived chondrocytes and oral stem cells for repair of cartilage lesions.

Genome-wide analysis of facial skeletal regionalization in zebrafish.

Patterning of the facial skeleton involves the precise deployment of thousands of genes in distinct regions of the pharyngeal arches. Despite the significance for craniofacial development, how genetic programs drive this regionalization remains incompletely understood. Here we use combinatorial labeling of zebrafish cranial neural crest-derived cells (CNCCs) to define global gene expression along the dorsoventral axis of the developing arches. Intersection of region-specific transcriptomes with expression changes in response to signaling perturbations demonstrates complex roles for Endothelin 1 (Edn1) signaling in the intermediate joint-forming region, yet a surprisingly minor role in ventralmost regions. Analysis of co-variance across multiple sequencing experiments further reveals clusters of co-regulated genes, with in situ hybridization confirming the domain-specific expression of novel genes. We then created loss-of-function alleles for 12 genes and uncovered antagonistic functions of two new Edn1 targets, follistatin a (fsta) and emx2, in regulating cartilaginous joints in the hyoid arch. Our unbiased discovery and functional analysis of genes with regional expression in zebrafish arch CNCCs reveals complex regulation by Edn1 and points to novel candidates for craniofacial disorders.

In vitro cementoblast-like differentiation of postmigratory neural crest-derived p75+ stem cells with dental follicle cell conditioned medium

Cranial neural crest-derived cells (CNCCs) play important role in epithelial–mesenchymal interactions during tooth morphogenesis. However, the heterogeneity of CNCCs and their tendency to spontaneously differentiate along smooth muscle or osteoblast lineages in vitro limit further understanding of their biological properties. We studied the differentiation properties of isolated rat embryonic postmigratory CNCCs, expressing p75 neurotrophin receptor (p75NTR). These p75NTR positive (p75+) CNCCs, isolated using fluorescence activated cell sorter, exhibited fibroblast-like morphology and characteristics of mesenchymal stem cells. Incubation of p75+ CNCCs in dental follicle cell conditioned medium (DFCCM) combined with dentin non-collagenous proteins (dNCPs), altered their morphological features to cementoblast-like appearance. These cells also showed low proliferative activity, high ALP activity and significantly increased calcified nodule formation. Markers related to mineralization or specific to cementoblast lineage were highly expressed in dNCPs/DFCCM-treated p75+ cells, suggesting their differentiation along cementoblast-like lineage. p75+ stem cells selected from postmigratory CNCCs represent a pure stem cell population and could be used as a stem cell model for in vitro studies due to their intrinsic ability to differentiate to neuronal cells and transform from neuroectoderm to ectomesenchyme. They can provide a potential stem cell resource for tooth engineering studies and help to further investigate mechanisms of epithelial–mesenchymal interactions in tooth morphogenesis.

Conditional ablation of Dlx3 in cranial neural crest-derived cells results in abnormal development of hair, teeth and craniofacial bone

Conditional ablation of Dlx3 in cranial neural crest-derived cells results in abnormal development of hair, teeth and craniofacial bone

Exploring the Chondrocytic Potential of Cranial Neural Crest Cells

Osteoarthritis is a degenerative joint disease where the joint cartilage becomes damaged. Despite the ability of cranial neural crest cells (CNCC) to form cartilage in the embryo, this population of cells has been overlooked when designing cell‐based therapies for osteoarthritis and associated diseases We have begun to explore the potential of CNCC in developing tissues. A LacZ‐encoded replication‐deficient retrovial vector was used to tag explants of CNCC excised from stage 10 chick embryos before injecting into the joint regions of developing limbs of stage 18‐28 embryos. Tagged chondrocytes were subsequently identified in the developing cartilage, particularly in the region of the developing joints. The results appear to demonstrate that cranial neural crest derived cells (CNCDC) retain their ability to give rise to cartilage when implanted into the developing limb and are restricted to particular regions of the limb. The chondrocytic potential of CNCC is now being refined to investigate the integrative properties of these cells in developing and regenerating cartilage. Funded by Brighton & Sussex Medical School.

Shox2-deficiency leads to dysplasia and ankylosis of the temporomandibular joint in mice

The temporomandibular joint (TMJ) is a unique synovial joint whose development differs from the formation of other synovial joints. Mutations have been associated with the developmental defects of the TMJ only in a few genes. In this study, we report the expression of the homeobox gene Shox2 in the cranial neural crest derived mesenchymal cells of the maxilla-mandibular junction and later in the progenitor cells and undifferentiated chondrocytes of the condyle as well as the glenoid fossa of the developing TMJ. A conditional inactivation of Shox2 in the cranial neural crest-derived cells causes developmental abnormalities in the TMJ, including dysplasia of the condyle and glenoid fossa. The articulating disc forms but fuses with the fibrous layers of the condyle and glenoid fossa, clinically known as TMJ ankylosis. Histological examination indicates a delay in development in the mutant TMJ, accompanied by a significantly reduced rate of cell proliferation. In situ hybridization further demonstrates an altered expression of several key osteogenic genes and a delayed expression of the osteogenic differentiation markers. Shox2 appears to regulate the expression of osteogenic genes and is essential for the development and function of the TMJ. The Shox2 conditional mutant thus provides a unique animal model of TMJ ankylosis.

Cranial neural crest-derived cells participate in craniofacial skeletal repair

Abstract Introduction: Similarities between skeletal formation and repair have led to the theory that adult fracture repair may parallel development. We hypothesize that since the mandible is derived from cranial neural crest (CNC) and forms via intramembranous ossification, non-stabilized mandibular fractures will heal in a similar fashion. Methods: Non-stabilized osteotomies were created in the right hemi-mandible of adult male mice. Mandibles were harvested at 7, 14, 21, and 28 days and processed for histology. Ossification was assessed and the presence of cartilage determined using histology and in situ hybridization (ISH). To determine the embryologic origins of the regenerate, transgenic mice were generated where CNC cells express GFP constitutively under the Wnt-1 promoter. Fractures were created as described above and processed for frozen section. CNC cells were localized using immunohistochemistry (IHC) for GFP. Closed, non-stabilized tibial fractures were also performed to determine the presence, if any, of CNC-derived cells in appendicular skeletal fractures. Results: In contrast to non-stabilized fractures of the appendicular skeleton, mandibular fractures healed predominantly via intramembranous ossification, with minimal and delayed cartilage production seen via histology and collagen2 ISH. IHC for GFP revealed that the mandibular fracture callous and regenerate was comprised primarily of GFP+, CNC-derived cells. In contrast, the tibial fracture callous was devoid of any CNC-derived cells. Conclusions: These data suggest that mandibular fractures heal by recapitulating their developmental program. These findings have important implications for designing appropriate cell-based approaches to craniofacial regeneration.

Temporospatial cell interactions regulating mandibular and maxillary arch patterning.

The cellular origin of the instructive information for hard tissue patterning of the jaws has been the subject of a long-standing controversy. Are the cranial neural crest cells prepatterned or does the epithelium pattern a developmentally uncommitted population of ectomesenchymal cells? In order to understand more about how orofacial patterning is controlled we have investigated the temporal signalling interactions and responses between epithelium and mesenchymal cells in the mandibular and maxillary primordia. We show that within the mandibular arch, homeobox genes that are expressed in different proximodistal spatial domains corresponding to presumptive molar and incisor ectomesenchymal cells are induced by signals from the oral epithelium. In mouse, prior to E10, all ectomesenchyme cells in the mandibular arch are equally responsive to epithelial signals such as Fgf8, indicating that there is no pre-specification of these cells into different populations and suggesting that patterning of the hard tissues of the mandible is instructed by the epithelium. By E10.5, ectomesenchymal cell gene expression domains are still dependent on epithelial signals but have become fixed and ectopic expression cannot be induced. At E11 expression becomes independent of epithelial signals such that removal of the epithelium does not affect spatial ectomesenchymal expression. Significantly, however, the response of ectomesenchyme cells to epithelial regulatory signals was found to be different in the mandibular and maxillary primordium. Thus, whereas both mandibular and maxillary arch epithelia could induce Dlx2 and Dlx5 expression in the mandible and Dlx2 expression in the maxilla, neither could induce Dlx5 expression in the maxilla. Reciprocal cell transplantations between mandibular and maxillary arch ectomesenchymal cells revealed intrinsic differences between these populations of cranial neural crest-derived cells. Research in odontogenesis has shown that the oral epithelium of the mandibular and maxillary primordia has unique instructive signaling properties required to direct odontogenesis, which are not found in other branchial arch epithelia. As a consequence, development of jaw-specific skeletal structures may require some prespecification of maxillary ectomesenchyme to restrict the instructive influence of the epithelial signals and allow development of maxillary structures distinct from mandibular structures.