Zebrafish Neural Crest: Lessons and Tools to Study In Vivo Cell Migration.

The study of cell migration has been greatly enhanced by the development of new model systems and analysis protocols to study this process in vivo. Zebrafish embryos have been a principal protagonist because they are easily accessible, genetically tractable, and optically transparent. Neural crest cells, on the other hand, are the ideal system to study cell migration. These cells migrate extensively, using different modalities of movement and sharing many traits with metastatic cancer cells. In this chapter, we present new tools and protocols that allow the study of NC development and migration in vivo.

Live Imaging of the Neural Crest Cell Epithelial-to-Mesenchymal Transition in the Chick Embryo.

Live embryo imaging may provide a wealth of information on intact cell and tissue dynamics, but can be technically challenging to sustain embryo orientation and health for long periods under a microscope. In this protocol, we describe an in vivo method to mount and image cell movements during the epithelial-to-mesenchymal transition (EMT) of neural crest cells within the chick dorsal neural tube. We focus on describing the collection of images and data preparation for image analysis throughout the developmental stages HH15-21 in the chick trunk. Trunk neural crest cell EMT is crucial to development of the peripheral nervous system and pigment cell patterning. The methods we describe may also be applied to other cell and tissue phenomena at various chick developmental stages with some modifications.

Avenues for Investigating the Neural Crest and Its Derivatives in Non-model (Unconventional) Vertebrates: A Craniofacial Skeleton Perspective.

One of the early, profound insights regarding the biology of the neural crest was the observation of its contribution to the skeletal structures of the cranium and jaws. The critical nature of these structures made the comparative analysis of the cranial neural crest and its derived structures essential investigative aims toward our understanding of the development and evolution of vertebrates and vertebrate-specific structures. Though classically applied to a relatively wide range of taxa in the nineteenth and early twentieth centuries, the application of traditional methodologies for complex comparative developmental and anatomical analyses subsequently become more limited by their time-consuming nature, resource scarcity, and a greater emphasis on the genetic and molecular regulation of patterning and morphogenesis in a select number of tractable model organisms. Recently, however, this trend has been reversed, and the value of genetic and molecular-based questions applied to non-model (unconventional) vertebrate organisms has been re-appreciated. This is particularly true of comparative investigations of cranial neural crest biology. Herein, we present methodologies for the analysis of the cranial neural crest and its structural derivatives employable in modern investigations of both model and unconventional vertebrate organisms.

A New Look at Etiological Factors of Idiopathic Scoliosis: Neural Crest Cells.

Idiopathic scoliosis is one of the most common disabling pathologies of children and adolescents. Etiology and pathogenesis of idiopathic scoliosis remain unknown. To study the etiology of this disease we identified the cells' phenotypes in the vertebral body growth plates in patients with idiopathic scoliosis. Materials and methods: The cells were isolated from vertebral body growth plates of the convex and concave sides of the deformity harvested intraoperatively in 50 patients with scoliosis. Cells were cultured and identified by methods of common morphology, neuromorphology, electron microscopy, immunohistochemistry and PCR analysis. Results: Cultured cells of convex side of deformation were identified as chondroblasts. Cells isolated from the growth plates of the concave side of the deformation showed numerous features of neuro- and glioblasts. These cells formed synapses, contain neurofilaments, and expressed neural and glial proteins. Conclusion: For the first time we demonstrated the presence of cells with neural/glial phenotype in the concave side of the vertebral body growth plate in scoliotic deformity. We hypothesized that neural and glial cells observed in the growth plates of the vertebral bodies represent derivatives of neural crest cells deposited in somites due to alterations in their migratory pathway during embryogenesis. We also propose that ectopic localization of cells derived from neural crest in the growth plate of the vertebral bodies is the main etiological factor of the scoliotic disease.

Atg7-Mediated Autophagy Is Involved in the Neural Crest Cell Generation in Chick Embryo.

Autophagy plays a very important role in numerous physiological and pathological events. However, it still remains unclear whether Atg7-induced autophagy is involved in the regulation of neural crest cell production. In this study, we found the co-location of Atg7 and Pax7+ neural crest cells in early chick embryo development. Upregulation of Atg7 with unilateral transfection of full-length Atg7 increased Pax7+ and HNK-1+ cephalic and trunk neural crest cell numbers compared to either Control-GFP transfection or opposite neural tubes, suggesting that Atg7 over-expression in neural tubes could enhance the production of neural crest cells. BMP4 in situ hybridization and p-Smad1/5/8 immunofluorescent staining demonstrated that upregulation of Atg7 in neural tubes suppressed the BMP4/Smad signaling, which is considered to promote the delamination of neural crest cells. Interestingly, upregulation of Atg7 in neural tubes could significantly accelerate cell progression into the S phase, implying that Atg7 modulates cell cycle progression. However, ß-catenin expression was not significantly altered. Finally, we demonstrated that upregulation of the Atg7 gene could activate autophagy as did Atg8. We have also observed that similar phenotypes, such as more HNK-1+ neural crest cells in the unilateral Atg8 transfection side of neural tubes, and the transfection with full-length Atg8-GFP certainly promote the numbers of BrdU+ neural crest cells in comparison to the GFP control. Taken together, we reveal that Atg7-induced autophagy is involved in regulating the production of neural crest cells in early chick embryos through the modification of the cell cycle.

Potential Involvement of Draxin in the Axonal Projection of Cranial Nerves, Especially Cranial Nerve X, in the Chick Hindbrain.

The appropriate projection of axons within the nervous system is a crucial component of the establishment of neural circuitry. Draxin is a repulsive axon guidance protein. Draxin has important functions in the guidance of three commissures in the central nervous system and in the migration of neural crest cells and dI3 interneurons in the chick spinal cord. Here, we report that the distribution of the draxin protein and the location of 23C10-positive areas have a strong temporal and spatial correlation. The overexpression of draxin, especially transmembrane draxin, caused 23C10-positive axon bundles to misproject in the dorsal hindbrain. In addition, the overexpression of transmembrane draxin caused abnormal formation of the ganglion crest of the IX and X cranial nerves, misprojection of some anti-human natural killer-1 (HNK-1)-stained structures in the dorsal roof of the hindbrain, and a simultaneous reduction in the efferent nerves of some motoneuron axons inside the hindbrain. Our data reveal that draxin might be involved in the fascicular projection of cranial nerves in the hindbrain.

How Tissue Mechanical Properties Affect Enteric Neural Crest Cell Migration.

Neural crest cells (NCCs) are a population of multipotent cells that migrate extensively during vertebrate development. Alterations to neural crest ontogenesis cause several diseases, including cancers and congenital defects, such as Hirschprung disease, which results from incomplete colonization of the colon by enteric NCCs (ENCCs). We investigated the influence of the stiffness and structure of the environment on ENCC migration in vitro and during colonization of the gastrointestinal tract in chicken and mouse embryos. We showed using tensile stretching and atomic force microscopy (AFM) that the mesenchyme of the gut was initially soft but gradually stiffened during the period of ENCC colonization. Second-harmonic generation (SHG) microscopy revealed that this stiffening was associated with a gradual organization and enrichment of collagen fibers in the developing gut. Ex-vivo 2D cell migration assays showed that ENCCs migrated on substrates with very low levels of stiffness. In 3D collagen gels, the speed of the ENCC migratory front decreased with increasing gel stiffness, whereas no correlation was found between porosity and ENCC migration behavior. Metalloprotease inhibition experiments showed that ENCCs actively degraded collagen in order to progress. These results shed light on the role of the mechanical properties of tissues in ENCC migration during development.

Quantitating membrane bleb stiffness using AFM force spectroscopy and an optical sideview setup.

AFM-based force spectroscopy in combination with optical microscopy is a powerful tool for investigating cell mechanics and adhesion on the single cell level. However, standard setups featuring an AFM mounted on an inverted light microscope only provide a bottom view of cell and AFM cantilever but cannot visualize vertical cell shape changes, for instance occurring during motile membrane blebbing. Here, we have integrated a mirror-based sideview system to monitor cell shape changes resulting from motile bleb behavior of Xenopus cranial neural crest (CNC) cells during AFM elasticity and adhesion measurements. Using the sideview setup, we quantitatively investigate mechanical changes associated with bleb formation and compared cell elasticity values recorded during membrane bleb and non-bleb events. Bleb protrusions displayed significantly lower stiffness compared to the non-blebbing membrane in the same cell. Bleb stiffness values were comparable to values obtained from blebbistatin-treated cells, consistent with the absence of a functional actomyosin network in bleb protrusions. Furthermore, we show that membrane blebs forming within the cell-cell contact zone have a detrimental effect on cell-cell adhesion forces, suggesting that mechanical changes associated with bleb protrusions promote cell-cell detachment or prevent adhesion reinforcement. Incorporating a sideview setup into an AFM platform therefore provides a new tool to correlate changes in cell morphology with results from force spectroscopy experiments.

Invadosomes in their natural habitat.

Podosomes and invadopodia (collectively known as invadosomes) are small, F-actin-rich protrusions that are located at points of cell-ECM contacts and endow cells with invasive capabilities. So far, they have been identified in human or murine immune (myelomonocytic), vascular and cancer cells. The overarching reason for studying invadosomes is their connection to human disease. For example, macrophages and osteoclasts lacking Wiskott-Aldrich syndrome protein (WASp) are not able to form podosomes, and this leads to altered macrophage chemotaxis and defective bone resorption by osteoclasts. In contrast, the ability of cancer cells to form invadopodia is associated with high invasive and metastatic potentials. While invadosome composition, dynamics and signaling cascades leading to their assembly can be followed easily in in vitro assays, studying their contribution to pathophysiological processes in situ remains challenging. A number of recent papers have started to address this issue and describe invadosomes in situ in mouse models of cancer, cardiovascular disease and angiogenesis. In addition, in vivo invadosome homologs have been reported in developmental model systems such as C. elegans, zebrafish and sea squirt. Comparative analyses among different invasion mechanisms as they happen in their natural habitats, i.e., in situ, may provide an outline of the invadosome evolutionary history, and guide our understanding of the roles of the invasion process in pathophysiology versus development.

Organization of the neuroepithelial actin cytoskeleton is regulated by heparan sulfation during neurulation.

Heparan sulfate and cytoskeletal actin microfilaments have both been shown to be important regulators of neural tube closure during embryonic development. To determine the functional relationship of these two molecules in formation of the spinal neural tube, we cultured ARC mouse embryos at embryonic day E8.5 in the presence of chlorate, a competitive inhibitor of glycosaminoglycan sulfation, and examined the effects on organization of actin microfilaments in the neuroepithelium. Compared against embryos cultured under control conditions, chlorate-treated embryos had shortened posterior neuropore, a loss of median hinge point formation and increased bending at the paired dorsolateral hinge points. Furthermore, apical organization of actin microfilaments in the neuroepithelial cells was absent, and this was associated with convex bending of the neuroepithelium. The results suggest that heparan sulfate is an important determinant of cytoskeletal actin organization during spinal neurulation, and that its biological action is dependent on sulfation of the heparan molecule.

Trophic and proliferative perturbations of in vivo/in vitro cephalic neural crest cells after ethanol exposure are prevented by neurotrophin 3.

Neural crest cells (NCCs), a transient population that migrates from the developing neural tube, distributes through the embryo and differentiates into many derivatives, are clearly involved in the damage induced by prenatal exposure to ethanol. The aim of this work was to evaluate alterations of trophic parameters of in vivo (in ovo) and in vitro NCCs exposed to teratogenic ethanol doses, and their possible prevention by trophic factor treatment. Chick embryos of 24-30h of incubation were treated during 10h with 100mM ethanol, or 40 ng/ml Neurotrophin 3 (NT3), or 10 ng/ml Ciliary Neurotrophic Factor (CNTF), or ethanol plus NT3 or CNTF, or defined medium; then the topographic distribution of NCC apoptosis was assessed using a whole-mount acridine orange supravital method. Cultures of cephalic NCCs were exposed to the same ethanol or NT3, or CNTF treatments, or ethanol plus one of both trophic factors, or N2 medium. A viability assay was performed using the calcein-ethidium test, apoptosis was evaluated with the TUNEL test, and proliferative capacity after BrdU labeling. After direct exposure of embryos to 100mM ethanol for 10h, a high level of NCC apoptosis was coincident with the abnormal closure of the neural tube. These anomalies were prevented in embryos exposed to ethanol plus NT3 but not with CNTF. In NCC cultures, high cell mortality and a diminution of proliferative activity were observed after 3h of ethanol treatment. Incubation with ethanol plus NT3 (but not with CNTF) prevented NCC mortality as well as a fall in NCC proliferation. The consequences of direct exposure to ethanol expand data from our and other laboratories, supporting current opinion on the potential risk of alcohol ingestion (even at low doses and/or during a short time), in any period of pregnancy or lactation. Our in vivo/in vitro model encourages us to examine the pathogenic mechanism(s) of the ethanol-exposed embryo as well as the use of trophic factors for the treatment and/or prevention of anomalies induced by prenatal alcohol.

Evidence on the neural crest origin of PEComas.

The perivascular epithelioid cell (PEC) has been proposed to be the proliferating cell type in a group of tumors known as PEComas. The histogenesis of PEComas is one of the most mysterious aspects of pathology. Hypothesis on its precursor are many, including a cell from blood vessel walls or the myoblast. In the current report, we review many morphologic, clinical, ultrastructural, molecular and genetic aspects that support the hypothesis of an origin of PEComas from the neural crest.

Characterization of nerve conduits seeded with neurons and Schwann cells derived from hair follicle neural crest stem cells.

In this study a tissue-engineered nerve conduit for repair of peripheral nerve defects was devised and characterized in vitro. To construct the nerve conduits, beagle sciatic nerves were acellularized with lysolecithin and seeded with neurons and Schwann cells, which were induced from rat hair follicle neural crest stem cells. The nerve constructs were cultured in vitro and characterized by multiple methods, including immnohistochemistry, electron microscopy, and electrophysiology at 1, 3, and 8 weeks. The same scaffolds injected with phosphate-buffered saline were used as control. We found that hair follicle neural crest stem cell-derived neurons could survive in the nerve constructs as long as 8 weeks, and the nerve constructs showed desirable electrophysiological features. This nerve construct could work as an alternative for the current standard autologous nerve transplantation, especially in peripheral nerves with large defects.

Rho-kinase and myosin II affect dynamic neural crest cell behaviors during epithelial to mesenchymal transition in vivo.

The induction and migration of neural crest cells (NCCs) are essential to the development of craniofacial structures and the peripheral nervous system. A critical step in the development of NCCs is the epithelial to mesenchymal transition (EMT) that they undergo in order to initiate migration. Several transcription factors are important for the NCC EMT. However, less is known about the effectors regulating changes in cell adhesion, the cytoskeleton, and cell motility associated with the EMT or about specific changes in the behavior of cells undergoing EMT in vivo. We used time-lapse imaging of NCCs in the zebrafish hindbrain to show that NCCs undergo a stereotypical series of behaviors during EMT. We find that loss of cell adhesion and membrane blebbing precede filopodial extension and the onset of migration. Live imaging of actin dynamics shows that actin localizes differently in blebs and filopodia. Moreover, we find that disruption of myosin II or Rho-kinase (ROCK) activity inhibits NCC blebbing and causes reduced NCC EMT. These data reveal roles for myosin II and ROCK in NCC EMT in vivo, and provide a detailed characterization of NCC behavior during EMT that will form a basis for further mechanistic studies.

Essential role of non-canonical Wnt signalling in neural crest migration.

Migration of neural crest cells is an elaborate process that requires the delamination of cells from an epithelium and cell movement into an extracellular matrix. In this work, it is shown for the first time that the non-canonical Wnt signalling [planar cell polarity (PCP) or Wnt-Ca2+] pathway controls migration of neural crest cells. By using specific Dsh mutants, we show that the canonical Wnt signalling pathway is needed for neural crest induction, while the non-canonical Wnt pathway is required for neural crest migration. Grafts of neural crest tissue expressing non-canonical Dsh mutants, as well as neural crest cultured in vitro, indicate that the PCP pathway works in a cell-autonomous manner to control neural crest migration. Expression analysis of non-canonical Wnt ligands and their putative receptors show that Wnt11 is expressed in tissue adjacent to neural crest cells expressing the Wnt receptor Frizzled7 (Fz7). Furthermore, loss- and gain-of-function experiments reveal that Wnt11 plays an essential role in neural crest migration. Inhibition of neural crest migration by blocking Wnt11 activity can be rescued by intracellular activation of the non-canonical Wnt pathway. When Wnt11 is expressed opposite its normal site of expression, neural crest migration is blocked. Finally, time-lapse analysis of cell movement and cell protrusion in neural crest cultured in vitro shows that the PCP or Wnt-Ca2+ pathway directs the formation of lamellipodia and filopodia in the neural crest cells that are required for their delamination and/or migration.

Disruption of actin cytoskeleton and anchorage-dependent cell spreading induces apoptotic death of mouse neural crest cells cultured in vitro.

In vertebrate embryos, neural crest cells emigrate out of the neural tube and contribute to the formation of a variety of neural and nonneural tissues. Some neural crest cells undergo apoptotic death during migration, but its biological significance and the underlying mechanism are not well understood. We carried out an in vitro study to examine how the morphology and survival of cranial neural crest (CNC) cells of the mouse embryo are affected when their actin cytoskeleton or anchorage-dependent cell spreading is perturbed. Disruption of actin fiber organization by cytochalasin D (1 microg/ml) and inhibition of cell attachment by matrix metalloproteinase-2 (MMP-2; 2.0 units/ml) were followed by morphologic changes and apoptotic death of cultured CNC cells. When the actin cytoskeleton was disrupted by cytochalasin D, the morphologic changes of cultured CNC cells preceded DNA fragmentation. These results indicate that the maintenance of cytoskeleton and anchorage-dependent cell spreading are required for survival of CNC cells. The spatially and temporally regulated expression of proteinases may be essential for the differentiation and migration of neural crest cells.

[Morphological study on the neural tube defects caused by passive smoking].

OBJECTIVE: To investigate the possible mechanisms of teratogenesis caused by passive smoking. METHODS: The stereomicroscope and electron microscope were used to observe the effects of passive smoking on the development of golden hamsters' neural tube and alternations of neuroepithelial ultrastructures. RESULTS: Passive smoking could induce teratogenesis during neurulation (chi 2 = 51.28, P < 0.01). The main forms of neural tube defects were spina bifida, exencephaly, and so on. In embryos of every passive smoking group, neuroepithelial cells arranged irregularly. The intercellular spaces became wide. The apical portion of many neuroepithelial cells bulged out into the lumen and many microvilli were shorted and swollen. A lot of vacuolation appeared in the cytoplasm. The cristae of mitochondria reduced even disappeared, and some mitochondria became elongate. Irregular nuclear, increased heterochromatin and karyopycnosis/karyorrhexis were observed easily. Perinuclear cisternae partially swollen and embraced tangible material. Some death cell's decomposed a lot of apoptotic bodies. Most of the apoptotic bodies were found within the cytoplasm of otherwise healthy-looking or healthy cells. CONCLUSION: These data prove that passive smoking can cause dymorphogenesis of the neural tube. And tobacco smoking might induce excess degeneration, death, and cells loss in the neural tube and mesenchyme, thereby resulting in failure of formation and differentiation of neural tube.

Antisense knockdown of the beta1 integrin subunit in the chicken embryo results in abnormal neural crest cell development.

Neural crest cells escape the neural tube by undergoing an epithelial to mesenchymal transition (EMT). This is followed by extensive migration along specific pathways that are lined with extracellular matrix (ECM). In this study, we have examined the roles of matrix receptors containing beta1 integrin subunits in neural crest cell morphogenesis using antisense morpholino oligos electroporated in ovo into avian neural crest cell precursors. Our results show that reduced levels of expression of beta1 integrin subunits in the dorsal neural tube results in an abnormal epithelial to mesenchymal transition. In approximately half of the experimental embryos, however, some neural crest cells filled with beta1 antisense are able to escape the neural tube and migrate ventrally, indicating that grossly normal migration of trunk neural crest cells can take place after beta1 integrin expression is reduced. This study shows the potential of this novel method for investigating the roles of genes that are required for the survival of early mouse embryos in later development events.

The effects of folic acid in the prevention of neural tube development defects caused by phenytoin in early chick embryos.

STUDY DESIGN: The effects of phenytoin and folic acid on the development of neural tube defects in early chick embryos were studies. OBJECTIVE: To investigate the effects of folic acid in the prevention of neural tube development defects. SUMMARY OF BACKGROUND DATA: Several studies have shown that phenytoin selectively inhibits neural tube closure. Folic acid supplementation has been reported to decrease the occurrence of neural tube defects. METHODS: This study shows the effects of folic acid in preventing neural tube development defects caused by phenytoin in chicks based on light microscopy, transmission electron microscopy, and histopathological examination. Forty-five fertile Hubbard Broil eggs, all at Stage 8 (four somite) of development, were divided into three equal groups: Group 1 embryos (n = 15), the control group, were explanted and grown for 18 hours in a nutrient medium (thin albumin). Group 2 embryos (n = 15) were explanted and grown for 18 hours in a nutrient medium containing 500 microg/mL of phenytoin. Group 3 embryos (n = 15) were explanted and grown for 18 hours in a nutrient medium containing 500 microg/mL of phenytoin and 0.4 microg/mL of folic acid. RESULTS: After the incubation period, 86.6% of the control embryos (Group 1) had intact neural tubes; 80% of Group 2 and 46.6% of Group 3 embryos showed neural tube defects. CONCLUSIONS: The results of this study suggest that phenytoin causes neural tube defects, whereas folic acid decreases the incidence of neural tube development defects caused by phenytoin in early chick embryos.

Origins and plasticity of neural crest cells and their roles in jaw and craniofacial evolution.

The vertebrate head is a complex assemblage of cranial specializations, including the central and peripheral nervous systems, viscero- and neurocranium, musculature and connective tissue. The primary differences that exist between vertebrates and other chordates relate to their craniofacial organization. Therefore, evolution of the head is considered fundamental to the origins of vertebrates (Gans and Northcutt, 1983). The transition from invertebrate to vertebrate chordates was a multistep process, involving the formation and patterning of many new cell types and tissues. The evolution of early vertebrates, such as jawless fish, was accompanied by the emergence of a specialized set of cells, called neural crest cells which have long held a fascination for developmental and evolutionary biologists due to their considerable influence on the complex development of the vertebrate head. Although it has been classically thought that protochordates lacked neural crest counterparts, the recent identification and characterization of amphioxus and ascidian genes homologous to those involved in vertebrate neural crest development challenges this idea. Instead it suggests thatthe neural crest may not be a novel vertebrate cell population, but could have in fact originated from the protochordate dorsal midline epidermis. Consequently, the evolution of the neural crest cells could be reconsidered in terms of the acquisition of new cell properties such as delamination-migration and also multipotency which were key innovations that contributed to craniofacial development. In this review we discuss recent findings concerning the inductive origins of neural crest cells, as well as new insights into the mechanisms patterning this cell population and the subsequent influence this has had on craniofacial evolution.