Cytoneme-mediated transport of active Wnt5b-Ror2 complexes in zebrafish.

Chemical signalling is the primary means by which cells communicate in the embryo. The underlying principle refers to a group of ligand-producing cells and a group of cells that respond to this signal because they express the appropriate receptors1,2. In the zebrafish embryo, Wnt5b binds to the receptor Ror2 to trigger the Wnt-planar cell polarity (PCP) signalling pathway to regulate tissue polarity and cell migration3,4. However, it remains unclear how this lipophilic ligand is transported from the source cells through the aqueous extracellular space to the target tissue. In this study, we provide evidence that Wnt5b, together with Ror2, is loaded on long protrusions called cytonemes. Our data further suggest that the active Wnt5b-Ror2 complexes form in the producing cell and are handed over from these cytonemes to the receiving cell. Then, the receiving cell has the capacity to initiate Wnt-PCP signalling, irrespective of its functional Ror2 receptor status. On the tissue level, we further show that cytoneme-dependent spreading of active Wnt5b-Ror2 affects convergence and extension in the zebrafish gastrula. We suggest that cytoneme-mediated transfer of ligand-receptor complexes is a vital mechanism for paracrine signalling. This may prompt a reevaluation of the conventional concept of characterizing responsive and non-responsive tissues solely on the basis of the expression of receptors.

Cancer-associated fibroblasts influence Wnt/PCP signaling in gastric cancer cells by cytoneme-based dissemination of ROR2.

Cancer-associated fibroblasts (CAFs) are a crucial component in the tumor microenvironment influencing cancer progression. Besides shaping the extracellular matrix, these fibroblasts provide signaling factors to facilitate tumor survival and alter tumor behavior. In gastric cancer, one crucial signaling pathway influencing invasion and metastasis is the Wnt/Planar Cell Polarity (PCP) signaling. The crucial PCP ligand in this context is WNT5A, which is produced by the CAFs, and gastric cancer cells react upon this signal by enhanced polarized migration. Why gastric cancer cells respond to this signal is still unclear, as their expression level for the central WNT5A receptor, ROR2, is very low. Here, we show that CAFs display long and branched filopodia that form an extensive, complex network engulfing gastric cancer cells, such as the gastric cancer cell line AGS. CAFs have a significantly higher expression level of ROR2 than normal gastric fibroblasts and AGS cells. By high-resolution imaging, we observe a direct transfer of fluorescently tagged ROR2 from CAF to AGS cells by signaling filopodia, known as cytonemes. Surprisingly, we find that the transferred ROR2 complexes can activate Wnt/JNK signaling in AGS cells. Consistently, blockage of ROR2 function in the CAFs leads to reduced paracrine Wnt/JNK signaling, cell polarization, and migration of the receiving AGS cells. Complementary, enhanced migration via paracrine ROR2 transfer was observed in a zebrafish in vivo model. These findings demonstrate a fresh role for cytoneme-mediated signaling in the tumor microenvironment. Cytonemes convey Wnt receptors from CAFs to gastric cancer cells, allowing them to respond to Wnt/PCP signals.

Vertebrate Wnt5a - At the crossroads of cellular signalling.

Wnt signalling is an essential pathway in embryogenesis, differentiation, cell motility, development, and adult tissue homeostasis in vertebrates. The Wnt signalling network can activate several downstream pathways such as the ß-catenin-dependent TCF/LEF transcription, the Wnt/planar cell polarity (PCP) pathway, and the Wnt/Calcium pathway. Wnt5a is a vertebrate Wnt ligand that is most often associated with the Wnt/PCP signalling pathway. Wnt5a/PCP signalling has a well-described role in embryogenesis via binding to a receptor complex of Frizzled and its co-receptors to initiate downstream activation of the c-Jun N-terminal kinase (JNK) signalling cascade and the Rho and Rac GTPases, Rho-Kinase (ROCK). This activation results in the cytoskeletal remodelling required for cell polarity, migration, and subsequently, tissue re-arrangement and organ formation. This review will focus on more recent work that has revealed new roles for Wnt5a ligands and consequently, an emerging broader function. This is partly due to our growing understanding of the crosstalk between the Wnt/PCP pathway with both the Wnt/ß-catenin pathway and other signalling pathways, and in part due to the identification of novel atypical receptors for Wnt5a that demonstrate a far broader role for this ligand.

Mechanisms of intercellular Wnt transport.

Wnt proteins are secreted glycoproteins that regulate multiple processes crucial to the development and tissue homeostasis of multicellular organisms, including tissue patterning, proliferation, cell fate specification, cell polarity and migration. To elicit these effects, Wnts act as autocrine as well as paracrine signalling molecules between Wnt-producing and Wnt-receiving cells. More than 40 years after the discovery of the Wg/Wnt pathway, it is still unclear how they are transported to fulfil their paracrine signalling functions. Several mechanisms have been proposed to mediate intercellular Wnt transport, including Wnt-binding proteins, lipoproteins, exosomes and cytonemes. In this Review, we describe the evidence for each proposed mechanism, and discuss how they may contribute to Wnt dispersal in tissue-specific and context-dependent manners, to regulate embryonic development precisely and maintain the internal steady state within a defined tissue.

Development of the electric organ in embryos and larvae of the knifefish, Brachyhypopomus gauderio.

South American Gymnotiform knifefish possess electric organs that generate electric fields for electro-location and electro-communication. Electric organs in fish can be derived from either myogenic cells (myogenic electric organ/mEO) or neurogenic cells (neurogenic electric organ/nEO). To date, the embryonic development of EOs has remained obscure. Here we characterize the development of the mEO in the Gymnotiform bluntnose knifefish, Brachyhypopomus gauderio. We find that EO primordial cells arise during embryonic stages in the ventral edge of the tail myotome, translocate into the ventral fin and develop into syncytial electrocytes at early larval stages. We also describe a pair of thick nerve cords that flank the dorsal aorta, the location and characteristic morphology of which are reminiscent of the nEO in Apteronotid species, suggesting a common evolutionary origin of these tissues. Taken together, our findings reveal the embryonic origins of the mEO and provide a basis for elucidating the mechanisms of evolutionary diversification of electric charge generation by myogenic and neurogenic EOs.

Modeling of Wnt-mediated tissue patterning in vertebrate embryogenesis.

During embryogenesis, morphogens form a concentration gradient in responsive tissue, which is then translated into a spatial cellular pattern. The mechanisms by which morphogens spread through a tissue to establish such a morphogenetic field remain elusive. Here, we investigate by mutually complementary simulations and in vivo experiments how Wnt morphogen transport by cytonemes differs from typically assumed diffusion-based transport for patterning of highly dynamic tissue such as the neural plate in zebrafish. Stochasticity strongly influences fate acquisition at the single cell level and results in fluctuating boundaries between pattern regions. Stable patterning can be achieved by sorting through concentration dependent cell migration and apoptosis, independent of the morphogen transport mechanism. We show that Wnt transport by cytonemes achieves distinct Wnt thresholds for the brain primordia earlier compared with diffusion-based transport. We conclude that a cytoneme-mediated morphogen transport together with directed cell sorting is a potentially favored mechanism to establish morphogen gradients in rapidly expanding developmental systems.

Studying molecular interactions in the intact organism: fluorescence correlation spectroscopy in the living zebrafish embryo.

Cell behaviour and function is determined through the interactions of a multitude of molecules working in concert. To observe these molecular dynamics, biophysical studies have been developed that track single interactions. Fluorescence correlation spectroscopy (FCS) is an optical biophysical technique that non-invasively resolves single molecules through recording the signal intensity at the femtolitre scale. However, recording the behaviour of these biomolecules using in vitro-based assays often fails to recapitulate the full range of variables in vivo that directly confer dynamics. Therefore, there has been an increasing interest in observing the state of these biomolecules within living organisms such as the zebrafish Danio rerio. In this review, we explore the advancements of FCS within the zebrafish and compare and contrast these findings to those found in vitro.

Pcdh18a regulates endocytosis of E-cadherin during axial mesoderm development in zebrafish.

The notochord defines the axial structure of all vertebrates during development. Notogenesis is a result of major cell reorganization in the mesoderm, the convergence and the extension of the axial cells. However, it is currently not fully understood how these processes act together in a coordinated way during notochord formation. The prechordal plate is an actively migrating cell population in the central mesoderm anterior to the trailing notochordal plate cells. We show that prechordal plate cells express Protocadherin 18a (Pcdh18a), a member of the cadherin superfamily. We find that Pcdh18a-mediated recycling of E-cadherin adhesion complexes transforms prechordal plate cells into a cohesive and fast migrating cell group. In turn, the prechordal plate cells subsequently instruct the trailing mesoderm. We simulated cell migration during early mesoderm formation using a lattice-based mathematical framework and predicted that the requirement for an anterior, local motile cell cluster could guide the intercalation and extension of the posterior, axial cells. Indeed, a grafting experiment validated the prediction and local Pcdh18a expression induced an ectopic prechordal plate-like cell group migrating towards the animal pole. Our findings indicate that the Pcdh18a is important for prechordal plate formation, which influences the trailing mesodermal cell sheet by orchestrating the morphogenesis of the notochord.

Delay-driven oscillations via Axin2 feedback in the Wnt/ß-catenin signalling pathway.

The Wnt signalling pathway plays an important role in development, disease, and normal tissue function. Mathematical models for Wnt signalling have predominantly focused on quantitatively predicting changes in steady-state ß-catenin concentrations (the main downstream protein regulated by canonical Wnt signalling). One of the genes targeted for expression by Wnt/ß-catenin signalling is the negative Wnt regulator Axin2. Recently, a number of authors have indicated a potential theoretical role of Axin2 feedback to induce oscillatory behaviour in the pathway and this has been observed in a number of detailed mathematical models. Due to the complexity of these models, the investigations to date have been limited to numerical experiments and parameter sensitivity analyses. In this manuscript, we study the fundamental structure of the dynamical system underlying the Wnt signalling mechanism with Axin2 feedback to gain some insight into why and when oscillations occur in models with this structure. We semi-rigorously analyse three simple models and, for these models, gain deep understanding of the characteristic set of conditions that are necessary and sufficient for oscillations to be induced. We discuss the possible biological consequences of these findings for Wnt signalling pathway oscillations. They include; to promote oscillations (1) Keeping all other parameters constant, the Wnt signal strength should neither be too high or too low but within a single finite window of values, (2) Wnt receptor complexes should fully deactivate Axin rather than temporarily deconstruct it from other scaffold proteins, (3) In the absence of stochastic effects or more complicated mechanisms, a critical delay in Axin2 feedback in the system is necessary, (4) Deactivation of Axin by the Wnt receptor complex needs to be critically efficient relative to ß-catenin removal by Axin, and (5) conditions necessary are less strict if Axin2 feedback occurs after a fixed time rather than a Poisson-distributed time with the same average.

The function of endocytosis in Wnt signaling.

Wnt growth factors regulate one of the most important signaling networks during development, tissue homeostasis and disease. Despite the biological importance of Wnt signaling, the mechanism of endocytosis during this process is ill described. Wnt molecules can act as paracrine signals, which are secreted from the producing cells and transported through neighboring tissue to activate signaling in target cells. Endocytosis of the ligand is important at several stages of action: One central function of endocytic trafficking in the Wnt pathway occurs in the source cell. Furthermore, the ß-catenin-dependent Wnt ligands require endocytosis for signal activation and to regulate gene transcription in the responding cells. Alternatively, Wnt/ß-catenin-independent signaling regulates endocytosis of cell adherence plaques to control cell migration. In this comparative review, we elucidate these three fundamental interconnected functions, which together regulate cellular fate and cellular behavior. Based on established hypotheses and recent findings, we develop a revised picture for the complex function of endocytosis in the Wnt signaling network.

From top to bottom: Cell polarity in Hedgehog and Wnt trafficking.

Spatial organization of membrane domains within cells and cells within tissues is key to the development of organisms and the maintenance of adult tissue. Cell polarization is crucial for correct cell-cell signalling, which, in turn, promotes cell differentiation and tissue patterning. However, the mechanisms linking internal cell polarity to intercellular signalling are just beginning to be unravelled. The Hedgehog (Hh) and Wnt pathways are major directors of development and their malfunction can cause severe disorders like cancer. Here we discuss parallel advances into understanding the mechanism of Hedgehog and Wnt signal dissemination and reception. We hypothesize that cell polarization of the signal-sending and signal-receiving cells is crucial for proper signal spreading and activation of the pathway and, thus, fundamental for development of multicellular organisms.

Role of cytonemes in Wnt transport.

Wnt signaling regulates a broad variety of processes during embryonic development and disease. A hallmark of the Wnt signaling pathway is the formation of concentration gradients by Wnt proteins across responsive tissues, which determines cell fate in invertebrates and vertebrates. To fulfill its paracrine function, trafficking of the Wnt morphogen from an origin cell to a recipient cell must be tightly regulated. A variety of models have been proposed to explain the extracellular transport of these lipid-modified signaling proteins in the aqueous extracellular space; however, there is still considerable debate with regard to which mechanisms allow the precise distribution of ligand in order to generate a morphogenetic gradient within growing tissue. Recent evidence suggests that Wnt proteins are distributed along signaling filopodia during vertebrate and invertebrate embryogenesis. Cytoneme-mediated transport has profound impact on our understanding of how Wnt signaling propagates through tissues and allows the formation of a precise ligand distribution in the recipient tissue during embryonic growth. In this Commentary, we review extracellular trafficking mechanisms for Wnt proteins and discuss the growing evidence of cytoneme-based Wnt distribution in development and stem cell biology. We will also discuss their implication for Wnt signaling in the formation of the Wnt morphogenetic gradient during tissue patterning.

Emerging role of contact-mediated cell communication in tissue development and diseases.

Cells of multicellular organisms are in continuous conversation with the neighbouring cells. The sender cells signal the receiver cells to influence their behaviour in transport, metabolism, motility, division, and growth. How cells communicate with each other can be categorized by biochemical signalling processes, which can be characterised by the distance between the sender cell and the receiver cell. Existing classifications describe autocrine signals as those where the sender cell is identical to the receiver cell. Complementary to this scenario, paracrine signalling describes signalling between a sender cell and a different receiver cell. Finally, juxtacrine signalling describes the exchange of information between adjacent cells by direct cell contact, whereas endocrine signalling describes the exchange of information, e.g., by hormones between distant cells or even organs through the bloodstream. In the last two decades, however, an unexpected communication mechanism has been identified which uses cell protrusions to exchange chemical signals by direct contact over long distances. These signalling protrusions can deliver signals in both ways, from sender to receiver and vice versa. We are starting to understand the morphology and function of these signalling protrusions in many tissues and this accumulation of findings forces us to revise our view of contact-dependent cell communication. In this review, we will focus on the two main categories of signalling protrusions, cytonemes and tunnelling nanotubes. These signalling protrusions emerge as essential structural components of a vibrant communication network in the development and tissue homeostasis of any multicellular organism.

Secreted Frizzled-related Protein 2 (sFRP2) Redirects Non-canonical Wnt Signaling from Fz7 to Ror2 during Vertebrate Gastrulation.

Convergent extension movements during vertebrate gastrulation require a balanced activity of non-canonical Wnt signaling pathways, but the factors regulating this interplay on the molecular level are poorly characterized. Here we show that sFRP2, a member of the secreted frizzled-related protein (sFRP) family, is required for morphogenesis and papc expression during Xenopus gastrulation. We further provide evidence that sFRP2 redirects non-canonical Wnt signaling from Frizzled 7 (Fz7) to the receptor tyrosine kinase-like orphan receptor 2 (Ror2). During this process, sFRP2 promotes Ror2 signal transduction by stabilizing Wnt5a-Ror2 complexes at the membrane, whereas it inhibits Fz7 signaling, probably by blocking Fz7 receptor endocytosis. The cysteine-rich domain of sFRP2 is sufficient for Ror2 activation, and related sFRPs can substitute for this function. Notably, direct interaction of the two receptors via their cysteine-rich domains also promotes Ror2-mediated papc expression but inhibits Fz7 signaling. We propose that sFRPs can act as a molecular switch, channeling the signal input for different non-canonical Wnt pathways during vertebrate gastrulation.

In vivo analysis of formation and endocytosis of the Wnt/ß-catenin signaling complex in zebrafish embryos.

After activation by Wnt/ß-Catenin ligands, a multi-protein complex assembles at the plasma membrane as membrane-bound receptors and intracellular signal transducers are clustered into the so-called Lrp6-signalosome [Corrected]. However, the mechanism of signalosome formation and dissolution is yet not clear. Our imaging studies of live zebrafish embryos show that the signalosome is a highly dynamic structure. It is continuously assembled by Dvl2-mediated recruitment of the transducer complex to the activated receptors and partially disassembled by endocytosis. We find that, after internalization, the ligand-receptor complex and the transducer complex take separate routes. The Wnt-Fz-Lrp6 complex follows a Rab-positive endocytic path. However, when still bound to the transducer complex, Dvl2 forms intracellular aggregates. We show that this endocytic process is not only essential for ligand-receptor internalization but also for signaling. The µ2-subunit of the endocytic Clathrin adaptor Ap2 interacts with Dvl2 to maintain its stability during endocytosis. Blockage of Ap2µ2 function leads to Dvl2 degradation, inhibiton of signalosome formation at the plasma membrane and, consequently, reduction of signaling. We conclude that Ap2µ2-mediated endocytosis is important to maintain Wnt/ß-catenin signaling in vertebrates.

Tyrosine phosphorylation of LRP6 by Src and Fer inhibits Wnt/ß-catenin signalling.

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) function as transmembrane receptors to transduce Wnt signals. A key mechanism for signalling is Wnt-induced serine/threonine phosphorylation at conserved PPPSPxS motifs in the LRP6 cytoplasmic domain, which promotes pathway activation. Conserved tyrosine residues are positioned close to all PPPSPxS motifs, which suggests they have a functional significance. Using a cell culture-based cDNA expression screen, we identified the non-receptor tyrosine kinases Src and Fer as novel LRP6 modifiers. Both Src and Fer associate with LRP6 and phosphorylate LRP6 directly. In contrast to the known PPPSPxS Ser/Thr kinases, tyrosine phosphorylation by Src and Fer negatively regulates LRP6-Wnt signalling. Epistatically, they function upstream of ß-catenin to inhibit signalling and in agreement with a negative role in regulating LRP6, MEF cells lacking these kinases show enhanced Wnt signalling. Wnt3a treatment of cells enhances tyrosine phosphorylation of endogenous LRP6 and, mechanistically, Src reduces cell surface LRP6 levels and disrupts LRP6 signalosome formation. Interestingly, CK1γ inhibits Fer-induced LRP6 phosphorylation, suggesting a mechanism whereby CK1γ acts to de-represses inhibitory LRP6 tyrosine phosphorylation. We propose that LRP6 tyrosine phosphorylation by Src and Fer serves a negative regulatory function to prevent over-activation of Wnt signalling at the level of the Wnt receptor, LRP6.

Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules.

It is widely accepted that tissue differentiation and morphogenesis in multicellular organisms are regulated by tightly controlled concentration gradients of morphogens. How exactly these gradients are formed, however, remains unclear. Here we show that Fgf8 morphogen gradients in living zebrafish embryos are established and maintained by two essential factors: fast, free diffusion of single molecules away from the source through extracellular space, and a sink function of the receiving cells, regulated by receptor-mediated endocytosis. Evidence is provided by directly examining single molecules of Fgf8 in living tissue by fluorescence correlation spectroscopy, quantifying their local mobility and concentration with high precision. By changing the degree of uptake of Fgf8 into its target cells, we are able to alter the shape of the Fgf8 gradient. Our results demonstrate that a freely diffusing morphogen can set up concentration gradients in a complex multicellular tissue by a simple source-sink mechanism.

Pax6 regulates the formation of the habenular nuclei by controlling the temporospatial expression of Shh in the diencephalon in vertebrates.

BACKGROUND: The habenula and the thalamus are two critical nodes in the forebrain circuitry and they connect the midbrain and the cerebral cortex in vertebrates. The habenula is derived from the epithalamus and rests dorsally to the thalamus. Both epithalamus and thalamus arise from a single diencephalon segment called prosomere (p)2. Shh is expressed in the ventral midline of the neural tube and in the mid-diencephalic organizer (MDO) at the zona limitans intrathalamica between thalamus and prethalamus. Acting as a morphogen, Shh plays an important role in regulating cell proliferation and survival in the diencephalon and thalamic patterning. The molecular regulation of the MDO Shh expression and the potential role of Shh in development of the habenula remain largely unclear. RESULTS: We show that deleting paired-box and homeobox-containing gene Pax6 results in precocious and expanded expression of Shh in the prospective MDO in fish and mice, whereas gain-of-function of pax6 inhibits MDO shh expression in fish. Using gene expression and genetic fate mapping, we have characterized the expression of molecular markers that demarcate the progenitors and precursors of habenular neurons. We show that the thalamic domain is shifted dorsally and the epithalamus is missing in the alar plate of p2 in the Pax6 mutant mouse. Conversely, the epithalamus is expanded ventrally at the expense of the thalamus in mouse embryos with reduced Shh activity. Significantly, attenuating Shh signaling largely rescues the patterning of p2 and restores the epithalamus in Pax6 mouse mutants, suggesting that Shh acts downstream of Pax6 in controlling the formation of the habenula. Similar to that found in the mouse, we show that pax6 controls the formation of the epithalamus mostly via the regulation of MDO shh expression in zebrafish. CONCLUSIONS: Our findings demonstrate that Pax6 has an evolutionarily conserved function in establishing the temporospatial expression of Shh in the MDO in vertebrates. Furthermore, Shh mediates Pax6 function in regulating the partition of the p2 domain into the epithalamus and thalamus.

Lhx2 and Lhx9 determine neuronal differentiation and compartition in the caudal forebrain by regulating Wnt signaling.

Initial axial patterning of the neural tube into forebrain, midbrain, and hindbrain primordia occurs during gastrulation. After this patterning phase, further diversification within the brain is thought to proceed largely independently in the different primordia. However, mechanisms that maintain the demarcation of brain subdivisions at later stages are poorly understood. In the alar plate of the caudal forebrain there are two principal units, the thalamus and the pretectum, each of which is a developmental compartment. Here we show that proper neuronal differentiation of the thalamus requires Lhx2 and Lhx9 function. In Lhx2/Lhx9-deficient zebrafish embryos the differentiation process is blocked and the dorsally adjacent Wnt positive epithalamus expands into the thalamus. This leads to an upregulation of Wnt signaling in the caudal forebrain. Lack of Lhx2/Lhx9 function as well as increased Wnt signaling alter the expression of the thalamus specific cell adhesion factor pcdh10b and lead subsequently to a striking anterior-posterior disorganization of the caudal forebrain. We therefore suggest that after initial neural tube patterning, neurogenesis within a brain compartment influences the integrity of the neuronal progenitor pool and border formation of a neuromeric compartment.