Vg1RBP phosphorylation by Erk2 MAP kinase correlates with the cortical release of Vg1 mRNA during meiotic maturation of Xenopus oocytes

Xenopus Vg1RBP is a member of the highly conserved IMP family of four KH-domain RNA binding proteins, with roles in RNA localization, translational control, RNA stability, and cell motility. Vg1RBP has been implicated in localizing Vg1 mRNAs to the vegetal cortex during oogenesis, in a process mediated by microtubules and microfilaments, and in migration of neural crest cells in embryos. Using c-mos morpholino, kinase inhibitors, and constitutely active recombinant kinases we show that Vg1RBP undergoes regulated phosphorylation by Erk2 MAPK during meiotic maturation, on a single residue, S402, located between the KH2 and KH3 domains. Phosphorylation temporally correlates with the release of Vg1 mRNA from its tight cortical association, assayed in lysates in physiological salt buffers, but does not affect RNA binding, nor self-association of Vg1RBP. U0126, a MAP kinase inhibitor, prevents Vg1RBP cortical release and Vg1 mRNA solubilization in meiotically maturing eggs, while injection of MKK6-DD, a constitutively activated MAP kinase kinase, promotes the release of both Vg1RBP and Vg1 mRNA from insoluble cortical structures. We propose that Erk2 MAP kinase phosphorylation of Vg1RBP regulates the protein:protein-mediated association of Vg1 mRNP with the cytoskeleton and/or ER. Since the MAP kinase site in Vg1RBP is conserved in several IMP homologs, this modification also has important implications for the regulation of IMP proteins in somatic cells.     

A Novel Role for VICKZ Proteins in Maintaining Epithelial Integrity during Embryogenesis

Background

VICKZ (IGF2BP1,2,3/ZBP1/Vg1RBP/IMP1,2,3) proteins bind RNA and help regulate many RNA-mediated processes. In the midbrain region of early chick embryos, VICKZ is expressed in the neural folds and along the basal surface of the neural epithelium, but, upon neural tube closure, is down-regulated in prospective cranial neural crest (CNC) cells, concomitant with their emigration and epithelial-to-mesenchymal transition (EMT). Electroporation of constructs that modulate cVICKZ expression demonstrates that this down-regulation is both necessary and sufficient for CNC EMT. These results suggest that VICKZ down-regulation in CNC cell-autonomously promotes EMT and migration. Reduction of VICKZ throughout the embryo, however, inhibits CNC migration non-cell-autonomously, as judged by transplantation experiments in Xenopus embryos.

Results and Conclusions

Given the positive role reported for VICKZ proteins in promoting cell migration of chick embryo fibroblasts and many types of cancer cells, we have begun to look for specific mRNAs that could mediate context-specific differences. We report here that the laminin receptor, integrin alpha 6, is down-regulated in the dorsal neural tube when CNC cells emigrate, this process is mediated by cVICKZ, and integrin alpha 6 mRNA is found in VICKZ ribonucleoprotein complexes. Significantly, prolonged inhibition of cVICKZ in either the neural tube or the nascent dermomyotome sheet, which also dynamically expresses cVICKZ, induces disruption of these epithelia. These data point to a previously unreported role for VICKZ in maintaining epithelial integrity.     

The KH domains of Xenopus Vg1RBP mediate RNA binding and self-association

Xenopus Vg1 mRNA is localized to the vegetal cortex during oogenesis in a process involving microtubules and microfilaments and proteins that specifically recognize the vegetal localization element (VLE) within the 3' untranslated region. One of the best characterized VLE-binding proteins is Vg1RBP or Vera. Primary sequence analysis of Vg1RBP and its homologs suggests that most of its open reading frame is occupied by RNA-binding modules, including two RRMs and four KH domains, arranged as three pairs of didomains. In the first detailed domain analysis of Vg1RBP, we show that the interaction of Vg1RBP with the VLE requires both KH didomains, but not the RRM didomain, and moreover that the KH didomains contribute cooperatively to RNA binding. In the full-length protein, individual KH domains display significant redundancy, and their relative importance appears to vary with the RNA target. We also demonstrate that the KH34 didomain mediates Vg1RBP self-association, which is stabilized by RNA, and occurs in vivo as well as in vitro. Altogether, our findings highlight the importance of multiple KH domains in mediating RNA-protein and protein-protein interactions in the formation of a stable complex of Vg1RBP and Vg1 mRNA.     

Cytoplasmic protein methylation is essential for neural crest migration

As they initiate migration in vertebrate embryos, neural crest cells are enriched for methylation cycle enzymes, including S-adenosylhomocysteine hydrolase (SAHH), the only known enzyme to hydrolyze the feedback inhibitor of trans-methylation reactions. The importance of methylation in neural crest migration is unknown. Here, we show that SAHH is required for emigration of polarized neural crest cells, indicating that methylation is essential for neural crest migration. Although nuclear histone methylation regulates neural crest gene expression, SAHH and lysine-methylated proteins are abundant in the cytoplasm of migratory neural crest cells. Proteomic profiling of cytoplasmic, lysine-methylated proteins from migratory neural crest cells identified 182 proteins, several of which are cytoskeleton related. A methylation-resistant form of one of these proteins, the actin-binding protein elongation factor 1 alpha 1 (EF1α1), blocks neural crest migration. Altogether, these data reveal a novel and essential role for post-translational nonhistone protein methylation during neural crest migration and define a previously unknown requirement for EF1α1 methylation in migration.     

VICKZ proteins mediate cell migration via their RNA binding activity

The highly conserved, RNA binding VICKZ proteins help regulate RNA localization, stability, and translation in many eukaryotes. These proteins are also required for cell migration in embryos and cultured cells. In adults, many tumors overexpress VICKZ homologs, and it has been hypothesized that the proteins can mediate cell motility and invasion. How these proteins facilitate cell movement and, in particular, whether their ability to bind RNA plays a role in their function remain unclear. Using HPLC and mass spectrometry to identify a region of Xenopus Vg1 RBP (xVICKZ3) that binds the vegetal localization element of Vg1 RNA, we generated a deletion construct that functions in a dominant-negative manner. The construct associates with full-length xVICKZ3 and severely reduces binding to target RNAs. This dominant-negative construct phenocopies the effect of down-regulating xVICKZ3 in Xenopus embryos. A corresponding deletion in the human homolog hVICKZ1 similarly functions in a dominant-negative fashion to reduce the ability of full-length hVICKZ protein to bind RNA. Expression of the dominant-negative construct in human carcinoma cells inhibits cell movement by several criteria. We conclude that the ability of VICKZ proteins to mediate cell migration, in vitro and in vivo, requires their RNA binding activity.     

The involvement of a conserved family of RNA binding proteins in embryonic development and carcinogenesis

Vg1 RBP is a member of a family of highly conserved proteins that appear to be involved in RNA localization, stability, and/or translational control in a wide variety of cell types and organisms. Over the last few years, the human homologs of these proteins have been found to be overexpressed in an increasing number of different kinds of cancers. Although the role of these proteins in neoplasia is not understood, results from several labs, including our own, are beginning to suggest that many of these proteins may be important in cell motility, a necessary requirement for metastasis. This paper will review these data and suggest a model for the role of Vg1 RBP and its homologs in embryonic development and carcinogenesis.     

Vg1 RNA localization in oocytes in the absence of xVICKZ3 RNA-binding activity

Vg 1 RNA becomes localized at the vegetal cortex of Xenopus oocytes in a process requiring both intact microtubules (MT) and microfilaments. This localization occurs during a narrow window of oogenesis, when a number of RNA-binding proteins associate with the RNA. xVICKZ3 (Vg1 RBP/Vera), the first Vg1 RNA-binding protein identified, helps mediate the association of Vg1 RNA with MT and is co-localized with the RNA at the vegetal cortex. Given the complexity of the Vg1 RNA ribonucleoprotein (RNP) complex, it has remained unclear how xVICKZ3 functions in Vg1 RNA localization. Here, we have taken a closer look at the process of xVICKZ3 localization in oocytes. We have made use of deletion constructs to perform a structure-function analysis of xVICKZ3. The ability of xVICKZ3-GFP constructs to vegetally localize correlates with their association to MT but not with Vg1 RNA-binding ability. We find that when the ability of xVICKZ3 to bind Vg1 RNA is inhibited by the injection of a construct that dominantly inhibits RNA binding, both the construct and Vg1 RNA still localize, apparently through their continued association with a Vg1 RNA-containing RNP complex. These results emphasize the importance of protein-protein interactions in both xVICKZ3 and Vg1 RNA localization.     

A novel role for MuSK and non-canonical Wnt signaling during segmental neural crest cell migration

Trunk neural crest cells delaminate from the dorsal neural tube as an uninterrupted sheet; however, they convert into segmentally organized streams before migrating through the somitic territory. These neural crest cell streams join the segmental trajectories of pathfinding spinal motor axons, suggesting that interactions between these two cell types might be important for neural crest cell migration. Here, we show that in the zebrafish embryo migration of both neural crest cells and motor axons is temporally synchronized and spatially restricted to the center of the somite, but that motor axons are dispensable for segmental neural crest cell migration. Instead, we find that muscle-specific receptor kinase (MuSK) and its putative ligand Wnt11r are crucial for restricting neural crest cell migration to the center of each somite. Moreover, we find that blocking planar cell polarity (PCP) signaling in somitic muscle cells also results in non-segmental neural crest cell migration. Using an F-actin biosensor we show that in the absence of MuSK neural crest cells fail to retract non-productive leading edges, resulting in non-segmental migration. Finally, we show that MuSK knockout mice display similar neural crest cell migration defects, suggesting a novel, evolutionarily conserved role for MuSK in neural crest migration. We propose that a Wnt11r-MuSK dependent, PCP-like pathway restricts neural crest cells to their segmental path.     

Vg1 RNA binding protein mediates the association of Vg1 RNA with microtubules in Xenopus oocytes.

Localized RNAs are found in a variety of somatic and developing cell types. In many cases, microtubules have been implicated as playing a role in facilitating transport of these RNAs. Here we report that Vg1 RNA, which is localized to the vegetal cortex of Xenopus laevis oocytes, is associated with microtubules in vivo. Because of the ubiquitous nature of tubulin, the association of specific RNAs with microtubules is likely to involve factors that recognize both RNA and microtubules. Vg1 RNA binding protein (Vg1 RBP), previously shown to bind with high affinity to the vegetal localization site in Vg1 RNA, appears to function in this capacity. Vg1 RBP is associated with microtubules: it is enriched in microtubule extracts of oocytes and is also co-precipitated by heterologous, polymerized tubulin. Furthermore, Vg1 RBP binding activity is required for the specific association of Vg1 RNA to microtubules in vitro. These data suggest a general model for how specific RNAs can be localized to particular sites via common cytoskeletal elements.     

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.     

A PTK7/Ror2 Co-Receptor Complex Affects Xenopus Neural Crest Migration

Neural crest cells are a highly migratory pluripotent cell population that generates a wide array of different cell types and failure in their migration can result in severe birth defects and malformation syndromes. Neural crest migration is controlled by various means including chemotaxis, repellent guidance cues and cell-cell interaction. Non-canonical Wnt PCP (planar cell polarity) signaling has previously been shown to control cell-contact mediated neural crest cell guidance. PTK7 (protein tyrosine kinase 7) is a transmembrane pseudokinase and a known regulator of Wnt/PCP signaling, which is expressed in Xenopus neural crest cells and required for their migration. PTK7 functions as a Wnt co-receptor; however, it remains unclear by which means PTK7 affects neural crest migration. Expressing fluorescently labeled proteins in Xenopus neural crest cells we find that PTK7 co-localizes with the Ror2 Wnt-receptor. Further, co-immunoprecipitation experiments demonstrate that PTK7 interacts with Ror2. The PTK7/Ror2 interaction is likely relevant for neural crest migration, because Ror2 expression can rescue the PTK7 loss of function migration defect. Live cell imaging of explanted neural crest cells shows that PTK7 loss of function affects the formation of cell protrusions as well as cell motility. Co-expression of Ror2 can rescue these defects. In vivo analysis demonstrates that a kinase dead Ror2 mutant cannot rescue PTK7 loss of function. Thus, our data suggest that Ror2 can substitute for PTK7 and that the signaling function of its kinase domain is required for this effect.     

Extracellular matrix and embryonal morphogenesis: role of fibronectin in cell migration

The neural crest provides a useful paradigm for cell migration and modulations in cell adhesion during morphogenesis. In the present review, we describe the major findings on the role of the extracellular matrix glycoprotein fibronectin and its corresponding integrin receptor in the locomotory behavior of neural crest cells. In vivo, fibronectin is associated with the migratory routes of neural crest cells and, in some cases, it disappears from the environment of the cells as they stop migrating. In vitro, neural crest cells show a great preference for fibronectin substrates as compared to other matrix molecules. Both in vivo and in vitro, neural crest cell migration can be specifically inhibited by antibodies or peptides that interfere with the binding of fibronectin to its integrin receptor. However, the migratory behavior of neural crest cells cannot result solely from the interaction with fibronectin. Thus, neural crest cells exhibit a particular organization of integrin receptors on their surface and develop a cytoskeletal network which differs from that of non-motile cells. These properties are supposed to permit rapid changes in the shape of cells and to favor a transient adhesion to the substratum. Recent findings have established that different forms of fibronectin may occur, which differ by short sequences along the molecule. The functions of most of these sequences are not known, except for 1 of them which carries a binding site for integrin receptors. We have demonstrated that this site is recognized by neural crest cells and plays a crucial role in their displacement. It is therefore possible that the forms of fibronectin carrying this sequence are not evenly distributed in the embryo, thus allowing migrating neural crest cells to orientate in the embryo. Fibronectin would then not only play a permissive role in embryonic cell motility, but have an instructive function in cell behavior.     

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.     

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.     

PTB/hnRNP I is required for RNP remodeling during RNA localization in Xenopus oocytes

Transport of specific mRNAs to defined regions within the cell cytoplasm is a fundamental mechanism for regulating cell and developmental polarity. In the Xenopus oocyte, Vg1 RNA is transported to the vegetal cytoplasm, where localized expression of the encoded protein is critical for embryonic polarity. The Vg1 localization pathway is directed by interactions between key motifs within Vg1 RNA and protein factors recognizing those RNA sequences. We have investigated how RNA-protein interactions could be modulated to trigger distinct steps in the localization pathway and found that the Vg1 RNP is remodeled during cytoplasmic RNA transport. Our results implicate two RNA-binding proteins with key roles in Vg1 RNA localization, PTB/hnRNP I and Vg1RBP/vera, in this process. We show that PTB/hnRNP I is required for remodeling of the interaction between Vg1 RNA and Vg1RBP/vera. Critically, mutations that block this remodeling event also eliminate vegetal localization of the RNA, suggesting that RNP remodeling is required for localization.     

Alterations in neural crest migration by a monoclonal antibody that affects cell adhesion

The possible role of a 140-kD cell surface complex in neural crest adhesion and migration was examined using a monoclonal antibody JG22, first described by Greve and Gottlieb (1982, J. Cell. Biochem. 18:221-229). The addition of JG22 to neural crest cells in vitro caused a rapid change in morphology of cells plated on either fibronectin or laminin substrates. The cells became round and phase bright, often detaching from the dish or forming aggregates of rounded cells. Other tissues such as somites, notochords, and neural tubes were unaffected by the antibody in vitro even though the JG22 antigen is detectable in embryonic tissue sections on the surface of the myotome, neural tube, and notochord. The effects of the JG22 on neural crest migration in vivo were examined by a new perturbation approach in which both the antibody and the hybridoma cells were microinjected onto neural crest pathways. Hybridoma cells were labeled with a fluorescent cell marker that is nondeleterious and that is preserved after fixation and tissue sectioning. The JG22 antibody and hybridoma cells caused a marked reduction in cranial neural crest migration, a build-up of neural crest cells within the lumen of the neural tube, and some migration along aberrant pathways. Neural crest migration in the trunk was affected to a much lesser extent. In both cranial and trunk regions, a cell free zone of one or more cell diameters was generally observed between neural crest cells and the JG22 hybridoma cells. Two other monoclonal antibodies, 1-B and 1-N, were used as controls. Both 1-B and 1-N bind to bands of the 140-kD complex precipitated by JG22. Neither control antibody affected neural crest adhesion in vitro or neural crest migration in situ. This suggests that the observed alterations in neural crest migration are due to a functional block of the 140-kD complex.     

Annexin A6 Modulates Chick Cranial Neural Crest Cell Emigration

The vertebrate neural crest is a population of migratory cells that originates in the dorsal aspect of the embryonic neural tube. These cells undergo an epithelial-to-mesencyhmal transition (EMT), delaminate from the neural tube and migrate extensively to generate an array of differentiated cell types. Elucidating the gene regulatory networks involved in neural crest cell induction, migration and differentiation are thus crucial to understanding vertebrate development. To this end, we have identified Annexin A6 as an important regulator of chick midbrain neural crest cell emigration. Annexin proteins comprise a family of calcium-dependent, membrane-binding molecules that mediate a variety of cellular and physiological processes including cell adhesion, migration and invasion. Our data indicate that Annexin A6 is expressed in the proper spatio-temporal pattern in the chick midbrain to play a potential role in neural crest cell ontogeny. To investigate Annexin A6 function, we have depleted or overexpressed Annexin A6 in the developing midbrain neural crest cell population. Our results show that knock-down or overexpression of Annexin A6 reduces or expands the migratory neural crest cell domain, respectively. Importantly, this phenotype is not due to any change in cell proliferation or cell death but can be correlated with changes in the size of the premigratory neural crest cell population and with markers associated with EMT. Taken together, our data indicate that Annexin A6 plays a pivotal role in modulating the formation of cranial migratory neural crest cells during vertebrate development.     

Cell surface beta 1,4-galactosyltransferase functions during neural crest cell migration and neurulation in vivo

Mesenchymal cell migration and neurite outgrowth are mediated in part by binding of cell surface beta 1,4-galactosyltransferase (GalTase) to N-linked oligosaccharides within the E8 domain of laminin. In this study, we determined whether cell surface GalTase functions during neural crest cell migration and neural development in vivo using antibodies raised against affinity-purified chicken serum GalTase. The antibodies specifically recognized two embryonic proteins of 77 and 67 kD, both of which express GalTase activity. The antibodies also immunoprecipitated and inhibited chick embryo GalTase activity, and inhibited neural crest cell migration on laminin matrices in vitro. Anti-GalTase antibodies were microinjected into the head mesenchyme of stage 7-9 chick embryos or cranial to Henson's node of stage 6 embryos. Anti-avian GalTase IgG decreased cranial neural crest cell migration on the injected side but did not cross the embryonic midline and did not affect neural crest cell migration on the uninjected side. Anti-avian GalTase Fab crossed the embryonic midline and perturbed cranial neural crest cell migration throughout the head. Neural fold elevation and neural tube closure were also disrupted by Fab fragments. Cell surface GalTase was localized to migrating neural crest cells and to the basal surfaces of neural epithelia by indirect immunofluorescence, whereas GalTase was undetectable on neural crest cells prior to migration. These results suggest that, during early embryogenesis, cell surface GalTase participates during neural crest cell migration, perhaps by interacting with laminin, a major component of the basal lamina. Cell surface GalTase also appears to play a role in neural tube formation, possibly by mediating neural epithelial adhesion to the underlying basal lamina.