Protein Tyrosine Phosphatase 4A3 (PTP4A3) Is Required for Xenopus laevis Cranial Neural Crest Migration In Vivo

Uveal melanoma is the most common intraocular malignancy in adults, representing between about 4% and 5% of all melanomas. High expression levels of Protein Tyrosine Phosphatase 4A3, a dual phosphatase, is highly predictive of metastasis development and PTP4A3 overexpression in uveal melanoma cells increases their in vitro migration and in vivo invasiveness. Melanocytes, including uveal melanocytes, are derived from the neural crest during embryonic development. We therefore suggested that PTP4A3 function in uveal melanoma metastasis may be related to an embryonic role during neural crest cell migration. We show that PTP4A3 plays a role in cephalic neural crest development in Xenopus laevis. PTP4A3 loss of function resulted in a reduction of neural crest territory, whilst gain of function experiments increased neural crest territory. Isochronic graft experiments demonstrated that PTP4A3-depleted neural crest explants are unable to migrate in host embryos. Pharmacological inhibition of PTP4A3 on dissected neural crest cells significantly reduced their migration velocity in vitro. Our results demonstrate that PTP4A3 is required for cephalic neural crest migration in vivo during embryonic development.     

A Src-Tks5 pathway is required for neural crest cell migration during embryonic development

In the adult organism, cell migration is required for physiological processes such as angiogenesis and immune surveillance, as well as pathological events such as tumor metastasis. The adaptor protein and Src substrate Tks5 is necessary for cancer cell migration through extracellular matrix in vitro and tumorigenicity in vivo. However, a role for Tks5 during embryonic development, where cell migration is essential, has not been examined. We used morpholinos to reduce Tks5 expression in zebrafish embryos, and observed developmental defects, most prominently in neural crest-derived tissues such as craniofacial structures and pigmentation. The Tks5 morphant phenotype was rescued by expression of mammalian Tks5, but not by a variant of Tks5 in which the Src phosphorylation sites have been mutated. We further evaluated the role of Tks5 in neural crest cells and neural crest-derived tissues and found that loss of Tks5 impaired their ventral migration. Inhibition of Src family kinases also led to abnormal ventral patterning of neural crest cells and their derivatives. We confirmed that these effects were likely to be cell autonomous by shRNA-mediated knockdown of Tks5 in a murine neural crest stem cell line. Tks5 was required for neural crest cell migration in vitro, and both Src and Tks5 were required for the formation of actin-rich structures with similarity to podosomes. Additionally, we observed that neural crest cells formed Src-Tks5-dependent cell protrusions in 3-D culture conditions and in vivo. These results reveal an important and novel role for the Src-Tks5 pathway in neural crest cell migration during embryonic development. Furthermore, our data suggests that this pathway regulates neural crest cell migration through the generation of actin-rich pro-migratory structures, implying that similar mechanisms are used to control cell migration during embryogenesis and cancer metastasis.     

Cellular Plasticity Among Axolotl Neural Crest-Derived Pigment Cell Lineages

Many of the factors and mechanisms guiding the migration/differentiation of neural crest cells that give rise to a number of distinguishable cell types, including all dermal and epidermal pigment cells, remain unknown. The axolotl possesses three pigment cell types that differentiate according to specific developmentally programmed sequences and contribute to pigment pattern in the adult. A single lineage of the crest that becomes restricted to one of three pigment cell types gives us the opportunity to examine the existence of a neural crest stem cell population and the potential for transdifferentiation events. Interpretations of experiments involving drug-treated and mutant axolotls implicate cellular plasticity leading to observed phenotypes. We present results from recent in vitro studies designed to identify parameters influencing differentiation events of individual neural crest-derived pigment cell lineages. We demonstrate that the differentiation of xanthophores is enhanced, while that of the melanophores are inhibited in guanosine-supplemented neural crest cell cultures. Data suggest that the increase in one pigment cell population is at the expense of another, indicative of cellular plasticity. Videomicroscopy used in this study agrees with an abundance of correlative evidence supporting the hypothesis of transdifferentiation events among neural crest-derived pigment cell populations. The embryonic neural crest-derived pigment cell system is an ideal model to study differentiation of multipotential stem cells that play critical roles in patterning.     

Regulation of neural crest cell populations: occurrence, distribution and underlying mechanisms

Regulation is a significant developmental event because successful cell proliferation and migration are critical to shaping young embryos. Regulation -- the replacement of undifferentiated embryonic cells by other cells in response to signals received from the environment -- is distinct from wound healing and regeneration. Investigations on regulation of neural crest cells span all vertebrates and have revealed that regulative ability varies both among classes (even species), and spatially and temporally within individuals. In general, there is greatest regulation for cranial neural crest cells, less for trunk, and virtually none forcardiac. Regulation also appears to be more complete at early embryonic stages. Fate-mapping studies have demonstrated that large regions of neural crest cells must be removed to generate missing or morphologically reduced structures. Recent studies reveal that less extensive neural crest cell extirpations result in normal morphology of cartilaginous and neuronal elements in the head, and normal development of pigmentation in the trunk. Ablation of cardiac neural crest cells frequently generates abnormalities of the heart, great vessels and parasympathetic nerve innervation. Decreased cell death, increased division, change in fate and altered migration are possible cellular mechanisms of regulation. In mostcases, the specific mechanisms of regulation are unknown, but a major premise underlying regulation is that cell potential is greater than cell fate. This concept was born from studies which demonstrated that some cells were able to express alternative fates if transplanted to a new environment. Among the potential cellular mechanisms for regulation, cell migration has received the most attention. Following ablation of neural crest cells, replacement neural crest cells migrate into gaps, most frequently from anterior/posterior locations. Cells from surrounding epidermal and neural ectoderm may have limited regulative ability, while compensation by cells from the ventral neural tube has been demonstrated to an even lesser extent. Regulation by such non-crest cells would require their transformation into neural crest cells. The potential for regulation of neural crest by placodal cells supports a closer relationship between neural crest and placodal ectoderm than previously recognized. Decreased cell death has been discussed primarily with reference to (1) cranial ganglia that have dual contributions from neural crest and placodal cells and (2) programmed cell death in rhombomeres three and five. Increased cell division in response to neural crest ablation is likely more common than has been reported, but this mechanism is difficult to interpret without a 3-D context for viewing how patterns of division differ from normal. Lastly, changes in cell fate may be the driving factor in regulation of embryonic cells. It has been repeatedly demonstrated thatcell potential is greaterthan cell fate. Once reliable mechanisms for assessing cell potential are established, we may find that fates are commonly altered in response to environmental signals. Regulation is therefore significant both as a basic developmental mechanism and as a mechanism for evolutionary change. The more labile the fate of embryonic cells, the more potential there is for maintaining existing characters and for generating new ones. According to Ettensohn (1992, p. 50), further analysis of such systems might . With regard to the neural crest, studies on regulation of this vital population of cells provide insight to the origin of the neural crest, to embryonic repair, and to the source of many craniofacial malformations, heart and other embryonic defects. (ABSTRACT TRUNCATED)     

Intercellular crosstalk in human malignant melanoma

Incidence of malignant melanoma is increasing globally. While the initial stages of tumors can be easily treated by a simple surgery, the therapy of advanced stages is rather limited. Melanoma cells spread rapidly through the body of a patient to form multiple metastases. Consequently, the survival rate is poor. Therefore, emphasis in melanoma research is given on early diagnosis and development of novel and more potent therapeutic options. The malignant melanoma is arising from melanocytes, cells protecting mitotically active keratinocytes against damage caused by UV light irradiation. The melanocytes originate in the neural crest and consequently migrate to the epidermis. The relationship between the melanoma cells, the melanocytes, and neural crest stem cells manifests when the melanoma cells are implanted to an early embryo: they use similar migratory routes as the normal neural crest cells. Moreover, malignant potential of these melanoma cells is overdriven in this experimental model, probably due to microenvironmental reprogramming. This observation demonstrates the crucial role of the microenvironment in melanoma biology. Indeed, malignant tumors in general represent complex ecosystems, where multiple cell types influence the growth of genetically mutated cancer cells. This concept is directly applicable to the malignant melanoma. Our review article focuses on possible strategies to modify the intercellular crosstalk in melanoma that can be employed for therapeutic purposes.     

The effect of tenascin and embryonic basal lamina on the behavior and morphology of neural crest cells in vitro

We have investigated the morphology and migratory behavior of quail neural crest cells on isolated embryonic basal laminae or substrata coated with fibronectin or tenascin. Each of these substrata have been implicated in directing neural crest cell migration in situ. We also observed the altered behavior of cells in response to the addition of tenascin to the culture medium independent of its effect as a migratory substratum. On tenascin-coated substrata, the rate of neural crest cell migration from neural tube explants was significantly greater than on uncoated tissue culture plastic, on fibronectin-coated plastic, or on basal lamina isolated from embryonic chick retinae. Neural crest cells on tenascin were rounded and lacked lamellipodia, in contrast to the flattened cells seen on basal lamina and fibronectin-coated plastic. In contrast, when tenascin was added to the culture medium of neural crest cells migrating on isolated basal lamina, a significant reduction in the rate of cell migration was observed. To study the nature of this effect, we used human melanoma cells, which have a number of characteristics in common with quail neural crest cells though they would be expected to have a distinct family of integrin receptors. A dose-dependent reduction in the rate of translocation was observed when tenascin was added to the culture medium of the human melanoma cell line plated on isolated basal laminae, indicating that the inhibitory effect of tenascin bound to the quail neural crest surface is probably not solely the result of competitive inhibition by tenascin for the integrin receptor. Our results show that tenascin can be used as a migratory substratum by avian neural crest cells and that tenascin as a substratum can stimulate neural crest cell migration, probably by permitting rapid detachment. Tenascin in the medium, on the other hand, inhibits both the migration rates and spreading of motile cells on basal lamina because it binds only the cell surface and not the underlying basal lamina. Cell surface-bound tenascin may decrease cell-substratum interactions and thus weaken the tractional forces generated by migrating cells. This is in contrast to the action of fibronectin, which when added to the medium stimulates cell migration by binding both to neural crest cells and the basal lamina, thus providing a bridge between the motile cells and the substratum.     

Cranial neural crest migration: New rules for an old road

The neural crest serve as an excellent model to better understand mechanisms of embryonic cell migration. Cell tracing studies have shown that cranial neural crest cells (CNCCs) emerge from the dorsal neural tube in a rostrocaudal manner and are spatially distributed along stereotypical, long distance migratory routes to precise targets in the head and branchial arches. Although the CNCC migratory pattern is a beautifully choreographed and programmed invasion, the underlying orchestration of molecular events is not well known. For example, it is still unclear how single CNCCs react to signals that direct their choice of direction and how groups of CNCCs coordinate their interactions to arrive at a target in an ordered manner. In this review, we discuss recent cellular and molecular discoveries of the CNCC migratory pattern. We focus on events from the time when CNCCs encounter the tissue adjacent to the neural tube and their travel through different microenvironments and into the branchial arches. We describe the patterning of discrete cell migratory streams that emerge from the hindbrain, rhombomere (r) segments r1-r7, and the signals that coordinate directed migration. We propose a model that attempts to unify many complex events that establish the CNCC migratory pattern, and based on this model we integrate information between cranial and trunk neural crest development.     

The impact of adhesion on cellular invasion processes in cancer and development

In this paper we consider a simple continuous model to describe cell invasion, incorporating the effects of both cell-cell adhesion and cell-matrix adhesion, along with cell growth and proteolysis by cells of the surrounding extracellular matrix (ECM). We demonstrate that the model is capable of supporting both noninvasive and invasive tumour growth according to the relative strength of cell-cell to cell-matrix adhesion. Specifically, for sufficiently strong cell-matrix adhesion and/or sufficiently weak cell-cell adhesion, degradation of the surrounding ECM accompanied by cell-matrix adhesion pulls the cells into the surrounding ECM. We investigate the criticality of matrix heterogeneity on shaping invasion, demonstrating that a highly heterogeneous ECM can result in a “fingering” of the invasive front, echoing observations in real-life invasion processes ranging from malignant tumour growth to neural crest migration during embryonic development.     

Origins of adult pigmentation: diversity in pigment stem cell lineages and implications for pattern evolution

Teleosts comprise about half of all vertebrate species and exhibit an extraordinary diversity of adult pigment patterns that function in shoaling, camouflage, and mate choice and have played important roles in speciation. Here, we review studies that have identified several distinct neural crest lineages, with distinct genetic requirements, that give rise to adult pigment cells in fishes. These lineages include post‐embryonic, peripheral nerve‐associated stem cells that generate black melanophores and iridescent iridophores, cells derived directly from embryonic neural crest cells that generate yellow‐orange xanthophores, and bipotent stem cells that generate both melanophores and xanthophores. This complexity in adult chromatophore lineages has implications for our understanding of adult traits, melanoma, and the evolutionary diversification of pigment cell lineages and patterns.     

RAC1 induces nuclear alterations through the LINC complex to enhance melanoma invasiveness

RHO GTPases are key regulators of the cytoskeletal architecture, which impact a broad range of biological processes in malignant cells including motility, invasion, and metastasis, thereby affecting tumor progression. One of the constraints during cell migration is the diameter of the pores through which cells pass. In this respect, the size and shape of the nucleus pose a major limitation. Therefore, enhanced nuclear plasticity can promote cell migration. Nuclear morphology is determined in part through the cytoskeleton, which connects to the nucleoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Here, we unravel the role of RAC1 as an orchestrator of nuclear morphology in melanoma cells. We demonstrate that activated RAC1 promotes nuclear alterations through its effector PAK1 and the tubulin cytoskeleton, thereby enhancing migration and intravasation of melanoma cells. Disruption of the LINC complex prevented RAC1-induced nuclear alterations and the invasive properties of melanoma cells. Thus, RAC1 induces nuclear morphology alterations through microtubules and the LINC complex to promote an invasive phenotype in melanoma cells.     

Metalloprotease ADAM9 cleaves ephrin-B ligands and differentially regulates Wnt and mTOR signaling downstream of Akt kinase in colorectal cancer cells

Ephrin-B signaling has been implicated in many normal and pathological processes, including neural crest development and tumor metastasis. We showed previously that proteolysis of ephrin-B ligands by the disintegrin metalloprotease ADAM13 is necessary for canonical Wnt signal activation and neural crest induction in Xenopus, but it was unclear if these mechanisms are conserved in mammals. Here, we report that mammalian ADAM9 cleaves ephrin-B1 and ephrin-B2 and can substitute for Xenopus ADAM13 to induce the neural crest. We found that ADAM9 expression is elevated in human colorectal cancer (CRC) tissues and that knockdown (KD) of ADAM9 inhibits the migration and invasion of SW620 and HCT116 CRC cells by reducing the activity of Akt kinase, which is antagonized by ephrin-Bs. Akt is a signaling node that activates multiple downstream pathways, including the Wnt and mTOR pathways, both of which can promote CRC cell migration/invasion. Surprisingly, we also found that KD of ADAM9 downregulates Wnt signaling but has negligible effects on mTOR signaling in SW620 cells; in contrast, mTOR activity is suppressed while Wnt signaling remains unaffected by ADAM9 KD in HCT116 cells. These results suggest that mammalian ADAM9 cleaves ephrin-Bs to derepress Akt and promote CRC migration and invasion; however, the signaling pathways downstream of Akt are differentially regulated by ADAM9 in different CRC cell lines, reflecting the heterogeneity of CRC cells in responding to manipulations of upstream Akt regulators.     

In vivo analysis reveals a critical role for neuropilin-1 in cranial neural crest cell migration in chick

The neural crest provides an excellent model system to study invasive cell migration, however it is still unclear how molecular mechanisms direct cells to precise targets in a programmed manner. We investigate the role of a potential guidance factor, neuropilin-1, and use functional knockdown assays, tissue transplantation and in vivo confocal time-lapse imaging to analyze changes in chick cranial neural crest cell migratory patterns. When neuropilin-1 function is knocked down in ovo, neural crest cells fail to fully invade the branchial arches, especially the 2nd branchial arch. Time-lapse imaging shows that neuropilin-1 siRNA transfected neural crest cells stop and collapse filopodia at the 2nd branchial arch entrances, but do not die. This phenotype is cell autonomous. To test the influence of population pressure and local environmental cues in driving neural crest cells to the branchial arches, we isochronically transplanted small subpopulations of DiI-labeled neural crest cells into host embryos ablated of neighboring, premigratory neural crest cells. Time-lapse confocal analysis reveals that the transplanted cells migrate in narrow, directed streams. Interestingly, with the reduction of neuropilin-1 function, neural crest cells still form segmental migratory streams, suggesting that initial neural crest cell migration and invasion of the branchial arches are separable processes.     

Directional Collective Cell Migration Emerges as a Property of Cell Interactions

Collective cell migration is a fundamental process, occurring during embryogenesis and cancer metastasis. Neural crest cells exhibit such coordinated migration, where aberrant motion can lead to fatality or dysfunction of the embryo. Migration involves at least two complementary mechanisms: contact inhibition of locomotion (a repulsive interaction corresponding to a directional change of migration upon contact with a reciprocating cell), and co-attraction (a mutual chemoattraction mechanism). Here, we develop and employ a parameterized discrete element model of neural crest cells, to investigate how these mechanisms contribute to long-range directional migration during development. Motion is characterized using a coherence parameter and the time taken to reach, collectively, a target location. The simulated cell group is shown to switch from a diffusive to a persistent state as the response-rate to co-attraction is increased. Furthermore, the model predicts that when co-attraction is inhibited, neural crest cells can migrate into restrictive regions. Indeed, inhibition of co-attraction in vivo and in vitro leads to cell invasion into restrictive areas, confirming the prediction of the model. This suggests that the interplay between the complementary mechanisms may contribute to guidance of the neural crest. We conclude that directional migration is a system property and does not require action of external chemoattractants.     

Mathematical and experimental insights into the development of the enteric nervous system and Hirschsprung's disease

The vertebrate enteric nervous system is formed by a rostro-caudally directed invasion of the embryonic gastrointestinal mesenchyme by neural crest cells. Failure to complete this invasion results in the distal intestine lacking intrinsic neurons. This potentially fatal condition is called Hirschsprung's Disease. A mathematical model of cell invasion incorporating cell motility and proliferation of neural crest cells to a carrying capacity predicted invasion outcomes to imagined manipulations, and these manipulations were tested experimentally. Mathematical and experimental results agreed. The results show that the directional invasion is chiefly driven by neural crest cell proliferation. Moreover, this proliferation occurs in a small region at the wavefront of the invading population. These results provide an understanding of why many genes implicated in Hirschsprung's Disease influence neural crest population size. In addition, during in vivo development the underlying gut tissues are growing simultaneously as the neural crest cell invasion proceeds. The interactions between proliferation, motility and gut growth dictate whether or not complete colonization is successful. Mathematical modeling provides insights into the conditions required for complete colonization or a Hirschsprung's-like deficiency. Experimental evidence supports the hypotheses suggested by the modeling.     

Integrin alpha5beta1 supports the migration of Xenopus cranial neural crest on fibronectin

During early embryonic development, cranial neural crest cells emerge from the developing mid- and hindbrain. While numerous studies have focused on integrin involvement in trunk neural crest cell migration, comparatively little is known about mechanisms of cranial neural crest cell migration. We show that fibronectin, but not laminin, vitronectin, or type I collagen can support cranial neural crest cell migration and segmentation in vitro. These behaviors require both the RGD and "synergy" sites located within the central cell-binding domain of fibronectin. While these two sites are sufficient for cranial neural crest cell migration, we find that the second Heparin-binding domain of fibronectin can provide additional support for cranial neural crest cell migration in vitro. Finally, using a function blocking monoclonal antibody, we show that cranial neural crest cell migration on fibronectin requires the integrin alpha5beta1.