Bioinformatic Analysis of Nematode Migration-Associated Genes Identifies Novel Vertebrate Neural Crest Markers

Neural crest cells are highly motile, yet a limited number of genes governing neural crest migration have been identified by conventional studies. To test the hypothesis that cell migration genes are likely to be conserved over large evolutionary distances and from diverse tissues, we searched for vertebrate homologs of genes important for migration of various cell types in the invertebrate nematode and examined their expression during vertebrate neural crest cell migration. Our systematic analysis utilized a combination of comparative genomic scanning, functional pathway analysis and gene expression profiling to uncover previously unidentified genes expressed by premigratory, emigrating and/or migrating neural crest cells. The results demonstrate that similar gene sets are expressed in migratory cell types across distant animals and different germ layers. Bioinformatics analysis of these factors revealed relationships between these genes within signaling pathways that may be important during neural crest cell migration.     

PVF1, a PDGF/VEGF homolog,is sufficient to guide border cells and interacts genetically with Taiman

The border cells of the Drosophila ovary undergo a well-defined and developmentally regulated cell migration. Two signals have previously been shown to control where and when the cells migrate. The steroid hormone ecdysone, acting through its receptor and a coactivator known as Taiman, contributes to regulating the timing of border cell migration. PVF1, a growth factor related to platelet-derived growth factor and vascular-endothelial growth factor, contributes to guiding the border cells to the oocyte. To probe the mechanisms controlling border cell migration further, we performed a screen for genes that exhibit dominant genetic interactions with taiman. We identified 14 genomic regions that interact with taiman. Within one region, we identified Pvf1 as the gene responsible for the interaction. Signaling by PVF1 has been proposed to guide the border cells to their proper target, but ectopic PVF1 has not been tested for its ability to redirect the border cells. We tested the ability of PVF1, as well as other factors such as Gurken, to guide the border cells to new targets. Our results demonstrate that ectopic expression of PVF1 is sufficient to redirect border cells in some egg chambers but that the other factors tested are not. These data suggest that the guidance of border cell migration is robust and that there are likely to be additional factors that contribute to long-range guidance of these cells. In addition, we find that taiman and Pvf1 regulate the dynamic localization of E-cadherin in the border cells, possibly accounting for the interaction between these two pathways.     

The P2Y6 receptor signals through Gαq/Ca2+/PKCα and Gα13/ROCK pathways to drive the formation of membrane protrusions and dictate cell migration

Cell migration is a ubiquitous process necessary to maintain and restore tissue functions. However, in cancer, cell migration leads to metastasis development and thus worsens the prognosis. Although the mechanism of cell migration is well understood, the identification of new targets modulating cell migration and deciphering their signaling events could lead to new therapies to restore tissue functions in diseases, such as inflammatory bowel disease, or to block metastatic development in different forms of cancer. Previous research has identified the G-protein-coupled P2Y₆ receptor as an innovative target that could dictate cell migration under normal and pathological conditions. Surprisingly, there is little information on the cellular events triggered by activated P2Y₆ during cell migration. Here, we demonstrated that P2Y₆ activation stimulated A549 human lung cancer cells and Caco-2 colorectal cancer cell migration. Activated P2Y₆ increased the number of filopodia and focal adhesions; two migratory structures required for cell migration. The generation of these structures involved Gα_q/calcium/protein kinases C (PKC) and Gα₁₃/RHO-associated protein kinase-dependent pathways that dictate the formation of the migratory structures. These pathways led to the stabilization of the actin cytoskeleton through a PKC-dependent phosphorylation of cofilin. These results support the idea that the P2Y₆ receptor represents a target of interest to modulate cell migration and revealed an intricate dialogue between two Gα-protein signaling pathways.     

Protein-protein interactions, cytoskeletal regulation and neuronal migration

Neuronal migration, like the migration of many cell types, requires an extensive rearrangement of cell shape, mediated by changes in the cytoskeleton. The genetic analysis of human brain malformations has identified several biochemical players in this process, including doublecortin (DCX) and LIS1, mutations of which cause a profound migratory disturbance known as lissencephaly ('smooth brain') in humans. Studies in mice have identified additional molecules such as Cdk5, P35, Reelin, Disabled and members of the LDL superfamily of receptors. Understanding the cell biology of these molecules has been a challenge, and it is not known whether they function in related biochemical pathways or in very distinct processes. The last year has seen rapid advances in the biochemical analysis of several such molecules. This analysis has revealed roles for some of these molecules in cytoskeletal regulation and has shown an unexpected conservation of the genetic pathways that regulate neuronal migration in humans and nuclear movement in simple eukaryotic organisms.     

Cell migration in invertebrates: clues from border and distal tip cells.

Recent studies in two invertebrate systems, border cells in Drosophila melanogaster and distal tip cells in Caenorhabditis elegans, have provided important insight into the mechanisms of directed cell migration. These migrating cells are guided by extracellular signals, such as EGF, TGF-beta and netrin. In addition, metalloproteases alter the extracellular matrix of the tissue through which these cells migrate. Along the migratory path, migrating cells respond to changes in guidance signals by altering the expression of receptor signaling pathways. Finally, Dock180, CrkII and the GTPase Rac link the extracellular signals to the cellular machinery that controls cell motility.     

The COUP-TF nuclear receptors regulate cell migration in the mammalian basal forebrain

Cells migrate via diverse pathways and in different modes to reach their final destinations during development. Tangential migration has been shown to contribute significantly to the generation of neuronal diversity in the mammalian telencephalon. GABAergic interneurons are the best-characterized neurons that migrate tangentially, from the ventral telencephalon, dorsally into the cortex. However, the molecular mechanisms and nature of these migratory pathways are only just beginning to be unravelled. In this study we have first identified a novel dorsal-to-ventral migratory route, in which cells migrate from the interganglionic sulcus, located in the basal telencephalon between the lateral and medial ganglionic eminences, towards the pre-optic area and anterior hypothalamus in the diencephalon. Next, with the help of transplantations and gain-of-function studies in organotypic cultures, we have shown that COUP-TFI and COUP-TFII are expressed in distinct and non-overlapping migratory routes. Ectopic expression of COUP-TFs induces an increased rate of cell migration and cell dispersal, suggesting roles in cellular adhesion and migration processes. Moreover, cells follow a distinct migratory path, dorsal versus ventral, which is dependent on the expression of COUP-TFI or COUP-TFII, suggesting an intrinsic role of COUP-TFs in guiding migrating neurons towards their target regions. Therefore, we propose that COUP-TFs are directly involved in tangential cell migration in the developing brain, through the regulation of short- and long-range guidance cues.     

Genetic screens for genes controlling motor nerve-muscle development and interactions

Motor growth cones navigate long and complex trajectories to connect with their muscle targets. Experimental studies have shown that this guidance process critically depends on extrinsic cues. In the zebrafish embryo, a subset of mesodermal cells, the adaxial cells, delineates the prospective path of pioneering motor growth cones. Genetic ablation of adaxial cells causes profound pathfinding defects, suggesting the existence of adaxial cell derived guidance factors. Intriguingly, adaxial cells are themselves migratory, and as growth cones approach they migrate away from the prospective axonal path to the lateral surface of the myotome, where they develop into slow-twitching muscle fibers. Genetic screens in embryos stained with an antibody cocktail identified mutants with specific defects in differentiation and migration of adaxial cells/slow muscle fibers, as well as mutants with specific defects in axonal pathfinding, including exit from the spinal cord and pathway selection. Together, the genes underlying these mutant phenotypes define pathways essential for nerve and muscle development and interactions between these two cell types.     

Vascular endothelial growth factor (VEGF) regulates cranial neural crest migration in vivo

The neural crest is an excellent model to study embryonic cell migration, since cell behaviors can be studied in vivo with advanced optical imaging and molecular intervention. What is unclear is how molecular signals direct neural crest cell (NCC) migration through multiple microenvironments and into specific targets. Here, we tested the hypothesis that the invasion of cranial NCCs, specifically the rhombomere 4 (r4) migratory stream into branchial arch 2 (ba2), is due to chemoattraction through neuropilin-1-vascular endothelial growth factor (VEGF) interactions. We found that the spatio-temporal expression pattern of VEGF in the ectoderm correlated with the NCC migratory front. RT-PCR analysis of the r4 migratory stream showed that ba2 tissue expressed VEGF and r4 NCCs expressed VEGF receptor 2. When soluble VEGF receptor 1 (sVEGFR1) was injected distal to the r4 migratory front, to bind up endogenous VEGF, NCCs failed to completely invade ba2. Time-lapse imaging revealed that cranial NCCs were attracted to ba2 tissue or VEGF sources in vitro. VEGF-soaked beads or VEGF-expressing cells placed adjacent to the r4 migratory stream caused NCCs to divert from stereotypical pathways and move towards an ectopic VEGF source. Our results suggest a model in which NCC entry and invasion of ba2 is dependent on chemoattractive signaling through neuropilin-1-VEGF interactions.     

Patterns of fibronectin gene expression and splicing during cell migration in chicken embryos

A variety of evidence suggests that fibronectin (FN) promotes cell migration during embryogenesis, and it has been suggested that the deposition of FN along migratory pathways may also play a role in cell guidance. In order to investigate such a role for FN, it is important to determine the relative contribution of migrating and pathway-forming cells to the FN in the migratory track, as any synthesis of FN by the migrating cells might be expected to mask guidance cues provided by the exogenous FN from pathway-forming cells. We have therefore used in situ hybridization to determine in developing chicken embryos the distribution and alternative splicing of FN mRNA during three different cell migrations known to occur through FN-rich environments; neural crest cell migration, mesenchymal cell migration in the area vasculosa and endocardial cushion cell migration in the heart. Our results show that trunk neural crest cells do not contain significant FN mRNA during their initial migration. In contrast, migrating mesenchymal cells of the area vasculosa and endocardial cushion cells both contain abundant FN mRNA. Furthermore, the FN mRNA in these migrating mesenchymal and endocardial cells appears to be spliced in a manner identical with that present in the cells adjacent to their pathways. This in vivo evidence for FN synthesis by migrating and pathway cells argues against a generalized role for exogenously produced FN as a guidance mechanism for cell migration.     

Tousled-like kinase regulates cytokine-mediated communication between cooperating cell types during collective border cell migration

Collective cell migration is emerging as a major contributor to normal development and disease. Collective movement of border cells in the Drosophila ovary requires cooperation between two distinct cell types: four to six migratory cells surrounding two immotile cells called polar cells. Polar cells secrete a cytokine, Unpaired (Upd), which activates JAK/STAT signaling in neighboring cells, stimulating their motility. Without Upd, migration fails, causing sterility. Ectopic Upd expression is sufficient to stimulate motility in otherwise immobile cells. Thus regulation of Upd is key. Here we report a limited RNAi screen for nuclear proteins required for border cell migration, which revealed that the gene encoding Tousled-like kinase (Tlk) is required in polar cells for Upd expression without affecting polar cell fate. In the absence of Tlk, fewer border cells are recruited and motility is impaired, similar to inhibition of JAK/STAT signaling. We further show that Tlk in polar cells is required for JAK/STAT activation in border cells. Genetic interactions further confirmed Tlk as a new regulator of Upd/JAK/STAT signaling. These findings shed light on the molecular mechanisms regulating the cooperation of motile and nonmotile cells during collective invasion, a phenomenon that may also drive metastatic cancer.     

FGF signals for cell proliferation and migration through different pathways

FGFs are pleiotropic growth factors that control cell proliferation, migration and differentiation. However, FGF transduction studies have so far focused primarily on the mitogenic effect of this growth factor family and it has been difficult to assess if the described intracellular signaling pathways are dedicated solely to cell proliferation, or whether they are equally important for the migratory activity often seen in responsive cells. We review here papers in which the migratory effects of this growth factor family were clearly discriminated from proliferative effects. In toto, these studies suggest that cells use different signaling pathways for migration, such as Src and p38 MAP kinase, from those for proliferation, which tend to upregulate the ERKs. Which signaling pathway a cell uses for proliferation or migration appears to depend on many factors, including the structure and the quantity of available FGF trapped in the basal lamina by heparan sulfate co-factors, the disposition of cognate high affinity receptors and the general environment of the cell. Thus the density of the cell population, the state of the cell cycle, the presence of other factors or receptors will modulate the migratory response of cells to FGF.     

Tumor cell locomotion: differential dynamics of spontaneous and induced migration in a 3D collagen matrix

Although great strides have recently been made in elucidating the factors initiating tumor cell migration and the relevant cellular pathways involved, the constituent components of migratory dynamics for individual tumor cell motion have still not been resolved. Utilizing a three-dimensional (3D) collagen assay and computer-assisted, continuous single cell tracking, we investigated the basic parameters for both the spontaneous and norepinephrine-induced migration of highly metastatic MBA-MB-468 breast, PC-3 prostate, and SW 480 colon carcinoma cells. We show that tumor cells do not migrate with uniform migrational structure and speed as previously thought, but rather, the induction of locomotion elicits significant increases in speed, break frequency, and total cell displacement, but decreases in break length and no change in the recruitment of nonlocomotory cells. We furthermore illustrate the corresponding morphological changes of induced tumor cell migration with emphasis on motion in a collagen matrix. These results demonstrate the complexity of tumor cell migration, and the compulsion for incorporating not only knowledge of intracellular pathways, but also fundamental parameters of migratory behavior into any expansive theory of tumor cell migration and metastasis formation. We furthermore establish the analytical methodology of investigating both the stimulation and potential pharmaceutical inhibition of tumor cell migration.     

Requirement for Par-6 and Bazooka in Drosophila border cell migration

Polarized epithelial cells convert into migratory invasive cells during a number of developmental processes, as well as when tumors metastasize. Much has been learned recently concerning the molecules and mechanisms that are responsible for generating and maintaining epithelial cell polarity. However, less is known about what becomes of epithelial polarity proteins when various cell types become migratory and invasive. Here, we report the localization of several apical epithelial proteins, Par-6, Par-3/Bazooka and aPKC, during border cell migration in the Drosophila ovary. All of these proteins remained asymmetrically distributed throughout migration. Moreover, depletion of either Par-6 or Par-3/Bazooka by RNAi resulted in disorganization of the border cell cluster and impaired migration. The distributions of several transmembrane proteins required for migration were abnormal following Par-6 or Par-3/Bazooka downregulation, possibly accounting for the migration defects. Taken together, these results indicate that cells need not lose apical/basal polarity in order to invade neighboring tissues and in some cases even require such polarity for proper motility.     

Cell migration in Drosophila.

Recent genetic studies in Drosophila have identified signals that direct cell movement, mechanisms that transduce such signals within migrating cells and some of the molecular machinery underlying cell motility. Activation of the fibroblast growth factor receptor signaling pathway is required for migration of the cells of the developing respiratory system and mesoderm. A signal dependent on 3-hydroxy-3-methylglutanyl Coenzyme A reductase attracts migrating primordial germ cells to the somatic gonad, whereas the phosphohydrolase, phosphatidic acid phosphatase type 2, repels germ cells. In the female germline, the migratory path of border cells is directed by the homophilic adhesion molecule E cadherin.