Wnt/planar cell polarity signaling controls morphogenetic movements of gastrulation and neural tube closure.

Gastrulation and neurulation are successive morphogenetic processes that play key roles in shaping the basic embryonic body plan. Importantly, they operate through common cellular and molecular mechanisms to set up the three spatially organized germ layers and to close the neural tube. During gastrulation and neurulation, convergent extension movements driven by cell intercalation and oriented cell division generate major forces to narrow the germ layers along the mediolateral axis and elongate the embryo in the anteroposterior direction. Apical constriction also makes an important contribution to promote the formation of the blastopore and the bending of the neural plate. Planar cell polarity proteins are major regulators of asymmetric cell behaviors and critically involved in a wide variety of developmental processes, from gastrulation and neurulation to organogenesis. Mutations of planar cell polarity genes can lead to general defects in the morphogenesis of different organs and the co-existence of distinct congenital diseases, such as spina bifida, hearing deficits, kidney diseases, and limb elongation defects. This review outlines our current understanding of non-canonical Wnt signaling, commonly known as Wnt/planar cell polarity signaling, in regulating morphogenetic movements of gastrulation and neural tube closure during development and disease. It also attempts to identify unanswered questions that deserve further investigations.

Emerging Roles of RNA-Binding Proteins in Inner Ear Hair Cell Development and Regeneration.

RNA-binding proteins (RBPs) regulate gene expression at the post-transcriptional level. They play major roles in the tissue- and stage-specific expression of protein isoforms as well as in the maintenance of protein homeostasis. The inner ear is a bi-functional organ, with the cochlea and the vestibular system required for hearing and for maintaining balance, respectively. It is relatively well documented that transcription factors and signaling pathways are critically involved in the formation of inner ear structures and in the development of hair cells. Accumulating evidence highlights emerging functions of RBPs in the post-transcriptional regulation of inner ear development and hair cell function. Importantly, mutations of splicing factors of the RBP family and defective alternative splicing, which result in inappropriate expression of protein isoforms, lead to deafness in both animal models and humans. Because RBPs are critical regulators of cell proliferation and differentiation, they present the potential to promote hair cell regeneration following noise- or ototoxin-induced damage through mitotic and non-mitotic mechanisms. Therefore, deciphering RBP-regulated events during inner ear development and hair cell regeneration can help define therapeutic strategies for treatment of hearing loss. In this review, we outline our evolving understanding of the implications of RBPs in hair cell formation and hearing disease with the aim of promoting future research in this field.

RBM24 in the Post-Transcriptional Regulation of Cancer Progression: Anti-Tumor or Pro-Tumor Activity?

RNA-binding proteins are critical post-transcriptional regulators of gene expression. They are implicated in a wide range of physiological and pathological processes by modulating nearly every aspect of RNA metabolisms. Alterations in their expression and function disrupt tissue homeostasis and lead to the occurrence of various cancers. RBM24 is a highly conserved protein that binds to a large spectrum of target mRNAs and regulates many post-transcriptional events ranging from pre-mRNA splicing to mRNA stability, polyadenylation and translation. Studies using different animal models indicate that it plays an essential role in promoting cellular differentiation during organogenesis and tissue regeneration. Evidence is also accumulating that its dysregulation frequently occurs across human cancers. In several tissues, RBM24 clearly functions as a tumor suppressor, which is consistent with its inhibitory potential on cell proliferation. However, upregulation of RBM24 in other cancers appears to promote tumor growth. There is a possibility that RBM24 displays both anti-tumor and pro-tumor activities, which may be regulated in part through differential interactions with its protein partners and by its post-translational modifications. This makes it a potential biomarker for diagnosis and prognosis, as well as a therapeutic target for cancer treatment. The challenge remains to determine the post-transcriptional mechanisms by which RBM24 modulates gene expression and tumor progression in a context- or background-dependent manner. This review discusses recent findings on the potential function of RBM24 in tumorigenesis and provides future directions for better understanding its regulatory role in cancer cells.

Rbm24 displays dynamic functions required for myogenic differentiation during muscle regeneration.

Skeletal muscle has a remarkable capacity of regeneration after injury, but the regulatory network underlying this repair process remains elusive. RNA-binding proteins play key roles in the post-transcriptional regulation of gene expression and the maintenance of tissue homeostasis and plasticity. Rbm24 regulates myogenic differentiation during early development, but its implication in adult muscle is poorly understood. Here we show that it exerts multiple functions in muscle regeneration. Consistent with its dynamic subcellular localization during embryonic muscle development, Rbm24 also displays cytoplasm to nucleus translocation during C2C12 myoblast differentiation. In adult mice, Rbm24 mRNA is enriched in slow-twitch muscles along with myogenin mRNA. The protein displays nuclear localization in both slow and fast myofibers. Upon injury, Rbm24 is rapidly upregulated in regenerating myofibers and accumulates in the myonucleus of nascent myofibers. Through satellite cell transplantation, we demonstrate that Rbm24 functions sequentially to regulate myogenic differentiation and muscle regeneration. It is required for myogenin expression at early stages of muscle injury and for muscle-specific pre-mRNA alternative splicing at late stages of regeneration. These results identify Rbm24 as a multifaceted regulator of myoblast differentiation. They provide insights into the molecular pathway orchestrating the expression of myogenic factors and muscle functional proteins during regeneration.

Collagen triple helix repeat containing 1a (Cthrc1a) regulates cell adhesion and migration during gastrulation in zebrafish.

Cell adhesion and migration are key cell behaviours during gastrulation in early embryos and metastasis in cancers. Cthrc1 is a secreted protein highly conserved among vertebrates; it is upregulated in injured and diseased arteries, as well as in malignant cancers. There is increasing evidence showing that its expression and activity are associated with cancer progression and inflammatory diseases. However, the mechanism by which it regulates cell migration, and its implication during early development remains unclear. Here we show that zebrafish Cthrc1a is expressed in hypoblast cells, and is required for cell adhesion and migration during gastrulation. Knockdown of cthrc1a in whole embryo inhibits epiboly and convergent extension movements, and reduces the elongation of anteroposterior axis. Cell adhesion assay indicates that Cthrc1a is necessary for mesendoderm cells to interact with fibronectin-coated substratum, and to extend polarised cellular protrusions. Moreover, secreted Cthrc1a proteins diffuse efficiently between blastoderm cells and are recruited by neighbouring cells in an integrin-dependent manner. Consistently, there exists a functional interaction between Cthrc1a and integrin ß1 in anteroposterior axis elongation. These results provide insight into the function of Cthrc1a in the regulation of cell adhesion and migration during embryonic axis elongation.

Transplantation of Zebrafish Cells by Conventional Pneumatic Microinjector.

Generating chimeric zebrafish by transplantation is extremely useful for live imaging in developmental, stem cell, and cancer biology, and to answer the questions of how cells acquire, keep, and/or change their fate. However, as it is technically challenging, the use of transplantation approach remains very limited by the zebrafish community. In this study, we show that this cell grafting operation can be easily achieved by using a conventional pneumatic microinjector normally used for microinjections. Compared with previously published protocols, which need additional transplantation apparatus, this alternative transplantation method works well, but needs a simpler experimental setup, and is more accessible to all investigators.

Identification of novel MYO18A interaction partners required for myoblast adhesion and muscle integrity.

The unconventional myosin MYO18A that contains a PDZ domain is required for muscle integrity during zebrafish development. However, the mechanism by which it functions in myofibers is not clear. The presence of a PDZ domain suggests that MYO18A may interact with other partners to perform muscle-specific functions. Here we performed double-hybrid screening and co-immunoprecipitation to identify MYO18A-interacting proteins, and have identified p190RhoGEF and Golgin45 as novel partners for the MYO18A PDZ domain. We have also identified Lurap1, which was previously shown to bind MYO18A. Functional analyses indicate that, similarly as myo18a, knockdown of lurap1, p190RhoGEF and Golgin45 by morpholino oligonucleotides disrupts dystrophin localization at the sarcolemma and produces muscle lesions. Simultaneous knockdown of myo18a with either of these genes severely disrupts myofiber integrity and dystrophin localization, suggesting that they may function similarly to maintain myofiber integrity. We further show that MYO18A and its interaction partners are required for adhesion of myoblasts to extracellular matrix, and for the formation of the Golgi apparatus and organization of F-actin bundles in myoblast cells. These findings suggest that MYO18A has the potential to form a multiprotein complex that links the Golgi apparatus to F-actin, which regulates muscle integrity and function during early development.

Identification of transmembrane protein 88 (TMEM88) as a dishevelled-binding protein.

Wnt signaling pathways are involved in embryonic development and adult tissue maintenance and have been implicated in tumorigenesis. Dishevelled (Dvl/Dsh) protein is one of key components in Wnt signaling and plays essential roles in regulating these pathways through protein-protein interactions. Identifying and characterizing Dvl-binding proteins are key steps toward understanding biological functions. Given that the tripeptide VWV (Val-Trp-Val) binds to the PDZ domain of Dvl, we searched publically available databases to identify proteins containing the VWV motif at the C terminus that could be novel Dvl-binding partners. On the basis of the cellular localization and expression patterns of the candidates, we selected for further study the TMEM88 (target protein transmembrane 88), a two-transmembrane-type protein. The interaction between the PDZ domain of Dvl and the C-terminal tail of TMEM88 was confirmed by using NMR and fluorescence spectroscopy. Furthermore, in HEK293 cells, TMEM88 attenuated the Wnt/ß-catenin signaling induced by Wnt-1 ligand in a dose-dependent manner, and TMEM88 knockdown by RNAi increased Wnt activity. In Xenopus, TMEM88 protein is sublocalized at the cell membrane and inhibits Wnt signaling induced by Xdsh but not ß-catenin. In addition, TMEM88 protein inhibits the formation of a secondary axis normally induced by Xdsh. The findings suggest that TMEM88 plays a role in regulating Wnt signaling. Indeed, analysis of microarray data revealed that the expression of the Tmem88 gene was strongly correlated with that of Wnt signaling-related genes in embryonic mouse intestines. Together, we propose that TMEM88 associates with Dvl proteins and regulates Wnt signaling in a context-dependent manner.

Discovery and characterization of a small molecule inhibitor of the PDZ domain of dishevelled.

Dishevelled (Dvl) is an essential protein in the Wnt signaling pathways; it uses its PDZ domain to transduce the Wnt signals from the membrane receptor Frizzled to downstream components. Here, we report identifying a drug-like small molecule compound through structure-based ligand screening and NMR spectroscopy and show the compound to interact at low micromolar affinity with the PDZ domain of Dvl. In a Xenopus testing system, the compound could permeate the cell membrane and block the Wnt signaling pathways. In addition, the compound inhibited Wnt signaling and reduced the levels of apoptosis in the hyaloid vessels of eye. Moreover, this compound also suppressed the growth of prostate cancer PC-3 cells. These biological effects suggest that by blocking the PDZ domain of Dvl, the compound identified in our studies effectively inhibits the Wnt signaling and thus provides a useful tool for studies dissecting the Wnt signaling pathways.

Identification of a specific inhibitor of the dishevelled PDZ domain.

The Wnt signaling pathways are involved in embryo development as well as in tumorigenesis. Dishevelled (Dvl) transduces Wnt signals from the receptor Frizzled (Fz) to downstream components in canonical and noncanonical Wnt signaling pathways. The Dvl PDZ domain is thought to play an essential role in both pathways, and we recently demonstrated that the Dvl PDZ domain binds directly to Fz receptors. In this study, using structure-based virtual ligand screening, we identified an organic molecule (NSC668036) from the National Cancer Institute small-molecule library that can bind to the Dvl PDZ domain. We then used molecular dynamics simulation to analyze the binding between the PDZ domain and NSC668036 in detail. In addition, we showed that, in Xenopus, as expected, NSC668036 inhibited the signaling induced by Wnt3A. This compound provides a basis for rational design of high-affinity inhibitors of the PDZ domain, which can block Wnt signaling by interrupting the Fz-Dvl interaction.

Identification of distinct genes with restricted expression in the somitic mesoderm in Xenopus embryo.

We have used whole-mount in situ hybridisation to identify genes expressed in the somitic mesoderm during Xenopus early development. We report here the analysis of eight genes whose expression pattern has not been described previously. They include the Xenopus homologues of eukaryotic initiation factor 2beta, methionine adenosyltransferase II, serine dehydratase, alpha-adducin, oxoglutarate dehydrogenase, fragile X mental retardation syndrome related protein 1, monocarboxylate transporter and voltage-dependent anion channel 1. Interestingly, these genes exhibit very dynamic expression pattern during early development. At early gastrula stages several genes do not show localised expression pattern, while other genes are expressed in the marginal mesoderm or in ectoderm. As development proceeds, the expression of these genes is gradually restricted to different compartments of somite. This study thus reveals an unexpected dynamic expression pattern for various genes with distinct function in vertebrates.

Murine Frizzled-1 behaves as an antagonist of the canonical Wnt/beta-catenin signaling.

Activation of the Wnt signaling cascade provides key signals during development and in disease. Wnt signals are transduced by seven-transmembrane Frizzleds (Fzs) and the single transmembrane low density lipoprotein receptor-related proteins 5 or 6. In the course of the analysis of genes regulated by bone morphogenetic protein 2 in mesenchymal cells we found a significant induction of murine Frizzled-1 (mFz1) gene expression. Unexpectedly overexpression of mFz1 dramatically repressed the induction of alkaline phosphatase mediated by either bone morphogenetic protein 2 or Wnt3a in these cells. Moreover mFz1 overexpression significantly repressed both beta-catenin translocation into the nucleus and T cell factor signaling mediated by Wnt3a. Importantly microinjection of mFz1 transcript in Xenopus embryo inhibited the ability of Wnt1 to induce the expression of the Wnt/beta-catenin target gene Siamois in animal cap assay and secondary axis formation in whole embryo. By using chimeric constructs in which N- and C-terminal segments of mFz1 were replaced by the corresponding parts of Xfz3 we demonstrated that the antagonistic activity resides in the cysteine-rich domain of the N-terminal part. The antagonist activity of mFz1 could be prevented by overexpression of Galphaq-(305-359), which specifically uncouples Gq-coupled receptors, suggesting that Galphaq signaling contributes to the inhibition of Wnt/beta-catenin pathway by mFz1. This is the first time that a Frizzled receptor has been reported to antagonize Wnt/beta-catenin.

Direct binding of the PDZ domain of Dishevelled to a conserved internal sequence in the C-terminal region of Frizzled.

The cytoplasmic protein Dishevelled (Dvl) and the associated membrane-bound receptor Frizzled (Fz) are essential in canonical and noncanonical Wnt signaling pathways. However, the molecular mechanisms underlying this signaling are not well understood. By using NMR spectroscopy, we determined that an internal sequence of Fz binds to the conventional peptide binding site in the PDZ domain of Dvl; this type of site typically binds to C-terminal binding motifs. The C-terminal region of the Dvl inhibitor Dapper (Dpr) and Frodo bound to the same site. In Xenopus, Dvl binding peptides of Fz and Dpr/Frodo inhibited canonical Wnt signaling and blocked Wnt-induced secondary axis formation in a dose-dependent manner, but did not block noncanonical Wnt signaling mediated by the DEP domain. Together, our results identify a missing molecular connection within the Wnt pathway. Differences in the binding affinity of the Dvl PDZ domain and its binding partners may be important in regulating signal transduction by Dvl.

Frizzled receptor dimerization is sufficient to activate the Wnt/beta-catenin pathway.

Wnt signaling has an important role in cell-fate determination, tissue patterning and tumorigenesis. Wnt proteins signal through seven-pass transmembrane receptors of the frizzled family to activate beta-catenin-dependent transcription of target genes. Using early Xenopus embryos, we show that frizzled receptors can dimerize and that dimerization is correlated with activation of the Wnt/beta-catenin pathway. Co-immunoprecipitation studies revealed that the receptor Xfz3 exists as a dimer when expressed in Xenopus embryos, and it has been shown to activate the Wnt/beta-catenin pathway as revealed by expression of the target gene siamois. Xfz3 dimerization requires intramolecular and/or intermolecular disulfide linkages, and the N-terminal extracellular region of the receptor, including the cysteine-rich domain (CRD), is sufficient for dimerization. The receptor Xfz7 behaves differently from Xfz3 when overexpressed in the embryo as Xfz7 is monomeric and is unable to directly activate the Wnt/beta-catenin pathway. However, activation of this pathway can be achieved by artificially forcing Xfz7 dimerization. These results provide the first direct evidence for the dimerization of frizzled receptors and suggest that dimerization contributes to transducing the Wnt/beta-catenin signal.

Zygotic Wnt/beta-catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus.

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/beta-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/beta-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/beta-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/beta-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/beta-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.